Standard Operating Procedures

and Regulatory Guidelines

BLOOD BANKING
Standard Operating Procedures
and Regulatory Guidelines
BLOOD BANKING

GP Saluja MBBS MD
Senior Consultant, Blood Bank
Alchemist Hospitals Ltd
Panchkula, Haryana, India

GL Singal M Pharm PhD LLB

State Drugs Controller
Department of Food and Drugs Administration
Panchkula, Haryana, India

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Standard Operating Procedures and Regulatory Guidelines–Blood Banking
First Edition: 2014
ISBN 978-93-5152-215-7
Printed at
 Dedicated to
Our families
and
our dear parents
whose inspiration, motivation, blessings
and moral support continue to contribute
a great deal to our academic endeavors
&
Everybody striving to contribute to
the blood safety
 Foreword

Minister of Health & Medical Education

Government of Haryana
Tel: 0172-2740278; Fax: 0172-2748043

I am extremely happy to know that Dr GP Saluja and Dr GL Singal

have come up with their book “Standard Operating Procedures and
Regulatory Guidelines–Blood Banking” that incorporates the latest
updated procedural guidelines regarding the operation of blood banks.
This book covers all the aspects of blood transfusion including regulatory
guidelines framed under the Drugs and Cosmetics Act, 1940 and Rules,
1945.
This book will serve both the student fraternity and the blood bank
professionals and will go a long way in achieving the accuracy and
proficiency in the various blood bank procedures.
I am also confident that this book will broaden the horizon of all
those working in blood banks and medical fraternity in understanding
the legal issues pertaining to safe blood transfusion.

(Rao Narinder Singh)

 Foreword

Additional Chief Secretary

Health Department
Government of Haryana
Tel: 0172-2711706; Fax: 0172-2771310

Blood transfusion plays an important role in the modern health system of

both developing and developed nations across the globe. These services
are well-organized in the developed countries; while in vastly populated
developing countries, these are yet to be brought at par with those
available in the developed countries. Blood transfusion is an essential
component of health care that underpins the management and care of
millions of patients each year in both emergency and routine situations.
Yet an unknown number of patients lose their lives unnecessarily every
day because they do not have access to safe blood transfusion. Therefore,
it is necessary to ensure the right quality of blood and its components for
safe transfusions.
Blood transfusion is a multi-step and multi-actor’s process and
desired quality results can only be achieved by following standard
operating procedures (SOPs). At every step, any lowering of quality
would reflect adversely on the final product, particularly when the
institutions/individuals do not have the proper knowledge about the
requirements for grant/renewal of license to operate the blood banks,
SOPs, quality and legal understanding of blood transfusions.
I am confident that this book will broaden the horizon of all those
working in blood banking, understanding the legal issues and providing
safe blood transfusion to the needy patients.

(Navraj Sandhu)
 Foreword

Commissioner, Food & Drugs Administration

Government of Haryana
Tel: 0172-2560124; Fax: 0172-2584066

It gives me immense pleasure to know that Dr GP Saluja and Dr GL Singal

have teamed up to write the book “Standard Operating Procedures and
Regulatory Guidelines–Blood Banking” for the blood bank professionals
in providing and using blood components to make sure that the right
blood/blood components is given to the right patient at the right time.
This can only be achieved when procedures for prescribing, ordering,
collecting, storing and administering blood components and clinical
policies are based on the Drugs and Cosmetics Act, 1940 and Rules, 1945
as well as National Blood Policy.
The Act sets legally-binding standards for quality and safety in
collection, testing, processing, storage and distribution of blood/blood
components for which Standard Operating Procedures (SOPs) are
required to be followed for ensuring safe blood transfusion. Besides,
some of the legal judgments included in the book will help those
associated with blood banking in understanding legal situation of the
transfusions.
I am sure that this book will be of immense benefit to one and all
working in this field and help them in achieving their objective of
providing adequate, safe, and quality blood/blood components.

(Dr Rakesh Gupta)

 Preface

This book “Standard Operating Procedures and Regulatory Guidelines–

Blood Banking” has been designed to provide blood bank technicians,
students undergoing various courses in medical technology, blood bank
specialists and residents with a concise and thorough practical and
simple procedural guide to all the procedures to check the quality and
safety of the blood donated for transfusion.
The first section of the book focuses on routine blood bank practices
including donor selection, phlebotomy procedure, sample collection,
component preparation, blood grouping—both routine and gel
technology, screening for the transfusion transmitted infections (TTIs),
compatibility testing, storage of blood/blood components, apheresis,
labeling, issue and transport of blood units, transfusion of the right blood
to the right patient, quality control/assurance, equipment maintenance
and bio-waste disposal.
The second section deals with the regulatory guidelines including
the Drugs and Cosmetics Act, 1940 and Rules, 1945, NACO guidelines
for the safety of blood/blood components and procedural details as well
as various documents required for processing of application for grant of
license under the said Act to operate the blood banks.
Some of the judicial pronouncements related to the blood safety
have also been included so as to create awareness amongst the blood
bank fraternity regarding the various legal issues involved in blood
transfusion.
The book is a culmination of the tremendous efforts of the dedicated
professionals because they care about the blood bank profession, and
it also aims at fostering improved patient care by providing the readers
with a basic understanding of the various blood bank procedures and
legal issues. We express our gratitude to all those associated including
Dr Gautam Wankhede, Director, Medical Affairs, Alliance Transfusion
(Pvt) Ltd, for contributing valuable inputs in compiling this publication.

GP Saluja
GL Singal
 Acknowledgments

We owe a great many thanks to a great many people who helped and
supported us during the writing of this book.
We express our thanks to Dr Baljeet Singh Dahiya, the then Director
General, Health Services, Government of Haryana, India, for the
support and encouragement in the publication of “Standard Operating
Procedures and Regulatory Guidelines” for the Health Department of
Haryana in 2004. We are also thankful to the blood bank professionals,
who had highly appreciated our efforts and encouraged us to write and
bring out this book.
Our thanks are extended to M/s Jaypee Brothers Medical Publishers
(P) Ltd, New Delhi, India, and their dedicated staff for professionally
designing and printing this book.
Our deepest thanks to Sh M Mitra, Former Deputy Drugs Controller,
CDSCO, Government of India, for guiding and providing his valuable
inputs in bringing out this book.
We express our gratitude to Dr Gautam Wankhede, Director, Medical
Affairs, Alliance Transfusion (Pvt) Ltd. for contributing his valuable
inputs in compiling this publication.
We would also thank our institutions for extending their support in
this endeavor.
 Contents

SECTION 1
Standard Operating Procedures
1. Standard Operating Procedure for Preparing, Revising
and Using Standard Operating Procedures (SOPs) 3
Scope and Application 3; Responsibility 3; Staff 3;
Contents of SOPs 6; Use of SOPs 7
2. Criteria for the Donor Selection 8
Scope and Application 8; Responsibility 8; Material
Required 8; Procedure 8; Annexure 2.1 11
3. Donor Screening 14
Scope and Application 14; Responsibility 14; Materials
Required 14; Medical Examination 14
4. Donor Screening for Hemoglobin
(Copper Sulfate Solution Method) 15
Scope and Application 15; Responsibility 15; Materials
Required 15; Procedure 16; Interpretation 16
5. Estimation of Hemoglobin of the Donor (Sahli’s Method) 17
Scope and Application 17; Responsibility 17; Materials
Required 17; Procedure 17; Interpretation 18
6. Estimation of Hemoglobin by Hemo-Control 19
Scope and Application 19; Responsibility 19; Materials
Required 19; Procedure 19; Interpretation 21
7. Preparation of Phlebotomy Site 22
Scope and Application 22; Responsibility 22; Materials
Required 22; Procedure 22
8. Selection of the Blood Bags 24
Scope and Application 24; Responsibility; 24 Materials
Required 24; Procedure 24
9. Venipuncture and Blood Collection 26
Scope and Application 26; Responsibility 26; Materials
Required 26; Procedure 26
10. Post-donation Care 29
Scope and Application 29; Responsibility 29; Materials
Required 29; Procedure 29
11. Management of Adverse Reactions in the Donors 31
Scope and Application 31; Responsibility 31; Materials
Required 31; Management of Adverse Reactions 32
xviii Standard Operating Procedures and Regulatory Guidelines-Blood Banking

12. Traceability of the Blood Units 34

Scope and Application 34; Responsibility 34; Materials
Required 34; Procedure 34
13. Blood Components Separation 36
Scope and Application 36; Responsibility 37; Equipment
and Materials Required 37; Procedure 38
14. Collection of Blood Sample for Grouping/Cross-matching 46
Scope and Application 46; Responsibility 46; Materials
Required 46; Pre-requisites for Blood Sample Collection 46;
Procedure 48; Troubleshooting Guidelines 51; Labeling
and Documentation 53
15. Preparation of Red Cell Suspension 55
Scope and Application 55; Responsibility 55; Materials
Required 55; Procedure 56
16. ABO Blood Grouping 57
Scope and Application 57; Responsibility 57; Equipment
57; Procedure 58; Interpretation 61
17. Resolution of ABO Group Discrepancy 63
Scope and Application 63; Responsibility 63;
Materials Required 63; Procedure 64; Resolving ABO
Discrepancies 65; Annexure 17.1 73; Annexure 17.2 74
18. Absorption and Elution (For Weak Subgroups
of A or B) 75
Scope and Application 75; Responsibility 75; Materials
Required 75; Procedure 76; Interpretation 77
19. Rh Blood Grouping 78
Scope and Application 78; Responsibility 78; Materials
Required 78; Procedure 80; Interpretation of Results 82
20. Rh Du Blood Grouping 83
Scope and Application 83; Responsibility 83; Materials
Required 83; Procedure 84; Interpretation 85
21. Antibody Screening 87
Scope and Application 87; Responsibility 87; Materials
Required 87; Procedure 88; Interpretation 90
22. Pre-transfusion Testing (Compatibility Testing) 91
Scope and Application 91; Responsibility 91; Materials
Required 91; Procedure 92; Interpretation 94
23. Direct Coomb’s Test/Direct Anti-globulin Test (DCT/DAT) 96
Scope and Application 96; Responsibility 96; Materials
Required 96; Procedure 97; Interpretation 98
24. Indirect Coomb’s Test (ICT) 100
Scope and Application 100; Responsibility 100; Materials
Required 100; Procedure 101; Interpretation 101
 Contents xix

25. Saline Addition and Replacement Technique 102

Scope and Application 102; Responsibility 102; Materials
Required 102; Procedure 102; Interpretation 103
26. Alternative Technologies in Blood Banking 104
Equipment 105; Principle 106; Interpretation of Results
107; Precautions to be Taken while Using ID Microtyping
System 108; Disposal of Used ID Cards 109
27. Confirmation of ABO and Rh Blood Grouping of the Donor 111
Scope and Application 111; Responsibility 111; Sample
and Materials Required 111; Procedure 111
28. Reverse Grouping of the Donor 113
Scope and Application 113; Responsibility 113; Sample
and Materials Required 113; Procedure 113
29. Forward and Reverse Grouping of the Patient 115
Scope and Application 115; Responsibility 115;
Sample and Materials Required 115; Procedure 115;
Annexure 29.1 117
30. Testing for Du Weak Antigen 119
Scope and Application 119; Responsibility 119; Sample
and Materials Required 119; Procedure 119
31. Antibody Screening in Coomb’s Phase 121
Scope and Application 121; Responsibility 121;
Sample and Materials Required 121; Procedure 121;
Annexure 31.1 123
32. Antibody Screening in Enzyme Phase 125
Scope and Application 125; Responsibility 125; Sample
and Materials Required 125; Procedure 125
33. Coomb’s Cross-match 127
Scope and Application 127; Responsibility 127; Materials
Required 127; Procedure 127; Minor Cross-match 128;
Interpretation 128; Annexure 33.1 130; Instructions for
Blood Transfusion 131
34. Coomb’s Test (Direct and Indirect) 132
Scope and Application 132; Responsibility 132; Materials
Required 132; Procedure 132; Annexure 34.1 134;
Annexure 34.2 136
35. ABO, Rh Grouping and DAT of Newborn 138
Scope and Application 138; Responsibility 138; Sample
and Materials Required 138; Procedure 138; Method 139;
Annexure 35.1 140
36. Detection of Cold Agglutinins 142
Scope and Application 142; Responsibility 142; Sample
and Materials Required 142; Procedure 142
37. Screening for Transfusion Transmitted Infections (TTIs) 143
xx Standard Operating Procedures and Regulatory Guidelines-Blood Banking

38. Screening for Syphilis (RPR Test) 146

Scope and Application 146; Responsibility 146; Materials
Required 146; Procedure 146; Interpretation 147
39. TPHA (Treponema pallidum Hemagglutination)
Test for Syphilis (Rapid TP Test) 148
Scope and Application 148; Responsibility 148; Materials
Required 148; Procedure 148; Interpretation 149
40. ELISA Test for Syphilis (Syphilis EIA II Total Antibody) 150
Scope and Application 150; Responsibility 150; Materials
Required 150; Procedure 150; Interpretation 152
41. Testing for Hepatitis B Surface Antigen (Rapid Test) 153
Scope and Application 153; Responsibility 153; Materials
Required 153; Procedure 153; Interpretation 154
42. Testing for Hepatitis B Surface Antigen (ELISA Method) 155
Scope and Application 155; Responsibility 155; Materials
Required 155; Procedure 155; Validation and
Interpretation 156
43. Anti-HIV Testing (Rapid TRI-DOT Method) 158
Scope and Application 158; Responsibility 158; Materials
Required 158; Procedure 158; Interpretation 159
44. Anti-HIV Testing (ELISA Method) 160
Scope and Application 160; Responsibility 160; Materials
Required 160; Procedure 160; Calculation of Results 161;
Interpretation of Results 162
45. Testing for HCV Antibodies (Rapid TRI-DOT Method) 163
Scope and Application 163; Responsibility 163; Materials
Required 163; Procedure 163
46. Testing for HCV Antibodies (ELISA Method) 166
Scope and Application 166; Responsibility 166; Materials
Required 166; Procedure 166; Calculation of Results 168;
Interpretation 168
47. Rapid Antigen Test for Malaria (Pan Malaria) 169
Scope and Application 169; Responsibility 169; Materials
Required 169; Procedure 169; Interpretation 170
48. Labeling of the Blood Bags 172
Scope and Application 172; Responsibility 172; Materials
Required 172; Procedure 173
49. Preservation of the Blood and Components 174
Scope and Application 174; Responsibility 174; Materials
Required 174; Procedure 174
50. Inventory of the Blood Units and Components 176
Scope and Application 176; Responsibility 176; Materials
Required 176; Procedure 176
 Contents xxi

51. Issue and Transport of Blood Units 178

Scope and Application 178; Responsibility 178; Materials
Required 178; Procedure 178
52. Return of Whole Blood/Packed Red Cells 181
Scope and Application 181; Responsibility 181; Materials
Required 181; Procedure 181
53. Transfusion of Right Blood to the Right Patient 183
Scope and Application 183; Responsibility 183; Materials
Required 183; Procedural Issues 183; Don’ts for Blood
Transfusion 188; Annexure 53.1 190; Annexure 53.2 192;
Annexure 53.3 194; Annexure 53.4 196
54. Investigation of Transfusion Reactions 198
Scope and Application 198; Responsibility 198;
Materials Required 199; Procedure 200;
Hemovigilance Program 209;
Annexure 54.1 211
55. Quality Control/Assurance of the Blood/Blood Components 213
Scope and Application 213; Responsibility 213; Scheme for
Quality Control/Assurance of Various Blood
Components 214
56. Storage of the Consumables, Reagents and Kits 218
Scope and Application 218; Responsibility 218; Materials
Required 218; Procedure 218
57. Equipment Maintenance 220
Scope and Application 220; Responsibility 220; Procedures
220
58. Quality Control of the Reagents 228
Scope and Application 228; Responsibility 228;
Quality Control of the Reagents 228
59. Assessing Suitability of the Donor for Platelet Apheresis 235
Scope and Application 235; Responsibility 235; Materials
Required 235; Procedure 235; Documentation 236;
Annexure 59.1 237
60. Plateletpheresis Using Blood Cell Separator (Dual Needle) 243
Scope and Application 243; Responsibility 243; Materials
Required 243; Procedure 244
61. Plateletpheresis Using Blood Cell Separator (Single Needle) 261
Scope and Application 261; Responsibility 261; Materials
Required 261; Procedure 262; End of Reinfusion 278
62. Donor Care during and after Apheresis 281
Scope and Application 281; Responsibility 281; Materials
Required 281; General Care during Procedure 282;
Complications 283; Post-donation Care 289
xxii Standard Operating Procedures and Regulatory Guidelines-Blood Banking

63. Bio-medical Waste Management 290

Scope and Application 290; Responsibility 290; Materials
Required 290; Types of Waste Generated, Segregation and
on-site Storage 290
64. The Management of Sharps/Needle Stick Incidents
and Other Exposure Incidents 294
Scope and Application 294; Responsibility 295; Procedure
and Guidelines 295; Consent and Cost (Source Person) 302;
Needle Stick Injury/Sharp Object/Body Fluid Exposure 303;
Training and Prevention 304; Record and Documentation
305; Safe Work Practices 305

SECTION 2
Regulatory Guidelines
65. Regulatory Requirements of Blood and/or
Its Components including Blood Products 311
National Blood Policy 312;
Scenario of Legal Framework 312
66. Drugs and Cosmetics Rules, 1945 (Part X-B) 314
67. Schedule F (Part XII B)Under the Drugs and Cosmetics
Rules, 1945 323
1. Blood Banks/Blood Components 323; A. General 323;
B. Accommodation for a Blood Bank 324; C. Personnel
324; D. Maintenance 325; E. Equipment 326; F. Supplies
and Reagents 327; G. Good Manufacturing Practices/
Standard Operating Procedures (SOPs) 328; H. Criteria for
Blood Donation 330; I. General Equipment and Instruments
332; J. Special Reagents 334; K. Testing of Whole Blood
334; L. Records 335; M. Labels 336;
2. Blood Donation Camps 337; 3. Processing of Blood
Components from Whole Blood by a Blood Bank 339
68. Storage Conditions, Expiry of Blood, Blood Components
and Blood Products as Per Schedule P of the Drugs
and Cosmetics Act, 1940, and Rules, 1945 345
69. Extract of Schedule K Under the Drugs and Cosmetics
Rules, 1945 347
70. Blood Storage Centers 350
Requirements 350; Staff 351; Storage 351; Issue of
Blood/Components 352; Blood Grouping 352; Cell
Grouping 353; Serum Grouping 353; Cross-matching 353;
Guidelines before Grant of Approval for Operation of Whole
Human Blood and/or Its Components Storage Centers Run by
 Contents xxiii

First Referral Unit, Comm­unity Health Center, Primary Health

Center or Any Hospital 355
71. Recent Amendments in the Drugs and Cosmetics
Rules, 1945, and Guidelines 358
I. Amendments 358; Part XII-D of Schedule-F of the Drugs
and Cosmetics Rules, 1945 365; II. Guidelines 375
72. Guidelines for the Preparation of Application for
Grant/Renewal of License to Operate Blood Bank 378
Annexure 72.1 382
Annexure 72.2 383
Annexure 72.3 384
Annexure 72.4 385
Annexure 72.5 386
Annexure 72.6 387
Annexure 72.7 388
Annexure 72.8 389
Annexure 72.9 390
Annexure 72.10 391
Annexure 72.11 392
Annexure 72.12 393
Annexure 72.13 394
Annexure 72.14 395
Annexure 72.15 396
73. Judicial Pronouncements–Blood Safety 410
Glossary 429
Bibliography 433
Index 435
 S E C T I O N 1
Standard Operating
Procedures
 C H A P T E R 1
Standard Operating Procedure
for Preparing, Revising and Using
Standard Operating Procedures
(SOPs)

SCOPE AND APPLICATION

Standard operating procedures are essential documents for ensuring
smooth, efficient and lawful functioning/operation of any blood bank.
These SOPs may be numerous in a particular blood bank defining
their functions but there are some SOPs which remain common for all
the blood banks, e.g. SOP for collection of blood, grouping and cross-
matching of blood and issuing procedures, etc,. These may also include
SOP for policies guiding the rational use of blood and blood components,
time-frame for availability of whole blood and its components and
instructions in case of established transfusion reaction. SOPs are critical
sub-elements of the quality system and are essential to ensure that
every procedure is undertaken in a standardized way for generation
of consistent results. Therefore individual blood bank must develop its
own blood bank specific SOPs based on infrastructure available, test
procedures to be followed and availability of reagents.

RESPONSIBILITY
It is the responsibility of the Management/Medical Officer/Technical
staff working in the blood bank to frame and follow the SOPs to yield the
desired quality results.

STAFF
Medical Officer
Key responsibilities
• Overall supervision of blood bank including administrative work.
4 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Supervision of all the functions like donor selection, phlebotomy,

grouping and cross-matching, TTI testing, storage, component
preparation, labeling, etc.
• Ensure timely indent of reagents, blood bags, gel cards, equipment
and other items required in the blood bank.
• Ensure blood safety measures.
• Ensure proper maintenance of records and reports and timely
submission of reports.
• Ensure proper licensing of the blood bank.
• Ensure timely preparation and submission of reports.
• As member secretary of Hospital Transfusion Committee (HTC) to
conduct the meetings of HTC to monitor the quality of blood and its
components.
• Conducting the proficiency tests for the technical staff.
• Preparation and ensuring use of standard operating procedures.
• Ensure proper handling of biomedical waste.
• Preparation of duty rota for the technician.
The following other categories of whole time competent technical
staff required is:

Blood Bank Technician(s)

Key responsibilities
• Registration and bleeding of donors.
• Preparing components.
• Maintenance of various statutory records and completion of
registers.
• Checking and recording temperature of all refrigerators, deep
freezer, platelet agitator and incubator.
• Preparation of pooled cells.
• Performing red cell serology-ABO and Rh test, Antibody screening,
antihuman globulin test, compatibility testing and testing in case of
adverse reaction of blood transfusion.
• Testing of blood units for HIV, HBsAg, HCV, VDRL and malarial
parasites.
• Issuing blood bags as per request received.
• Discarding of pilot tubes and patient samples after 7 days.
• Disposing of TTI positive bags.
• Maintaining of file of all the requisitions received.
• Carrying out all procedures.
• Maintaining mandatory records.
• Segregation of biomedical waste at the time of its generation.
 Standard Operating Procedure for Preparing, Revising and Using... 5

• Recording and maintaining of temperature of different blood bank

refrigerators.
• Informing technical supervisor immediately in the event of
breakdown of equipment or any discrepancy in results of tests.
• Contact to other blood banks for supply of blood at the time of
emergency.
• Any other work assigned from time to time by the management or
the medical officer in charge of blood bank.

Registered Nurse(s)
Key responsibilities
• Registration and bleeding of donors.
• Assisting in pre-donation medical check up.
• Post-donation care.
• Monitoring the refreshment for the donors.
• Completion of registers.
• Maintaining stocks of emergency medicines.
• Maintaining of file of all the blood donor registration forms.
• Maintaining mandatory records in the department.
• Segregation of biomedical waste at the time of its generation.
• Informing Blood Bank Officer immediately in the event of
breakdown of equipment in the donor room.
• Recording and maintaining of temperature of different blood bank
refrigerators.
• Any other work assigned from time to time.

Technical Supervisor (Where Blood Components are Manufactured)

Key responsibilities
• Supervising and coordinating the blood bank activities.
• Registration and bleeding of donors.
• Preparing components.
• Completion of registers.
• Check and record temperature of all refrigerators, deep freezers,
platelet agitator and incubator.
• Preparation of pooled cells as per SOP.
• Performing red cell serology-ABO and Rh test, antibody screening,
antihuman globulin test, compatibility testing and testing in case of
adverse reaction of blood transfusion.
• Testing of blood bags for HIV, HBsAg, HCV, VDRL and malarial
parasites.
6 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Issuing blood bags as per request received.

• Discarding pilot tubes and patient samples after 7 days.
• Disposing of TTI positive bags.
• Maintaining of file of all the requisitions received.
• Carrying out all procedures.
• Maintaining mandatory records in the department.
• Segregation of biomedical waste at the time of its generation.
• Informing Blood Bank Officer immediately in the event of
breakdown of equipment or any discrepancy in results of tests.
• Contact to other blood banks for supply of blood at the time of
emergency.
• Preparation and timely submission of reports.
• Maintaining inventory of blood bank consumables and stationery.
• Any other work assigned from time to time.

CONTENTS OF SOPs
Technical
The technical contents of SOP should include the following:
• Scope and application.
• Responsibility.
• Materials required to perform the procedure.
• Various steps of procedure to be performed.
• Interpretation of results.
• Quality assurance of reagents/chemicals.
• Documentation required to be maintained.

General
The header of SOP should contain
• Title of SOP.
• SOP number for each SOP.
• Date of issue of SOP.
• Date from which it will be effective.
• Page numbering.
• Revision date and number (The SOPs shall be reviewed/Updated at
least once a year).
The specimen of the header is given below:
 Standard Operating Procedure for Preparing, Revising and Using... 7

Name & Address of Blood SOP No.

Bank: …………………………
Version No./Date of issue
………………………………….
Drug License No: …………. Effective Date
Valid up to ………………….. Review Date
Page No. Page–of–

The footer should contain the signatures of the authorized person(s)

who has prepared it and the person from the management who can
authorize for the use of SOP.
The specimen of the footer is given below:

Prepared & Issued By Approved By

Each of the SOPs needs to be validated. It includes setting up criteria

for acceptance of results generated by SOPs, report of a workshop
formulating a protocol, undertaking testing using SOPs, comparing the
observed results with the predefined criteria and, if matched, declaring
that the SOPs have been validated.

USE OF SOPs
• Once prepared and approved for implementation, SOPs become
the roadmaps for operationalizing the Blood Bank.
• Every staff member must have access to all SOPs that affect actions
and areas of responsibility.
• SOP’s should be followed as approved and maintained by a
particular blood bank and if any amendment is required, it has to
be documented and must be made by authorized persons only after
following proper procedure.
• Any deviation from the SOP must be documented and got approved
from authorized person.
• All SOPs including the outdated ones are to be retained in the blood
bank for the period as per blood bank policy.
• Their use is mandatory by all the staff members of the Blood Bank
every time they perform any activity in the blood bank.
• The licensing and accreditation procedures also demand
compulsory use of SOPs.
• The SOPs are also used for capacity building in the quality
management of blood transfusion services in which emphasis is
laid on ensuring consistency in performing various activities so that
the safety and quality of blood is guaranteed.
 C H A P T E R 2
Criteria for Donor Selection

SCOPE AND APPLICATION

This Standard Operating Procedure (SOP) describes the criteria for a
donor to be accepted or rejected for blood donation, for ensuring safety
of donor as well as recipient. The purpose of donor selection is to identify
the factors that might make an individual unsuitable as a donor, either
temporarily or permanently.

RESPONSIBILITY
The Medical Officer is responsible for determining the suitability of
donor for blood donation. He/she should confirm that the criteria are
fulfilled after evaluation of Blood Donor Questionnaire and Informed
Consent Form duly filled by the donor and medical examination
including the results of pre donation screening tests.

MATERIAL REQUIRED
• Donor Questionnaire and Informed Consent Form (Annexure 2.1).

PROCEDURE
Criteria for Selection of Blood Donor
Accept only voluntary/replacement non-remunerated blood donors if
following criteria are fulfilled—
1. General: No person shall donate blood and no blood bank shall
draw blood from a person, more than once in three months. The
donor shall be in good health, mentally alert and physically fit and
shall not be inmate of jail, person having multiple sex partner and
drug-addict. The donors shall fulfill the following requirements,
namely:
 Criteria for Donor Selection 9

a. The donor shall be in the age group of 18 to 65 years.

b. The donor shall not be less than 45 kilograms.
c. Temperature and pulse of the donor shall be (As per Transfusion
Medi­cine Technical Manual; Govt. of India, the oral temperature
should not exceed 37.5°C and pulse should be 80–100/min and
re­gular);
d. The systolic and diastolic blood pressures are within normal
limits without medication (As per Transfusion Medicine
Technical Manual; Govt. of India, the systolic and diastolic
pressures should be bet­ween 100–180 and 50–100 mm of Hg
respectively).
e. Hemoglobin should not be less than 12.5 grams.
f. The donor shall be free from acute respiratory diseases.
g. The donor shall be free from any skin diseases at the site of
phle­botomy.
h. The donor shall be free from any disease transmissible by blood
transfusion, in so far as can be determined by history and
exam­ination indicated above.
i. The arms and forearms of the donor shall be free from skin
punctures or scars indicative of professional blood donors or
addiction of self-injected narcotics.
2. Additional qualifications of a donor: No person shall donate
blood, and no blood bank shall draw blood from a donor, in the
conditions mentioned in column (1) of the Table 2.1 given below
before the expiry of the period of deferment mentioned in the
column (2) of the said Table.
3. No person shall donate blood and no blood bank shall draw blood
from a person, suffering from any of the diseases mentioned below,
namely:
a. Cancer
b. Heart disease
c. Abnormal bleeding tendencies
d. Unexplained weight loss
e. Diabetes-controlled on Insulin
f. Hepatitis infection
g. Chronic nephritis
h. Signs and symptoms suggestive of AIDS
i. Liver disease
j. Tuberculosis
k. Polycythemia vera
l. Asthma
m. Epilepsy
10 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

n. Leprosy
o. Schizophrenia
p. Endocrine disorders
Table 2.1: Deferment of blood donors
Sr No Conditions Period of Deferment
1. Abortions 6 months
2. History of blood transfusion 6 months
3. Surgery 12 months
4. Typhoid 12 months after recovery
5. History of malaria and duly treated 3 months (Endemic)
3 years (Non-endemic area)
6. Tattoo 6 months
7. Breast-feeding 12 months after delivery
8. Immunization (Cholera, Typhoid, 15 Days
Diphtheria, Tetanus, Plague,
Gamma globulin)
9. Rabies vaccination 1 year after vaccination
10. Hepatitis in family or close contact 12 months
11. Hepatitis immunoglobulin 12 months

Private interview
A detailed sexual history should be taken to exclude any risky behavior.

Informed consent
Provide information regarding:
1. Need for blood
2. Need for voluntary donation
3. Regarding transfusion transmissible infections
4. Need for questionnaire and honest answers
5. Safety of blood donation
6. How the donated blood is processed and used
7. Tests carried out on donated blood.

Note
Request the donors to sign the Donor Questionnaire and Informed Consent
Form indicating that he/she is donating voluntarily. This will give the donor an
opportunity to give his/her consent if they feel them­selves as safe donors.
 Criteria for Donor Selection 11

Annexure 2.1: Blood Donor Questionnaire and Informed

Consent Form
Hospital/Blood
Bank Logo Blood Bank ……….…………. (License No …………………)

Blood Unit & Segment No. ______________ Donor’s Name: _______________________

Starting Time of Phlebotomy: ____________ Father’s Name:______________________
Duration of Phlebotomy:_______________ Age/DOB:________________Sex:_______
Ending Time of Phlebotomy:_________Min
Phlebotomy Site: Right/Left Antecubital
Fossa
Weight of Blood Unit: _____________ gms
BLOOD DONOR QUESTIONNAIRE
CONFIDENTIAL
[√] Tick wherever applicable
Please answer the following questions correctly. This will help to protect you and the patient who receives
your blood.
Occupation:______________Address:_______________________________________________________
Tel. No:_____________________ Mobile No:_______________________E-mail:_____________________
Type of Donor: Voluntary Replacement, if Replacement Patient Name__________MR No:____
Would you like us to call you on your mobile: Yes No
Have you donated previously: Yes No
If yes, how many occasions:______ When Last:_____
Did you have any discomfort during/ after donation? Yes No
Your Blood Group:_____________ Time of last meal:______________

[√] the appropriate answer:

1. Do you feel well today?

Yes No
2. Did you have something to eat in the last 4 hours?
Yes No
3. Did you sleep well last night?
Yes No
4. Have you any reason to believe that you may be infected by Hepatitis, Malaria, HIV/
AIDS, and/or venereal disease?
Yes No
5. In the last 6 months, have you had any history (Signs & symptoms of AIDS) of the
following:
Unexplained weight loss
Repeated Diarrhea
Swollen glands
Continuous low-grade fever
Sexually Transmitted diseases
6. In the last 6 months have you had any:
Tattooing Ear Piercing
Dental Extraction
7. Are you taking or have taken any of these in the past 72 hours?
Antibiotics Aspirin Alcohol
Steroids Vaccinations
Dog Bite/Anti-rabies vaccine (1 yr.)
Contd...
12 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

8. Is there any history of surgery or blood transfusion in the past 6 months?

Major Minor
Blood Transfusion
9. Do you suffer from or have suffered from any of the following diseases?
Heart Disease
Diabetes
Leprosy
Endocrine Disorders
Chronic Nephritis
Cencer/Malignant Disease
Tuberculosis
Abnormal bleeding tendency
Hepatitis B/C
Asthma
Liver Disease
Polycythemia Vera
Malaria (6 months)
Typhoid (last 1 yr)
Epilepsy
Schizophrenia
10. For women donors
a. Are you menstruating?
Yes No
b. Are you pregnant?
Yes No
c. Have you had an abortion in the last 6 months?
Yes No
d. Do you have a child less than one year old/Are you breastfeeding?
Yes No
11. Would you like to be informed about any abnormal test result at the address furnished
by you?
Yes No
I have read and understood all the information presented and answered all the
questions truthfully, as any incorrect statement or concealment may affect my
health or may harm the recipient.
Yes No

CONSENT FOR BLOOD DONATION

I understand that:

(a) Blood donation is a totally voluntary act and no inducement or remuneration has been offered

(b) Donation of blood/components is a medical procedure and that by donating voluntarily, I accept
the risks associated with this procedure which is safe and most people tolerate giving blood very
well. Reactions are rare but can happen, especially if a person has not eaten or is tired or nervous.
Reactions that may occur include bruising around the vein, pain, infection (caused by needle stick);
dizziness, fainting, nausea, vomiting (caused by low blood volume, pain or the sight of blood); muscle
spasms (caused by anxiety and rapid breathing or fainting), or an allergic reaction (caused by arm
scrub or tape)

Contd...
 Criteria for Donor Selection 13

(c) My blood will be tested for Hepatitis B, Hepatitis C, Malarial parasite, HIV/AIDS and venereal diseases
in addition to any other screening tests required to ensure blood safety. The medical and personal
information and results of testing will be held by the Blood Bank in strict confidence and will not
be disclosed to anyone unless specifically authorized by me in writing or as required by the Govt.
authority or legal process.

I acknowledge that I have read and understood the information provided on this form about Blood Donation.
I have truthfully, completely and accurately answered all the questions on this form.
I hereby voluntarily consent to donate my blood/blood components to be used as directed by the Blood
Bank as per policy of the Govt. for the blood safety.
Date & Time___________ Donor’s Sign:_________

General Physical Examination

Weight ________ Kg. Height: ________ cms Hb: ________ gms%
Temp: ________ Pulse: ________ BP: _____/_____ mmHg
Accept   Defer   Reject   Reason:______________________________________

Signature of the Medical Officer:________________________Date:______________________________

Blood safety begins with a Healthy Donor ...

 C H A P T E R 3
Donor Screening

SCOPE AND APPLICATION

This SOP describes performance of physical examination of the donor
for confirming fulfillment of the criteria which ensure safety of the donor
as well as of the recipient.

RESPONSIBILITY
It is the responsibility of the Medical Officer to perform the physical
exam­ination of the donor.

MATERIALS REQUIRED
• Weighing scale and height measuring scale.
• Sphygmomanometer.
• Stethoscope.
• Clinical thermometer.
• CuSO4 solution in Coplin’s jar.
• Sahli hemoglobinometer.
• Capillaries.
• Lancet.
• Donor Questionnaire and Informed Consent Form.

MEDICAL EXAMINATION
• Record as to whether the donor appears in good health, mentally
alert and physically fit. Also examine the donor as to whether he/
she is under the influence of drugs/alcohol, and responds reliable
answers to the medical history.
• Check and record height and weight of the donor.
• Record BP, pulse and temperature of the donor.
• Estimate and record the hemoglobin of the donor.
Donors are accepted/rejected/deferred on the basis of findings
in Donor Questionnaire and Informed Consent Form by the Medical
Officer. Record for the deferral donors is to be maintained separately.
 C H A P T E R 4
Donor Screening for Hemoglobin
(Copper Sulfate Solution Method)

SCOPE AND APPLICATION

This SOP describes the method for estimation of hemoglobin of blood
donors by copper sulfate solution before blood donation for assessing
their suitability in order to ensure their safety as well as product quality.
Copper sulfate solution is used for testing the hemoglobin con­cen­
tration of blood donors before blood donation.

RESPONSIBILITY
It is the responsibility of the Medical Officer/Technician to perform the
testing of hemoglobin of the donor.

MATERIALS REQUIRED
Equipment
• Urinometer.

Reagents
• Copper sulfate solution.
• Distilled water.
• EDTA blood samples of known hemoglobin concentration.

Glassware
• Coplin jar.
• Test tubes.

Miscellaneous
• Tissue paper.
• Weighing scale.
• Crystalline CuSO4.5H2O.
• Copper sulfate record book.
• Test tube stand.
16 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

PROCEDURE
i. Dissolve 170 gms crystalline CuSO4.5H2O in 1000 ml distilled water.
ii. Dilute 51 ml of stock solution with distilled water to make it
100 ml. Label it as working solution.
iii. Check and adjust specific gravity of the working solution using
urinometer to 1.053 by adding stock solution or distilled water.
Copper sulfate solution is checked to ensure that a drop of blood
sample of predetermined hemoglobin value reacts as expected
(sinks/floats).
iv. Transfer 30 ml copper sulfate working solution in a Coplin jar.
v. Clean the fingertip with a spirit swab thoroughly. Allow it to dry.
vi. Puncture the fingertip with a sterile disposable lancet to ensure
good free flow of blood. Do not squeeze the finger repeatedly to
avoid dilution of blood with excess tissue fluid.
vii. Wipe out the first drop of blood. Allow second drop of blood to fall
gently from the finger from a height of about 1 cm above the surface
of the copper sulfate solution, into the Coplin jar.
viii. Observe the drop of blood for 15 seconds.

Note
• The working solution is prepared fresh every morning and changed after
every 25 tests.
• The Coplin jar is kept covered when not in use.
• The lancet and capillaries are disposed off in a container with 1% sodium
hypochlorite solution.

INTERPRETATION
• If the drop of blood sinks within 15 seconds, it shows that the
donor’s hemoglobin is more than 12.5 gm/dl.
• However, if the blood drop floats for more than 15 seconds or sinks
mid­way, it shows that the donor’s hemoglobin is less than 12.5 gms/
dl.
• Test the hemoglobin of the donor using Sahli’s method in case the
drop sinks slowly, hesitates and then goes to the bottom of the jar.
The donors having hemoglobin 12.5 gm/dl and more are accepted
for blood donation.
The results are entered in the Donor Questionnaire and Informed
Consent Form/Donor Register.
 C H A P T E R 5
Estimation of Hemoglobin of the
Donor (Sahli’s Method)

SCOPE AND APPLICATION

This SOP describes the method for hemoglobin estimation of blood
donors before blood donation by Sahli’s hemoglobinometer. This
method is used to re-determine the hemoglobin status of those donors
who fail in the copper sulfate solution method.

RESPONSIBILITY
It is the responsibility of the Medical Officer/Technician working in the
donor area.

MATERIALS REQUIRED
• Sahli hemoglobinometer.
• 0.1 N hydrochloric acid (HCl).
• Distilled water.
• Pasteur pipettes.
• Lancet.
• Sterile cotton swabs.

PROCEDURE
i. Fill the graduated tube to the mark 20 with 0.1 N HCl using Pasteur
pipette.
ii. Clean the fingertip with a spirit swab thoroughly. Allow it to dry.
iii. Puncture the fingertip with a sterile disposable lancet to ensure
good free flow of blood. Do not squeeze the finger repeatedly to
avoid dilution of blood with excess tissue fluid.
iv. Draw blood up to 20 ul mark in the Hb-pipette. Adjust the column
carefully without bubbles. Wipe excess of the blood on the sides of
the pipette by using a dry piece of cotton.
18 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

v. Transfer blood to the graduated tube containing 0.1 N HCL. Rinse

the pipette well, mix the reaction mixture and allow the tube to
stand for at least 10 minutes.
vi. Add distilled water drop by drop in the tube and ensure mixing after
each addition, until the color of the solution matches with the color
of the standards of the hemoglobinometer when observed against
the natural light.
vii. Record the level of the fluid at its lower meniscus. This reading
expressed in g/dl indicates the hemoglobin status of the donor.

INTERPRETATION
The donors having hemoglobin 12.5 gm/dl and more are accepted for
blood donation.
The results are entered in the Donor Questionnaire and Informed
Consent Form/Donor Register.
 C H A P T E R 6
Estimation of Hemoglobin by
Hemo-Control

SCOPE AND APPLICATION

This SOP describes the method of testing hemoglobin by Hemo-Control
photometer for those donors who fail in copper sulfate and Sahli’s
hemoglobinometer methods.

RESPONSIBILITY
It is the responsibility of the Medical Officer/technician working in the
donor area.

MATERIALS REQUIRED
• Hemo-Control photometer.
• Microcuvettes.
• Control cuvette.
• Sterile gauze/cotton, spirit.

PROCEDURE
Principle
The recognized reference method for total Hb determination is the
cyanmethemoglobin method which is also known as cyanhemoglobin
method. The blood sample is diluted 1:251 with a reagent buffering
solution. The erythrocytes are hemolyzed and the bivalent iron in oxy-
and deoxyhemoglobin are oxidized by the reagent potassium hexa­
cyanoferrate and stable, colored complex-cyanhemoglobin is formed.
This has a wide absorption maximum at 540 nm. This absorption is pro­
portional to total Hb concentration.
In 1966, Vanzetti replaced KCN by NaN3 and thus reduced the
toxicity of the reagent mixture and this Vanzetti’s method is also known
as the Azide Methemoglobin Method.
20 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

In the Hemo-Control photometer, however, the use of microcuvettes

with short light pathways makes it possible to analyse undiluted blood.
In microcuvette, sodium deoxycholate dissolves and disperses the
cell walls of RBC’s. The bivalent iron in oxy-and deoxyhemoglobin
so released is oxidized by Sodium Nitrite (NaNO2) to methemoglobin
which combines with azide from Sodium Azide (NaN3) to form a colored
complex having maximal absorption at 540 nm and 575 nm and is
measured quantitatively with Hemo-Control photometer.
The proportionality factor of the absorption and the Hb
concentration against the cyanmethemoglobin method has been
determined by the manufacturer and is contained in the Hemo-Control
photometer as an individual parameter.

Method
i. Take out microcuvette from the container and close it again.
ii. Make the donor sit comfortably. Lightly massage the fingers to
stimulate the circulation.
iii. The fingertip is cleaned thoroughly with a spirit swab and allowed
to dry.
iv. The finger is punctured firmly near the tip with a sterile disposable
lancet. A good free flow of blood is ensured. The finger is not to
be squeezed repeatedly since it may dilute the drop of blood with
excess tissue fluid and give false low results.
v. The first drop of blood is wiped and once a drop of blood is large
enough to fill the microcuvette completely hold the tip of the
microcuvette in the middle of the drop of blood and let the cavity fill
in one step.
vi. Remove the surplus blood from outside of the microcuvette to avoid
the contamination of the cuvette holder.
vii. Open the cuvette holder as far as it will go. The Hemo-Control
photometer ascertains the optical performance of the measuring
system. This process takes approx. 1–2 seconds.
viii. Release the cuvette holder and do not touch it again until the
process is finished and an acoustic signal occurs.
ix. Insert the microcuvette into the cuvette holder.
x. Push lightly on the cuvette holder until it closes itself.
xi. Read off the results.
xii. Open the cuvette holder; take out the used cuvette and dispose of
this in the correct way.
 Estimation of Hemoglobin by Hemo-Control 21

Note
The microcuvettes are used only once and are sensitive to moisture. Therefore,
the microcuvettes are to be taken out only immediately before use.
Reading of the microcuvettes is taken immediately or within 10 min­utes at
the latest.

Quality Control
The Hemo-Control photometer provides an integrated algorithm to
check the optical and electronic components. This self-check will be
performed automatically without any user action.
Carry out a blank reading whenever the cuvette holder is removed.
A control cuvette is used to run a simple, economic check of the
mea­surement quality.
The Hemo-Control photometer has been calibrated against
the cyanmethemoglobin reference and hence determines the
proportionality constant K. The calibration has been done against the
NCCLS standard procedure at the manufacturer level and gives results
comparable to ICSH (1995). A maximum deviation of 0.3 g/dl is tolerated
at 15.0 g/dl during the calibration process.

INTERPRETATION
The donors having hemoglobin 12.5 gm/dl or more are accepted
for blood donation. In case the hemoglobin is lower than 12.5 g/dl,
hematinics are prescribed to build up Hb.
The results are entered in the Donor Questionnaire and Informed
Consent Form/Donor Register.
 C H A P T E R 7
Preparation of Phlebotomy Site

SCOPE AND APPLICATION

This SOP describes preparation of the skin at the phlebotomy site before
vene-puncture to ensure aseptic collection of blood.

RESPONSIBILITY
The phlebotomist collecting the blood unit from the donor is responsible
for preparation of phlebotomy site.

MATERIALS REQUIRED
• Sterilizing tray.
• Demethylated spirit.
• Povidone iodine solution 5% w/v.
• Sterile cotton/gauze/swabs.
• Artery forceps.
• BP apparatus.

PROCEDURE
i. Select the vene-puncture site in the anti-cubital area of the arm.
ii. Apply blood pressure cuff, inflate to 50–60 mm of Hg and select a
large vein in the area that is free of any skin lesion.
iii. Deflate the blood pressure cuff and thoroughly clean the selected
site of vene-puncture with spirit, povidone-iodine solution and
finally with spirit swab. Start disinfection of the skin on an area of
5 cm diameter from the center to periphery in a circular motion.
iv. Scrub the providone-iodine swab vigorously for at least 30 seco­nds
or till froth is formed.
v. Do not touch the site so prepared for vene-puncture. Should it be
necessary, touch the skin away from the point of insertion of needle?
If the puncture site is touched, repeat skin preparation procedure as
detailed earlier.
 Preparation of Phlebotomy Site 23

vi. Discreetly check the used swab. If it is physically observed soiled,

take a new swab and repeat skin preparation procedure as detailed
earlier.
vii. Dispose off used swab(s) into a waste bin meant for bio-hazardous
materials.
viii. Allow the skin to air dry. Do not wipe the area with cotton wool, fan
or blow on it.
 C H A P T E R 8
Selection of the Blood Bags

SCOPE AND APPLICATION

This SOP describes the type of blood bag to be used for blood collection
of the donor to optimize the availability of blood and its components.

RESPONSIBILITY
The Medical Officer/Laboratory Technician of the blood bank is res­
ponsible for deciding the type of blood bags to be used to optimize the
availability of blood and its components.

MATERIALS REQUIRED
• Different types of blood bags.

PROCEDURE
• Single blood bag is to be used for collection of blood unit in case the
end product is whole human blood.
• Select the type of blood bags to be used for preparation of blood
components as per Table 8.1.

Note
• Check the blood bag visually before collection of blood.
• Do not use in case of puncture or discoloration or suspended particulate
matter in the bag.
• Check the expiry date of the blood bag.
• Record the details of blood bag, i.e. type and segment no. in the “Donor
Questionnaire and Informed Consent Form” and donor register.
 Selection of the Blood Bags 25

Table 8.1: Selection of blood bags for blood components

Donor Components Blood Bag

Weight Aspirin Required Type Qty.(ml) of

Intake Blood

>55 Kg No PRBCs + FFP + PLT Triple or 450 ml

Quadruple

>55 Kg Yes PRBCs +FFP Double 350/450 ml

PRBCs + FVIIID + Triple
CRYO

45–55 Kg No PRBCs + FFP Double 350 ml

PRBCs – PLT
PRBCs + FVIIID + Triple
CRYO

45–55 Kg Yes PRBCs + FFP Double 350 ml

PRBCs + FVIIID
PRBCs + FVIIID + Triple
CRYO
PRBCs: Packed Red Blood Cells; FFP: Fresh Frozen Plasma; PLT: Platelets,
F VIII DP: Factor VIII Deficient Plasma; CRYO: Cryo-precipitate
 C H A P T E R 9
Venipuncture and
Blood Collection

SCOPE AND APPLICATION

This SOP describes procedure for blood collection from the donor
aseptically.

RESPONSIBILITY
The phlebotomist is responsible for blood collection from the donor after
verifying the donor screening details, checking the blood unit number to
be allotted and preparing the phlebotomy site.

MATERIALS REQUIRED
• Sterile cotton/Gauze swabs.
• Artery forceps.
• Sterile disposable syringes (2 ml).
• Sterile disposable hypodermic needles (26 gauge).
• Pilot tubes: Plain and EDTA.
• Oxygen cylinder with accessories.
• First aid tray.
• Tube sealer.
• Needle destroyer.
• Blood collecting CPD-A bags.
• Scissors.
• Adhesive tapes.
• Blood collection monitor.
• Donor couch.

PROCEDURE
i. Make the donor recline in comfortable donor couch. Loosen tight
garments.
ii. Identify the donor by name. Enter the bag and segment number on
the donor questionnaire and informed consent form.
 Venipuncture and Blood Collection 27

iii. Ask the donor if he/she is in a comfortable position. Give the donor
a hand roller/squeezer to hold.
iv. Clean the venipuncture site.
v. Set the blood collection monitor for the required volume of blood
(350/450 ml) to be collected and place the blood bag on it. Route
the tube through the clamp.
vi. Apply the blood pressure cuff on donor arm.
vii. Clamp the bleed line of the blood bag using artery forceps to
ensure that no air enters the tubing or bag upon removal of the
needle cover.
viii. Keep the level of the needle facing upward and the shaft at an angle
of 15° to the arm. Once the needle is beneath the skin, release the
artery forceps. Insert the blood bag needle into the vein for about
1 to 1.5 cms by a bold single prick to ensure smooth flow of blood
and secure on the arm with adhesive tape.
ix. Advise the donor to gently squeeze the ball/roller with the hand to
improve blood flow.
x. If the venipuncture is unsuccessful, do not make further attempt in
the same arm. Take the donor’s permission for a second attempt.
Use a new bag and discard the previous bag.
xi. Once blood enters the bag tubing, press the “start key” of the blood
collection monitor which automatically tares the bag weight and
the weight of CPD-A solution so that the “display” shows only the
volume of blood being collected. It also gives gentle agitation to the
blood bag for proper mixing of blood with CPD-A solution to avoid
clotting.
xii. When the collected blood volume reaches “programmed volume
minus 5 ml”, the rocking motor stops its agitation and when col­­
le­c­ted blood volume reaches “programmed volume minus
3 ml”, the clamp automatically gets activated ensuring no further
entry of blood into the blood bag. The collected volume and the
duration of the bleeding are displayed in the first line of the LCD of
the monitor scale.
xiii. Pick up the bag from the tray of the blood collection monitor scale
to release the clamp at the end of collection.
xiv. Clamp the bloodline of the blood bag at 2 different sites and cut in
the middle. Collect blood in the pilot tubes from the tubing so that
blood flows directly into the tubes from the donor arm.
xv. Deflate the BP cuff and remove the needle gently from the donor’s
vein pressing the phlebotomy site. Place the sterile swab at the
venipuncture site and apply pressure over the swab. Ask the donor
to elevate arm and fold it so that swab may not fall.
28 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

xvi. Seal the blood bag tubing using the tube sealer. Hold the tube at
both the ends and place the tube in between the sealing electrodes
through the slot provided in the electrode cover. The tube detection
lever gets depressed in this process. Moving electrode pushes the
tube against the fixed electrode. ‘Seal’ LED turns ON; ‘Ready’ LED
turns OFF. Tube melts and the sealed pattern forms. Do not stretch
the tube while sealing. This may cause leaking.
xvii. After a time delay, the electrode will release, ‘SEAL’ LED goes OFF
and the ‘READY’ LED turns ON. Take out the sealed tube. In this
way, make couple of segments in the tubing and that are used for
compatibility testing.
xviii. The needle of the bag along with the cut portion of the tubing is
discarded in the red capped containers.
xix. The blood unit is stored in the blood bank refrigerator meant
for storage of untested blood units at 4 ± 2°C immediately after
collection.

Note
The procedure described above may be modified keeping in view the make and
type of blood collection monitor.
 C H A P T E R 10
Post-donation Care

SCOPE AND APPLICATION

This SOP describes about the observation of the donor during post-
donation period so as to take care any immediate adverse reaction.

RESPONSIBILITY
The Medical Officer is responsible for the post-donation care of the
donor.

MATERIALS REQUIRED
• Sterile swabs.
• Adhesive tape.
• Thrombophob ointment.
• Leaflet for post-donation instructions.

PROCEDURE
a. Direct the donor not to get up from the donor couch for 5 minutes
even if it feels perfectly all right to prevent adverse reactions like
giddiness.
b. Observe the donor for another 10 minutes in the refreshment area
whilst having coffee.
c. Inspect the venepuncture site before the donor leaves the donor
room. Apply an adhesive tape only after oozing stops. If there is
persistent oozing at the site of venepuncture, apply pressure with a
dry, sterile cotton swab. If there is hematoma, apply thrombophob
ointment gently over the area after 5 minutes. Inform the donor
about the expected change in skin color. If the pain persists, ask
him/her to apply ice.
d. Instruct the donor to–
• Drink adequate fluid in the day.
• Avoid strenuous activities.
30 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Don’t smoke for next 30 minutes.

• Avoid climbing steps for next 30 minutes.
• Resume all normal activities if no symptoms occur.
• Remove bandage next day.
 C H A P T E R 11
Management of Adverse
Reactions in the Donors

SCOPE AND APPLICATION

This SOP describes about the care of the donor in the immediate post-
donation period.

RESPONSIBILITY
The Medical Officer of the blood bank is responsible for managing the
adverse reaction of the donor.

MATERIALS REQUIRED
Following materials are required to attend to any emergency arising in
the post-donation period.

Oral Medication
• Analgesic tablets, e.g. Paracetamol tablet IP 500 mg.
• Calcium and Vitamin C tablets.
• Electrolyte replacement fluid (ORS).

Injection
• Epinephrine (Adrenaline).
• Atropine sulfate.
• Pheniramine maleate.
• Diazepam.
• Glucocorticoid.
• Glucose (Dextrose 25% w/v).
• Furesemide.
• Metoclopramide.
• Prochlorperazine maleate.
• Sodium bicarbonate.
• Glucose saline (Sodium chloride and Dextrose 500 ml).
32 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Antiseptics
• Savlon solution.

Miscellaneous
• Betnovate ointment.
• Bandages/Dressings.
• Band-aids.
• Heparin and benzyl nicotinate ointment.
• Spirit of ammonia.
• Analgesic balm.
• Tongue depressor.
• Disposable syringes (2/5 ml) and needles 22 G.
• Clinical thermometer.
• Oxygen cylinder.
• Infusion set.
• Paper bag.

MANAGEMENT OF ADVERSE REACTIONS

1. Giddiness/Syncope (Vasovagal Syndrome)
• Raise feet and lower head end.
• Loosen tight clothing (belt, tie, etc.)
• Ensure adequate airway.
• Check pulse and blood pressure.
• Apply cold compresses to forehead and back.
• Administer inhalation of spirit of ammonia if needed. The
donor should respond by coughing which will elevate the blood
pressure.
• If there is bradycardia and hypotension–
■ Administer atropine Injection 1 ml IM, if bradycardia continues
for more than 20 minutes.
■ Administer IV normal saline or dextrose saline infusions if hy­
po­tension is prolonged.
2. Convulsions: Keep the head tilted to the side; prevent the tongue
bite; keep the airway patent by inserting a tongue depressor or
gauze between the teeth.
3. Vomiting: Usually vomiting provides relief if the donor feels
nauseous or if vomiting is severe, give Stemetil Injection. Usually
vomiting subsides on its own.
 Management of Adverse Reactions in the Donors 33

4. Tetany/muscularspasm/twitching: These are usually due to hyper-

ventilation in an apprehensive donor. Ask the donor to breath in a
paper bag, which provides prompt relief. Do not give oxygen.
5. Hematoma: Release the tourniquet/pressure cuff immediately.
Apply pressure on the venepuncture site and withdraw the needle
from the vein. Raise the arm above the head for a few minutes.
Apply thrombophob ointment gently around the phlebotomy site
after about 5 minutes. Advise the donor to apply ice if there is pain
and inform about the expected change in skin color.
6. Eczematous reactions of the skin around venepuncture site:
Apply steroid ointment, e.g. Betnovate ointment.
7. Delayed syncope: These may occur as late as 30 minutes to 1 hour
after donation (usually after the donor has left the blood bank).
Permanently defer any donor who gives history of such attacks
more than twice.
 C H A P T E R 12
Traceability of the Blood Units

SCOPE AND APPLICATION

This SOP describes about the allotment of unique ID number to the
donor and labeling of blood units and pilot tubes after donor verification
for accurately relating the blood product to the donor. This is essential
for tracebility of blood donor.

RESPONSIBILITY
It is the responsibility of the phlebotomist collecting the blood unit to
ensure proper labeling.

MATERIALS REQUIRED
• Sticker labels with pre-printed serial number.
• Donor Questionnaire and Informed Consent Form.

PROCEDURE
• Allot each donor a unique ID number and identify him/her by that
number only.
• Do not write donor’s name on his/her blood bag or sample tube to
maintain the donor’s confidentiality.
• Affix pre-printed ID number labels on the primary bag on both
sides, on all the satellite bags in case of multiple bags and the three
pilot tubes (two plain and one with CPD-A anticoagulant).
• Verify the donor’s identity with the Donor Questionnaire and
Informed Consent Form. Affix the unit ID number label on it.
• Cross check the ID numbers on the blood unit, pilot tubes and
donor Questionnaire and Informed Consent Form to ensure donor
identity. Record it in the donor register using the same ID number.
• Transcribe this ID number on all records henceforth for storage,
testing, issue records and transfusion records.
 Traceability of the Blood Units 35

Note
Make sure that–
• The ID number is written clearly on all records.
• There are no transcription errors.
This will help in tracing any product to the donor of the blood and vice
versa.
 C H A P T E R 13
Blood Components Separation

SCOPE AND APPLICATION

Modern blood transfusion envisages the optimal use of every blood
donation by way of component therapy as per the need rather than
using whole human blood. The whole human blood collected in double
bags, is separated into Packed Cells and Fresh Frozen Plasma (FFP) or
Factor VIII (F-VIII) deficient plasma. From blood collected in triple bags,
Packed cells, FFP and Platelets or Packed Cells, FVIII deficient plasma
and cryoprecipitate are separated. When the plasma frozen at –80°C,
it is then thawed at 4°C, a cryoglobulin remains as a precipitate which
is called cryoprecipitate. It contains mainly F-VIII and fibrinogen. The
process of separating Blood Components is shown in Figure 13.1.

Fig. 13.1: Flowchart for component preparation

 Blood Components Separation 37

RESPONSIBILITY
It is the responsibility of the Technical Supervisor and Medical Officer
to separate components from whole human blood collected in multiple
bags.

EQUIPMENT AND MATERIALS REQUIRED

• Tube sealer.
• Sterile connecting device.
• Water distillation still.
• Transfer bags.
• Laminar flow bench.
• Refrigerated centrifuge.
• Plasma expresser.
• Electronic weighing scale.
• Double pan weighing balance.
• Cryoprecipitate thawing bath.
• Double bags (350 ml) or triple bags with SAGM solution (450 ml).
• Manuals of all equipment for reference regarding use and main­
tenance of each equipment.
• Dry rubber balancing material.
• Deep freezer –40°C and –80°C.
• Platelet agitator with incubator.
• Blood bank refrigerator.

General Precautions
• Donors taking anti-platelet drugs (e.g. Aspirin), donors who have
consumed alcohol during the preceeding 12 hours and donors
who give history of bleeding tendencies should not be selected for
preparation of platelet concentrates.
• Donors selected for component separation should be given a serial
number after checking the Blood Donor Register. Write this number
on (1) Donor Form; (2) Blood Bags, including all satellite bags; and
(3) two clean glass tubes selected as ‘Pilot Tubes’.
• If phlebotomy is traumatic or if blood collection takes more than
10 minutes, record this on the donor form. Blood bags from which
platelet concentrates are to be prepared should not be refrigerated.
Keep such blood bags between 20°C and 24°C (either in the platelet
incubator or in a room where air-conditioner is on).
38 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 13.2: Color coding for components

• Before starting component separation make appropriate entries in

the “Component work register”. Make sure that component sepa­
ration is completed with six hours after blood collection.
• Color coding depicting various components as shown in Fig. 13.2.

PROCEDURE
Preparation of (1) Packed Red Cells (PRC), and (2) Platelet-rich
Plasma (PRP)/FFP using Double Bags (Fig. 13.3)
• The whole blood is collected in bag no 1.
• Keep the blood units vertical on the laminar flow bench for 30 to 45
minutes.
• Note the weight of the primary bag and record in the daily work register.
• Keep the bags in the buckets of the refrigerated centrifuge and
balance them using dry rubber. Keep the equally balanced buckets
with blood bags diagonally opposite in the refrigerated centrifuge
ensuring that the position of the blood bags in buckets is parallel to
the direction of the spin.
• Spin the blood bags in refrigerated centrifuge after balancing the
opposing buckets carefully at 5000 × g (heavy spin) for 5 minutes at
4°–6°C.

Fig. 13.3: Double bag for preparation of PRC and FFP

 Blood Components Separation 39

Fig. 13.4: Blood bag contains RBC below and PRP on the top

• After centrifugation, gently remove the blood bags from the bucket.
The blood bag 1 has RBCs below and platelet-rich-plasma (PRP) on
the top (Fig. 13.4).
• Place blood bag 1 on the expresser stand and bag 2 on weighing
balance under the laminar flow bench. Break the integral seal of
the tube connecting it to the satellite bag 2 manually and express
the supernatant plasma into the satellite bag 2 (Fig. 13.5). In case of

Fig. 13.5: Packed RBCs in bag 1 and platelet rich plasma in bag 2
40 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

double bag, leave 50–60 ml of plasma back along with the red cells
in the primary bag 1 and this component is Packed Red Cells (PRC).
Label the plasma in the satellite bag 2, as Fresh Frozen Plasma (FFP
if separated within 6 hours of collection and stored immediately
below –30°C in Deep Freezer.
• If plasma is separated after 6 hours of collection label it as Factor
VIII deficient plasma (F-VIIID).
• Cut the tubing connecting bag 2 containing FFP/F-VIIID bags from
bag 1 with the help of sealer.
• The bag number 1 containing PRCs is then sealed and stored at
2–6°C in blood bank refrigerator. This has a shelf-life of 35 days.
• The bag 2 containing FFP/F-VIIID plasma is then kept at –30°C in
deep freezer. This has shelf-life of 1 year.

Preparation of Packed Cells, Platelet Concentrates and FFP Using

Triple Bag System with or without Additive Solution (SAGM)
(Fig. 13.6)
• The whole human blood is collected in bag 1.
• Keep the units vertical on the laminar flow bench for 30 to 45 minutes.
• Note the weight of the primary bag and record in daily work register.
• Keep the blood bags in the buckets and balance them using dry
rubber.
• Keep the equally balanced buckets with blood bags diagonally
opposite in the refrigerated centrifuge ensuring that the position of
the bags in buckets is parallel to the direction of the spin.
• Spin the blood bags in refrigerated centrifuge after balancing the
opposing buckets carefully at 2000 × g (light spin) for 3 minutes at
20–22°C. (Adjust the speed and duration of centrifugation as per
manufacturer’s guidelines).

Fig. 13.6: Triple bag system for preparation of packed cells, platelet
concentrates and FFP
 Blood Components Separation 41

Fig. 13.7: Centrifugation

• After centrifugation, gently remove the blood bags (Fig. 13.7) from
the bucket and place them on the expresser stand under the laminar
flow bench. Break the integral seal of the tube connecting it to the
satellite blood bag 2 manually and express the supernatant plasma
into the satellite bag 2 (Fig. 13.8) leaving 50–60 ml of plasma back
along with the red cells in the primary bag 1 and this component is
Packed Red Cells (PRC).
• If the blood bag with additive solution is used, remove all plasma
in satellite bag 2 before clamping. Remove the clamp of the bag
3 containing additive solution and let the additive solution slowly
pass into the primary bag 1 containing PRC (Fig. 13.9).
• Mix the contents of bag 1 thoroughly and seal the tubing between
bag 1 and bag 2 using dielectric sealer and detach the bag 1
containing packed red cells with additive solution in the blood bank
refrigerator for quarantine.
• After carrying out mandatory testing of PRC, label the bag 1,
transfer it to blood bank refrigerator for tested blood and take it on
the inventory.

Fig. 13.8: PRP to bag 2

42 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 13.9: SAGM to bag 1

• Spin the satellite bag 2 containing platelet rich plasma (PRP) and
connecting bag 3 (from which additive solution was emptied), at
20–22°C in refrigerated centrifuge at 5000 × g for 5 minutes after
balancing the buckets (Fig. 13.10).
• Place the bag 2 containing PRP on the expresser stand.
• Express the supernatant platelet poor plasma into the empty bag
3 leaving 50–60 ml plasma along with the platelets in bag 2 (Fig.
13.11).
• Seal the tubing using dielectric sealer and cut the tubing of the
plasma bag 3 leaving approximately 1” tubing with the bag to avoid
breakage during storage.
• Mix the contents of bag 3 and prepare a small segment (approxi­
mately 8 cms) of tube containing platelets for quality control test­
ing.

Fig. 13.10: Bag No. 2 and 3 after Fig. 13.11: Platelet poor plasma
centrifuge
 Blood Components Separation 43

• Leave the bag 2 containing platelet concentrates on the laminar

flow bench for 30 minutes, keeping the label side down. Mix the
contents of the bag 2 manually and transfer the bag for quarantine
storage in the platelet agitator/ incubator at 22°–24°C. The product
has shelf life of 5 days from the date of preparation.
• Transfer the unit to the upper shelf of the agitator after mandatory
tasting and labeling.
• Keeps the bag 3 containing platelet poor plasma in the deep
freezer under quarantine and transfer to the compartment of deep
freezer (–30°C) containing tested units after mandatory testing and
labeling.

Note
For preparation of cryo-precipitate from Platelet Poor Plasma, the units are to
be stored in the deep freezer at –80°C to preserve the activity of Factor VIII in it.

Preparation of Cryo-precipitate
1. The basic material is platelet poor fresh frozen plasma (PPP). The
plasma should be free of red cell. Use PPP frozen at –65°C or below
within 1 hour of preparation and stored at –30°C or below. The PPP
thus stored can be used for preparation of cryo-precipitate within
1 year.
2. The bag to be used for preparation of cryo-precipitate must have
longer segment of the tubing.
3. Fill the cryo-precipitate thawing bath with double distilled water.
4. Maintain the temperature of water in continuous circular motion at
4°C.
5. Keep the frozen plasma bags in this cryo-precipitate thawing bath
till the plasma is thawed. Now place the bags in centrifuge buckets
and balance the buckets on weighing scale.
6. Keep the position of the bags in buckets parallel.
7. Spin the buckets at 5000 × g (heavy spin) for 5 minutes at 4°C.
8. Under laminar flow bench, connect empty transfer bag to the bag
containing plasma and cryo-precipitate using sterile connecting
device.
9. Place the plasma bag on expresser and separate plasma into the
transfer bag leaving approximately 15–25 ml plasma and whitish
jelly-like precipitate called “cryo-precipitate” in the original bag.
The cryo-precipitate is rich in factor VIII.
10. Seal the tubing and separate the bags containing cryo-precipitate
and the cryo-poor plasma bag.
44 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

11. Weigh the cryo-precipitate and the cryo-poor plasma bags and
record in daily work register.
12. The cryo-precipitate poor plasma separated is F-VIII deficient
plasma but rich in factor IX.
13. Quarantine these products at –30°C in deep freezer till the tests are
completed.
14. Label and transfer to the compartment of deep freezer meant for
storage of tested units after the mandatory testing is completed.
These products have a shelf-life of one year when stored at –30°C.
After thawing at room temperature in water bath (37°C), these products
can be administered intravenously.
For the treatment of hemophilia as a whole, the plasma (FFP) is the
main therapeutic material and for specific treatment for hemophilia A
(factor VIII deficiency), cryo-precipitate should be the drug of choice,
whereas, in hemophilia B (factor IX deficiency), cryo-poor-plasma
(CCP) is the drug of choice.

Washed Packed Red Cell

1. Undertake the washing procedure only if there is compatibility
between the donor and the patient.
2. If a single bag is used for blood collection attach a transfer bag using
sterile connecting device.
3. Balance the blood bag in the centrifuge bucket with another empty
bucket.
4. Spin the bag at 2000 × g (light spin) for 3 minutes at 4°C.
5. Remove the supernatant plasma completely in a transfer bag using
expresser under laminar flow bench.
6. Carry out compatibility tests and in case the PRC is compatible,
proceed further for washing only.
7. The bag containing PRC is balanced in the centrifuge bucket with
another empty bucket. The buckets are centrifuged as per program
detailed above.
8. The bag containing PRC is removed and supernatant plasma is
com­pletely transferred in a transfer bag using an expresser under
laminar flow bench.
9. Connect the bag containing PRC with a sterile 0.9% saline bag using
sterile connecting device.
10. Check and record batch number and expiry date of saline bag
before use.
11. Transfer approximately 250 ml of saline solution into the packed
cell bag and mix thoroughly. Centrifuge the bag again using heavy
spin (5000 × g for 5 minutes).
 Blood Components Separation 45

12. Transfer the supernatant saline with some plasma into the transfer
bag using the expresser under laminar flow bench.
13. Disconnect the transfer bag, seal and discard.
14. Repeat the washing procedure using saline twice more (total three
times) exactly in the same manner as described above. In the end
keep 25–30 ml saline with the red cells in the bag.
15. Seal the bag containing finally washed red cells.
16. Weigh the bag and record details in the register.
17. Store the washed packed red cell unit in the blood bank refrigerator
and use within 24 hours of washing.
18. Use this washed packed red cell unit only for the patient for which
req­ue­sted. If not used, discard it after 24 hours with standard
dis­posal protocol, after subjecting small sample for bacteriological
exam­ination.
 C H A P T E R 14
Collection of Blood Sample for
Grouping/Cross-matching

SCOPE AND APPLICATION

The prime objective of this SOP is to describe the method for collection
of blood sample of the patient for blood grouping and cross-matching.
Further; it aims at developing clear communication between clinician
and blood bank in ensuring the safe blood transfusion.

RESPONSIBILITY
It is the responsibility of the clinician in-charge/RMO and the staff nurse
on duty.

MATERIALS REQUIRED
Equipment
• Disposable 5 ml syringe with needle (0.55 × 25 mm).
• Tourniquet.
• Spirit wipes.
• Gauze sponges.
• Vacutainers.
These tubes are designed to fill with a predetermined volume of
blood by vacuum. The rubber stoppers are color coded according to
the additive that the tube contains. The purple (Fig. 14.1) and Red Top
(Fig. 14.2) vacutainers are used for collection of samples for blood bank.

PRE-REQUISITES FOR BLOOD SAMPLE COLLECTION

Patient Relations and Identification
The phlebotomist’s role requires a professional, courteous, and under­
st­anding manner in all contacts with the patient. Greet the patient and
identify yourself and indicate the procedure that will take place. Effective
communication—both verbal and nonverbal—is essential.
 Collection of Blood Sample for Grouping/Cross-matching 47

Fig. 14.1: Purple top vacutainer

Fig. 14.2: Red top vacutainer

Proper patient identification is Mandatory. If the patient is able to

respond, ask for a full name. Using the requisition for reference, ask the
patient to provide additional information such as a surname or birth
date.
If possible, chat with the patient during the process. The patient
who is at ease will be less focused on the procedure. Always thank the
patient and excuse yourself courteously when finished.

Venipuncture Site Selection

Although the larger and fuller median cubital and cephalic veins of the
arm are used most frequently, the basilic vein on the dorsum of the arm
or dorsal hand veins are also acceptable for venipuncture. Foot veins are
a last resort because of the higher probability of complications.
Avoid the following sites for collection of blood samples:
• Extensive scars from burns and surgery—it is difficult to puncture
the scar tissue and obtain a blood sample.
48 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• The upper extremity on the side of a previous mastectomy—test

results may be affected because of lymphedema.
• Hematoma—may cause erroneous test results. If another site is not
available, collect the specimen distal to the hematoma.
• Intravenous therapy (IV)/blood transfusions—fluid may dilute the
specimen, so collect from the opposite arm if possible. Otherwise,
satisfactory samples may be drawn below the site of IV therapy by
following these procedures:
■ Turn off the IV infusion for at least 2 minutes before venipuncture.
■ Apply the tourniquet below the IV site. Select a vein other than
the one with the IV infusion.
• Perform the venipuncture. Draw 5 ml of blood and discard before
cannula/fistula/heparin lock-hospitals have special policies regar­
ding these devices. In general, blood should not be drawn from
an arm with a fistula or cannula without consulting the attending
physician.
• Edematous extremities-tissue fluid accumulation alters test results.

PROCEDURE
Vein Selection
• Palpate and trace the path of veins with the index finger. Arteries are
most elastic, have a thick wall and pulsate. Thrombosed veins lack
resilience, feel cord-like and roll easily.
• If superficial veins are not readily apparent, you can force blood into
the vein by massaging the arm from wrist to elbow, tap the site with
index and second finger, apply a warm, damp washcloth to the site
for 5 minutes, or lower the extremity over the bedside to allow the
veins to fill.

Performance of a Venipuncture
• Approach the patient in a friendly, calm manner. He should be
made to feel comfortable and gain the patient’s cooperation.
• Identify the patient correctly.
• Fill the requisition form for blood/components properly.
• Check for any allergy to antiseptics, adhesives, or latex by observing
for armbands and/or by asking the patient.
• Position the patient. (The patient should sit either in a chair or lie
down or sit up in bed). Hyperextend the patient’s arm.
 Collection of Blood Sample for Grouping/Cross-matching 49

• Apply the tourniquet 3–4 inches above the selected puncture site.
Do not place too tightly or leave not more than 2 minutes.
• The patient should make a fist without pumping the hand.
• Select the venipuncture site.
• Prepare the patient’s arm using spirit swab. Cleanse in a circular
fashion, beginning at the site and working outward. Allow to air dry.
• Grasp the patient’s arm firmly using your thumb to draw the skin
taut and anchor the vein. The needle should form a 15 to 30 degree
angle with the surface of the arm as shown in Figure 14.3. Swiftly
insert the needle through the skin and into the lumen of the vein.
Avoid trauma and excessive probing drawing the blood sample for
testing.
• When the blood sample is drawn into the syringe, remove the tour­
niquet.
• Remove the needle from the patient’s arm using a swift backward
motion.
• Press the gauze once the needle is out of the arm, applying adequate
pressure to avoid formation of a hematoma.
• Put 2 ml of blood sample into the purple top vacutainer and 2 ml
into the red top vacutainer.
• Invert the tube with purple top 8 times to mix the blood with EDTA
for prevention of clotting and platelet clumping.
• Label tubes at the patient bedside.
• Dispose of used materials/supplies in designated containers.
• Deliver blood specimens promptly to the blood bank after making
proper entry in the record.

Safegaurds/Preventions during Blood Sampling

Prevention of hematoma
• Puncture only the uppermost wall of the vein.

Fig. 14.3: Needle angle with the surface of the arm

50 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Remove the tourniquet before removing the needle.

• Use the major superficial veins.
• Ensure that the needle penetrates the upper most wall of the vein
fully. (Partial penetration may lead to blood leakage into the soft
tissue surrounding the vein).
• Apply pressure to the venipuncture site.

To prevent hemolysis
• Mix blood sample tube with red top containing anticoagulant gently
8 times.
• Do not draw blood from a hematoma.
• Do not draw the plunger back too forcefully, if using a needle and
syringe.
• Avoid frothing of the sample.
• Ensure the dryness of venipuncture site.
• Avoid probing and traumatic venipuncture.

Safety and infection control

Because of contacts with sick patients and their specimens, it is
important to follow safety and infection control procedures.

Protect Yourself
• Practice universal precautions:
■ Wear gloves and a lab coat or gown when handling blood/body
fluids.
■ Change gloves after each patient or when soiled with blood.
■ Wash hands using antiseptic detergent preparation or disinfect
with alcohol.
■ Dispose of used items in appropriate containers.
• Dispose of needles immediately upon removal from the patient’s
vein. Do not bend, break, recap, or resheath needles to avoid
accidental needle puncture or splashing of contents.
• Clean up any blood spills with a disinfectant (freshly made 10%
bleach solution).
• If you stick yourself with a contaminated needle:
■ Remove your gloves and dispose of them properly.
■ Squeeze puncture site to promote bleeding.
■ Wash the area well with soap and water.
■ Record the patient’s name and ID number.
■ Follow institution’s guidelines regarding treatment and follow-
up.
 Collection of Blood Sample for Grouping/Cross-matching 51

Note
The use of prophylactic Tab zidovudine following needle stick injury on exposure
to HIV infected patient has shown effectiveness (about 79%) in preventing
seroconversion.

Protect the Patient

• Place blood collection equipment away from patients, especially
children and psychiatric patients.
• Practice hygiene for the patient’s protection. When wearing gloves,
change them between each patient and wash your hands frequently.
Always wear a clean lab coat or gown.

TROUBLESHOOTING GUIDELINES
If an Incomplete Collection or No Blood is Obtained–
• Change the position of the needle as it may not be in the lumen.
Move it forward (Fig. 14.4).
• Or move it backward as it may have penetrated too far (Fig. 14.5).

Fig. 14.4: Needle not in the lumen of the vein

Fig. 14.5: Needle penetrated too far

52 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 14.6: Bevel of the needle against the vein wall

• Adjust the angle since the bevel may be against the vein wall (Fig.
14.6).
• Loosen the tourniquet. It may be obstructing blood flow.
• Try another sample tube. There may be no vacuum in the one being
used.
• Re-anchor the vein. Veins sometimes roll away from the point of the
needle and puncture site.

If Blood Stops Flowing into the Tube–

• The vein might have collapsed (Fig. 14.7); resecure the tourniquet to
increase venous filling. If this is not successful, remove the needle,
take care of the puncture site, and try again.
• The needle might have been pulled out of the vein when switching
tubes. Hold equipment firmly and place fingers against patient’s
arm, using the flange for leverage when withdrawing and inserting
tubes.

Problems Other than an Incomplete Collection

• A hematoma forms under the skin adjacent to the puncture site
(Fig. 14.8). Release the tourniquet immediately and withdraw the
needle. Apply firm pressure.

Fig. 14.7: Collapsed vein

 Collection of Blood Sample for Grouping/Cross-matching 53

Fig. 14.8: Hematoma under the skin

Hematoma Formation is a Problem in Older Patients

• The blood being drawn for sample is bright red (arterial) rather than
venous (Fig. 14.9). Apply firm pressure for more than 5 minutes.

LABELING AND DOCUMENTATION

Label both the sample tubes clearly and accurately with the following
information at the bed side of the patient immediately after the collection
of the sample.
• Patient’s name and family’s name.
• Age and Sex.
• OPD No.
• IPD No.
• Name of patient’s ward.
• Date of admission.
• Signature of the staff drawing the sample and date.

Fig. 14.9: Arterial puncture

54 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

The name and other details of the patient must be cross-checked

with the indoor file and the requisition form. In case of the unconscious
patient, ask his/her relative or a second member of the staff to verify the
patient’s identity.
Do not label the sample tubes before collec­ting the blood sample
because of the risk of putting the patient’s blood into the wrong tube.
Fresh sample is essential to ensure that the patient does not
receive incompatible blood.
In case of newborn baby up to 4 months, send the mother’s
sample also.
The requisition form must be completed and signed by the clinician-
in-charge/RMO. All the details requested on the requisition form must
be completed accurately and legibly.
 C H A P T E R 15
Preparation of Red Cell
Suspension

SCOPE AND APPLICATION

This SOP describes the method of preparation of red cell suspension and
applies to all tests requiring use of red cell preparation.

RESPONSIBILITY
It is the responsibility of Laboratory Technician to prepare the red cell
suspension for the different tests. Suspensions of A, B and O red cell are
prepared daily in the morning.

MATERIALS REQUIRED
Equipment
• Table top centrifuge machine.

Reagents
• Low-ionic strength saline solution (LISS).

Specimen
• Anti-coagulated blood specimen of donor.
• Anti-coagulated blood specimen of patient.
• Anti-coagulated blood specimens of known groups.

Glassware
• Test tubes.

Miscellaneous
• Micropipettes.
• Test tube stand.
56 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

PROCEDURE
Principle
The ratio of serum to red cells may dramatically affect the sensitivity of
agglutination tests. Consistent preparation of 1 to 5% red cell suspension
is critical to any agglutination test.

Pooled Cell Suspension

1. Label tubes with A, B, and O groups.
2. Put 1 drop of red cells each from 3 of A group sample tubes or seg­
ment into the A labeled tube.
3. Put 1 drop of red cells each from 3 of B group sample tubes or seg­
ment into the B labeled tube.
4. Put 1 drop of red cells each from 3 of O group sample tubes or seg­
ment into the O labeled tube.
5. Fill all the tubes 3/4th full with 0.9% saline to resuspend the cells.
6. Centrifuge the tubes for 2 minutes at 2000 rpm. Decant the super­
natant fluid.
7. Repeat steps 5 and 6 twice and this gives properly washed cells. The
3rd washing should be in low-ionic strength saline solution; (LISS)
8. Now with the use of semi-automatic micropipette, take 10 ul of the
packed RBCs and mix in 1000 ul of LISS to make 1% suspension. For
making 5% suspension of RBCs, take 25 ul of the packed RBCs and
mix in 500 ul of LISS.
9. Test the suspension of pooled cells thus prepared using the anti-
sera (anti-A, B, AB and D).
10. Donor/Patients’ Cell Suspension: Centrifuge the blood sample at
2000 rpm for 10 minutes. Take 10 ul of packed RBCs and mix in 1000
ul of LISS to make 1% suspension. For making 5% suspension of
RBCs, take 25 ul of the packed RBCs and mix in 500 ul of LISS.
11. The cells should be kept at 4±2°C in the refrigerator when not in use.

Note
Hemolysis of the red blood cells from improper washing may result in false
results. A cell suspension that is too heavy or too light may produce false positive
or false negative results.
Cells are washed in isotonic saline to remove all traces of plasma or serum.
They must then be accurately diluted to give a standard sus­pension in saline/
low-ionic strength saline solution (LISS).
 C H A P T E R 16
ABO Blood Grouping

SCOPE AND APPLICATION

This SOP describes the method to determine the correct ABO group
of an individual and ensure the reliability of the result. This procedure
describes the method of detection of ABO antigens on the red cell and
the reciprocal antibodies in the serum (Landsteiner’s Law). It provides
guidance for the use of blood grouping reagents (anti-sera and standard
red cells) in order to detect weak variants, acquired antigens, Bombay
(Oh) blood group and irregular red cell antibodies.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the blood bank
under the supervision of the Medical Officer to perform the ABO grouping
of donors and patients. Both red cell testing (Forward Grouping) and the
serum testing (Reverse Grouping) must be performed; except umbilical
cord blood and peripheral blood typing for infants less than 4 months
of age and RBC unit confirmation, where in only a forward grouping is
performed. Reaction with the reverse grouping cells may be obtained
if these cells contain an antigen for which a cold-active alloantibody
is present in the patient’s plasma other than anti-A or anti-B. Where
possible, the reverse group should be repeated at a higher temperature
or using reverse grouping cells that lack the implicated antigen. When
the interpretation of the forward and reverse reactions differs, the “ABO
discrepancy” must be resolved by further testing.

EQUIPMENT
• Refrigerator to store blood samples and reagents/Diagnostic kits at
2–6°C.
• Table top centrifuge.
• Microscope.
• Incubator.
58 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Specimen
• Clotted and anti-coagulated blood samples of donor/patient.
• Test red cells suspension.

Reagents
• Anti-A, Anti-B, Anti-AB anti-sera.
• Group A, B and O pooled cells.
• 0.9% saline.
• Distilled water.

Glassware
• Serum tubes.
• Micro-tubes.
• Pasteur pipettes.
• Glass slides.

Miscellaneous
• Rubber teats.
• Disposal box.
• Sticks.
• 2 plastic beakers.
• Test tube racks.

PROCEDURE
Principle
ABO system is the only system in which there is a reciprocal relationship
between the antigen on the red cells and the naturally occurring anti­
bodies in the serum. Routine blood grouping of donors and patients
must therefore include both RBC and serum tests, each serving as check
on the other. The procedure is based on the principle of agglutination of
antigen positive red cells in the presence of antibody directed towards
the antigens.
Three manual methods can be used for blood grouping:
• Glass slide or white tile.
• Glass test tube.
• Microwell plate or Microplate.
Glass slide or white tile method may be used for emergency ABO
grouping tests or preliminary grouping particularly in an outdoor camp;
 ABO Blood Grouping 59

however it should always be supplemented with a cell and serum

grouping either by tube or microplate technique. It is not recommended
for routine use because it is not reliable for:
i. Weakly reactive antigens on cells
ii. Serum grouping with low titre anti-A or anti-B
Besides there is drying effect which can cause aggregation of cells,
giving false positive results and weaker reactions are difficult to interpret.

Glass Slide or White Tile Method

1. Place 1 drop of anti-A and 1 drop of anti-B reagent separately on a
labeled slide or tile.
2. Add 1 drop of 20% test red cell suspension to each drop of the typing
antiserum (the suspension may be prepared by adding 20 parts of
red cells to 80 parts of normal saline).
3. Mix the cells and reagent using a clean stick. Spread each mixture
evenly on the slide over an area of 10–15 mm diameter.
4. Tilt the slide and leave the test for 2 minutes at room temperature
(22°–24°C). Then rock again and look for agglutination (Fig. 16.1).
5. Record the results.

Interpretation: Agglutination Indicates a Positive Result

Fig. 16.1: Slide method for blood grouping

60 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Glass Test Tube Method

Forward grouping
a. Set the table and prepare the workbook.
b. Cross-check the identity of the blood sample on the labels,
requisition form and the work book.
c. Prepare 5% suspension of red blood cells.
d. Label 3 tubes for Anti-A, Anti-B, Anti-AB.
e. Add one drop each of the anti-sera to the marked tubes.
f. Add one drop of the red cell suspension to each of the tubes contain­
ing anti-sera.
g. Put up control in the fourth tube (Add one drop of serum and red
cell suspension of the sample to be tested).
h. Mix thoroughly by shaking the tubes.
i. Incubate at room temperature (RT) for 10 minutes.
j. Centrifuge the tubes at 1000 rpm for 1 minute, remove the tubes
and observe for hemolysis in the supernatant. Then, shake the tube
to find out if there is agglutination.
k. If there is no visible agglutination, transfer a part of the contents of
the tube to a microscope slide and examine under a low power lens
for agglutination.
l. Record the grading of reactions on the appropriate worksheet.
m. Do not discard the tubes (They may be required to be checked later).

Reverse grouping
a. Set the table and prepare the workbook.
b. Mark three tubes for A-Cells, B-Cells and O-Cells.
c. Add one drop of the unknown serum to be tested to each of these
tubes.
d. Add one drop of “Pooled Cells” from the panel of A1-cells, B-cells
and O-cells to the respective tubes.
e. Mix well by shaking the tubes
f. Incubate at RT for 30 minutes
g. Centrifuge the tubes at 1000 rpm for 1 minute, remove the tubes
and observe for hemolysis in the tubes. Then, shake the tubes to
find out if there is agglutination.
h. If there is no visible agglutination, transfer a part of the contents of
the tube to a microscope slide and examine under a low power lens
for agglutination.
i. Record the grading of reactions on the appropriate worksheet.
j. Do not discard the tubes (They may be required to be checked later).
 ABO Blood Grouping 61

Defining the Strength of Reaction (Grading of Agglutination)

To record the difference in the strength of reaction, it is necessary to
have a system of grading or scoring the reactions, as depicted in the
table. (Table 16.1)
Table 16.1: Grading of agglutination
Grades Description of agglutination
4+ One solid agglutinate
3+ Several large agglutinate
2+ Medium size agglutinate, clear background
1+ Small agglutinate, turbid background
1+w Very small agglutinate, turbid background
W+ or +/- Barely visible agglutination, turbid background
Zero/Negative No agglutination
mf Mixtures of agglutinated and unagglutinated red cells
H Complete hemolysis (positive reaction)
Ph Partial hemolysis, some red cells remain

INTERPRETATION
The interpretation of blood group is done as given in Table 16.2
Table 16.2: Interpretation of blood group
Cell grouping (Forward Forward Serum Reverse
Grouping) interpretation grouping inter-
(Reverse pretation
Grouping)
Anti-A Anti-B Anti-AB Ac Bc Oc
+ – + A – + – A
– + + B + – – B
– – – O + + – O
+ + + AB – – – AB
– – – O + + + Oh or any
other
irregular
antibody
62 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Discrepancies Between Cell and Serum Results

If there is a discrepancy between the forward and reverse grouping,
when the reactions in the forward grouping do not match the reactions
in the reverse grouping or where expected reaction grading are less
than grade 3 in the forward group and grade 2 in the reverse group or
where there is discrepancy between historical results and current test
results, an interpretation must not be concluded but the results must be
recorded and the “ABO discrepancy” must be resolved by further testing.

Precautions
Never rely on your memory.
In case you have been distracted halfway through the test and do
not remember where you had left it, run the entire test again, from the
beginning.
Always put serum in the tubes first, check for its presence in the
tube before you add cell suspension to the same tube.
Whenever possible, let someone else check your results as well as
record.
 C H A P T E R 17
Resolution of ABO Group
Discrepancy

SCOPE AND APPLICATION

To resolve an ABO grouping discrepancy when the reactions in the
forward grouping do not match the reactions in the reverse grouping or
where expected reaction gradings are less than grade 3 in the forward
group and grade 2 in the reverse group or where there is discrepancy
between historical results and current test results.
ABO interpretations must be delayed until the discrepancy is
resolved. If emergency transfusion is indicated, give group O RBCs of
the appropriate D type.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell
serology laboratory to resolve the ABO discrepancy under guidance of
the Blood Bank Medical Officer.

MATERIALS REQUIRED
Equipment
• Table top centrifuge.
• Microscope.
• Test tubes.
• Rack for test tubes.
• Incubator and refrigerator.

Specimen
• Clotted or anti-coagulated whole blood samples.

Reagents
• Screening panel and/or donor cells.
• Anti-A, Anti-B, Anti-AB.
64 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Anti-A1 lectin.
• A1, A2 and B cells.
• Low-ionic strength saline solution (LISS).

PROCEDURE
General Considerations
i. ABO grouping is not reported in case:
• If the strength of reactions is less than grade 3+ in the forward
group and grade < 2+ in the reverse group.
• If the previous and current ABO group does not correspond.
• If any ABO discrepancy is found.
ii. Technical errors causing false negative reactions may be caused by:
• Failure to add serum or antiserum to a test.
• Failure to identify hemolysis as a positive reaction.
• Not using the appropriate serum (or reagent) to cell ratio.
• Improper centrifugation.
• Incubation of tests at temperatures above 20–25 C.
• Use of inactive reagents.
• Failure to interpret or record test results correctly.
iii. Technical errors causing false positive reactions may be caused
by—
• Over-centrifugation.
• Use of contaminated reagent antibodies, RBCs or saline.
• Use of dirty glassware.
• Incorrect interpretation or recording of test results.
• Over-centrifugation.
iv. Problems associated with testing red blood cells (forward type)
• False Positive.
■ Acquired antigens (B, A).
■ Poly-agglutination.
■ B (A) phenomenon.
■ Post-transfusion/transplantation of different Blood Groups.
■ DAT positive sample/Auto Abs.
■ Abs (Antibodies) to dyes in typing reagents.
• False Negative.
■ Antigen expression decrease (e.g.AML).
■ A subgroups (A3, Ax).
v. Problems associated with the serum testing (reverse type).
• False Positive.
■ Small fibrin clots.
■ Rouleaux.
 Resolution of ABO Group Discrepancy 65

■ Patients with abnormally high levels of abnormal serum

proteins or who have received plasma expanders.
■ Antibodies other than anti-A or anti-B.
■ Antibodies to chemical constituents of the diluents used to
preserve the reverse cells.
■ Bone marrow transplant from ABO non-identical donor.
■ Very weak or negative reactions from patients with immunod­
eficiency due to disease, therapy, depressed immunoglobulin
levels, elderly patients, or patients who have received large
amounts of IV fluids.
■ Unexpected reactions when patient receives sufficient vol­umes
of blood components containing plasma of an ABO group other
than their own.
• False Negative.
■ Negative or weak reactions on specimens from infants 4–6
months of age.
■ Very weak or negative reactions from patients with immuno­
deficiency due to disease, therapy, depressed immunoglobulin
levels, elderly patients, or patients who have received large
amounts of IV fluids.

RESOLVING ABO DISCREPANCIES

i. When a discrepancy occurs, important factors to be considered are:
• Patient’s age.
• Diagnosis.
• Transfusion history.
• The specimen used for testing and technique.
ii. Next course of action should always be to repeat ABO group testing
since washed red cells may reduce false positive results associated
with Rouleaux or auto-antibodies and to ascertain the technique.
iii. Recheck the suitability of specimen, e.g. specimen contaminated
with I/V fluids may give weak reactions in the reverse grouping. If
the sample is hemolyzed, interpretation cannot be made because
it is difficult to see reactions if they are weak or if a hemolyzing
antibody is present. Using a fresh, unhemolyzed specimen in this
situation is best, so that proper interpretation can be carried out.
In case of any doubt about the identity or the quality of the
specimen, collect a new specimen and repeat the test.
iv. If discrepancy persists:
a. If the patient appears to be group A test the RBCs with anti-A1
lectin and serum with A2 cells. If the patients are negative with
66 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

the lectin they are of the subgroup A2, and if the serum is negative
with the A2 cells, this shows they have anti-A1 in their serum.
• Most commonly encountered discrepancy.
• Test with additional A2 cells to confirm specificity due to anti-A1.
b. Incubate at RT for 30 minutes for detection of weakened
antibodies or antigens. It is frequently needed for elderly patients.
Can also incubate at 4°C but must run an auto-control. Cold
antibodies are frequently encountered in tests performed at 4°C.
c. Test the serum against group O adult, group O cord and a patient
auto-control (Patient serum plus patient cells) to determine if
cold reactive antibodies are interfering with testing (also fairly
frequently encountered).
d. Wash the patient and reagent RBCs several times.
e. Obtain a new specimen.
v. Identify whether the problem is in forward or reverse grouping.
Consider the following:
• ABO reactions are usually strong. Suspect that weak reactions are
questionable.
• Problems in reverse grouping are more common.
• More than one problem may exist, e.g. a weak subgroup of A may
have anti-A1 in serum.
The various possibilities are:
a. Forward Grouping has expected positive weak (< 3+) or Missing
reaction(s)
b. Forward Grouping has unexpected or extra reaction(s)
c. Reverse Grouping has expected positive weak (< 2+) or Missing
reaction(s)
d. Reverse Grouping has an unexpected or extra reaction(s).
a. Forward Grouping has expected positive weak (< 3+) or missing
reaction(s)
This means that cell typing indicates weak reaction but match the
reaction in reverse grouping the various possibilities are:
• Subgroups of A or B.
• Sepression in H substrate production.
• Depression of antigen production associated with disease.
• Presence of 2 cells population (chimeras or in-patients receiving
high amount of type compatible blood).
• Check if the recipient has been transfused with non-group
specific RBC components in the last 3 months (or history of
transplantation).
 Resolution of ABO Group Discrepancy 67

• Check for cloudiness of the supernatant in the anti-A and/or

anti-B tubes. Read the ABO tubes microscopically checking for
mixed field reaction(s).
If mixed field is noticed and the recipient has been transfused with
non-group specific RBC components in the last 3 months:
• Perform an ABO grouping on a specimen collected before the
transfusion of non-group specific donor unit(s), if available.
• If a pre-transfusion specimen is not available and the discrepancy
is still not resolved, a report should be sent stating “ABO cannot
be determined at this time”. If blood components are required,
group O cellular and group AB plasma components should be
transfused.
• Record ABO group performed on both specimens.
• Report the ABO group performed on the pre-transfusion
specimen.
• When a recipient has been transfused with non-group specific
blood, group specific blood may not be compatible due to
passive ABO antibodies. In these cases, non-group specific, group
compatible blood should be cross-matched until anti-A and/or
anti-B is no longer demonstrable by IAT cross-match.
If mixed field agglutination is noticed and the recipient has not
been transfused in the last 3 months, review the diagnosis.
The possible causes are:
• Recent transfusion with non-group specific donor units.
• ABO grouping from recipients who have received an allogeneic
bone marrow or hematopoietic stem cell transplant may give
mixed field results during the transplant period. For some
recipients, the mixed field will remain indefinitely.
• Feto-maternal hemorrhage.
• Weak subgroups (e.g., A3); other subgroups of A or B may not react
as mixed field with anti-A and/or anti-B.
• Altered expression of A and/or B antigens due to disease when the
previous ABO grouping does not agree with the current grouping
and the recipient diagnosis suggests a reason for the change.
• Poly-agglutinable red cells such as Tn-activated cells. The reverse
grouping fails to confirm the cell grouping.
• Twin: Chimerism (very rare).
If there is no mixed field agglutination, wash the recipients cells
2 times and repeat the forward grouping on the washed cell
suspension.
• If the discrepancy is resolved by additional washing, excess blood
group substance in recipient serum or plasma neutralizing anti-A
or anti-B (e.g. mucin producing adenocarcinomas) may be the
possible cause.
68 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• If the discrepancy is not resolved, a report should be sent stating

“ABO cannot be determined at this time”. If blood components
are required, group O cellular and group AB plasma components
should be transfused.
b. Forward Grouping has an unexpected or extra reaction(s) with
anti-A and/or anti-B:
• Repeat blood grouping after washing the red cells twice. If a cold-
reactive agglutinin is suspected, use saline warmed to 37°C to
wash the cells.
• Re-suspend the cells to 3%.
• Repeat the ABO grouping on the 3% washed cells suspension.
• Record that the second ABO grouping has been done on a 3%
washed cell suspension (and, if applicable, with saline warmed to
37°C).
This will resolve the discrepancy if it is due to:
■ Strong cold auto-agglutinin (if washed with 37°C saline).
■ Rouleaux.
■ Wharton’s jelly.
■ Arti-factual - fibrin, other debris.
■ Presence of anticoagulant (anti-complementary).
If the problem remains and the recipient appears to be group
AB, consider acquired B antigen. In this case:
• Antigen types the recipient cells using anti-A1 lectin. Acquired B
antigen red cells type as A1.
• Prepare acidified anti-A and anti-B reagent:
– Add 1 drop of 0.1 N HC1 to 4 drops of commercial anti-A.
– Check pH.
– Adjust to pH 4.5 by adding 0.1 N HC1 or anti-A.
• Repeat grouping with acidified (pH below 6.0) anti-A and anti-B.
Extra reaction in the forward grouping should be resolved if
acquired B antigen.
• Set up an auto-control. The auto-control shall be negative to
confirm the presence of an acquired B antigen.
• If acquired B antigen has been excluded or if the recipient does
not appear to be group AB, try repeating the ABO group using one
of the following:
– Anti-sera from a different manufacturer.
– Monoclonal anti-sera.
– Eluted recipient cells.
• If the discrepancy is still not resolved, a report should be sent
stating “ABO cannot be determined at this time”. If blood
 Resolution of ABO Group Discrepancy 69

components are required, group O cellular and group AB plasma

components should be transfused.
c. Reverse Grouping with weak or missing reactions: Cau­­ses for
weak (i.e., weaker than 2+) or missing reactions in the rev­erse
grouping:
• Neonatal recipients sometimes do not demonstrate anti-A and/or
anti-B until 4 to 6 months of age.
• If the recipient is elderly or immune suppressed, suspect hypo-
gammaglobulinemia or agamma-globulinemia.
• The possibility that the recipient has received a bone marrow
or hematopoietic stem cell transplant (e.g., a group A recipient
who has received a group O transplant will type as group O in
the forward grouping but may not have an anti-A). In this case,
follow the transfusion protocol of the facility where the transplant
occurred.
Perform the following enhancement method(s).
Enhancement Method #1: (addition of more serum/plasma).
• Add two additional drops of recipient serum or plasma to the A1
and B cells tubes.
• Mix and centrifuge for optimal time. Re-suspend, read macro­sco­
pi­cally, grade and record results.
• If reaction is not enhanced to at least 2+, increase the incubation
time to 30 minutes at room temperature. Mix and centrifuge for
optimal time. Re-suspend, read macroscopically.
• Grade and record results.
If the reaction(s) is enhanced to at least 2+:
• Record the enhancement method on the request form (i.e., 4
drops of serum/plasma).
• Confirm the ABO of donor units.
• Interpret the ABO group.
• If the reaction is still weaker than 2+: Do not discard the tubes (A1
and B cells) and proceed with enhancement method #2.
Enhancement Method #2: (incubation at 40°C).
• Set up an auto-control and antibody screen cells.
• Label tubes with recipient name, auto, and name of the screen
cell, as appropriate.
• Add 2 drops of serum or plasma to all tubes. If using the A1 and B
tubes from enhancement method #1 that contain 4 drops of serum
or plasma, add 4 drops serum or plasma to the auto-control and
antibody screen cell tubes.
• Add 1 drop of the appropriate cell suspension to each tube.
• Mix.
70 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Incubate the tubes for A1, B, auto-control, and antibody screen

cells at 40°C for 15 minutes.
• After incubation, centrifuge for optimal time. Re-suspend the
cells, read macroscopically, grade and record results.
a. If the auto-control and screen cells are negative and the expected
reactions with A1 and/or B cells are Enhanced to at least 2+ (i.e.,
forward agrees with reverse grouping):
• Record the enhancement method on the request form (i.e. 40°C
incubation).
• Confirm the ABO of donor units.
• Interpret the ABO group.
b. If the auto-control, A1, B, and all screen cells are positive and the
diagnosis is viral or mycoplasma pneumonia or cold agglutinin
disease, suspect an auto-anti-I.

Note
Many normal people have an auto-anti-I that reacts at 40°C but only rarely at RT
(22°C), and not usually above 15°C.

Repeat the reverse grouping at 37°C.

• If the expected reaction(s) with A1 and/or B cells at 37°C is 2+ or
stronger and the ABO discrepancy is resolved:
• If the reaction(s) with A1 and/or B cells at 37°C is weak or 1+ and/
or the ABO discrepancy still exists: Repeat the reverse grouping
using cold auto-adsorbed serum or plasma.
• If the discrepancy is still not resolved, a report should be sent
stating “ABO cannot be determined at this time”. If blood
components are required, group O cellular and group AB plasma
components should be transfused.
c. If extra reactions occur in the reverse grouping and one or more
of the screen cells (auto-control is negative).
Perform Antibody Identification of Cold Reactive Antibodies
If the antibody identification demonstrates a specific cold reactive
allo-antibody (e.g., Anti-M, - Lea, -Leb, -Pi, -N), repeat the reverse
grouping at 37ºC. If the expected reaction(s) with A1 and/or B cells
is 2+ or stronger at 37ºC, and the discrepancy is resolved, interpret
the ABO group.
If the expected reaction(s) with A1 and/or B cells is weaker than 2+
at 37ºC, repeat the reverse grouping using Ai and B cells that are
lacking the antigen to the identified antibody (37°C not necessary).
Reverse grouping cells lacking the antigen maybe selected by
 Resolution of ABO Group Discrepancy 71

antigen typing group A and B donor units with the corresponding

commercial anti-sera.

Note
ABO group should not be reported when expected positive reactions with A1
and/or B cells are weak or 1+ by saline replacement or pre-warm technique. If
the discrepancy is still not resolved a report should be sent stating “ABO cannot
be determined at this time”. If blood components are required, group O cellular
and group AB plasma components should be transfused.

d. Reverse Grouping with Unexpected or Extra Reactions: If the

reverse grouping has an unexpected or extra reaction(s) with A1
and/or B cells, suspect
• Rouleaux.
• Cold-reactive auto-antibody.
• Anti-A1.
• Irregular cold allo-antibody: Anti-M, -N, -Pi, -Lea, etc.
If the recipient’s forward group suggests group A or AB with
an unexpected reaction in the reverse group with A1 cells, the
most likely cause of a discrepancy in the reverse group is anti-A1,
therefore, it should be investigated first.
■ Type the recipient’s red cells with anti-A1 lectin.
■ If the reaction is positive with anti-A1 lectin the recipient is group
A1 and cannot have anti-A1 - proceed to set up an auto-control
and antibody screen cells.
■ If the reaction is negative with anti-A1 lectin, set up the following
by saline room temperature method: Setting up 3 A1 and 3 A2 cells
will provide an acceptable level of probability that the antibody is
anti-A1 should all A1 cells and no A2 cells react.
■ Label tubes with recipient name, auto, and name of the screen
cell, as appropriate.
• Add 2 drops of serum or plasma to all tubes.
• Add 1 drop of the appropriate (3 A1 and 3 A2 cells, auto-control and
antibody screen cells) cell suspension to each tube.
• Mix and incubate for 30 minutes at room temperature.
• After incubation, centrifuge for optimal time, re-suspend, and
read macroscopically.
• Grade and record results.
■ If anti-A1 has not been identified (auto is negative and one or
more screen cells are positive suspect a cold allo-antibody.
Perform antibody identification of Cold Reactive Antibodies.
If the antibody identification demonstrates a specific cold
reactive. Allo-antibody (e.g., anti-M, - Lea, -Leb, -Pi, -N), repeat
72 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

the reverse grouping at 37°C. If the expected reaction(s) with A1

and/or B cells is 2+ or stronger at 37°C, and the discrepancy is
resolved, interpret the ABO group.
■ If the expected reaction(s) with A1 and/or B cells is weaker than
2+ at 37°C, repeat the reverse grouping using Ai and B cells that
are lacking the antigen to the identified antibody (37°C not
necessary). Reverse grouping cells lacking the antigen may be
selected by antigen typing group A and B donor units with the
corresponding commercial anti-sera.

Note
ABO group should not be reported when expected positive reactions with A1
and/or B cells are weak or 1+ by saline replacement or pre-warm technique. If
the discrepancy is still not resolved a report should be sent stating “ABO cannot
be determined at this time”. If blood components are required, group O cellular
and group AB plasma components should be transfused.

The flow chart for resolving ABO discrepancies is given at Annexure

17.1 and examples of ABO discrepancies along with their possible
resolution is given at Annexure 17.2.
Nowadays, employment of molecular blood group diagnostics for
the genetically exact definition of blood group variants (e.g. RHD and
ABO), clarification of original genotypes in poly-transfused sample
material, or antibody-masked erythrocytes are generally accepted.
 Annexure 17.1: Resolving ABO Discrepancies Chart
Perform clerical check. Check patient history, age, medications and diagnosis. Check for technical problems–
Repeat Testing
If discrepancy remains–
Unexpected Negative Unexpected Positive
Reverse Forward Reverse Forward
Incubate at room temperature for 15–30 Perform antibody screen w/ Wash cells w/warm saline and repeat testing
minutes auto-control
Incubate at 4°C All cells positive Al+(unexpected Screen- Resolved? Unresolved?
for 15–30 minutes Screen-
include auto-control Auto) Auto-
Increase Switch to Rouleaux Auto- Subgroup of Allo- Rouleaux Mixed field Cells+w/all
serum different Saline antibody Aw/Anti-A1 antibody Wharton’s Reactions? antisera?
cell ratio anti-serum replace- Cold Test RBCs w/ Panel to Jelly Transfusion Polyagglutinable
ment Auto- A1 lectin ID Cold or HPC cells
adsorption agglutinin Transplant Verify w/control
Prewam Repeat Repeat & lectins
Read microscopically Grouping w/A2 reverse Check by Switch to
cells grouping Acquired B monoclonal
and antigen “Autologous“ reagents
Treat RBCs with neg RBCs
enzymes anti-B
Acidified
Absorption anti-B
/eluticn
Secretor
Resolution of ABO Group Discrepancy

studies
73
 Annexure 17.2: ABO Discrepancies and Possible Resolution 74

Forward Grouping Reverse ‘O’ Auto- Possible cause Resolution

Grouping cells control
Patient Anti-A Anti-B A1 calls B call
1 0 0 0 0 0 0 Group O newborn; elderly patient; Incubate tests at 4°C, check age of
Very low immunoglobulin levels patient
2 4+ 4+ 2+ 2+ 2+ 2+ Rouleaux; cold Wash RBCs and repeat testing test
autoantibody for cold antibodies
3 4+ 0 1+ 4+ 0 0 Probable A2 subgroup with anti A1 Test with anti-A1 and anti-H lectins
and A2 cells
4 4+ 4+ 1+ 0 0 0 Probable A2-B sub-group with Test with anti-A1 and anti-H lectins
anti-A1 and A2 cells
5 0 0 4+ 4+ 4+ 0 Probable O2 (Bombay) Test with anti-H lectin may be sent
to reference lab for confirmation
6 4+ 2+ 0 4+ 0 0 Group A with an acquired B Check history for lower GIT problem
phenotype or septicemia; Test with acidified
Anti-B serum test serum with
autologous cells
7 0 4+ 4+ 1+ 1+ 1+ Group B with cold antibody Test for cold antibodies and identify
if appropriate
8 4+ 4+ 2+ 0 2+ 0 Group AB with alloantibody Test for cold antibodies and identify
if appropriate
9 4+ 0 0 4+ 3+ 0 A1 with potent anti-H Confirm A1 with anti-A1 lectin test
additional A2, O and A1 cells and an
oh if available
10 0 0 2+ 4+ 0 0 A subgroup probably Ax with Perform saliva studies or absorption
elution
Standard Operating Procedures and Regulatory Guidelines-Blood Banking

anti-A1
 C H A P T E R 18
Absorption and Elution (For Weak
Subgroups of A or B)

SCOPE AND APPLICATION

This is a method for confirming the presence of weaker antigens–A
and B on the surface of red cells. The red cells are incubated with the
appropriate anti-sera to absorb the antibody, i.e. known antibody
is incubated with unknown RBCs to see if the cells will take up the
antibody. The antibody on the red cells and the antibody present in the
antiserum form antigen-antibody complex in other words the part of
antibody is absorbed on the red cells. After absorption and subsequent
washing away of unbound serum, the combined antibody is eluted,
usually by raising the temperature to about 56°C. The elute is then tested
with indicator cells of the appropriate group; agglutination indicates the
presence of an antigen of the same specificity as that of the indicator
cells.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell
serology laboratory to perform the absorption and elution test for
detection of weak antigens of A or B group under direct supervision of
Medical Officer.

MATERIALS REQUIRED
Equipment
• Refrigerator to store samples and reagents at 2–6°C.
• Table top centrifuge.
• Microscope.
• Incubator/dry bath.
• Water bath.

Specimen
• Clotted or anti-coagulated blood samples.
76 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Reagents
• Anti A1/Anti B.
• Pooled O group red cells.
• Pooled A1 cells/Pooled B cells.
• Normal saline.

Glassware
• Serum tubes.
• Microtubes.
• Pasteur pipettes.
• Glass slides.

Miscellaneous
• Rubber teats.
• Disposal box.
• 2 plastic beakers.
• Wooden block to hold microtubes.
• Aluminium racks to hold serum tubes.

PROCEDURE
Absorption and Elution
i. Wash 1 ml of cells to be tested at least 3 times with saline. Discard
the supernatant after last wash.
ii. Add 1 ml of anti-A1 to red cells if weak variant of A suspected or 1 ml
of anti-B if weak variant of B is suspected.
iii. Mix the cells with anti-sera and incubate at room temperature for
one hour.
iv. Centrifuge the mixture and discard the supernatant anti-sera.
v. Centrifuge the mixture and discard the supernatant for a minimum
of 5 times with a large volume of saline (10 ml or more). Save the
supernatant of the fifth wash to test for free antibody.
vi. Add an equal volume of saline to the washed and packed cells and
mix.
vii. Elute the adsorbed antibody by placing the tube at 56°C in water
bath for 10 minutes and mix the red cell saline mixture at least once
during this period.
viii. Centrifuge and remove the cherry colored elute and discard the
cells.
 Absorption and Elution (For Weak Subgroups of A or B) 77

Testing the Elute

i. If anti-A was used, test the elute against three different samples of
A1 cells and 3 group O cells at room temp at 37°C.
ii. If anti-B was used, test the elute against three different samples of B
cells and 3 group O cells at room temp at 37°C.
iii. Test the fifth saline wash in the same manner to show that the
washing has removed all antibody not bound to the cells.

INTERPRETATION
If the elute agglutinates or reacts with specific A or B cells and does
not react with O, cells tested have active A or B antigen on their surface
capable of binding with specific antibody.
If the elute also reacts with O cells, it indicates nonspecific reactivity
in the elute and the results are not valid.
If fifth saline wash material is reactive with A and B cells, the results
of the test made on elute are not valid because it indicates that active
antibody was present in the medium unattached to the cells being
tested.
 C H A P T E R 19
Rh Blood Grouping

SCOPE AND APPLICATION

Routine Rh grouping of red cells involves D-antigen testing in patients
and D & Du antigen testing in donors.
However, tests for other important Rh antigens, e.g. C, c, E and e
may be done for Rh genotyping when there are specific reasons.
The method of Rh grouping mainly depends on the type of reagents
available, for which the manufacturers’ instructions have to be strictly
followed. However, this SOP provides guidance for the use of anti-D
blood grouping reagent.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the blood bank
under supervision of Medical Officer to perform the D typing of donors
and patients using one monoclonal and one bi-clonal reagent. If a
discrepancy is encountered between the two batches of anti-D, the test
should be repeated by the other technician. If the discrepancy persists,
the Medical Officer should be informed. If results of D typing of a blood
donor are negative, the technician should proceed with Du typing
procedure.

MATERIALS REQUIRED
Equipment
• Refrigerator to store samples and reagents at 2–6°C.
• Table top centrifuge.
• Microscope.
• Incubator.

Specimen
• Clotted or anti-coagulated blood samples of donors.
 Rh Blood Grouping 79

• Clotted blood sample of patients.

• Red cells suspension.

Reagents
Different types of anti-Rh (D) sera
i. Polyclonal human anti-D serum (IgG): Potentiating or enhancing
substances such as albumin, enzymes and AHG reagents are used
to bring about agglutination with human IgG anti-D.
• Anti-D serum (IgG) for saline or rapid tube test (high protein
medium). This contains macromolecular additives and gives
reliable results.
• Anti-D for saline tube test are of 2 types.
■ Anti-D IgM.
■ Anti-D IgG-Chemically modified.
ii. Monoclonal anti-D reagents:
• IgM anti-D monoclonal reagent.
• IgM and IgG anti-D monoclonal reagent.
• Blend of IgM monoclonal + IgG polyclonal reagent.
These antibodies are highly specific, react equally well at 20°C
as well as 37°C and are reliable for slide and rapid test tube
technique.
IgM anti-D monoclonal reagent cannot be used for Du testing
by indirect antiglobulin test (IAT) while IgM + IgG monoclonal
reagent and blend of IgM monoclonal and IgG polyclonal can be
used for Du testing.
It is preferable to use potent anti-D reagents from two different
firms following the manufacturers’ recommended technique.
iii. Controls for Rh (D) grouping: Known 0 Rh (D) positive and 0 Rh
(D) negative cells may be used as controls with monoclonal anti-D
reagent.
Alternatively, AB serum or diluent control provided with the anti-D
reagent or 22% bovine serum albumin may be used as negative
control with the test cells.
iv. 0.9% saline.
v. Distilled water.
vi. Glassware:
• Test tubes.
• Pasteur pipettes.
• Glass slides.
• Clean wooden sticks.
80 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

vii. Miscellaneous:
• Rubber teats.
• Plastic beakers.
• Wooden block to hold microtubes.
• Test tube rack.
• Rh view box.

PROCEDURE
Three manual methods can be used for blood grouping:
• Glass slide or white tile.
• Glass test tube.
• Microwell plate or Microplate.

Slide Technique
This technique may be used in emergency for Rh (D) typing if a
centrifuge is not available. The slide test is not recommended for routine
test as it may not pick up weak reactions, thus giving negative results.
IgM monoclonal anti-D reagents work well for the slide technique.
1. Take three slides and label as positive control, negative control and
Test
2. Place 1 drop of anti-D (monoclonal) reagent to each slide
3. Add 1 drop of 20% test red cell suspension to each drop of the typing
anti-serum.
(The suspension may be prepared by adding 20 parts of red cells to
80 parts of normal saline).
4. Mix the cells and reagent using a clean stick. Spread each mixture
evenly on the slide over an area of 10–15 mm diameter.
5. Place the slides on a view box surface (lighted), tilt gently and
continuously for two minutes.
6. Observe for agglutination (Fig. 19.1) and record the results. All
negative results must be confirmed under microscope.

Glass Test Tube Technique

The method depends upon the type of the anti-D reagent available.
Monoclonal anti-D will work in saline at room temperature while others
need to be incubated at 37ºC. Therefore, instructions provided with the
reagents by the manufacturers are to be followed.

Using Monoclonal Anti-D/Saline Agglutination Test

1. Place 1 drop of anti-D (Dl) in clean tube labelled Dl and place 1
drop of anti-D (D2) from a different manufacturer in another clean
tube labelled D2.
 Rh Blood Grouping 81

Fig. 19.1: Slide method for Rh blood grouping

2. Place 1 drop of 22% bovine albumin/control reagent in 3rd tube

labeled C.
3. Add 1 drop of 2–5% test cell suspension to each tube.
4. Mix well and centrifuge at 1000 rpm for 1 minute (in case of using
IgG anti-D, incubate at 37°C for 10 minutes and centrifuge).
5. Re-suspend the cell button and look for agglutination. All negative
results must be confirmed under microscope.

Interpretation
Positive test: Agglutination in anti-D (both tubes) and smooth
suspension in control tube.
Negative test: Smooth suspension of RBC button in all the tubes (test
and control) is a negative test result.
Test is invalid if both test and control tubes show a positive reaction
or conflicting results in tubes Dl and D2. In such case, the test should be
repeated using saline IgM anti-D.
For all microscopically negative reactions in donor grouping, Du
testing must be performed.

Albumin Technique for Rh (D) Typing

Principle
Albumin increases the dielectric constant of the medium and thus
reduces the zeta potential. Due to this effect, the electrical repulsion
82 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

between the red blood cells is less and the cells agglutinate. Mostly 22%
bovine albumin is used, as higher concentrations can cause Rouleaux
formation.

Procedure
1. Place 1 drop of anti-D in a labeled tube.
2. Add 1 drop of 2–5% test red cell saline suspension.
3. Incubate at 37°C for 45–60 minutes.
4. Allow 1 drop of 22% albumin to run down the inside wall of the tube.
Albumin will form a layer on top of the red cells. Do not mix.
5. Incubate further at 37°C for 15–20 minutes.
6. Examine for agglutination after gentle shaking and confirm all
negative results under microscope.

INTERPRETATION OF RESULTS (TABLE 19.1)

The anti-sera are prepared to give a 2+ to 4+ reaction with the anti-D
either after immediate spin or a weak D test, and negative reaction (O)
with any controls needed. Further investigation and testing is necessary
for any test that does not result in a 2+ to 4+ reaction.
Table 19.1: Immediate spin interpretation
Anti-D Rh control Interpretation Comment
+* 0 Rh positive
0 0 Rh negative Continue with weak D test
+ + Invalid test See discrepancies (below)

*If the strength of reaction with the test cells is not 2+ or greater, perform a weak
D test on the cells.
 C H A P T E R 20
Rh Du Blood Grouping

SCOPE AND APPLICATION

After the A and B antigens, serologic determination of D antigens status
is the most important in transfusion practice and sometimes become
problematic due to various factors like the use of different methods,
different reagents of various manufacturers and the variability of the D
antigen expression on some RBCs leading to typing discrepancies. The
variants of the D antigen can be week D (due to reduced number of D
antigen sites on RBCs but D antigen has all the epitopes) and partial D
(qualitative variant, i.e. D antigen is characterized by the absence of one
or more epitopes but there is no difference in the number of sites on
RBCs). It is important to identify a donor as partial D or weak D as the
red cells of these donors could elicit an immune response if transfused
to a negative recipient. But a recipient of partial D or weak D can be
safely considered as RhD negative and Rh negative units can be used
for transfusion. But at the same time it is advisable to determine the
correct RhD status of patients because discrepancy in RhD grouping will
arise when these patients may become donors. At blood banking level it
may be difficult to differentiate between partial D and weak D and they
generally termed as weak D. This SOP describes the method of testing
all RhD negative individuals to find out their accurate RhD status as to
whether they are weak D or really RhD negative.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell
serology laboratory to perform the Du typing of donors and patients by
anti-globulin testing using blend of IgM+IgG monoclonal reagent.

MATERIALS REQUIRED
Equipment
• Refrigerator to store samples and reagents at 2–6°C.
84 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Table top centrifuge.

• Microscope.
• Incubator/dry bath.

Specimen
• Clotted or anti-coagulated blood samples of donors.
• Clotted blood sample of patients.
• Test red cells suspended in native serum/plasma or saline.

Reagents
• Blend of IgM+IgG monoclonal reagent.
• Anti-human globulin reagent (AHG-Coombs’ reagent).
• Known IgG sensitized control cells.

Glassware
• Serum tubes.
• Microtubes.
• Pasteur pipettes.
• Glass slides.

Miscellaneous
• Rubber teats.
• Disposal box.
• 2 plastic beakers.
• Wooden block to hold microtubes.
• Racks to hold serum tubes.

PROCEDURE
i. Take 1 drop of anti-D (IgM+IgG monoclonal reagent) in a clean
labeled test tube (T).
ii. Take 1 drop of appropriate diluent control in another tube (C).
iii. Add 1 drop of 2–5% washed test red cell suspension to both the
tubes.
iv. Mix and incubate at 37°C for 45–60 minutes.
v. Centrifuge at 1000 rpm for 1 minute.
vi. Gently suspend the cell button and look for agglutination, if positive
test, no need to proceed as the sample is Rh (D) positive.
vii. If negative, wash the cells 3–4 times with saline and decant the last
washing.
 Rh Du Blood Grouping 85

viii. Add 1–2 drop of anti-human globulin reagent (AHG-Coombs’

reagent). Mix gently and centrifuge at 1000 rpm for 1 minute.
ix. Re-suspend the cell button gently, examine for agglutination. Grade
and record the results.

INTERPRETATION
For valid typing the weak D control must be negative. Interpret results as
follows (Table 20.1).
Table 20.1: Interpretation of Rh Du grouping
Anti-D Control Interpretation
0 0 Rh Negative
2+ to 4+ 0 Rh Positive
1+ 0 Unable to determine
Additional tests required

Positive Positive Unable to determine

Additional tests required

Note
All negative reactions should be confirmed by microscopic examination and
by adding known IgG sensitized control cells, re-centrifuge and re-examine
for agglutination. The presence of agglutination confirms and validates the test
result indicating that the AHG serum added was capable of reacting.

Discrepancies and Problems

i. Improper and inadequate washing of the red cell suspension
may cause pseudo-agglutination due to the presence of serum
macromolecules in the suspension.
ii. The presence of strong autoagglutinins in the patient’s or donor’s
serum may cause agglutination. Proper washing and the control are
designed to prevent or detect this problem.
iii. Antibody coating of the red cell (positive DAT) can cause a false
positive reaction, particularly in the weak D test. A DAT will detect
this occurrence.
iv. A false negative reaction may be seen due to the “blocking
phenomenon”. This occurs most commonly in HDFN due to anti-D.
Since the red cell’s antigen sites are heavily coated with maternal
antibody, they may not react with the antiserum.
86 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

v. False positive or negative reactions may occur in the Rh test due to

technical errors.
vi. RBCs that react with anti-D reagent of a particular make but not
with another may have a partial D antigen.

Resolution
i. If transfusion is required before resolution of discrepant results,
issue Rh negative RBCs or whole blood.
ii. If the immediate spin results are invalid due to a positive Rh control,
repeat the test using a new suspension of RBCs washed twice with
warm saline.
iii. If the weak D test results are invalid due to a positive DAT and a
weak D determination is necessary, treat the patient’s RBCs with
chloroquine or glycine-EDTA and repeat the DAT. If the DAT is
negative the weak D test can be repeated.
iv. Patients with weak D reactions less than 2+ are to be reported as
Rh positive. Such patients should be transfused with Rh negative
cellular components pending serologic and clinical evaluation. In
particular, patients who react with anti-D reagent of a particular
make but not with another may have a partial D antigen. Intimate
immediate supervisor or blood bank medical officer.
v. Donors with weak D test reactions less than 2+ are labeled as Rh
positive.
 C H A P T E R 21
Antibody Screening

SCOPE AND APPLICATION

The objective of antibody testing is to ensure that enough red cells will
survive when transfused for sufficient length of time and the blood is
transfused with safety. Blood grouping/cross-matching done alone,
may not be a suitable way to detect compatibility as this method hardly
detects any incompatibility other than major mismatches. Undetected
antibodies account for the adverse transfusion reactions which could
manifest as immediate/delayed hemolytic transfusion reactions or
could give rise to Hemolytic Disease of Newborn. Therefore, this SOP
describes the method to detect clinically significant antibodies at the
earliest to give sufficient time for the blood bank to find compatible
blood.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell
serology laboratory to perform the antibody screening of the donors and
the patients. If any unexpected blood group antibody is detected, inform
the Medical Officer of the Blood Bank.

MATERIALS REQUIRED
Equipment
• Refrigerator to store samples and reagents at 2–6°C.
• Deep freezer to store enzyme papine cystein in frozen state.
• Tabletop centrifuge.
• Automated cell washer (for patient pre-transfusion and prenatal
testing).
• Microscope.
• Dry bath/Incubator.
88 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Specimen
Clotted blood sample of donor/patient.

Reagents
• Group O pooled cells/Antibody-screening reagent red blood cells
(two or three cells)–commercial reagent source.
• Papain cystein.
• 22% bovine albumin.
• Antihuman globulin reagent (anti-IgG+anti-C3d).
• IgG sensitized control cells.
• 0.9% saline.
• Distilled water.

Glassware
• Serum tubes.
• Coombs’ tubes (for patient pre-transfusion and prenatal testing).
• Microtubes.
• Pasteur pipettes.
• Glass slides.

Miscellaneous
• Rubber teats.
• Disposal box.
• 2 plastic beakers.
• Wooden blocks to hold microtubes.
• Racks to hold serum and Coombs’ tubes.

PROCEDURE
Principle
In this test, pooled O cells or the antibody-screening reagent red blood
cells are combined with serum under investigation. The addition of a
potentiating medium enzyme/albumin helps to promote the interaction
of red cells and antibodies allowing antibody/antigen reactions to occur.
Positive reactions (hemolysis or agglutination) in any tests indicate
the presence of alloantibody or autoantibody in the serum. The saline
test at room temperature (20–25ºC) identifies cold antibodies (anti-M,
anti-N, anti-Lea, anti-Leb, anti P, etc.) The enzyme technique enhances
the reaction of Rh, Lewis and Kidd antibodies but weaken or inactivate
 Antibody Screening 89

certain antigens, i.e. M, N, S, Fya and Fyb. The indirect Anti-globulin Test
(IAT) identifies the warm reacting IgG and complement binding-allo
and auto-antibodies.
The IAT using red cells suspended in low-ionic strength saline sol­
ution (LISS) is considered to be the most suitable for the detection of
clinically significant antibodies, because of its speed, sensitivity and
spec­i­fi­city.

Method
i. Label tubes with donor/patient and test identification.
ii. Add two drops of test serum to each tube.
iii. Add 1 drop of papain cysteine to all tubes labelled ‘enzyme’ (if
enzyme method is being followed).
iv. To each of the tubes labelled ‘saline’ or ‘enzyme/albumin’, add 1
drop of 2% pooled O red cell suspension (or 2% suspension of the
antibody-screening reagent red cells).
v. Add 1 drop of 22% abovine albumin to tubes labelled ‘albumin’ (if
albumin method is being followed).
vi. Add 1 drop of 5% pooled O red cell suspension (or 5% suspension
of antibody-screening reagent red cells) to tubes labelled ‘IAT’,
followed by 2 drops of 22% bovine albumin.
vii. Mix the contents of the tubes gently and incubate for 1 hour for
saline at RT and IAT at 37ºC and incubate for 45 minutes for
enzyme and albumin at 37°C.
viii. Centrifuge saline, enzyme and albumin tests at 1000 rpm for 1
minute.
ix. Examine for hemolysis.
x. Gently re-suspend the red cell button and examine for aggluti­
nation.
xi. Examine all visually negative tests microscopically.
xii. Grade and record test results immediately.
xiii. Proceed to perform anti-globulin phase of the indirect anti-
globulin test on tubes labelled ‘IAT’.
xiv. Wash the cells 3 times with saline. Decant completely after last
wash (washing can be done manually or using automated cell
washer).
xv. Add 2 drops antihuman globulin reagent to the dry cell button.
xvi. Mix well and centrifuge at 1000 rpm for 1 minute.
xvii. Read and record results.
xviii. Add drop IgG sensitized cells to all negative results. This shows a
positive agglutination.
90 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

INTERPRETATION
i. Hemolysis or agglutination in any test may indicate the presence
of an unexpected antibody. A positive antibody screen must be
investigated further for identification of antibody.
ii. No agglutination or hemolysis of the screening cells at any phase of
the test is a negative test result and indicates the absence of a blood
group antibody in the serum or plasma specimen.
iii. After addition of IgG-sensitized cells to a negative test, the presence
of agglutination indicates that the AHG serum added was capable of
reacting and that the negative anti-globulin test is valid.
iv. If IgG-sensitized cells added to confirm the activity of the anti-IgG
show only weak or no agglutination after centrifugation, the test is
invalid and must be repeated.

Note
If tests with all reagent red cells are reactive, the possibility of spontaneous
agglutination should be considered. A control of cells washed three to four times
added to two drops of saline must be non-reactive.
 C H A P T E R 22
Pre-transfusion Testing
(Compatibility Testing)

SCOPE AND APPLICATION

The purpose of pre-transfusion testing is to minimize the risk of blood
transfusion to a patient by selecting blood and blood components
that will have acceptable survival when transfused and will not cause
destruction of the recipient’s red cells.
The term compatibility testing or pre-transfusion and cross-
matching is sometimes used interchangeably; however they should be
clearly differentiated. A cross-match is only part of compatibility testing
which refers to a set of procedures required before blood is issued as
being compatible:
• Review of patient’s past blood transfusion history and records (ABO
and Rh blood group).
• ABO and Rh typing of recipient and donor.
• Antibody screening of recipient and donor’s serum.
• Selection of blood for the patient.
• Cross match.
The cross-match is the final check of ABO compatibility between
donor and patient. This also ensures that there are no antibodies present
in patient’s serum that will react with donor cells when transfused.
This procedure is applied for compatibility testing of all patients
requiring blood transfusion.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician and the Medical
Officer of the blood bank to perform the cross-match to find out the
compatible blood for the patient.

MATERIALS REQUIRED
Equipment
• Refrigerator to store samples and reagents at 2–6°C.
92 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Deep freezer to store enzyme papaine/cysteine in frozen state.

• Tabletop centrifuge.
• Microscope.

Specimen
Clotted blood samples of donors/patients.

Reagents
• Group O pooled cells/Antibody-screening reagent red blood cells
(two or three cells).
• Papain/cysteine.
• 22% Bovine albumin.
• Antihuman globulin reagent (anti-IgG+anti-C3d).
• IgG sensitized control cells.
• 0.9% saline.
• Distilled water.

Glassware
• Serum tubes.
• Pasteur pipettes.
• Glass slides.

Miscellaneous
• Rubber teats.
• 2 plastic beakers.
• Racks to hold serum and Coombs’ tubes.

PROCEDURE
The cross-match testing procedures have been divided into two parts:
• Major cross-match which consists of mixing of donor’s red cells
with recipient’s (patient’s) serum.
• Minor cross-match which consists of mixing of donor’s plasma with
recipient’s (patient’s) red cells.
Most of the blood banks have given up the minor cross-match
because the donor samples are screened before hand for antibodies.
The major cross-match techniques are:
• Saline technique.
• Albumin technique.
• Enzyme technique.
• Indirect anti-globulin technique.
 Pre-transfusion Testing (Compatibility Testing) 93

Saline Technique
i. Mark two small tubes as ‘major’ and ‘minor’.
ii. Add one drop of the recipient’s serum in the tube marked ‘major’.
iii. Add one drop of the donor’s serum in the tube marked ‘minor’. Add
one drop of the donor’s red cells (washed and suspended in saline
to form 2 to 5% suspension). in the tube marked ‘major’.
iv. Cut off one tubing segment from a type donor unit to be cross-
matched. Cut open the segment and add one drop of red cells into
the tube labeled only with the donor number and prepare the cell
suspension. Inspect the donor unit at this time. Any unit which
appears contaminated (i.e., cloudy or discolored) is not to be used.
v. Add one drop of recipient’s red cells (washed and suspended in
saline to form 2 to 5% suspension) in the tube marked ‘minor’.
vi. Mix well and incubate at RT for 90 minutes (or at 37°C for 30
minutes).
Or
vii. For immediate spin method, incubate at RT for 5–10 minutes,
centrifuge at 1000 rpm for 1 minute.
viii. Look for agglutination. No agglutination should occur if the donor’s
and recipient’s blood are compatible.

Interpretation
Do not proceed further if agglutination occurs since it indicates ABO
mismatch. Regroup the recipient and the donor.
If no agglutination occurs in the saline technique and no antibody
screening has been performed, proceed to the anti-globulin cross-
match.

Bovine Albumin Technique

i. The test is similar to the ‘Saline Technique’ with the following
difference:
ii. To the tube containing the recipient’s serum add one drop of (10 to
20%) Bovine Albumin (to form a layer above the serum).
iii. Then add the suspension of donor’s red cells to this tube. The red
cells are allowed to sediment through the albumin before they
come in contact with the serum.
iv. Incubate and read as for ‘saline technique’.

Enzyme Technique
i. The test is similar to the Bovine Albumin Technique with the
difference that instead of Bovine albumin, one drop of papain
cysteine reagent is added.
94 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

ii. Incubate at 37°C for 1 hour.

iii. Look for agglutination. No agglutination should occur if the donor’s
and recipient’s blood is compatible.

Note
Auto-control should be put up.

Indirect Anti-Globulin Technique (IAT)

Carry out the ‘major’ matching as described under ‘saline technique’
with the following additions:
i. After incubating the mixture of donor’s cells and recipient’s serum,
wash the red cells in normal saline at least four times.
ii. Re-suspend the cells in one drop of normal saline.
iii. Add one drop of Coomb’s serum (Antihuman Globulin Serum).
iv. Incubate at RT for 10 minutes, centrifuge at 1000 rpm for 1 minute.
v. Look for agglutination by naked eye as well as under a microscope.
vi. If the test is negative, add 1 drop of control IgG coated cells.
Centrifuge again at 1000 rpm for 1 minute.
vii. Look for hemolysis or agglutination. If no agglutination, the test is
invalid and the test is to be repeated.

INTERPRETATION
Negative reactions at all test phases indicate that the unit is serologically
compatible with the patient and may be reserved for the patient if other
criteria are met.
Hemolysis or agglutination at any stage of the test procedure
indicates incompatibility between donor red cells and patient’s serum.
Further investigation is necessary and units may not be released until
the problem is resolved.
After addition of IgG-sensitized cells to a negative test, the presence
of agglutination indicates that the AHG serum added was capable of
reacting and that the negative anti-globulin test is valid.

Causes of an incompatible cross-match in the presence of a negative

antibody screen
i. Error in ABO grouping of either donor or patient.
ii. Presence of an antibody in the patient’s serum directed against a low
incidence antigen or another antigen not present on the screening
cells (e.g. anti-Kpa or anti A1 respectively).
iii. Donor has a positive DAT.
 Pre-transfusion Testing (Compatibility Testing) 95

iv. An antigen present in double dose (homozygous donor) on the

donor cells but single dose on the antibody detection (screening)
cells. In this case the patient’s antibody may not be strong enough
to react with the single dose screening cells (“dosage effect”).
v. Rouleaux
vi. A cold allo-or autoantibody.

Resolution of Incompatibility
The most likely cause of a positive IS cross-match after a negative
antibody screen is a cold allo- or autoantibody. However, an ABO
incompatibility due to a patient typing or unit labeling error must be
immediately ruled out.
If the IS cross-match is positive in a patient with a negative antibody
screen, proceed as follows:
i. Perform a clerical check of the unit and patient specimen, and then
confirm the unit typing and repeat the patient ABO typing, both
forward and reverse (consider incubating the reverse type if anti-A1
is a possibility).
ii. Examine the tube for Rouleaux and, if negative, complete the cross-
match through to the AHG phase. Initiate a second pre-warmed
AHG cross-match(s).
iii. Perform a DAT on the donor RBCs. If the donor’s DAT is positive,
repeat the cross-match with a new unit.
If there is no discrepancy in the ABO, the DAT on the donor cells
is negative, there is no Rouleaux, proceed with investigation as for a
positive antibody screen including tests to identify cold antibodies.

Note
If an ABO incompatibility is ruled out and both the antibody detection test and
an IS cross-match with pre-warmed elements is negative; units may be issued
prior to completion of the workup without requiring an emergency blood request
to be signed.

If the antibody screen is positive after an immediate spin crossmatch,

was performed, resolve the antibody problem and complete all cross-
match(s) on the patient through the anti-globulin phase.
If an IAT cross-match(s) is positive in a patient with a blood group
antibody using donor RBCs lacking the corresponding antigen:
i. Review the antibody identification procedure for errors.
ii. Initiate an investigation for an additional antibody directed against
a low incidence antigen.
After the above investigations are complete, select antigen negative
units and/or an appropriate cross-match technique (e.g. use of auto-
adsorbed specimen, or pre-warmed technique), and repeat the cross-
match(s).
 C H A P T E R 23
Direct Coomb’s Test/Direct Anti-
globulin Test (DCT/DAT)

SCOPE AND APPLICATION

Anti-human globulin serum (AHG) recognizes IgG and/or C3d
determinates bound to red cells as antigen and agglutinates RBCs
sensitized with them. In the direct anti-globulin test washed RBCs are
tested against AHG to determine if such sensitization exists. Poly-specific
AHG is directed against both human IgG and complement, and is the
most sensitive reagent for detecting RBC sensitization. Alternatively,
mono specific anti-sera may be used which detects only IgG or C3.
When a DAT is requested, initial testing is done with poly-specific AHG.
If this is positive, it is repeated with both mono-specific reagents (anti-
IgG and anti-C3) as a “reflex” test.
The presence of human serum in a cell suspension will neutralize
the AHG resulting in false negative reactions. Therefore, prior to the
addition of AHG, the RBC suspension must be washed repeatedly with
saline to remove all serum. Proper and complete washing is verified by
the addition of IgG coated Coomb’s control cells (“check cells”) to all
negative AHG reactions.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell serol­
ogy laboratory to perform Direct Coomb’s Test and document the results.

MATERIALS REQUIRED
Equipment
• Table top centrifuge.
• Incubator.
• Microscope.
 Direct Coomb’s Test/Direct Anti-globulin Test (DCT/DAT) 97

Sample
• Clotted blood sample of the patient.
• EDTA/CPDA blood sample of the patient.

Reagent
• Low-ionic strength saline solution (LISS).
• Control cells (IgG coated cells).
• 2% suspension of reagent O cells.
• AHG reagent (Coomb’s serum).

Glassware
• Glass test tubes.
• Glass slide.

PROCEDURE
• Set the table and label the tubes. Prepare record books.
• Add a drop of AHG reagent (Coomb’s serum) to a small tube after
appropriately labeling it.
• Check the identity of the specimen.
• Make a 2% suspension of the red cells of the specimen in LISS after
washing the cells three times.
• Add 1 drop of the red cells to be tested to it. Mix by shaking.
• Spin at 1000 rpm for 1 minute.
• Observe for agglutination with the naked eye. If no agglutination is
seen, read the contents under a microscope (Low power).
• If the test is negative, add 1 drop of control cells (IgG coated cells).
• Mix and spin at 1000 rpm for 1 minute and observe for agglutination.
Observe for agglutination with the naked eye.
If no agglutination is seen, the result is invalid. Repeat the test
procedure.
If the DAT is positive with poly-specific AHG, for all but cord blood
workups, repeat the test with mono-specific reagents as below.

Procedure using mono-specific anti-IgG and anti-C3

• Properly label 10×75 or 12×75 mm test tubes with patient identifier
and “IgG” and “C3”.
• Add one drop of the red cell suspension to each tube and wash 4
times with saline.
98 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Add 1 or 2 drops of anti-IgG to the tube marked “IgG” and 1 or 2

drops of anti-C3 to the tube marked “C3” each according to the
manufacturer’s instructions.
• Mix and centrifuge at the appropriate speed and time for the sero-
fuge in use.
• Examine the tubes for agglutination. If a reaction appears equivocal
or mixed field macroscopically, examine it microscopically. Record
the results.
• Add one drop of check cells as appropriate to any tube(s) with
a negative reaction. Centrifuge and examine for agglutination.
Record the results.

INTERPRETATION (TABLE 23.1)

A negative DAT is invalid if the Coomb’s control cells (“Check cells”) are
non-reactive. Reasons for a negative reaction with check cells include:
A. Incomplete washing so that remaining serum neutralized the AHG
serum added.
B. Failure to add AHG serum to the test.
C. Improper function of the AHG serum.

Table 23.1: Interpretation of DCT/DAT

Polyspec AHG Anti-IgG Anti-C3 Interpretation
0 Negative (no repeat with mono-
specific reagents)
+ + 0 Positive reaction due to IgG coated
RBCs. Seen in HDN, WAIHA, some
drug antibodies, passively acquired
ABO antibodies.
+ 0 + Positive reaction due to complement
coated RBCs. Seen with cold
agglutinins (common) and some drug
antibodies.
+ + + Positive reaction due to IgG and C
coated RBCs. May be seen in WAIHA
and some drug antibodies.
mf mf mf or 0 Positive reaction due to IgG coating
of one RBC population. Classically
associated with a delayed hemolytic
transfusion reaction.
 Direct Coomb’s Test/Direct Anti-globulin Test (DCT/DAT) 99

• Hemolytic disease of the fetus or newborn.

• Autoimmune hemolytic anemia or benign auto-antibodies.
DCT/DAT is positive in cases of:
• Drug-related antibodies sensitizing RBCs.
• Hemolytic transfusion reactions and delayed serologic
transfusion reactions.
• Passive transfer of blood group antibodies (e.g. by transfusion of
group O platelets to a group A person, maternal RhIG bound to
cord RBCs, treatment with IVIG or RhIG for ITP).
• Passive adsorption of antibody not directed against RBCs in
patients with polyclonal hyper gamma-globulinemia (e.g.
patients with renal failure or HIV infection).
 C H A P T E R 24
Indirect Coomb’s Test (ICT)

SCOPE AND APPLICATION

It detects antibodies against RBCs that are present unbound in the
patient’s serum. In this case, serum is extracted from the blood, and the
serum is incubated with RBCs of known antigenicity. If agglutination
occurs, the indirect Coomb’s test is positive. It is used to screen donor
as well as recipient for atypical antibodies, screen pregnant women for
IgG antibodies that are likely to pass through the placenta into the fetal
blood and cause hemolytic disease of the newborn and to test a sample
of the recipient’s serum against a sample of the blood donor’s RBCs, i.e.
Cross-matching.

RESPONSIBILITY
It is the responsibility of the technician in the red cell serology laboratory
to perform Indirect Coomb’s Test and document the results.

MATERIALS REQUIRED
Equipment
• Table top centrifuge.
• Incubator.
• Microscope.

Sample
• Clotted blood sample of the patient.
• EDTA/CPDA blood sample of the patient.

Reagent
• Low ionic strength saline solution (LISS).
• Control cells (IgG coated cells).
• 2% suspension of reagent O cells.
• AHG reagent (Coomb’s serum).
 Indirect Coomb’s Test (ICT) 101

• 22% bovine albumin.

• Papain/Cysteine.

Glassware
• Glass test tubes.
• Glass slide.
• Pasteur pipettes.

Miscellaneous
• Test tube racks.
• Beakers.

PROCEDURE
i. Take 1 drop of serum to be tested in a pre-labeled tube.
ii. Add 1 drop of 2% suspension of reagent O red cells.
iii. Incubate at 37°C for 45–60 minutes (for saline/albumin/enzyme)
and the incubation time for LISS suspended cells will be 10–15
minutes.
iv. Look for hemolysis or agglutination. Agglutination or hemolysis at
this stage indicates presence of saline reacting antibody.
v. If no hemolysis or agglutination, wash the cells four times in saline.
vi. Add 1 drop of AHG reagent to the washed cells and mix.
vii. Spin at 1000 rpm for 1 minute.
viii. Observe for agglutination with the naked eye. If no agglutination is
seen, read the contents under a microscope (low power).
ix. If the test is negative, add 1 drop of control cells. (IgG coated cells).
x. Mix and spin at 1000 rpm for 1 minute and observe for agglutination.
If no agglutination is seen, the test is invalid and the test needs to be
repeated.

Note
Auto-control should be kept with IAT

INTERPRETATION
IAT/ICT is used to detect the presence of incomplete antibodies and
complement-binding antibodies in the serum, after coating on red cells
in vitro in:
• Compatibility testing.
• Screening and detection of atypical antibodies in serum.
• Detection of red cell antibodies not detected by other techniques
(lea, K,FYa, FYb, JKa, JKb, etc.).
 C H A P T E R 25
Saline Addition and
Replacement Technique

SCOPE AND APPLICATION

The saline addition or replacement technique is used to differentiate
rouleaux from true agglutination. The procedure may be used when
rouleaux is suspected with any test performed at 4°C, room temperature
or 37°C by direct agglutination. Rouleaux will disperse with the addition
of or replacement with saline, whereas true agglutination will remain as
such. Tests that are negative following saline addition or replacement
are considered to be negative.

RESPONSIBILITY
It is the responsibility of the technician in the red cell serology laboratory
to perform Saline Addition and Replacement Technique for differ­enti­
ation between true agglutination and Rouleaux formation.

MATERIALS REQUIRED
• Centrifuge.
• Transfer pipettes.
• 0.9% w/v saline.

PROCEDURE
a. Saline addition technique:
• Add one drop of saline to the tube, mix gently.
• Centrifuge for 15 seconds at 3,400 rpm.
• Re-suspend each tube and read macroscopically.
• Grade and record results.
b. If the Rouleaux persists, use the saline replacement technique:
• Centrifuge the tube(s) for 15 seconds at 3,400 rpm.
• Remove the plasma with a pipette.
• Replace plasma with an equal volume of saline.
 Saline Addition and Replacement Technique 103

• Mix the tubes and centrifuge for 15 seconds at 3,400 rpm.

• Re-suspend each tube and read macroscopically.
• Grade and record results.

INTERPRETATION
• Tests that are non-reactive following saline addition or replacement
are considered to be negative.
• Tests that are reactive following saline addition or replacement are
considered to be positive. Further investigation is required.

Note
When red cells are suspended in plasma or anti-sera, a false agglutination called
rouleaux may be observed. This may be caused by the administration of plasma
expanders or by protein abnormalities. Macroscopically this agglutination cannot
be distinguished from true agglutination. Microscopically, it may appear as the
characteristic “stack of coins” form or as large, shiny rosettes.
 C H A P T E R 26
Alternative Technologies
in Blood Banking

In response to the pressures of current good manufacturing practices,

two alternative technologies, the gel test and solid phase assays, have
emerged to provide accurate, reproducible blood bank testing. This gel
based test is named the ID-Micro Typing System being marketed by
Ortho-Clinical Diagnostics and Bio-rad.
The Solid-Phase Technology is being manufactured under the
trade name of CAPTURE by IMMUCOR. The priniciple of solid-phase
immunoassay is based on solid phase red cell adherence (SPRCA)
technology using microplates.
The gel test, as developed by Dr Lapierre in 1988, is an innovative
approach to blood group serology. This technology addresses the issue of
standardization and incorporates sensitivity, specificity and efficiency.
The Bio-rad ID Micro Typing System utilizes a sephadex gel to capture
agglutinates in a semi-solid medium. This enhances visualization of
agglutination as compared to the traditional tube techniques. In the
latter, the agglutinate, particularly in weak reactions, mixes with the free
cells at the bottom of the tube, making visualization difficult.
The capacity of the gel test to separate RBCs from their surrounding
fluid permits an anti-globulin test to be performed without washing.
At the beginning of centrifugation, the RBCs are pulled away from the
suspension medium (unbound serum globulin) and enter the gel first.
The surrounding medium remains above the gel and the characteristics
of the gel prevent the medium from interfering with the anti-globulin
reaction. Sensitized RBCs agglutinate as they come in contact with the
anti-globulin reagent in the gel and are trapped. Un-sensitized RBCs are
not agglutinated and pass through the gel to pellet at the bottom of the
microtube.
The gels may also contain other elements: preservatives such as
sodium azide, sedimenting agents such as bovine serum albumin, and,
in some cases, specific reagents such as anti-IgG or other RBC-specific
anti-sera (ABO and D). When gels are to contain specific reagents, the
manufacturer adds the reagents to the gel during preparation, before the
microtube is filled. Thus, the reagent is dispersed throughout the length
 Alternative Technologies in Blood Banking 105

of the gel column. The gel column is about 75 percent packed gel and 25
percent liquid. Six of these microtubes are embedded in a plastic card
to allow ease of handling, testing, reading, and disposal (Malyska and
Weiland 1994).
The Bio-rad ID system can be used for any immuno-hematological
test that has hem-agglutination as its endpoint such as:
• ABO and RH Typing.
• Typing for Other Blood Group Systems.
• Compatibility testing including cross-matching.
• Antibody Screening and Identification.

EQUIPMENT
The basic pieces of equipment and materials required for the gel test are
the dedicated incubator, centrifuge and RBC diluents. Accessories are
also available to make the testing run smoothly. These include specially
designed workstations, micropipettes with disposable tips.

The ID-incubator (Fig. 26.1)

Incubator is required for certain applications for maintaining
temperature of 37°C.

Fig. 26.1: ID-Incubator 37 SI

106 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 26.2: ID-Centrifuge 6S

The ID-centrifuge (Fig. 26.2)

The only special equipment that is required for the gel test is the
centrifuge.
The centrifuge ensures the correct centrifugation parameters.
Centrifugation plays a key role in the gel test and the centrifugation
criteria are extremely important. The axis of the microtube during
centrifugation must be held strictly in line with the direction of the
centrifugal force. In addition, the speed and time of centrifugation are
critical. If the microtubes are centrifuged too long or too fast, the weakly
agglutinated RBCs may be driven through the gel, thereby causing false-
negative results.
Alternatively, if centrifugation is too slow or too short, all of the
unagglutinated RBCs may not reach the bottom of the microtube,
potentially leading to a false-positive result. The optimal centrifugation
parameters are a relative centrifugal force of 80 applied over 10 minutes.

PRINCIPLE
The basic principle of the gel test is that, instead of a test tube, the serum
and cell reaction takes place in a microtube consisting of a reaction
chamber that narrows to become a column about 15 mm long and 4 mm
wide.
Six microtubes are held together in a plastic card about the same size
as a credit card, thus minimizing labeling and handling. Each microtube
 Alternative Technologies in Blood Banking 107

within a card may contain the same gel or a variety of different gels,
depending on the application. The cards are filled with the respective
gels at the point of manufacture and then sealed.
The gel test uses the principle of controlled centrifugation of red
blood cells through a dextran-acrylamide gel and appropriate reagents
pre-dispensed in a specifically designed microtube. Measured volumes
of serum or plasma and/or red blood cells are dispensed into the reaction
chamber of the microtube. The “card” is incubated and then centrifuged.
If agglutination is present, the red cells are trapped in the gel and cannot
travel through the gel during centrifugation. Agglutinated red cells that
are present remain fixed or suspended in the gel. Unagglutinated red
cells travel unimpeded through the length of the microtube, forming
a pellet at the bottom. Unlike agglutination observed with traditional
test tube hem-agglutination methods, the gel test reactions are stable,
allowing observation or review over an extended period of time.

INTERPRETATION OF RESULTS
After centrifugation, positive reactions are indicated by RBC agglutinates
trapped anywhere in the column of the gel. Positive reactions can be
graded from 0 to 4+ (Fig. 26.3).

Fig. 26.3: Interpretation of results

108 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

A 4+ reaction is indicated by a solid band of RBCs on top of the gel.

A 3+ reaction displays agglutinated RBCs in the upper half of the gel
column.
A 2+ reaction is characterized by RBC agglutinates dispersed
throughout the length of the column.
A1+ reaction is indicated by RBC agglutinates mainly in the lower
half of the gel column with some unagglutinated RBCs pelleted at the
bottom.
Negative reactions display a pellet of RBCs at the bottom of the
microtube and no agglutinates within the matrix of the gel column.
Mixed-field reactions can also be observed in the gel test. These
reactions are more commonly encountered during RBC typing
procedures, rather than during serum or plasma testing methods, but
in either case they are easy to recognize. Antigen positive RBCs, in this
case, are completely agglutinated by the specific anti-sera present and
they lie at the top of the gel, whereas the remainder of the RBCs that are
antigen negative do not agglutinate and pellet at the bottom.

PRECAUTIONS TO BE TAKEN WHILE USING ID

MICROTYPING SYSTEM
DO’s
i. Centrifuge unused cards for 1 cycle (10 minutes) in the ID centrifuge
before use.
ii. Do not use if cards show drying/cracking or damage to the gel.
iii. Cards should always be stored upright in the racks provided at
temperature between 18–25°C.
iv. Open foil seal of only as many microtubes as required. Use half used
cards first before opening new cards.
v. ID-Diluent 2(LISS) and test cells should always be stored at 2–8°C.
vi. Whenever the test cells are required, preferably use only bio-rad
cells.
vii. Cell suspensions to be made in ID-diluent 2 (LISS) only.
viii. Prepare cell suspensions as per protocols provided. Too heavy or
too weak cell suspensions can cause aberrant results.
ix. Always pipette cells before serum/plasma.
x. Cells should always be pipetted into the well at an angle of 45°.
xi. Serum/plasma should always be added straight from the top.
xii. When serum samples are used, the serum should be well cleared
by centrifugation for 10 minutes at 1500 g before use (to avoid fibrin
residues interfering with the reaction pattern).
 Alternative Technologies in Blood Banking 109

xiii. To clean the instruments, please use a soft cloth/cotton ball/gauze

dipped in a mild soap solution like Dettol/Savlon to wipe the
chamber and rotor head (in case of centrifuge).

Don’ts
i. Do not place cards horizontally (lying down) on any surface. Cards
are to be kept upright in racks provided.
ii. Do not open the foil seal of cards unless they are to be used
immediately or maximum, within an hour of opening.
iii. Do not use gel cards if the gel matrix is absent or the liquid level in
the microtube is at or below the top of the gel matrix.
iv. Do not store cards near any direct source of heat–on top refrigerators,
incubators, etc.
v. Do not use cards in which the gel shows any sign of drying, cracking
or bubbles entrapped in the gel.
vi. Do not use hemolyzed samples for testing.
vii. Do not use normal saline as cell diluent to prepare cell suspensions.
viii. Do not touch the tip of the pipette with the serum or test cells in the
microtube to prevent any carryover of sample.
ix. Do not reduce/change incubation time, where necessary, when
doing tests. Perform as per prescribed protocols.
x. Do not use acid/other cleaning agents to clean or disinfect machines.

DISPOSAL OF USED ID CARDS

Discard the used ID cards in the bin with red colored bag only.

Advantages of the Gel Test

Advantages of the gel test include small sample size, decreased variation
in volume delivery, greater uniformity between repeat tests, no cell
washing step and decreased technique dependence.
Applicable to a broad range of tests routinely performed in the blood
bank, such as antibody screening, antigen typing, and cross-matching,
gel test procedures are easy to perform and yield clear-cut end points
that are stable; besides, they can be reviewed at a later time.
The gel test is also easy to learn, since there are relatively few steps
in the procedure and it provides a clear, easy-to-read, stable endpoint.
Easier to training students, evaluate student progress and assess
competency. When the microtubes are covered and refrigerated, the gel
cards can be read with accuracy for at least 24 hours after testing.
110 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Sensitivity and specificity of gel testing have been found to be

comparable to the tube LISS-IAT. However, there are a few disadvantages.
For reference laboratories, batch testing is less of an option because of
urgency, timing and variation in samples. Therefore, the efficiency made
possible by batch testing is often lost.
Complexity of antibody identification usually requires multiple runs
of selected cells. Although a 0.8 percent panel of RBCs is commercially
available, any other test cell that needs to be tested must be prepared as
a 0.8 percent concentration in the appropriate diluent prior to use. This
is more cumbersome than adding the cells right from the vial into a test
tube.
The ABO and Rh typing may also have a disadvantage, depending
on the laboratory’s needs. The RBCs must be incubated in diluent 1 for
10 minutes prior to centrifugation in the appropriate card. Therefore, it
takes a minimum of 20 minutes (10 minutes incubation and 10 minutes
centrifugation) to perform an ABO and Rh test. The test-tube method
still wins on this one with ease and speed. Although direct anti-globulin
tests (DATs) can be done on the gel system, only a DAT using anti-IgG is
available.
Finally, rouleaux and incompletely clotted samples may cause
patterns that resemble positive reactions.
 C H A P T E R 27
Confirmation of ABO and Rh
Blood Grouping of the Donor

SCOPE AND APPLICATION

To confirm the ABO group of the donors by gel technology to ensure the
reliability of the results.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell serology
laboratory to perform confirmation of ABO and Rh blood grouping of
the Blood Unit and Patient (recipient) by the gel card technology.

SAMPLE AND MATERIALS REQUIRED

• Blood sample in EDTA.
• ID-Diluent-2(LISS) at room temperature.
• Micropipettes.
• ID-Centrifuge 6S.
• ID-Incubator 37 SI.
• Disposable pipette tips.
• Diaclon ABD-Confirmation for Donors (ID No 51051) Cat No
001134.
• Table top centrifuge.

PROCEDURE
The Gel Card-Diaclon ABD-Confirmation for Donors (ID No 51051) Cat
No 001134 is positive for Epitope of D antigen which is most immuno­
geneic and donor should be D-negative. Two donors can be typed in
one card.

Sample Preparation
• Centrifuge the sample of the donor at 2000 rpm for 10 minutes.
• Put 25 ul of packed RBCs of the donor into another tube.
112 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Add 500 ul of ID-Diluent (LISS) to the above tube.

• Mix well and this gives 5% suspension of red cell.

Method
• Add 10 ul of the 5% suspension of red cell in the 3 wells of the card.
• Centrifuge the card for 10 minutes.
• Read the result.
• Enter the results of the donor grouping in the donor grouping regi­
ster.

Note
The ID/catalogue numbers of gel cards and ID of the centrifuge as well as
incubator should be as per manufacturer’s catalogue.
 C H A P T E R 28
Reverse Grouping of the Donor

SCOPE AND APPLICATION

To confirm the cell grouping results with those obtained in the forward
group­ing.

RESPONSIBILITY
It is the responsibility of the technician in the red cell serology laboratory
to perform reverse grouping of the donor by the gel card technology.

SAMPLE AND MATERIALS REQUIRED

• Blood sample in EDTA.
• ID-Diluent (LISS) at room temperature.
• Micropipettes.
• ID-Centrifuge 6S.
• ID-Incubator 37 SI.
• Disposable pipette tips.
• ID Diacell A1 (ID No 06012) Cat No 3620.
• ID Diacell B (ID No 06032) Cat No 3622.
• ID Diacell O (ID No 06042) Cat No 3623.
• Diaclon NaCl/Enzyme/Cold agglutinin Card (ID No 50520) Cat No
005014.

PROCEDURE
The Gel Card-Diclon NaCl/Enzyme/Cold agglutination Card (ID No
50520) Cat No 005014 is used for reverse typing of two donors.
• Mark three wells as A, B, O.
• Make 1% suspension of pooled A1, B, O cells (1000 ul LISS + 10 ul
Packed cells).
• Add 50 ul of the above suspension of red cell in the corresponding
wells of the card.
• Add 50 ul of the donor plasma/serum.
114 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Incubate at RT for 10 minutes.

• Centrifuge the card for 10 minutes.
• Read the result.
• Enter the results of the patient grouping in the patient grouping
register/blood requisition form.
The Gel Card-Diclon NaCl/Enzyme/Cold agglutination Card (ID No
50520) Cat No 005014 is used for for reverse typing of two donors.

Note
The ID/catalogue numbers of Gel cards and ID of the centrifuge as well as
Incubator should be as per manufacturer’s catalogue.
 C H A P T E R 29
Forward and Reverse
Grouping of the Patient

SCOPE AND APPLICATION

This procedure describes the method of detection of ABO antigens on
the red cells and the reciprocal antibodies in the serum, thereby ensuring
correct ABO group of the patient and ensure the reliability of the result.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell sero­
logy laboratory to perform forward and reverse grouping of the patient
by the gel card technology.

SAMPLE AND MATERIALS REQUIRED

• Blood sample in EDTA.
• ID-Diluent-2 (LISS) at room temperature.
• Micropipettes.
• ID-Centrifuge 6S
• ID-Incubator 37 SI.
• Disposable pipette tips.
• Table top centrifuge.
• Diaclon ABO/D+ reverse grouping card (A-B-D (vi-)-ctl/A1-B
I(ID50092) Cat No.001234.
• D Diacell A1 (ID No 06012) Cat No 3620.
• ID Diacell B(ID No 06032) Cat No 3622.

PROCEDURE
The control in Diaclon ABO/D+ reverse grouping card (A-B-D (vi-)-
ctl/A1-B (ID50092) Cat No. 001044 is very important as in this case
when RBCs are coated with antibodies or any interfering proteins, the
control will be positive and the forward grouping will be invalid. The
forward grouping is to be repeated after washing the cells.
116 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

First of all, the reverse grouping is to be processed as it will save time

when during incubation, the cell suspension for the forward grouping is
prepared:
• Make 1% suspension of pooled A1, and B cells (1000 ul LISS +
10 ul packed cells).
• Add 50 ul of A1 cells (1% suspension) in the 5th well and 50 ul of
B cells (1% suspension) in the 6th well of the card.
• Add 50 ul of plasma/serum in both the above wells.
• Incubate at RT for 10 minutes.
• Make 5% suspension of the patient’s packed RBCs (500 ul LISS
+ 25 ul packed cells).
• Add 10 ul of the above suspension in the first 4 wells, i.e. A, B, D (vi-)
and Ctl.
• Centrifuge the card for 10 minutes.
• Read the result.
• Enter the results of the Patient/Recipient grouping in the requisition
form.

Discrepancies between Cell and Serum Results

If there is a discrepancy between the forward and reverse grouping,
when the reactions in the forward grouping do not match the reactions
in the reverse grouping or where expected reaction gradings are less
than grade 3 in the forward group and grade 2 in the reverse group or
where there is discrepancy between historical results and current test
results, an interpretation must not be entered but the results must be
recorded and the “ABO discrepancy” must be resolved by further testing.
The format for reporting is given in Annexure 29.1.

Note
The ID/catalogue numbers of Gel cards and ID of the centrifuge as well as
Incubator should be as per manufacturer’s catalogue.
 Forward and Reverse Grouping of the Patient 117

Annexure 29.1: Format of Report for Blood Grouping with

Reverse Grouping
Name of Blood Bank: …………………………………….

MR No: 12345 Result Number:

IP Number: Result Date:
Patient Name: ABC Result Time:
Patient Age: Sample Date:
Patient Sex: Sample Time:
Clinic:
Specimen: Blood
Referred By:

Test Name: Blood Grouping with Reverse Grouping

Method: Gel Card Technology

Observation

Fig. 29.1: Scanned copy of gel card for observation of test results

Result: ‘AB’ Rh Positive

Technologist Consultant I/C Blood Bank

Interpretation of Results
Positive reactions in the micro-tube are indicated by RBC agglutinates
trapped anywhere in the column of the gel.
Positive reactions can be graded from 1+ to 4+.
118 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

A 4+ reaction is indicated by a solid band of RBCs on top of the gel.

A 3+ reaction displays agglutinated RBCs in the upper half of the gel
column.
A 2+ reaction is characterized by RBC agglutinates dispersed
throughout the length of the column.
A 1+ reaction is indicated by RBC agglutinates mainly in the lower
half of the gel column with some un-agglutinated RBCs pelleted at the
bottom.
Negative reactions display a pellet of RBCs at the bottom of the
micro-tube and no agglutinates within the matrix of the gel column.
Mixed-field reactions can also be observed in the gel test. These
reactions are more commonly encountered during RBC typing
procedures, rather than during serum or plasma testing methods, but
in either case they are easy to recognize. Antigen positive RBCs, in this
case, are completely agglutinated by the specific anti-sera present and
they lie at the top of the gel, whereas the remainder of the RBCs that are
antigen negative do not agglutinate and pellet at the bottom.

Note
This report represents only an opinion. If clinical findings of the patient do not
correlate with the opinion given/inferred in the report, then the patient/clinician
may please contact the Blood Bank immediately. This report is not valid for
medico-legal purposes.
 C H A P T E R 30
Testing for Du Weak Antigen

SCOPE AND APPLICATION

To confirm the status of Du weak antigen in cases where, Rh grouping
reaction results are either 2+ or 1+. Such reaction results (2+ or 1+) are
indicative of weak or partial D antigen. This test is also done in Rh-ve
cases.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell
serology laboratory to perform testing for Du weak antigen by the gel
card technology.

SAMPLE AND MATERIALS REQUIRED

• Blood sample in EDTA.
• ID-Diluent (LISS) at room temperature.
• Micropipettes.
• ID-Centrifuge 6S
• ID-Incubator 37 SI.
• Disposable pipette tips.
• Diaclon anti-IgG card (ID No 50540) Cat No 004024.
• Table top centrifuge.
• ID Diaclon anti-D (ID No 09410) Cat No 007531 for verification of D
weak.

PROCEDURE
The Gel Card-Diaclon Anti-IgG Card (ID No 50540) Cat No 004024 is
used for this test and is done using one well.
• Make 1% suspension of patient’s packed RBCs (1000 ul LISS +
10 ul packed cells).
• Take Coombs’ anti-IgG card.
120 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Add 50 ul of the above suspension of red cell in one well of the card.
• Add 25 ul of anti-D (verification for D weak) reagent.
• Incubate at RT for 15 minutes.
• Centrifuge the card for 10 minutes.
• Read the result.
4+ reactions confirm D weak antigen.
3+ to 1+ reactions confirm D variant.
Enter the results of the donor/patient grouping register, and requi­
sition form.

Note
The ID/catalogue numbers of Gel cards and ID of the centrifuge as well as
Incubator should be as per manufacturer’s catalogue.
 C H A P T E R 31
Antibody Screening
in Coomb’s Phase

SCOPE AND APPLICATION

This procedure applies to all testing that requires antibody screening,
including donor units, patient’s pre-transfusion blood grouping and
pren­atal specimens.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell
serology laboratory to perform the antibody screen using 3 cell panels
by gel technology. One technician performs all tests. If any unexpected
blood group antibody is detected, inform the Consultant Blood Bank for
fur­ther investigations.

SAMPLE AND MATERIALS REQUIRED

• Clotted blood sample.
• ID-Diluent (LISS) at room temperature.
• Micropipettes.
• Table Top Centrifuge.
• ID-Centrifuge 6S.
• ID-Incubator 37 SI.
• Disposable pipette tips.
• LISS/Coomb’s cards (ID No 50531) Cat No. 004014 for Patients.
• Coomb’s anti-IgG cards (ID No 50540) Cat No 004024 for antenatal
cases.
• ID-Diacell ABO/I, II, III Cell Panel ID No 45184 Cat No 4304 (Ready
to use 1% suspension).

PROCEDURE
• Add 50 ul of each ID Dia Cell I, II, and III in three wells of the res­pec­
tive card.
122 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Add 25 ul of patient’s serum/ante-natal mother’s serum in all the

3 wells.
• Incubate at 37°C for 15 minutes.
• Centrifuge the card for 10 minutes.
• Read the result.
A positive reaction is recorded when red cells are retained in or
above the gel column after centrifugation.
A negative reaction is recorded (as 0) when a distinct button of
cells sediment to the bottom of the column after centrifugation.
The positive/negative reactions obtained are entered in the antigen
table provided in the reagent kit and following the reaction pattern
the particular antibody or antibodies can be suspected and further
investigation to identify the antibody specificity should be performed.
In case of the positive reaction for antibody screening in Coombs’ phase,
screening of antibody is done in enzyme phase.

Note
1. If antibody screening is positive, then put up the auto-control in the Coombs’
card.
2. The ID/catalogue numbers of gel cards and ID of the centrifuge as well as
incubator should be as per manufacturer’s catalogue.

The format for reporting of antibody screening in pre-natal mothers

is given at Annexure 31.1.
 Antibody Screening in Coomb’s Phase 123

Annexure 31.1: Format of Report for Antibody Screening-

Antenatal Mother
Name of Blood Bank: …………………

MR No: 45678 Result Number:

IP Number: Result Date:
Patient Name: DEFH Result Time:
Patient Age: Sample Date:
Patient Sex: Sample Time:
Clinic:
Specimen: Blood (Serum)
Referred By:
Test Name Antibody Screening-Antenatal Mothers

Method: Gel Card Technology

Observation

Fig. 31.1: Scanned copy of gel card for observation of test results
124 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Rh-hr Rh-hr Kell Duffy Kidd Lewis P MNS Luth Xg Result

D C E c e Cw K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P M N S s Lua Lub Xga  LISS/
% Coombs

I CwCD.ee R1wR1 150-1 + + 0 0 + + 0 + 0 + nt + + 0 0 + 0 0 + + 0 + 0 0 + 0 NEG

II ccD.EE R 2R 2 150-2 + 0 + + 0 0 + + 0 + nt + 0 + 0 + 0 + + 0 + + 0 0 + + NEG

III ccddee rr 150-3 0 0 0 + + 0 0 + + + nt + + + + 0 + 0 + + + 0 + 0 + + NEG

Result
Negative LISS COOMB’S in SC I, II and III as evidenced by presence of
distinct pellet of RBC’s at the bottom of micro-tube suggest the absence
of atypical antibody.
Technologist Technical Supervisor Pathologist
(Consultant Blood Bank)

Interpretation of results
Positive reactions in the micro-tube are indicated by RBC agglutinates
trapped anywhere in the column of the gel.
Positive reactions can be graded from 1+ to 4+.
• A 4+ reaction is indicated by a solid band of RBCs on top of the gel.
• A 3+ reaction displays agglutinated RBCs in the upper half of the gel
column.
• A 2+ reaction is characterized by RBC agglutinates dispersed
throughout the length of the column.
• A 1+ reaction is indicated by RBC agglutinates mainly in the lower
half of the gel column with some unagglutinated RBCs pelleted at
the bottom.
Negative reactions display a pellet of RBCs at the bottom of the
micro-tube and no agglutinates within the matrix of the gel column.

Note
The objective of antibody screening in the antenatal screening in the antenatal
mothers are essentially to minimize the incidence and severity of Hemolytic
disease (HDN) of newborn by identifying clinically significant atypical antibodies
to red cell antigens and assisting in the diagnosis and management of HDN both
during pregnancy and following delivery. Any atypical antibody detected should
be fully identified.
The commonly associated antibodies with clinically HDN are anti-D, - c,
- E,- e, - C, - K, and Anti-k.
 C H A P T E R 32
Antibody Screening
in Enzyme Phase

SCOPE AND APPLICATION

This procedure is applicable in case of the positive reaction for antibody
screening in Coomb’s phase for donor units, patient’s pre-transfusion
blood grouping and prenatal specimens.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell
serology laboratory to perform the antibody screen using ID Diacell pool
by gel technology. In case of positive reaction, inform the consultant
blood bank for further investigations.

SAMPLE AND MATERIALS REQUIRED

• Clotted/EDTA blood sample.
• ID-Diluent (LISS) at room temperature.
• Micropipettes.
• Table top centrifuge.
• ID-Centrifuge 6S.
• ID-Incubator 37 SI.
• Disposable pipette tips.
• Diaclon NaCl/Enzyme/Cold agglutinin card (ID No 50520) Cat No.
005014.
• II D- Diacell ABO/ I, II, III cell panel ID No 45184 Cat No. 4304
(Ready to use 1% suspension).

PROCEDURE
• Add 50 ul of each ID Dia Cell I, II, III in three wells of the card.
• Add 25 ul of enzyme (Papain) in all the wells.
• Add 25 ul of patient’s serum/Ante-natal mother’s serum in all the 3
wells.
126 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Incubate at 37°C for 15 minutes.

• Centrifuge the card for 10 minutes.
• Read the result.
A positive reaction is recorded when red cells are retained in or
above the gel column after centrifugation. The reaction is graded.
A negative reaction is recorded (as 0) when a distinct button of
cells sediment to the bottom of the column after centrifugation.
1. If the reaction is enhanced in enzyme phase in comparison to
Coomb’s phase, Rh, Kell, Kidd, Lewis and P1 system antibodies may
be considered.
2. If the reaction is diminished in enzyme phase in comparison to
Coomb’s phase, Duffy, MNS, and Xg system antibodies may be
considered.
The results of the donor/patient are entered in grouping register
and requisition form.

Note
The ID/catalogue numbers of gel cards and ID of the centrifuge as well as
incubator should be as per manufacturer’s catalogue.
 C H A P T E R 33
Coomb’s Cross-match

SCOPE AND APPLICATION

This procedure is applied for detecting incompatibility between donor
and patient requiring transfusion.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell sero­
logy laboratory to perform cross-match and document the results. If any
unexpected antibody is detected, the consultant blood bank should be
informed.

MATERIALS REQUIRED
• Clotted blood sample of the patient.
• EDTA/CPDA blood sample of the donor.
• ID-Diluent (LISS) at room temperature.
• Micropipettes.
• Table top centrifuge.
• ID-Centrifuge 6S.
• ID-Incubator 37 SI.
• Disposable pipette tips.
• LISS/Coomb’s cards (IgG + C3 D) ID No 50531 Cat No. 004014.

PROCEDURE
Major Cross-match

Principle
The major cross-match is used to detect unexpected blood group
antibodies in patient’s serum against antigens on donor cells. Positive
reaction in any test indicates incompatibility.
128 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Method
• Make 1% suspension of donor packed RBCs (1000 ul LISS +
10 ul packed cells).
• Add 50 ul of the above suspension of red cell in one well of the card.
• Add 25 ul of patient’s serum.
• Incubate at 37°C for 15 minutes.
• Centrifuge the card for 10 minutes.
• Read the result.
A positive reaction is recorded when red cells are retained in or
above the gel column after centrifugation. The reaction is graded.
A negative reaction is recorded (as 0) when a distinct button of
cells sediment to the bottom of the column after centrifugation.

MINOR CROSS-MATCH
Principle
This compares donor serum to recipient erythrocytes and checks for
preformed antibodies in donor serum that could hemolyze recipient
red cells. This cross-match is less important as usually the donor serum
is markedly diluted after transfusion and is unlikely to produce a
significant transfusion reaction.
In case of FFP, minor cross-match is necessary.

Method
• Make 1% suspension of patient’s packed RBCs (1000 ul LISS + 10 ul
packed cells).
• Add 50 ul of the above suspension of red cell in one well of the card.
• Add 25 ul of donor’s plasma/serum.
• Incubate at 37°C for 15 minutes.
• Centrifuge the card for 10 minutes.
• Read the result.
A positive reaction is recorded when red cells are retained in or
above the gel column after centrifugation The reaction is graded.
A negative reaction is recorded (as 0) when a distinct button of
cells sediment to the bottom of the column after centrifugation.

INTERPRETATION
Positive reaction indicates incompatibility of the donor blood with
the recipient due to presence of antibodies directed against antigens
 Coomb’s Cross-match 129

on the donor red cells. Further investigation to identify the antibody

identification and specificity should be performed.
Negative reaction indicates compatibility of the donor blood with
the recipient and the format of compatibility report is given at Annexure
33.1.

Note
1. In case blood bank is doing donor screening for antibodies, then the minor
cross-match depends upon the decision of the consultant Blood Bank. It
has been recognized that patients with a negative antibody screen and
no history of red cell antibodies do not require a complete 20–30 minute
cross-match. The chances of a clinically significant red cell antibody being
missed in a patient with a negative antibody screen (false negative) are
1–4/10,000. Approximately, 95% of transfusions occur in patients with a
negative antibody screen. Such patients can undergo cross-match testing
in which only ABO compatibility of the unit needs to be established.
Results are entered in cross-match register and compatibility report form.
All records are initialed by technician who performed the test.
2. The ID/catalogue numbers of Gel cards and ID of the centrifuge as well as
Incubator should be as per manufacturer’s catalogue.
130 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Annexure 33.1: Format of Report for Compatibility Report

Name of Blood Bank: ……….............
MR No: 12456 IP Number:
Patient Name: XYZ Investigation No:
Patient Age: Patient Sex:
Consultant: Dr Cross-match Date:
Blood Group:

Cross-match Method: Gel LISS Coomb’s Technique

Observation

Fig. 33.1: Scanned copy of gel card for observation of test results

Report
The cross-match put up in the Gel Card as shown above displays negative
reaction as evidenced by the presence of a distinct pellet of RBCs at the
bottom of the micro-tube indicates that the following donor unit/s is/
are compatible with the blood sample of the patient and is/are suitable
for the transfusion.
 Coomb’s Cross-match 131

Important Note
1. Cross-matched blood will be kept in reserve for the said patient
only up to 72 hrs unless specific request is received.
2. Fresh blood sample of patients requiring repeated blood transfusion
must be sent every 72 hrs for antibody screening/cross-matching
to avoid blood transfusion reaction due to atypical antibodies
developed on account of previous transfusion.

Unit Blood Segment Expiry Date and time of Sign. of

No. group No. issue technician
1396
1405

Signature of Technologist

Instructions for Blood Transfusion

• Start transfusion immediately.
• Store continuously at 4 to 6°C before use.
• Check the blood group on label and recipient’s blood group before
administration.
• Use fresh disposable transfusion set with integral filter for
transfusion.
• Change the blood administration set every 12 hrs during Blood/
Blood component transfusion.
• Do not add any other medication to the blood unit.
• Administer without warming.
• Shake gently before use.

Note
Blood once issued from the Blood Bank, will not be taken back after 30 minutes
from the time of issue.
 C H A P T E R 34
Coomb’s Test
(Direct and Indirect)

SCOPE AND APPLICATION

This procedure is applied for detection of coating of red cells in vivo
with antibody (IgG) or C3d (Direct Antiglobulin Test, i.e. DAT/DCT) and
presence of incomplete anti-bodies/complement-binding antibodies in
serum (Indirect Antiglobulin Test i.e. IAT/ICT).

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell sero­
logy laboratory to perform Direct and Indirect Coomb’s test and docu­
ment the results.

MATERIALS REQUIRED
• Clotted blood sample of the patient.
• EDTA/CPDA blood sample of the patient.
• Table top centrifuge.
• ID-Diluent (LISS) at room temperature.
• Micropipettes.
• ID-Centrifuge 6S.
• ID-Incubator 37 SI.
• Disposable pipette tips.
• LISS/Coomb’s cards (IgG +C3D) ID No 50531 Cat No 004014.
• ID Diacell O (ID No 06042) Cat No 03630.

PROCEDURE
A. Direct Anti-globulin Test, i.e. DAT/DCT
• Make 1% suspension of patient’s packed RBCs (1000 ul LISS +
10 ul packed cells).
• Add 50 ul of the above suspension of red cell in one well of the card.
• Centrifuge the card for 10 minutes.
• Read the result.
The reporting format is given at Annexure 34.1
 Coomb’s Test (Direct and Indirect) 133

B. Indirect Anti-globulin Test, i.e. IAT/ICT

• Make 1% suspension of pooled O cells or use ID-Diacell Pool which
are ready to use.
• Add 50 ul of the above suspension of red cell in one well of the card.
• Incubate at 37°C for 15 minutes.
• Centrifuge the card for 10 minutes.
• Read the result.
The reporting format is given at Annexure 34.2.
A positive reaction is recorded when red cells are retained in or
above the gel column after centrifugation. The reaction is graded.
A negative reaction is recorded (as 0) when a distinct button of cells
sediment to the bottom of the column after centrifuge.
DAT/DCT is positive in cases of:
• Hemolytic disease of newborn (HDN).
• Autoimmune hemolytic anemias (AIHA).
• Drug induced red cell sensitization.
• Hemolytic transfusion reactions.
IAT/ICT is used to detect the presence of incomplete antibodies
and complement-binding antibodies in the serum, after coating on red
cells in vitro in:
• Compatibility testing.
• Screening and detection of atypical antibodies in serum.
• Detection of red cell antibodies not detected by other techniques
(lea, K, FYa, FYb, JKa, JKb, etc).

Note
The ID/catalogue numbers of Gel cards and ID of the centrifuge as well as
incubator should be as per manufacturer’s catalogue.
134 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Annexure 34.1: Format of Report for Direct Coomb’s Test

(DCT/DAT)
Name of Blood Bank: ………………
MR No: 123 Result Number:
IP Number: Result Date:
Patient Name: XYZ Result Time:
Patient Age: Sample Date:
Patient Sex: Sample Time:
Clinic:
Specimen: Blood (Serum)
Referred By:
Test Name Direct Coomb’s Test (DCT/DAT)

Method: Gel Card Technology

Observation

Fig. 34.1: Scanned copy of gel card for observation of test results

Result: Positive
Technologist Consultant I/C BLOOD BANK

Interpretation of Results
Positive reactions in the micro-tube are indicated by RBC agglutinates
trapped anywhere in the column of the gel. Positive reactions can be
graded from 1+ to 4+.
 Coomb’s Test (Direct and Indirect) 135

• A 4+ reaction is indicated by a solid band of RBCs on top of the gel.

• A 3+ reaction displays agglutinated RBCs in the upper half of the gel
column.
• A 2+ reaction is characterized by RBC agglutinates dispersed
throughout the length of the column.
• A 1+ reaction is indicated by RBC agglutinates mainly in the lower
half of the gel column with some un-agglutinated RBCs pelleted at
the bottom.
Negative reactions display a pellet of RBCs at the bottom of the
micro-tube and no agglutinates within the matrix of the gel column.
Direct Coomb’s Test is used to detect in-vivo sensitization (coating)
of red cells with immune antibody (IgG) or the complement generally
C3d in cases of hemolytic disease of newborn (HDN), autoimmune
hemolytic anemia (AIHA), drug induced red cell sensitization and
hemolytic transfusion reactions.

Note
This report represents only an opinion. If clinical findings of the patient do not
correlate with the opinion given/inferred in the report, then the patient/clinician
may please contact the lab immediately. Not for medicolegal purpose.
136 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Annexure 34.2: Format of Report for Indirect

Coomb’s Test (ICT)
Name of Blood Bank: ……………
MR No: 123 Result Number:
IP Number: XYZ Result Date:
Patient Name: Result Time:
Patient Age: Sample Date:
Patient Sex: Sample Time:
Clinic:
Specimen: Blood (Serum)
Referred By:
Test Name Indirect Coomb’s Test (ICT )
Method: Gel Card Technology

Observation

Fig. 34.2: Scanned copy of gel card for observation of test results

Result: Negative
Technologist Consultant I/C Blood Bank

Interpretation of Results
Positive reactions in the micro-tube are indicated by RBC agglutinates
trapped anywhere in the column of the gel.
Positive reactions can be graded from 0 to 4+.
 Coomb’s Test (Direct and Indirect) 137

A 4+ reaction is indicated by a solid band of RBCs on top of the gel.

A 3+ reaction displays agglutinated RBCs in the upper half of the gel
column.
A 2+ reaction is characterized by RBC agglutinates dispersed
throughout the length of the column.
A 1+ reaction is indicated by RBC agglutinates mainly in the lower
half of the gel column with some unagglutinated RBCs pelleted at the
bottom.
Negative reactions display a pellet of RBCs at the bottom of the
micro-tube and no agglutinates within the matrix of the gel column.
Indirect Coomb’s Test is used to detect presence of incomplete
Antibodies and complement binding antibodies in the serum after
coating on red cells in-vitro in compatibility testing for the blood to be
transfused, for screening and identification of unexpected antibodies in
the serum and for detection of red cell antigens using specific antibodies
reacting only in anti-globulin test Fya,fyb,K,JKa,JKb, etc.

Note
This report represents only an opinion. If clinical findings of the patient do not
correlate with the opinion given/inferred in the report, then the patient/clinician
may please contact the lab immediately. Not for medicolegal purpose.
 C H A P T E R 35
ABO, Rh Grouping and
DAT of Newborn

SCOPE AND APPLICATION

To confirm the ABO group of the newborn and to detect the presence of
antibodies on the surface of red cells by gel technology.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell sero­
logy laboratory to perform confirmation of ABO Rh blood grouping and
DAT of the newborn by the gel technology.

SAMPLE AND MATERIALS REQUIRED

• Blood sample in EDTA or cord blood.
• ID-Diluent (LISS) at room temperature.
• Micropipettes.
• ID-Centrifuge 6S.
• Table top centrifuge.
• ID-Incubator 37 SI.
• Disposable pipette tips.
• Diaclon ABO/Rh for newborns (ID No 50071) Cat No. 001027.

PROCEDURE
Sample Preparation
• Centrifuge the sample of blood at 2000 rpm for 10 minutes.
• Put 25 ul of packed RBCs into another tube.
• Add 500 ul of ID-Diluent (LISS) to the above tube.
• Mix well and this gives 5% suspension of red cell.
In case of cord blood sample, wash the cells three times and then
prepare the suspension as stated above.
 ABO, Rh Grouping and DAT of Newborn 139

METHOD
• Add 10 ul of the 5% suspension in all the wells.
• Centrifuge the card for 10 minutes.
• Read the result.
A positive reaction is recorded when red cells are retained in or
above the gel column after centrifugation. The reaction is graded.
A negative reaction is recorded (as 0) when a distinct button of
cells sediment to the bottom of the column after centrifugation.
Enter the results of the donor grouping in the donor grouping
register.
The format for reporting is given at Annexure 35.1.

Note
The ID/ catalogue numbers of gel cards and ID of the centrifuge as well as
incubator should be as per manufacturer’s catalogue.
140 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Annexure 35.1: Format of Report for ABO/Rh/DAT

of Newborn
Name of Blood Bank: ……….

MR No: 4567 Result Number:

IP Number: Result Date:
Patient Name: Baby of XYZ Result Time:
Patient Age: Sample Date:
Patient Sex: Sample Time:
Clinic:
Specimen: Clotted Blood and EDTA Blood
Referred By:

Fig. 35.1: Scanned copy of gel card for observation of test results

Result
1. Blood Group: ‘O’ Rh Positive
2. DAT: Negative.
Technologist Consultant I/C Blood Bank
 ABO, Rh Grouping and DAT of Newborn 141

Interpretation of Results
Positive reactions in the micro-tube are indicated by RBC agglutinates
trapped anywhere in the column of the gel.
Positive reactions can be graded from 0 to 4+.
A 4+ reaction is indicated by a solid band of RBCs on top of the gel.
A 3+ reaction displays agglutinated RBCs in the upper half of the
gel column.
A 2+ reaction is characterized by RBC agglutinates dispersed
throughout the length of the column.
A 1+ reaction is indicated by RBC agglutinates mainly in the lower
half of the gel column with some unagglutinated RBCs pelleted at the
bottom.
Negative reactions display a pellet of RBCs at the bottom of the
micro-tube and no agglutinates within the matrix of the gel column.
Group A and B antigens are not fully developed at birth. Weaker
reactions may occur with red cells of newborns than of adults and
sub-groups often cannot be identified. The serum of adults contains
antibodies directed against the A and B antigens absent from their own
red cells. Both antibodies appear after the first 4 to 6 months of life. As
a result, reverse grouping is not usually undertaken on newborn blood
samples.
Confirmation of of the newborn’s blood group is indicated when the
A and B antigen expression is fully developed (2-4 years). The D antigen
as well as weak D is fully developed at birth. The determination of the
RhD status of the newborn’s blood group is important if the mother is
RhD negative.
Direct Coomb’s Test on newborn blood samples has become a
standard procedure, since it is of importance to know if the newborn’s
red cells have been coated with maternal antibodies (IgG) in-utero or
the complement generally C3d in cases of hemolytic disease of new-
born (HDN). It is also done in cases of autoimmune hemolytic anemia
(AIHA), drug-induced red cell sensitization and hemolytic transfusion
reactions to detect antibody coating of red cells.

Note
This report represents only an opinion. If clinical findings of the patient do not
correlate with the opinion given/inferred in the report, then the patient/clinician
may please contact the lab immediately. Not for medicolegal purpose.
 C H A P T E R 36
Detection of Cold Agglutinins

SCOPE AND APPLICATION

Testing of the blood sample for cold agglutinins by using gel technology.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician in the red cell sero­
logy laboratory to perform cold agglutinin test by the gel technology.

SAMPLE AND MATERIALS REQUIRED

• Clotted blood sample of the patient.
• ID-Diluent (LISS) at room temperature.
• Micropipettes.
• ID-Centrifuge 6S.
• Table top centrifuge.
• ID-Incubator 37 SI.
• Disposable pipette tips.
• Diaclon NaCl/enzyme/cold agglutinin card (ID No 50520) Cat No
005014.
• ID Diacell O (ID No 06042) Cat No. 03630.

PROCEDURE
• Add 50 ul of the 1% suspension of the pooled O cells in one well.
• Add 25 ul of the serum or plasma of the patient.
• Incubate at 4°C for 30 minutes.
• Centrifuge the card for 10 minutes.
• Read the result.
A positive reaction is recorded when red cells are retained in or
above the gel column after centrifugation. The reaction is graded.
A negative reaction is recorded (as 0) when a distinct button of
cells sediment to the bottom of the column after centrifugation.
Enter the results of the test on the blood requisition form and the
com­puter.
Note
The ID/catalogue numbers of gel cards and ID of the centrifuge as well as
Incubator should be as per manufacturer’s catalogue.
 C H A P T E R 37
Screening for Transfusion
Transmitted Infections (TTIs)

Transfusion Transmitted Infections (TTIs) is a major challenge to the

transfusion services all over the world. The problem of TTIs is directly
proportionate to the prevalence of the infection in the blood donor
community. With every unit of blood there is 1% chance of transfusion
associated problems including transfusion transmitted diseases. In
India, hepatitis B/C, HIV, malaria, syphilis, cytomegalovirus, parvovirus
B-19 and bacterial infections are important causes of concern. Hepatitis
B and C infections are prevalent in India and carrier rate is about 1–5%
and 1%, respectively.
It is mandatory for the blood banks to test each and every unit of
donor blood for antibodies to HIV-1 and 2, syphilis, hepatitis C, hepatitis
B surface antigen. ELISA is recommended and preferred technique for
such screenings in the blood banks. Many blood banks in India still
do not have this facility and they use rapid, easy to perform and user-
friendly kits. Most of these rapid assays use recombinant proteins from
the prototype virus alone, specifically for HCV. Variants of HCV may
differ substantially in nucleotide sequence from one another and show
varied geographical and epidemiological distributions. This shows that
the rapid tests are inferior compared to ELISA. Not only have that, lot of
discrepancies been reported in the results of rapid tests as compared to
those of ELISA. Even at times control does not appear at all, making the
test invalid. Therefore, ELISA testing should be preferred for screening
of TTIs.
The main types of assay used for blood screening are:
• Immunoassays (IAs):
■ Enzyme immunoassays (EIAs).
■ Chemiluminescent immunoassays (CLIAs).
■ Hemagglutination (HA)/particle agglutination (PA) assays.
■ Rapid/simple single-use assays (rapid tests).
• Nucleic acid amplification technology (NAT) assays.
Enzyme and chemiluminescent immunoassays are currently the
most commonly used assays for screening donated blood for TTIs. EIAs
144 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

and CLIAs are suitable for the screening of large numbers of samples
and require a range of specific equipment.
Most EIAs and CLIAs have greater sensitivity and specificity than
particle agglutination assays or rapid tests. Their manufacture and
performance are generally more reliable and consistent and have better
outcomes for blood screening. High quality particle agglutination assays
are not available commercially for all the routine markers for which
blood is screened.
The use of rapid/simple assays is generally not recommended
for blood screening as they are designed for the immediate and rapid
testing of small number of samples, mainly for diagnostic purposes.
They may also be appropriate when a laboratory needs to screen specific
donations on an emergency basis for immediate release of products
due to a critically low blood inventory or when rare blood is required
urgently. In such emergency situations, the use of the rapid/simple
assay should be followed up with repeat testing using an EIA, CLIA or
particle agglutination assay if these assays are routinely used.
The introduction of NAT should be considered only when an
efficient and effective program based on antibody/antigen testing is
in place and there is a clear, evidenced, additional benefit. Although
NAT reduces the window period of infection, in countries with a low
incidence of infection, the incremental gain is minimal as the number
of donors in the window period at the point of donation is generally very
low. However, in countries with a high incidence of infection there are
likely to be significant numbers of window period donations that can
be identified by NAT. Thus although the risk of transfusing a blood unit
collected during the window period may be decreased using NAT, the
actual benefit in most populations has first to be determined and should
be balanced against the complexity and high cost of performing NAT,
including the infrastructure required.
Malaria is a real problem for India due to the lack of a simple and
sensitive screening test. This is done by examination of stained PBF and
is gold standard method of diagnosis of malaria. Although this method
is not suitable for screening large number of donations because of
difficulty in finding parasites in short time especially if the density of
parasites is less than 100 per microliter of blood, yet this is considered the
best method for diagnosis in suspected cases. The standards for blood
banks and blood transfusion services adopted by NACO describe that
all blood units should be tested for malarial parasites using a validated
and sensitive antigen test.
A separate laboratory for screening of TTIs is mandatory under the
Drugs and Cosmetics Act 1940 and Rules 1945 which should be located
 Screening for Transfusion Transmitted Infections (TTIs) 145

within the blood bank but should have independent air handling unit
(AHU) to prevent cross contamination.
Incidence of bacterial contamination is greatly reduced due
to improved collection/preservation techniques. However, proper
vigilance and quality control is needed to prevent this problem.
 C H A P T E R 38
Screening for Syphilis (RPR Test)

SCOPE AND APPLICATION

Test for screening of syphilis is mandatory for each blood unit before it is
transfused. This is carried out on all donor units’ samples.

RESPONSIBILITY
It is the responsibility of Laboratory Technician under supervision of
Medical Officer to carry out the test of all blood units.

MATERIALS REQUIRED
• Micropipettes.
• Disposable tips.
• Test tubes.
• Glass slides.
• Timer.
• Centrifuge.
• Test tubes.
• Rotator set at 100 rpm.
• Test kit.

PROCEDURE
Principle
• The test reagent is an antigenic cardio-lipin based emulsion for
dete­c­ting syphilis reagins (antibodies) in serum, plasma and spinal
fluid.
• The test reagent emulsion contains cardio-lipin, lecithin and cho­
lesterol as its active components which produce a flocculation reaction
with serum or plasma that contains syphilis antibodies (regains).
• The test reagent emulsion also contains carbon particles to improve
the visual reading of the result.
 Screening for Syphilis (RPR Test) 147

Reagent Storage
Reagent is stable at 2–8°C. Bring to room temperature before use and
gently stir the reagent.
The test is carried out as per kit manufacturer’s instructions.

Method
i. Samples do not need inactivation.
ii. Centrifuge the samples to be tested and collect clear serum. Fresh
serum samples are required.
iii. Dispense one drop (50 ul) of the serum sample to be tested on a
glass slide.
iv. Spread the sample over the entire area of the test circle using the flat
ends of the stirrer.
v. Discard the tip and the stirrer.
vi. Attach a new tip to the pipette and deliver one drop (50 ul) of the
2nd sample.
vii. Repeat this step for all the samples.
viii. Note the position of the samples added.
ix. Lastly add the negative and positive controls.
x. Mix the test reagent emulsion and add one drop to all the test
samples, negative and positive controls contained in a VDRL slide.
xi. Rotate the VDRL slide at 100 rpm for 8 minutes.
xii. Examine macroscopically and microscopically for flocculation.
Repeat reactive and doubtful results again along with controls.
Test results are reported by comparing it with positive control and
negative control results.

INTERPRETATION
• Reactive: Presence of flocculation indicates the presence of anti-
lipoidal antibodies in test samples.
Strength of flocculation depends upon the degree of positivity of the
test samples.
• Non-reactive: Absence of flocculation indicates the absence of
anti-lipid antibodies in test samples.
Strength of flocculation depends upon the degree of positivity of the
test samples.
The results of the tested blood units are recorded in the stock
register.
 C H A P T E R 39
TPHA (Treponema pallidum
Hemagglutination) Test for
Syphilis (Rapid TP Test)

SCOPE AND APPLICATION

Test for screening of syphilis is mandatory for each blood unit before it is
transfused. This is carried out on all donor units’ samples.

RESPONSIBILITY
It is the responsibility of Laboratory Technician under supervision of
Medical Officer to carry out the test of all blood donor units.

MATERIALS REQUIRED
• Test strips.
• Disposable specimen droppers.
• Pipette and its tips.
• Positive and negative controls.
• Package insert.
• Serum/Plasma sample.
Store as packaged in the sealed pouch or closed canister at 2°–8°C.
The kit is stable up to the expiration date as mentioned on the labels and
kit box.

PROCEDURE
Principle
The rapid TP (Treponema pallidum/Syphilis) test uses a double antigen
“sandwich principle” for the detection of Treponema pallidum antibody
(IgM and IgG) in human whole blood, plasma or serum. It is a one step,
visual, qualitative and immunochromatic assay for screening of sexually
transmitted diseases (STDs) among high-risk group of people, antenatal
 TPHA (Treponema pallidum Hemagglutination) Test for Syphilis... 149

screening, regular health examinations and field screen test for injection
drug users and blood bank.
The test is carried out as per kit manufacturer’s instructions.

Method
• Allow the test strip, specimen, buffer and/ or controls to attain RT
(15–30°C) prior to testing.
• Remove the test strip from the sealed foil pouch or closed canister.
• Identify the strip with patient or sample ID.
• Apply 80 ul of the specimen to the sample pad behind the (↓ ↓ ↓)
mark at the bottom of the test strip using a pipette.
• As the sample flows, purple color is seen moving across the result
window in the center of the test strip.
• Results are read at 15 minutes.

Note
Do not interpret the results after 15 minutes.

INTERPRETATION
Positive
Two distinct color bands appear–One line in the control region (C) and
another line in the test region (T).

Negative
One band appears in the control region(C)–No apparent band appears
in the test region (T).
The details of the test done each day are entered in the register:
• The date on which the test is run.
• The name of the kit used.
• Lot number and expiry date of the kit.
• Initials of the technologist who performed it.
Thereafter, the results of blood units tested are recorded in the
Master Record for Blood Stock register.
 C H A P T E R 40
ELISA Test for Syphilis
(Syphilis EIA II Total Antibody)

SCOPE AND APPLICATION

Screening of syphilis is mandatory for each blood unit before it is
transfused. This is carried out on all donor units’ samples.

RESPONSIBILITY
It is the responsibility of Laboratory Technician under supervision of
Medical Officer to carry out the test.

MATERIALS REQUIRED
• Elisa reader.
• Elisa washer.
• Micropipettes and disposable tips.
• Timer.
• Disposable gloves.
• Disposal container with sodium hypochlorite solution.
• Absorbent tissue.
• Distilled water.
• Kit: Plate (12 strips of 8 wells coated with T. pallidum, Ag Wash
solution, negative control, positive control, conjugate, substrate
and stop solution).
• Serum or plasma may be used.

PROCEDURE
Principle
Syphilis EIA II total antibody uses three recombinant antigens in a
sandwich test to produce a test that is both highly specific and sensitive.
The antigens will detect Treponema pallidum-specific IgG, IgM and IgA;
enabling the test to detect antibodies during all stages of infection.
 ELISA Test for Syphilis (Syphilis EIA II Total Antibody) 151

The kit is stored at 2–8°C. The test is carried out as per kit
manufacturer’s instructions. The samples should be stored at 2–8°C
if the testing is to be carried out within 7 days or stored at –20°C for a
longer period.

Method
• Remove reagents from the fridge 30 minutes prior to testing. Mix the
reagents gently by inverting the vials without foaming.
• Bring samples to room temperature before testing.
• Dilute wash buffer 1 in 20 with distilled water prior to use.
• Discard all disposable tips in a container with hypochlorite solution.
• Arrange the samples so that well 1A, 1 B, 1C are negative controls,
1D and 1E are positive control and thereafter samples to be tested
are arranged.
• Dispense 50 ul of the controls as well undiluted samples in the
corresponding wells.
• Add 50 ul of conjugate to the wells.
• Mix the plate gently and cover with a seal.
• Incubate at 37°C for 30 minutes.
• Remove seal and wash 5 times with the buffer ensuring soak time of
about 30 seconds between each cycle.
• Invert the plate and tap on a clean paper towel to remove excess
wash buffer.
• Dispense 50 ul of substrate/chromogen to each well.
• Cover the plate with sealer and incubate at 37°C for 30 minutes.
• Add 50 ul of stop solution to each well. Blue color will change to
yellow.
• Remove moisture from bottom of plate and read OD at 450 nm
using a plate reader within 30 minutes of stopping the reaction.

Validation
Each negative control OD should be lower or equal to 0.080. If one of the
control values is above the value, the reading is ignored and the cut-off
is calculated using the remaining two.
Each positive control OD should be greater than or equal to 1.000.

Cut-off Value
It is calculated as the mean of the negative control values plus 0.100
Negative control (NC)1 + NC2 + NC3
i.e. + 1.000
3
152 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

INTERPRETATION
Samples with OD less than the cut-off value are considered negative by
syphilis EIA II Total Antibody.
Results just below the cut-off value (CO-10 %< OD<CO) should
however, be interpreted with caution. It is advisable to retest the
corresponding samples.
Samples with OD greater than or equal to the cut-off value are
considered positive by Syphilis EIA II Total Antibody.
 C H A P T E R 41
Testing for Hepatitis B Surface
Antigen (Rapid Test)

SCOPE AND APPLICATION

HBsAg is a mandatory test for blood unit screening before it is transfused.
This is carried out on all donor units.

RESPONSIBILITY
It is the responsibility of Laboratory Technician under supervision of
Medical Officer from to carry out the test.

MATERIALS REQUIRED
• Test kit.
• Package insert.
• Clotted blood sample.
• Pipette.

PROCEDURE
Principle
One step HBsAg Test is based on the principle of antigen capture,
or+ sandwich immunoassay for determination of HBsAg in serum.
Mono­clonal antibodies conjugated to colloidal gold and polyclonal
antibodies immobilized on a nitrocellulose strip in a thin line. The test
sample is introduced to and flows laterally through an absorbent pad
where it mixes with the signal reagent. If the sample contains HBsAg,
the colloidal gold-antibody conjugate binds to the antigen, forming an
antigen-antibody-colloidal gold complex. The complex then migrates
through the nitrocellulose strip by capillary action. When the complex
meets the line of immobilized antibody (Test line) “T”, the complex is
trapped forming an antibody-antigen-antibody-colloidal gold complex.
This forms a pink band indicating the sample is reactive for HBsAg. To
serve as a procedural control, an additional line of anti-mouse antibody
154 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

(control) “C” has been immobilized at a distance from the test line on the
strip. If the test is performed correctly, this will result in the formation of
a pink band upon contact with the conjugate.

Storage
The test is carried out as per kit manufacturer’s instructions. Store the
test kit at 2–8°C in the sealed pouch till its expiry.

Method
• Bring the sealed pouch and serum to room temperature.
• Open the sealed pouch by tearing along the notch.
• Remove the test device from the pouch.
• Label the test card with patient’s name or identification number.
• Add 70 ul (2 drops) of serum/plasma sample using the dropper and
dispense into the sample well of the device.
• Wait for 20 minutes and read the results.

Note
It is important that the background is clear before the results are read. Do not
read the results after more than 30 minutes.

INTERPRETATION
Reactive: One distinct pink colored band in the test region (T) and a
pink colored band in the control (C) region.
Non-reactive: Only one pink colored band in the control (C) region.
no apparent band in the test (T) region.
Invalid: A total absence of color in either regions or no colored line
appears in the control (C) region is an indication of procedural error
and/or test reagent deterioration due to improper storage.
Record the results in blood stock register.
 C H A P T E R 42
Testing for Hepatitis B
Surface Antigen (ELISA Method)

SCOPE AND APPLICATION

HBsAg is a mandatory test for blood unit screening before it is transfused.
This is carried out on all donor units.

RESPONSIBILITY
It is the responsibility of Laboratory Technician under supervision of
Medical Officer to carry out the test of all blood units.

MATERIALS REQUIRED
• Elisa reader.
• Elisa washer.
• Incubator.
• Micropipettes and disposable tips.
• Timer.
• Disposable gloves.
• Disposal container with sodium hypochlorite.
• Absorbent tissue.
• Distilled water.
• 1 mol/liter sulfuric acid.
• Hepatitis kit.

PROCEDURE
Principle
In the monoclonal EIA procedures microplate wells are coated with
monoclonal antibody to hepatitis B Surface Antigen (Anti-HBs) are
incubated with serum or plasma and Anti-HBs peroxidase (Horse
radish) conjugate in one step assay. During the incubation period
HBsAg if present is bound to the conjugate (Anti-HBs-HRPO). Unbound
material is aspirated and washed away. On the addition of substrate
156 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

color develops in proportion to the amount of HBsAg which is bound.

The enzyme reaction is stopped by the addition of stop solution.
The test is carried out as per kit manufacturer’s instructions given in
the package insert.

Method
i. Remove reagents from the fridge 30 minutes prior to testing. Mix the
reagents gently by inverting the vials without foaming.
ii. Bring reagents and samples to room temperature before testing.
iii. Arrange all donor unit test tube samples, apheresis samples, serially
in ascending order in a test tube rack. Add required number of
internal kit controls and external lab controls.
iv. Discard all disposable tips into hypochlorite solution.
v. Place the tray in front of the test tube rack.
vi. Arrange the samples so that well A1, B1, C1, D1 are negative controls,
and E1 is positive control and thereafter samples to be tested are
arranged from F1onwards.
vii. Add 100 ul of controls and samples to the corresponding wells.
viii. Add 50 ul of conjugate to the wells.
ix. Mix the plate gently and cover with a seal.
x. Incubate at 37°C for 30 minutes.
xi. Remove seal and wash 5 times with the buffer ensuring soak time of
about 30 seconds between each cycle.
xii. Invert the plate and tap on a clean paper towel to remove excess
wash buffer.
xiii. Dispense 100 ul substrate/chromogen to each well.
xiv. Cover the plate with sealer and incubate at 37°C for 30 minutes.
xv. Add 100 ul of stop solution to each well. On adding this, pink color
of the substrate disappears or turns from blue to yellow (for positive
samples).
xvi. Remove moisture from bottom of plate and read optical density
(OD) at of 450 nm using a plate reader within 4–30 minutes of
stopping the reaction.

VALIDATION AND INTERPRETATION

Cut-off OD is automatically calculated by the ELISA reader.
Calculate the cut-off value by adding 0.050 to the mean absorbance
(NCx) of the negative control.
Cut off = NCx + 0.050
 Testing for Hepatitis B Surface Antigen (ELISA Method) 157

All the values of the negative controls should be lower or equal to

0.080 unit of optical density.
The positive control value (OD) should be over or equal to 1.000.
The test must be redone if all control values are out of these norms.

Check the printout carefully for absorbance value

Divide the sample absorbance by the cut-off value to find out the ratio
between the OD of the sample and cut-off value.
Positive: Ratio absorbance/cut off ≥ 1.0
Negative: Ratio absorbance/cut off < 0.080
Equivocal: Ratio absorbance/cut off ≥ 0.9 < 1.0

Equivocal results are re-analyzed

Paste the printout in the HBsAg register and record the results in blood
stock register.
 C H A P T E R 43
Anti-HIV Testing
(Rapid TRI-DOT Method)

SCOPE AND APPLICATION

Anti-HIV antibodies testing is carried out on all samples of blood units
before these are released for transfusion. This test is carried out on all
bag samples in case the number of blood units is small.

RESPONSIBILITY
It is the responsibility of Laboratory Technician under supervision of
Medi­cal Officer to carry out the test.

MATERIALS REQUIRED
• HIV 1 and 2 test kit.
• Kit package inserts.
• Disposable sample dropper.
• Dilution buffer.
• Clotted/EDTA blood sample.

PROCEDURE
Principle
The HIV antigens are immobilized on a porous immune filtration
membrane. Sample and the reagents pass through the membrane and
are absorbed into the underlying absorbent. As the patient’s sample
passes through the membrane, HIV antibodies, if present, bind to
the immobilized antigens. Conjugate binds to the Fc portion of the
HIV antibodies to give distinct pinkish purple DOT(s) against a white
background.

Storage
Store the test kit at 2–8°C in the sealed pouch till expiry. The test device
is sensitive to humidity as well as to heat. Perform the test immediately
after removing the test device from the foil pouch.
 Anti-HIV Testing (Rapid TRI-DOT Method) 159

The test is carried out as per kit manufacturer’s instructions.

Method
• Bring all the reagents and specimen to RT before beginning the test.
• Remove the test device from the foil and place it on a flat dry surface.
• Add 3 drops of buffer solution to the center of the device.
• Add 1 drop of sample slowly into the sample well holding the sample
dropper vertically above the well of test device.
• Add 2 drops of liquid conjugate directly from the conjugate vial.
• Add 5 drops of buffer solution and read the results.

Note
The procedural sequence of the reagent addition should be strictly adhered to
avoid any discrepant results.

Validation
If no DOT appears after the test is complete, either with clear background
or with complete pinkish/purple background, the test indicates error.
This may indicate a procedural error or deterioration of specimen/
reagents or particulate matter in the sample. The sample should be
tested on a new device.
The performance of the test with reference to sensitivity and
specificity has been determined by National HIV Reference Centers of
Govt. of India and WHO, Geneva, using various testing panels.

INTERPRETATION
Reactive
• If two DOTs, one for the control and other for HIV-1 appear, the
specimen is reactive for antibodies to HIV-1.
• If two DOTs, one for the control and other for HIV-2 appear, the
specimen is reactive for antibodies to HIV-2.
• If all the three DOTs, one for the control, other for HIV-1 and the
third for HIV-2 appear, the specimen is reactive for antibodies to
HIV-1 and 2.
Record the results in HIV and blood stock register.
 C H A P T E R 44
Anti-HIV Testing (ELISA Method)

SCOPE AND APPLICATION

Anti-HIV antibodies testing is carried out on all samples of blood units
before these are released for transfusion.

RESPONSIBILITY
It is the responsibility of Laboratory Technician under supervision of
Medical Officer to carry out the test.

MATERIALS REQUIRED
• Reagent kit.
• Mirco-pipettes and disposable pipette tips.
• Timer.
• ELISA reader.
• ELISA washer.
• Incubator 37°C.
• Vortex mixer.
• Glassware.
• Distilled water.

PROCEDURE
Principle
Human serum or plasma diluted in specimen diluent and incubated
with the proteins of HIV 1 HIV 2, coated auto microplate wells and
incubated. If the HIV antibodies are present in the test sample, it will
bind with the proteins coated on the microwell. After washing off the
unbound analyte, horseradish peroxidase conjugated with anti-human
IgG antibodies is added. Enzyme conjugate binds through the antigen
antibody complex if present. Unbound analyte is washed and substrate
solution is added. Color will develop in proportion to the amount of HIV
 Anti-HIV Testing (ELISA Method) 161

antibodies present in the specimen. Stopping solution is added at the

end of the incubation to stop the reaction. The reaction is read by ELISA
reader.
The test is carried out as per kit manufacturer’s instructions.

Method
Use the negative, positive and cut-off controls for each series of
determinations to validate the results.
i. Remove reagents from the fridge 30 minutes prior to testing. Mix the
reagents gently by inverting the vials without foaming.
ii. Bring the samples to room temperature before testing.
iii. Carefully establish the sample distribution and identification plan.
iv. Prepare the dilute washing solution.
v. Take the carrier tray and the strips out of the protective pouch.
vi. Apply directly, without prior washing of the plate and in succession:
25 ul of the diluents in each well
75 ul of the Ag positive control serum in well A1
75 ul of Ab positive serum in well B1
75 ul of negative control serum in wells C1, D1 and E1
75 ul of specimen 1 in well F1
75 ul of specimen 2 in well G1, etc…….
vii. Incubate the microplate at 37°C for 30 ± 5 minutes.
viii. Remove seal and wash 5 times with the buffer ensuring soak time of
about 30 seconds between each cycle.
ix. Invert the plate and tap on a clean paper towel to remove excess
wash buffer.
x. Add 100 ul of conjugate solution in each well.
xi. Incubate the microplate at 37°C for 30 ± 5 minutes.
xii. Remove seal and wash 5 times with the buffer ensuring soak time of
about 30 seconds between each cycle.
xiii. Add 80 ul of substrate solution after reconstituting in each well.
xiv. Incubate the microplate at RT for 30 ± 5 minutes in dark place.
xv. Add 100 ul of stop solution.
xvi. Read absorbance at 450–620 nm within 4–30 minutes in ELISA
reader.

CALCULATION OF RESULTS
The presence or absence of antibodies to HIV-1 and/or HIV-2 is
determined by comparing the absorbance measured for each sample to
that of the calculated cut-off value.
i. Calculate the mean absorbance (NCx) of the negative controls.
ii. Calculate the cut-off value (CO) by adding 0.200 to NCx.
162 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Assay Validation
iii. The absorbance of the each negative control serum should be less
than 0.170:NCx < 0.170.
iv. The absorbance of the HIV Ab positive should be greater than
0.9:OD of Ab positive control > 0.9.

INTERPRETATION OF RESULTS
Negative: Samples with absorbance value less than the cut-off value
Positive: Samples with absorbance value equal to or greater than the
cut-off value
Samples with absorbance value within 10% below the cut-off should
be considered suspect for the presence of antibodies and/or antigen and
should be retested in duplicate.
Paste the printout in the HIV register and record the results in blood
stock register.
 C H A P T E R 45
Testing for HCV Antibodies
(Rapid TRI-DOT Method)

SCOPE AND APPLICATION

Anti-HCV is a mandatory test for all blood units before these are trans­
fused.

RESPONSIBILITY
It is the responsibility of Laboratory Technician under supervision of
Medi­cal Officer to carry out the test.

MATERIALS REQUIRED

The Test Kit

The components of the Kit are:
• HCV TRI-DOT Device.
• Buffer solution.
• Protein-A conjugate.
• Sample dropper.

Clotted/EDTA Blood Sample

PROCEDURE
Principle
4th generation HCV TRI-DOT is a rapid, visual, sensitive and qualitative
in vitro test for detection of antibodies to Hepatitis C virus in human
serum/plasma. It has been developed using modified HCV antigens
representing the immunodominent regions of HCV antigen. The
device (an immunofiltration membrane) includes two test dots “T1”
and “T2” and a built-in quality control dot “C” which always develops
color during the test, thereby confirming the proper functioning of the
164 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

device. HCV antigens are immobilized on the membrane. Sample and

the reagents pass through the membrane and are absorbed into the
underlined absorbent pad. HCV antibodies if present in serum/plasma,
bind to the immobilized antigens. During the washing step, unbound
serum/plasma proteins are removed. Protein-A conjugate when added,
binds to the Fc portion of the antibodies to give distinct pinkish purple
dot against a white background at the test region.
The test is carried out as per kit manufacturer’s instructions.

Method
• Bring all the reagents and samples to room temperature before
beginning the test.
• Place the required no. of HCV TRI-Dot test devices at the working
area.
• Cut open the pouch and take out the device for performing the test.
• Write the sample ID no. to be tested on the device.
• Add 3 drops of buffer solution to the center of the device.
• Using separate sample dropper for each sample, add 1 drop of the
sample.
• Add 5 drops of buffer solution.
• Add 2 drops of protein-A conjugate.
• Add 5 drops of buffer solution.
• Read the results immediately and discard the device considering it
to be potentially infectious.

Note
It is important to allow each solution to soak in the test device before adding the
next solution.

Validation
This test has built-in quality control dot “C” which always develops color
during the test, thereby confirming the proper functioning of the device
reagent and correct procedural application.
The assay is only validated for serum/plasma from individual
patients/donors and not for pools of serum/plasma or any other body
fluid.
The performance of 4th Generation HCV TRI-DOT has passed phase
1 of the WHO, Geneva. The sensitivity and specificity of the test are 100%
and 98.9% respectively.
• Appearance of only one dot at the control region “C” indicates that
the sample is non-reactive for antibodies to HCV.
 Testing for HCV Antibodies (Rapid TRI-DOT Method) 165

• Appearance of 2/3 dots at the test region “T1” and control region
“C”/at the test region “T2” and control region “C” and at the test
region “T1, “T2” and control region “C” indicates that the sample is
Reactive for antibodies to HCV.
Record the results in the blood stock register.
 C H A P T E R 46
Testing for HCV Antibodies
(ELISA Method)

SCOPE AND APPLICATION

Anti-HCV is a mandatory test for all blood units before these are tran­s­
fused.

RESPONSIBILITY
It is the responsibility of blood bank technician under supervision of
Medical Officer to carry out the test.

MATERIALS REQUIRED
• ELISA reader.
• ELISA washer.
• Microshaker.
• Incubator.
• Micropipettes and disposable tips.
• Timer.
• Disposable gloves.
• Disposable container with sodium hypochlorite solution.
• Absorbent tissue.
• Distilled water.
• 1 mol/liter sulphuric acid.
• HCV test kit.

PROCEDURE
Principle
In HCV ELISA, the microwell is coated with recombinant hepatitis
C virus encoded antigens as the solid phase. If the HCV antibody is
present, it becomes bound to the solid phase and can be detected by
a complementary anti-human IgG conjugated to an enzyme (capable
of acting on a chromogenic substrate). When substrate is added to
 Testing for HCV Antibodies (ELISA Method) 167

the bound complex, the presence of antibody can be detected by

development of a colored end product.
The test is carried out as per kit manufacturer’s instructions.

Method
Remove reagents from the refrigerator 30 minutes prior to testing. Mix
the reagents gently by inverting the vials without foaming.
i. Bring samples to room temperature before testing.
ii. Prepare wash buffer solution as per pack insert.
iii. Prepare the substrate solution as per pack insert.
iv. Discard all disposable tips in a container with hypochlorite
solution.
v. Prepare the record (Plate Map) identifying the placement of
controls, calibrators and specimens in the microwells. Arrange
the assay control/calibrator so that well A1 is the reagent blank.
As per configuration of the software, arrange all the controls and
calibrators.
• Reagent Blank………… A1.
• Negative Calibrator…… B1.
• Negative Calibrator……..C1.
• Negative Calibrator… D1.
• Positive Control……….E1.
• Positive Control………. F1.
vi. Add 200 ul of specimen diluents to all wells including A1.
vii. Add 20 ul of the controls, calibrators and specimens to the
appropriate wells
viii. Mix the plate gently and cover with a seal.
ix. Incubate at 37°C for 60±5 minutes.
x. Remove seal and wash 5 times.
xi. Invert the plate and tap on a clean paper towel to remove excess
wash buffer.
xii. Dispense 200 ul conjugate to all wells.
xiii. Cover the plate with sealer and incubate at 37°C for 60±5 minutes.
xiv. Remove seal and wash 5 times.
xv. Prepare substrate 10 minutes prior to use and add 200 ul of
substrate solution to all the wells including A1.
xvi. Incubate at RT in dark for 30 minutes.
xvii. Add 50 ul of 4N sulfuric acid to all wells including A1 and mix.
xviii. Read absorbance at 450–620 nm within 60 minutes in ELISA
Reader.
168 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

CALCULATION OF RESULTS
Individual negative calibrator values must be less than or equal to 0.120
and greater than or equal to –0.005.
A plate is considered valid if the value of the blank is greater than or
equal to –0.020.
A plate is considered valid if both positive control values are greater
than or equal to 0.800.
Calculate the cut-off value by adding 0.600 to the mean absorbance
(NCx) of the negative control.
Cut off = NCx + 0.600

INTERPRETATION
Negative: Samples with absorbance value less than the cut-off value.
Positive: Samples with absorbance value equal to or greater than the
cut-off value.
Specimens with absorbance values less than –0.025 should be
retested in a single well.
Paste the printout in the HCV register and record the results in
blood stock register.
 C H A P T E R 47
Rapid Antigen Test for Malaria
(Pan Malaria)

SCOPE AND APPLICATION

Screening for malaria parasite is a mandatory test for each blood
unit before it is transfused. The standards for blood banks and blood
transfusion services adopted by NACO describe that all blood units
should be tested for malarial parasites using a validated and sensitive
antigen test.

RESPONSIBILITY
It is the responsibility of Laboratory Technician under supervision of
Medi­cal Officer to carry out the test.

MATERIALS REQUIRED
The Test Kit
The components of the kit are:
• Malaria test device.
• Sample loop.
• Nozzle and Nozzle cap.
• Assay buffer.
EDTA Blood Sample

PROCEDURE
Principle
Pan malaria card is an immunoassay based on the “sandwich” principle.
The method uses monoclonal anti-pan specific pLDH (parasite
Lactate Dehydrogenase) antibody conjugated to colloidal gold and
another monoclonal anti-pan specific pLDH antibody immobilized
on a nitrocellulose strip in a thin line. The test sample is added in the
sample well “A”, followed by addition of assay buffer in the buffer well
170 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

“B”. If the sample contains P. falciparum/P. vivax/P. malariae/P. ovale,

the colloidal gold conjugate complexes the pan specific pLDH in the
lysed sample. This complex migrates through the nitrocellulose strip by
capillary action. When the complex meets the line of the immobilized
antibody, the complex is trapped forming a purplish pink band which
confirms a reactive test result. Absence of colored band in the test region
indicates a non-reactive test result. To serve as a procedural control an
additional line of anti-mouse antibody has been immobilized on the
strip as control.
The test is carried out as per kit manufacturer’s instructions.

Method
• Bring the complete kit and the specimen to be tested to room tem­
perature prior to testing.
• Remove the test card from the foil pouch prior to use. The test
should be performed immediately after removing the test card from
the foil pouch.
• Label the test card with Donor’s ID.
• Place the nozzle on the assay buffer vial in such a way (as per kit) to
avoid the leakage.
• Mix the anti-coagulated blood sample evenly by gentle swirling
to make it homogeneous before use. Dip the sample loop into the
sample and make sure that the loop is full of sample. Blot the blood
ion to the sample pad in the sample well “A”.
• Add 5 drops of the assay buffer in the buffer well “B”.
• Allow the reaction to occur for 20 minutes.
• Read the result at 20 minutes.

Validation
This test has built-in quality control line “C” which always develops
color during the test, thereby confirming the proper functioning of the
device, reagent and correct procedural application. The test can detect
parasitemia level of 200 parasites per µl of blood. The sensitivity and
specificity of the test are 100 percent and 98.21 percent respectively.
Record the results in blood stock register.

INTERPRETATION
• Appearance of two purplish pink colored lines—one each in test
(T) and Control (C) regions indicate that the sample is reactive for
P. falciparum/P. vivax/P. malariae/P. ovale.
 Rapid Antigen Test for Malaria (Pan Malaria) 171

• Appearance of one purplish line in the region of control indicates

that the sample is non-reactive for all Plasmodium species.
• If no line appears after completion of the test either with clear
background or with complete pinkish/purplish background, the
test is considered invalid and should be repeated with a new card.
 C H A P T E R 48
Labeling of the Blood Bags

SCOPE AND APPLICATION

The blood after it is collected, remains in quarantine and is released
for transfusion only after testing for all mandatory tests required as
per provisions of Drugs and Cosmetic Act 1940 and Rules 1945. Before
blood units are taken on inventory for use, these are labeled as per the
provisions under the Drugs and Cosmetics Act 1940 and Rules 1945.
Labeling of blood units is a three-step process. The first step,
performed by the nursing staff, involves the appropriate labeling of the
primary collection bag. The second step, performed by the laboratory
staff, occurs after the blood is tested, and involves (among other
processes) demonstrating the ABO/Rh type of the donor and the expiry
date of the components made from the whole blood unit. Finally, the
third step, also performed by the laboratory staff, is done when the blood
is ordered for transfusion. It involves recording such data as the name of
the intended transfusion recipient, the results of the cross-match test,
etc. on the cross-match label.
All labeling steps must be performed with precise attention to detail.
Mix up of blood units with one another during labeling can potentially
lead to a patient dying from an ABO-incompatible transfusion reaction.
The label is required for identification and retrieval of blood units
for use, disposal and follow up in case of adverse reactions.

RESPONSIBILITY
It is the responsibility of the staff nurse and Laboratory Technician as
well as the Medical Officer of the blood bank to ensure proper labeling
of the blood units.

MATERIALS REQUIRED
• Unit number stickers.
• One yellow-top and one purple-top vacutainers.
 Labeling of the Blood Bags 173

• Pre-printed adhesive labels confirming to regulatory requirement

with color coding as per blood groups. Group A has yellow labels,
Group B pink labels; Group O, blue labels and Group AB have white
labels for both positive and negative Rh grouping.

PROCEDURE
• Immediately prior to collection of the donor’s blood, unit number
sticker is attached on top of the donor base label. Care must be
taken to ensure that this unit number has never been used before
by the Blood Bank.
• Write the collection date on the donor base label.
• Paste one unit number sticker on the donor form.
• And, finally, unit number stickers are attached to the blood sample
tubes.
• At this time, the blood bag is ready for the blood collection process.
• After collection and processing whole blood, the blood units remain
in quarantine in a separate blood storage refrigerator.
• Once all the reports of blood group and TTI testing are ready, place
the bags on a table in chronological order.
• Segregate those which are found reactive for any TTI or found
unsuitable for use and are disposed as per guidelines of bio-medical
waste. Retain those found suitable for transfusion on the bench for
labeling.
• Mention clearly the unit number, date of collection and expiry and
the volume on each label as per the grouping register records.
• Date of collection and date of expiry is very important. The expiry
date of blood collected in CPDA-1 bag is 35 days from the day of
blood collection which is considered as the zero days.
• After the bags are labeled, second technician must cross-check the
number and group on the bags tallying them with the records.
• Enter all labeled bags group-wise in the stock register.
• After checking, the labeled blood units are kept and stored in the
other blood bank refrigerator meant for tested blood units.
• All labeled tested blood bags are entered in the inventory for use.
• After receiving the requisition for blood/blood components, units
are cross-matched. Thereafter the details of the recipient, blood
group, blood/unit details, etc. are entered on the cross-match label
which is then stuck on the unit before its issue.
 C H A P T E R 49
Preservation of the Blood and
Components

SCOPE AND APPLICATION

Blood and blood components are stored in conditions designed to pre­
serve optimal viability and function during the storage period.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician to store the units
in the quarantine storage till mandatory tests are completed and those
found suitable for transfusion, after labeling, are transferred in their
respective storage areas.

MATERIALS REQUIRED
• Blood bank refrigerator maintaining temperature 4 ± 2°C.
• Deep freezers.
• Platelet incubator cum agitator.

PROCEDURE
• All untested units are kept in the quarantine in blood bank
refrigerator meant for untested blood.
• After testing is over, release the fully tested. Transfer those found
suitable for clinical use from quarantine area to the stock area
(Blood bank refrigerator meant for tested blood) after labeling.
• Label those found unsuitable for use with a biohazard label and
keep for disposal separately.
• Store whole blood and red cell concentrates in trays in the Blood
Bank Refrigerator maintaining temp. 4 ± 2°C. Each tray is reserved
for a particular group having its label pasted on the outer side.
Arrange the blood bags in chronological order, group-wise and
according to the expiry dates in trays and then stack the trays in the
refrigerator. This makes it very easy for the laboratory technicians
on duty to remove the bags for issuing, whenever required.
 Preservation of the Blood and Components 175

• Store blood collected in CPDA-1 up to 35 days and the red cells

separately in closed system up to 35 days. Red cells suspended in
additive solutions up to 42 days.
• Fresh frozen plasma, cryoprecipitates and FVIII deficient plasma bags
in the deep freezer at –40°C and the shelf-life of all these compo­
nents is 1 year.
• Platelets are stored in platelet incubator at 20–22°C on an agitator.
The platelets are stored up to 5 days.
• Maintain sterility of the blood units by taking all necessary precau­
tions.
• Ensure maintenance of storage conditions mandatory for blood
and its components throughout their storage in the blood bank.
Monitor the temperature of all storage areas with continuous
graphic recorder. Change the charts every week, and preserve them.
Check the alarm system every month.
Blood Cold Chain from Collection to Transfusion
 C H A P T E R 50
Inventory of the Blood Units
and Components

SCOPE AND APPLICATION

In order to avoid outdating and make optimum use of available blood,
it is important to maintain a day to day inventory of tested blood units
which helps in selection of blood units to be cross-matched for patients
requiring transfusion.

RESPONSIBILITY
The Laboratory Technician checks the records and transfers all the units
which are serologically negative and labeled to inventory.

MATERIALS REQUIRED
Inventory register.

PROCEDURE
• Inventory is maintained on a day to day basis. After labeling the
units, enter the numbers of whole blood unit numbers group-wise
on the right hand page of the inventory register kept in the serology
laboratory. The inventory bears columns for A group, B group, AB
group, O group as well as negative groups of these four groups.
Enter the units group-wise and according to the date of collection
in the inventory register (daily stock). The laboratory technician
on night duty is responsible for physical checking of the printed
number tag with the hand written number on the label and enters
in the inventory.
• After labeling the FFP, enter the donor unit numbers group-wise in
the stock register of FFP similar to blood units.
• Enter FVIII deficient plasma units labeled group-wise in the stock
register similar to plasma register.
• Enter the labeled cryoprecipitate unit numbers in the register.
 Inventory of the Blood Units and Components 177

• Clearly mark the inventory of bags that have less volume of

blood collected or are reserved for specific patients with specific
instructions.
• All unit numbers are entered group-wise and expiry date-wise in
the inventory register.
 C H A P T E R 51
Issue and Transport of
Blood Units

SCOPE AND APPLICATION

The blood and blood components are used as per the need of the pati­
ents. These are issued against the prescription of registered medical
practitioner (RMP) after ensuring the compatibility and testing results.
The Blood Bank must ensure that the blood is not wasted and made
available to needy patients following first-in-first-out (FIFO) policy.

RESPONSIBILITY
It is the responsibility of the Laboratory Technician to issue the blood
against the requisition of treating Physician/Surgeon (Registered
Medical Practitioner) after receiving the acknowledgment in writing on
the understanding that the whole blood/packed red blood cells (PRBC)
will be transfused within 30 minutes.

MATERIALS REQUIRED
• Issue register.
• Issue slip.
• Master record for blood (Stock register).
• Requisition form duly filled and signed.
• Compatibility report.
• Thermal box/Blood transport box.

PROCEDURE
A. Check List:
• In order to avoid outdating, implement FIFO policy.
• Carry out compatibility testing using SOP described earlier.
• Ensure that the compatible units are tested for TTI and found
suitable for use.
B. Remove the correct unit from blood bank refrigerator and keep it in
the thermal box /Blood Transport Box.
 Issue and Transport of Blood Units 179

C. At the time of issue of blood, observe the following:

• Cross-check the identification of the patient and the blood unit to
be issued.
• Check the expiry date to avoid issuing outdated blood.
• Inspect the unit to ensure that it does not have abnormal color or
appearance.
• Time of issue must always be recorded.
D. Keep blood unit in the thermal box for transport.
It is essential that the blood must be stored within the temperature
range of 2–8°C during transportation.
There must be system in place to monitor the temperature during
transportation. This can be achieved using specially designed blood
transport boxes. If these are not available, sturdy, well-insulated
containers may be used only after they have been evaluated and
validated to ensure that they can reliably maintain temperatures at
+2°C to +10°C for the planned journey, using appropriate coolants
or ice packs. The refrigerant recommended for most shipments is
wet ice in leak proof containers, such as plastic bags. Wet ice from
commercial ice-making machines is satisfactory. Super-cooled
cubed ice, canned ice or dry ice should not be used for shipping or
storing whole blood or red cells, because they can create very low
local temperatures which may cause red cells in their immediate
vicinity to freeze and undergo hemolysis. In an insulated container,
the temperature can be considered to be in the +2°C to +10°C range
so long as un-melted ice is still present on arrival at destination.
E. During transport, frozen plasma and cryoprecipitate must be main­
tained at or below the required storage temperature to be achieved
with a suitable quantity of dry or wet ice in well-insulated containers
or cartons lined with insulating material such as plastic air bubble
packaging or dry packaging fragments. During transportation,
frozen components must be maintained in frozen state throughout
the transport. That ensures they will remain frozen.
F. Every effort must be made to ensure that platelets are maintained
at temperatures between +20°C and +24 °C during transportation. A
well-insulated container without added ice is often sufficient.
G. Laboratory technician shall provide the following documents with
each blood unit issued from blood bank:
• Patient’s details on the cross-match label and compatibility report.
• Patient’s ABO and Rh group.
• Unique donation no (Unit No).
• Blood group of the blood unit.
• Blood reaction form.
180 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

H. Make entries in the issue register.

I. Instruct the individual to take the unit straight to Operation theatre
(OT)/Ward for transfusion.
J. Once blood unit is issued from the blood bank, It shall not be taken
back into the stock of the blood bank. However, if a unit of blood is
returned to the blood bank, the following checklist should be used
to decide whether it should be put back into stock or discarded.
• Check that the unit has been returned to the blood bank within 30
minutes of issue.
• Verify that the unit has not been opened, by squeezing it gently
and looking for blood at the entry port.
• Check the temperature by hand or by folding the unit around a
thermometer.
• After mixing the unit gently, keep it in the upright position while
it settles out’ in the refrigerator and look for signs of hemolysis or
other signs of deterioration in the plasma and red cells.
K. The unit must be discarded if:
• It has been out of the refrigerator for more than 30 minutes, or.
• There is any sign that the pack has been opened, or.
• There is any sign of hemolysis, or.
• If the temperature is over +10°C.
 C H A P T E R 52
Return of Whole Blood/
Packed Red Cells Unit

SCOPE AND APPLICATION

The Laboratory Technicians have the duty to see that the blood is not
wasted and made available to another patient of the same group to
ensure the safe blood transfusion.

RESPONSIBILITY
It is the responsibility of the Technical Supervisor and Laboratory Tech­
nicians to decide as to whether the returned blood unit should be put
back into the stock or discarded.

MATERIALS REQUIRED
• Issue register.
• Thermometer.
• Master record for blood (stock register).

PROCEDURE
Ideally, the blood should be requested only at the time from the blood
bank, when it is intended to be administered. If the transfusion cannot
be initiated promptly, the whole blood/packed RBCs unit is returned to
the blood bank. On receipt of blood unit, the laboratory technician will
perform the following checks for accepting the blood unit for further
use.
• Check that the unit has been returned to the blood bank within 30
minutes of issue.
• Verify that the whole blood/Packed RBCs unit has not been opened,
by squeezing it gently and looking for blood at the entry port.
• Check the temperature by hand or by folding the unit around the
thermometer.
182 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• After mixing the unit gently, keep it in the upright position while it
settles out in the refrigerator and look for signs of hemolysis or other
signs of deterioration in the plasma and red cells.
• It must be ensured that at least one sealed segment of integral do­
nor tubing is attached to the blood unit. In case the segment/s is/
are found detached from the tubing of the blood unit, then deta­
ched segments should be reattached after confirming the tube
iden­tification numbers.
• The whole blood/Packed RBCs unit is discarded if:
■ The blood unit has been out of refrigerator for longer than 30 min­
utes, or.
■ If the seal is broken, or.
■ There is any sign that the unit has been opened, or.
■ There is any sign of hemolysis, or.
■ If the temperature is over +10°C.
• Make entries in the issue register and master blood stock register
regarding the date and time of return and observations on the above
checks performed.
 C H A P T E R 53
Transfusion of Right Blood
to the Right Patient

SCOPE AND APPLICATION

The prime objective of this SOP is to minimize the risk of a patient
receiving a wrong blood/blood component unit and to provide the
guidelines for monitoring the patient during the blood transfusion so
as to identify the reaction if any, at the earliest for proper management.

RESPONSIBILITY
It is the responsibility of the Staff Nurse on duty and Consultant In-
charge/RMO to provide right blood to right patient once the blood unit
has been received from the blood bank.

MATERIALS REQUIRED
The following are needed for administering the blood/blood component:
• Blood/Blood component unit.
• Sterile blood administration set with filter/pressure line with filter
(in case of newborns).
• Tourniquet.
• Spirit wipes.
• Gauze sponges.

PROCEDURAL ISSUES
(a) Getting Blood from Blood Bank
The blood/blood components are processed for the patients on receipt
of the properly filled and signed requisition form (Annexure 53.1) from
the Consultant Doctor. Incomplete forms are not to be entertained. The
blood bank must provide the following while issuing the unit:
• Patient’s details on the cross-match label and compatibility report.
• Patient’s ABO and Rh group.
184 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Unique donation no (Unit No.).

Blood group of the blood unit.
• Blood Reaction Form (Annexure 53.2).
If all the information on the various documents as stated above
and the blood/blood component unit correlates, the blood/blood
component unit is issued after getting receipt thereof from the person
receiving the unit.

(b) Informed Consent

Clinicians are responsible for explaining and obtaining informed
consent (Annexure 53.3) prior to transfusion and for explaining to the
patient or legal guardian the reason for the transfusion as well as the
benefits expected, the risks and alternatives as outlined on the consent
form. The patient or legal guardian must sign the consent form.
This form must be appropriately dated and witnessed.

(c) Checking the Blood Unit (Fig. 53.1)

The blood unit should always be inspected for signs of deterioration:
• On arrival in the ward or operating room.
• Before transfusion, if it is not used immediately.
Discoloration or signs of any leakage may be the only warning that
the blood has been contaminated by bacteria and could cause a severe
or fatal reaction when transfused.

Fig. 53.1 Physical checking of blood unit

• Any sign of hemolysis in the plasma indicating that the blood has
been contaminated, allowed to freeze or become too warm.
• Any sign of hemolysis on the line between the red cells and plasma.
 Transfusion of Right Blood to the Right Patient 185

• Any sign of contamination, such as a change of color in the red cells,

which often look darker or purple/black when contaminated.
• Any clots, which may mean that the blood was not mixed properly with
the anticoagulant when it was collected or might also indi­cate bac­t­erial
contamination due to the utilization of citrate by proli­ferating bacteria.
• Any signs that there is a leak in the pack or that it has already been
opened.
Do not administer the transfusion if the blood unit appears
abnormal or damaged or it has been (or may have been) out of the
refrigerator for longer than 30 minutes.

(d) Storing and Transporting Blood Products Prior to Tran­s­fusion

Once issued by the blood bank, the transfusion of whole blood, red
cells and thawed fresh frozen plasma should be commenced within 30
minutes of their removal from refrigeration. If the transfusion cannot
be started within this period, they must be stored in an approved blood
bank refrigerator at a temperature of 2°C to 6°C. The upper limit of 6°C is
essential to minimize the growth of any bacterial contamination in the
unit of blood.
The lower limit of 2°C is essential to prevent hemolysis, which can
cause fatal bleeding problems or renal failure. If the ward or operating
room does not have a refrigerator that is appropriate for storing blood,
the blood should not be released from the blood bank until immediately
before transfusion.
It is essential that the blood must be kept within the temperature
range of 2–8°C during transportation. There must be a system in place
to monitor the temperature during transportation. This can be achieved
using blood transport containers.
All unused/residual blood products should be safely disposed of as
per guidelines of biomedical waste.

(e) Checking the Patient’s Identity and the Blood Unit before
Transfusion (Annexure 53.4)
The final check at the patient’s bedside is the last opportunity to detect an
identification error and prevent a potentially incompatible transfusion,
which may be fatal.
Before administering blood, two staff members (one of whom must
be a doctor or staff nurse) must check:
• The patient’s full identity.
• The blood unit compatibility report and cross-match label.
• The blood unit for signs of hemolysis or leakage from the pack.
186 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Any discrepancies means that the blood must not be transfused and
that the Blood Bank must be informed.

(f) Time Limits for Starting Transfusion

There is a risk of bacterial proliferation or loss of function in blood and
its products once they have been removed from the correct storage
conditions.
Time limits for transfusion
Start transfusion Complete transfusion
Whole blood/Packed RBC Within 30 minutes of Within 4 hours
remov­ing blood unit from
refri­gerator
Platelet concentrates Immediately Within 20 minutes
FFP and Cryo-precipitate As soon as possible Within 20 minutes

(g) Warming Blood

Blood should never be warmed in a bowl of hot water as this could lead
to hemolysis of the red cells which could be life-threatening.
Blood should only be warmed in a blood warmer and warmed
blood is most commonly required in:
• Large volume rapid transfusions.
• Adults: greater than 50 ml/kg/hour.
• Children: greater than 15 ml/kg/hour.
• Exchange transfusion in infants.
• Patients with clinically significant cold agglutinins.

(h) Pharmaceuticals and Blood Products

• Do not add any medicines or any infusion solutions other than
normal saline (sodium chloride 0.9%) to any blood component.
• Separate IV line if an intravenous fluid other than normal saline has
to be given at the same time as blood components.

(i) Monitoring the Transfused Patient

It is essential to take baseline observations and to ensure that the
patient is being monitored during and after the transfusion as per Blood
Transfusion Record/Reaction Form given at the Annexure 53.2 in order
to detect any adverse event as early as possible. This will ensure that
potentially life-saving action can be taken quickly.
Before commencing the transfusion, it is essential to:
 Transfusion of Right Blood to the Right Patient 187

• Encourage the patient to notify a nurse or doctor immediately if he

or she becomes aware of any reactions such as shivering, flushing,
pain or shortness of breath or begins to feel anxious.
• Ensure that the patient is in a setting where he or she can be directly
observed.
Begin transfusion at a rate of 20 cc/hr for the first 15 minutes.
Severe reactions most commonly present during the first 15 minutes
of a transfusion. All patients and, in particular, unconscious patients,
should be monitored during this period and for the first 15 minutes of
each subsequent unit as most ABO incompatibilities occur in the first
fifteen minutes.
• After the first 15 minutes have passed and no reaction noted.
• Take and record a second set of vital signs.
• Increase the rate of infusion depending on recipient’s hemodynamic
status.
• If hemodynamically stable, transfuse over 2 hours.
• If hemodynamically unstable, transfuse over 4 hours.
This time limit is empirical based on the time it takes the blood bag
to reach room temperature. Since blood is an excellent culture media,
keeping the blood bag at room temperature for longer duration could
result in bacterial overgrowth. In case, medical condition of recipient
demands transfusion over a longer period, ask for split units of blood
from blood bank and give each over 4 hours.
i. For each unit of blood transfused, monitor the patient:
• Before starting the transfusion.
• As soon as the transfusion is started.
• 15 minutes after starting the transfusion.
• At least every 30 minutes during transfusion.
• On completion of the transfusion.
• Hours after completing the transfusion.
ii. At each of these stages, the staff nurse will record the following vital
information on the blood administration record of the patient:
• Patient’s general appearance.
• Temperature.
• Pulse.
• Blood pressure.
• Respiratory rate.
• Fluid balance:
■ Oral and IV fluid intake.
■ Urinary output.
iii. Records:
• Time the transfusion is started.
188 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Time the transfusion is completed.

• Volume and type of all products transfused.
• Unique donation numbers of all products transfused.
• Any adverse effects.
The transfusion of each unit of the blood or blood component
should be completed within four hours of the pack being punctured. If a
unit is not completed within four hours, discontinue its use and dispose
of the remainder through the biomedical waste system.
When the transfusion is complete, take a full set of vital signs,
document on the Blood Administration Record/Reaction form, and
discontinue the blood tubing. Dispose of the used blood bag and IV
tubing into a red biohazard bag and place the red bag (Blood bags and
tubing are the exception to the hazardous waste rule and are considered
hazardous waste).

(j) Transfusion Reactions

If the patient appears to be experiencing an adverse reaction, the
following immediate actions may be taken:
• Stop the transfusion.
• Keep intravenous (IV) line open with normal saline.
• Check all blood component(s) labels, forms, patient identity for
errors.
• Notify patient’s physician as appropriate.
• Treat reaction.
• Notify blood bank; submit work-up specimens; submit report
forms.
In the case of a suspected transfusion reaction, do not discard the
blood pack and infusion set, but return the blood bag, administration set
along with 5 ml of patient’s post-transfusion blood (EDTA and clotted)
in the sterile tubes and sample of post-transfusion specimen of urine of
patient to the blood bank for investigation.
Record the clinical details and actions taken in the patient’s case-
notes.

DON’TS FOR BLOOD TRANSFUSION

i. Don’t use blood from unlicensed blood bank.
ii. Don’t delay initiation of blood transfusion.
iii. Don’t warm blood.
iv. Don’t use routine pre-transfusion medication.
v. Don’t infuse over more than 4 hrs.
vi. Don’t leave patients unmonitored.
 Transfusion of Right Blood to the Right Patient 189

vii. Don’t add any medication to blood bag.

viii.Discard blood if not utilized.
ix. Don’t ask for all the blood bags at one time.
x. Don’t use unmonitored refrigerator for storage.
xi. Don’t use the same transfusion set for more than one blood bag.
xii. Do not wet outlet port of blood.
xiii.Don’t store platelet in refrigerator.
xiv. Don’t be complacent while checking identifying information.
xv. Don’t insist for immediate relative’s blood and directed donation.
Before administering blood components, it is important to write the
reason for transfusion in the patient’s case-notes. If the patient later has
a problem that could be related to the transfusion, the records should
show who ordered the components and why. This information is also
useful for conducting an audit of transfusion practice.
The record you make in the patient’s case-notes is your best pro­
tection if there is any medicolegal challenge later on.
The following information should be recorded in the patient’s notes.
i. Whether the patient and/or relatives have been informed about the
proposed transfusion treatment.
ii. The reason for transfusion.
iii. Signature of the prescribing clinician.
iv. Pre-transfusion checks of:
• Patient’s identity
• Blood pack
• Compatibility label
• Signature of the person performing the pre-transfusion iden­tity
check.
v. The transfusion:
• Type and volume of each product transfused
• Unique donation number of each unit transfused
• Blood group of each unit transfused
• Time at which the transfusion of each unit commenced
• Signature of the person administering the blood component
• Monitoring of the patient before, during and after the trans­fusion.
vi. Any transfusion reactions.
190 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Annexure 53.1: Requisition Form for Blood/ Blood

Components
Name of Hospital
with Logo Paste patient ID label

Date: ............................. Received on .............. Al.............

Request No: ...................... Signature ....................................
Patient Patient’s Name: ......................... MR No:........ IP No.:........ Age:........ Sex:.........
Details Father’s/Husband’s Name:..................... Address:................................................
Ward/Department................................ Consultant in-charge:...............................

Clinical diagnosis with short history: .....................................................................................

Is the patient Newborn? Yes No
Hb gm/dl ............................ Platelet count......................... PT/PTT......................................
Previous History of:
Any Transfusion : Yes No Blood Group, if Known
Any Reaction : Yes No Antibodies (if any) detected earlier:...................
Allergy : Yes No
IN CASE OF FEMALES: No. of Pregnancies: ................... Stillbirth/Miscarriage.................
Hemolytic Disease of Newborn.................................

Blood or Blood Products Required

(Number of Units)

Whole Packed Platelet Single Donor Fresh Frozen Cryo-precipitate Others

Blood RBCs conc. Platelet Plasma (FFP) (Cryo.ppt)
(WB) (PRBC) (RANDOM) Apheresis (SOP)

Indication for Transfusion: ....................................................................................

Type of Request:    Routine    Emergency    Group and Save
Request on: ........................ Time: ............ am/pm    Donors Provided: Yes No

Specimen Collected and Labeled by: ........... Sign and ID # ........... Date ........... Time ...........
Certified that the blood samples and details in the requisition form are correct. I have
explained the necessity of Blood Transfusion or any procedure and the risk associated with
it to the patient/relatives. The informed consent for this has been taken from patient/relative.

Name of the doctor I/C and ID#: ........................................ Signature ....................................

Consent for Investigations (Rapid Method)
I am fully aware that the fresh whole blood is needed for my patient's treatment urgently. It
has to be tested for infectious diseases like HIV, HCV, VDRL, HbsAg, MP by Rapid method
which have slightly lower sensitivity and may miss such infections, if present, in the donor
even after testing. Keeping in view the requirement, I am prepared to take the risk involved
for endangering the patient to such infections.

Signature of the attending Physician ........

Name of Doctor (Assuming Responsibility) ...........................................................................
Date and Time: ................................. Signature: .............................. Emp ID: .........................

Contd...
 Transfusion of Right Blood to the Right Patient 191

INSTRUCTIONS
1. Send 2 ml blood in purple-top vacutainer and 4 ml blood in red-top
vacutainer with the requisition form; These must be labeled with
the following information:
a. Patient’s name and Family name
b. Age and Sex
c. MR. No.
d. IP No.
e. Patient’s Ward
f. Date
g. Signature of the staff taking the sample
2. In case of newborn baby up to 4 months old, send the mother’s
sample also.
3. All requests must accompany replacement donors.
4. The requisition form will not be accepted if it is not signed or any
section is left blank.
5. Blood and its products must be taken when required for definite
use, normally once issued it will not be taken back.
6. Cross matched blood will be kept reserve for 72 hrs only.
7. Blood must be used as soon as received.
8. Blood will be issued after about 2 hours of receiving the requisition
and blood sample.
9. Please ensure appropriate and rational use of blood.
FOR THE USE OF BLOOD BANK
Sl. Unit Blood Segment Date of Cross- Cross- Date and Name and Remarks
No. No. Group No. Expiry match match Time of Signature of
Method Report cross- Laboratory
matching Technician
1
2
3
4
5
6
7
8
9
10
11
12
13
14
192 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Annexure 53.2: Blood Transfusion Record/Reaction Form

(Return the form duly completed after the transfusion)

PATIENT’S PARTICULARS

Name Age and Sex IPD No

MR No Ward Bed No
Clinical diagnosis Patient’s Blood Group

Date of Transfusion: ................. Time Started: ............ Time Stopped: ..................

Rate of Transfusion: ................. Time of onset of reaction: ....................................

Blood Unit No: .......................... Blood Bag Segment No: ......................................

Blood Group: ............................ Date of Issue of Blood Unit: .................................

Issue time: ................................ Whether blood has been warmed?

How long blood unit was out of Refrigerator: ……………………………………

Clerical Check (please circle)

Patient ID correct Yes/No

Blood pack correct Yes/No

Temperature

Blood Transfusion Record correct Yes/No FEBRILE

AFEBRILE

Observation during transfusion:

Period of observation Vital Signs
Time Temp. Respiration BP Pulse
Pre-transfusion
During 1st 15 minutes
After next 30 minutes
After next 30 minutes
After next 30 minutes
After next 30 minutes
After next 30 minutes
After next 30 minutes
After next 30 minutes
After next 30 minutes
Contd...
 Transfusion of Right Blood to the Right Patient 193

After 4 hrs of
Completion of Blood
Transfusion
At the time of reaction

In Case of Reaction:
Signs and Symptoms Noticed—Please Tick
Fever Lower Back Pain Skin Pallor
Chills
Nausea/Vomiting Chest Pain Dark Urine
Itching Anxiety Dyspnea
Headache Bleeding from Wound Or IV Site

Any other symptoms (please note)……………………………………………………

……….......................................…………….......................................………………
…………………………………………………………………………

Name, Signature and ID of Consultant/RMO

INSTRUCTIONS:
• In case of no reaction, send this form duly completed. No sample is
required.
• In case of reaction, send this form duly completed along with the
following samples to the blood bank:
1. Used Blood Bag along with the administration set.
2. 5 ml of patient's post-transfusion blood (EDTA and clotted) in the
sterile tubes.
3. Sample of post-transfusion specimen of urine of the patient.
194 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Annexure 53.3
Name of Hospital and Logo Place patient identification label or write
below:
Name:___________________________
MR Number:_________IP Number:_________
Age/DoB:__________ Sex: _______________

INFORMED CONSENT: TRANSFUSION OF BLOOD/BLOOD

PRODUCTS
My/The Patient’s Doctor ___________________ has advised me that due
to my/The patient’s medical condition, the chances for my/The patient’s
improvement or recovery will be significantly helped by receiving blood/
blood products such as packed red cells, fresh frozen plasma, platelets or
cryoprecipitate by transfusions.
The doctor has explained the benefits that are expected from my/the patient
being transfused and as well the risk. I understand that although the blood/
blood products to be administered have been prepared and tested in
accordance with the mandatory provisions under the Drugs and Cosmetics
Act 1940 and Rules 1945 as amended from time to time yet, there is still a
very small (one in a thousand) chance that the blood/blood products will
be incompatible with my/the patient’s body and a transfusion reaction
(Hemolytic Transfusion Reaction) can occur. Although transfusion reactions
can be treated successfully. I understand that on very rate occasions they
can be fatal (one in two hundred fifty thousand transfusions). I understand
that allergic reactions like itching and fever to blood/blood products are
more common but can be treated and may not require the transfusion to
be stopped. In understand that even with testing by the most up-to-date
methods, there is a small chance that blood/the blood products may contain
a virus that will enter my/the patient’s system and may not be recognized
as an infection for many months or years. Even with proper testing, my/the
patient’s chances for contracting viral Hepatits may be approximately 30
in every 1 million transfusions or of contracting HIV in three of 1 million
transfusions and of contracting bacterial infections in one of one million
transfusions.
I have had an opportunity to ask questions regarding transfusion of blood/
blood products for myself/for the patients and with my signatures; I give
 Transfusion of Right Blood to the Right Patient 195

consent to administering blood/blood products for myself/for the patient

for the number of units on the date and time as per details mentioned below:
Sr Date Blood/Blood No. of Patient’s/Surrogate’s Doctor's
No. and Components Units (along with relation) Signature
Time Name and Address

Emergent/Life-threatening Circumstances
Informed Consent has not been obtained because of a life-threatening/
emergent medical condition. I have not provided the patient with
information sufficient to be considered Informed consent and I have
proceeded with ordering blood/blood products to be administered
in sufficient quantity to alter, improve or reverse a life-threatening/
emergent medical condition.
Time: ........................ Patient Name: ........................
Date : ........................ Consultant Signature: ........................
 Annexure 53.4: Pre-transfusion Checks 196

Paste patient identification label or write below:

Hospital/Blood Name:___________________________
Bank logobank MR Number:_________ IP Number:_________
Age/DOB:__________ Sex: _______________

Date: _______________ Ward/Dept: _______________ Bed No.: _______________

Patient’s Patient’s Blood Unit Compatibility Label Compatibility Blood/Blood
Check for: Must Match
Medical Wrist Label-Primary on the Blood/ Report Component Unit
what is on the
Record Band Label on the Component Unit
File Product

Physician’s Order
Patient’s First and Last name
Patient’s MR No
Patient’s ABO/Rh
Donor’s ABO/Rh
Expiry Date and Time of Blood/Blood Component
Type of Component Ordered for Transfusion
Blood/Blood Unit No and Segment No
Pack Integrity
Presence of Large Blood Clots

for
Evidence of Hemolysis in the Plasma or at the

Must look
Interface of Red Cells and Plasma
Contd...
Standard Operating Procedures and Regulatory Guidelines-Blood Banking
Contd...

(1st Checker)
Name of S/N: _____________________________ Sign: _____________________________ ID No: _____________________________
(2nd Checker)
Name of Doctor: _____________________________ Sign: _____________________________ ID No: _____________________________
Note:
• Before beginning the transfusion, it is extremely important to correctly identify the patient and the Blood product. A registered Staff Nurse and a second
Doctor must carry out the pre-transfusion checks.
• The pre-transfusion checks must be carried out at the bed side of the patient.
• Utilize the format given above while carrying out the pre-transfusion checks.
• Inform the Blood Bank immediately of any discrepancy. Do not start the transfusion until the discrepancy is resolved.
• To complete the process of pre-transfusion checks, both the Checkers must sign at the appropriate place on the format.

Note: This format may be suitably designed as per processes adopted by the hospital and data must be filled up in unshaded areas.
Transfusion of Right Blood to the Right Patient
197
 C H A P T E R 54
Investigation of
Transfusion Reactions

SCOPE AND APPLICATION

This SOP provides the protocol to be followed in case of transfusion
reaction for:
a. Diagnosis
b. Selection of appropriate therapy
c. Transfusion management
d. Prevention of future transfusion reaction.
Investigations should include correlations of clinical data with
laboratory result.
Important clinical data are:
a. Diagnosis
b. Medical history of pregnancies, transplant, and previous
transfusion.
c. Current medication.
d. Clinical signs and symptoms of the reaction.
The following questions related to the transfusion are also taken
into consideration:
• Amount of blood transfused to cause the reaction.
• How fast, how long?
• The use of blood warmer.
• Any filter used? Other solutions.
• Any drugs given at the time of transfusion.

RESPONSIBILITY
It is responsibility of the Laboratory Technician to accept the blood/
component implicated in the transfusion reaction which is returned
from the ward/operation theater. It is the duty of the same technician
to ensure that there is documented evidence of the nature of reaction
either on the transfusion request form or on a separate letter addressed
to blood bank, along with the post-transfusion blood sample (both EDTA
and clotted) and urine specimen, if necessary. The direct antiglobulin
 Investigation of Transfusion Reactions 199

test (DAT) should be performed on the post-transfusion EDTA sample

immediately on receipt before refrigeration. The blood unit and samples
should be preserved properly for detailed investigations.

MATERIALS REQUIRED
Equipment
• Refrigerator to store samples and reagents at 2–6°C.
• Table top centrifuge.
• Micropipettes.
• Microscope.
• Disposable pipette tips.
• Incubator.

Specimen
• Blood/component bag returned room ward/OT.
• Patient’s pre-transfusion blood sample (clotted).
• Patient’s post-transfusion blood sample (EDTA and clotted).
• Patient’s post-transfusion urine sample.

Reagents
• Anti-A, Anti-B anti-sera.
• Group A, B and O pooled cells.
• Papain-cysteine/22 percent bovine albumin.
• Antihuman globulin reagent (anti-IgG anti-C3d).
• IgG sensitized control cells.
• 0.9 percent saline.
• Distilled water.
• 30 g/l sulfosalicylic acid solution.
• Ammonium sulfate (NH4)2SO4.

Glassware
• Serum tubes.
• Coombs’ tubes (for patient grouping only).
• Microtubes.
• Pasteur pipettes.
• Glass slides.
200 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Small funnel.
• 20 ml test tubes.
• 5 ml pipette.

Miscellaneous
• Rubber teats.
• Disposal box.
• 2 plastic beakers.
• Whatman No.1 filter paper.
• 5 ml plastic vial with screw cap.
• Test tube rack.

PROCEDURE
The first step in the investigation of a reported transfusion reaction is to
rule out an acute hemolytic reaction (e.g. when the patient has received
blood from the wrong ABO group). The investigations are to be followed
in four steps called phases:

A. Phase I (Immediate Procedures)

• Clerical checks.
• Visual inspection of serum and plasma for free hemoglobin (pre
and post-transfusion).
• Direct anti-globulin test (post-transfusion EDTA sample).

B. Phase II
• ABO grouping and Rh typing of pre and post-transfusion sample of
patient and donor unit.

C. Phase III
• Major compatibility testing, pre and post-transfusion.
• Antibody screening test, pre and post-transfusion.
• Alloantibody identification.
• Antigen typing.
• Gram stain/Culture of donor blood unit.
• Free hemoglobin in first voided urine post-transfusion.

D. Phase IV
• Serum bilirubin (unconjugated bilirubin 5–7 hours post-
transfusion).
 Investigation of Transfusion Reactions 201

• Creatinine.
• Blood urea nitrogen (BUN).
• Quantitative serum hemoglobin.
• Serum haptoglobin, pre and post-transfusion.
• Peripheral blood film.
• Coagulation and renal output study.
• Urine hemosiderin.

A. Phase I (Immediate Procedures)

• Clerical check: Unfortunately, many transfusion reactions occur
because of preventable clerical errors. Mislabeling the product
and misidentifying the recipient must be ruled out by carefully
reviewing the records. These include patient identification, blood
component labels, type and cross-match data and requisition
forms. The transfusionist (Staff Nurse/Doctor) checks patient and
donor unit identification at the bedside while the investigating
Blood Bank technician and the doctor checks the same elements on
container specimens and records.
• Visual inspection: The patient’s post-reaction serum or plasma
should be inspected for evidence of hemolysis and compared to
pre-transfusion samples, if available. A pink or reddish hue present
only in the post-transfusion specimen is indicative of hemolysis
and hemoglobinemia. As little as 2.5 ml of hemolysis can be
visible. Presence of 0.2 g/L of Hb imparts pink color and in case of
presence of Hb upto 1g/L or more produces red color. However,
free myoglobin released from muscle can also cause a red or pink
discoloration and should be distinguished from hemoglobin if the
patient has crush injuries. If the sample was collected at least three
to six hours after the hemolytic event occurred, if icteric (bright
yellow discoloration), bilirubin is present. Heart bypass machines
and medications or other solutions “piggybacked” on blood lines
are common causes of non-immune intravascular hemolysis.
• Direct antiglobulin test (DAT): The DAT is used to demonstrate
the presence of antibodies or complements bound to red blood
cells on EDTA sample before refrigeration immediately on receipt.
A positive DAT in only the post-transfusion specimen indicates
red blood cells expressing antigens to which the patient has been
previously sensitized have been transfused. However, the DAT can
202 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

be paradoxically negative if the incompatible red blood cells have

been rapidly destroyed. If the DAT is positive, an elution should be
performed to identify the specificity of the antibody or antibodies
present.
These initial procedures are used to determine the likelihood that a
hemolytic transfusion reaction has occurred. Once acute hemolysis
has been ruled out, the treating physician/consultant is notified
about the results with the advice that subsequent transfusion may
be given. If the Phase 1 finding is positive, however, a Phase II
investigation is launched.

Results of phase I testing

i.
•All clerical checks match.
•Pre-transfusion and post-transfusion samples match in.
•Degree of hemolysis (no hemolysis exists).
•Pre-transfusion and post-transfusion samples have negative DAT
or same DAT result.
No evidence of hemolytic transfusion reaction. No further testing
required.
ii.
• Post-transfusion clotted sample shows visible signs of hemolysis
that is greater than pre-transfusion.
• Sample (check with collector to ensure sample was drawn without
difficulty).
• DAT from post-transfusion EDTA is positive (if pre-transfusion
sample is also positive, the post-transfusion DAT would be greater
than the pre-transfusion DAT in the case of a recent transfusion).
B. Phase II
If any of the 3 tests (clerical check, hemolysis, and DAT) in Phase I has
positive or suspicious results, the following tests are required to be
carried out:
• ABO grouping and Rh typing of pre and post-transfusion sample of
patient and donor blood unit.
• Check the blood bag, tubing and segments for hemolysis.
Red cell transfusions may have sensitized the patient to non-
ABO blood group antigens, such as those in the Rh, Kell, Duffy, and
Kidd systems. Transfusions of non-ABO-compatible plasma, whether
 Investigation of Transfusion Reactions 203

in platelet pools, in platelet apheresis units, as fresh frozen plasma,

or as cryoprecipitate, are often overlooked as a cause of a weak post-
transfusion DAT.
In case of any suspicious findings from Phase II, the technologist is
directed to carry out the tests of Phase III.

C. Phase III
The Phase III includes the following test:
• Antibody screening test, pre and post-transfusion.
• Alloantibody identification.
• Antigen typing.
• Free hemoglobin in post-transfusion first voided urine sample for
detection of intravascular hemolysis.
Antiglobulin cross-matches are performed with both pre- and post-
transfusion serum samples and donor unit red cells. A positive DAT on
the donor unit in Phase II would explain an incompatible cross-match
in Phase III. If the donor unit has an unexpected red cell alloantibody—a
rare occurrence, since blood suppliers screen all donor plasma for un-
expected allo-antibodies—the results of a minor cross-match between
donor plasma and patient pre-transfusion red cells are recorded.
The positive antibody screen test in post-reaction sample that was
not there before could be due to—
• Clerical or technical error.
• Pre-transfusion: screening cells represented a single dose.
• Passive transfer of antibody from a recently transfused component.
• Amnestic response: Appearance of alloantibodies can occur within
hours of exposure. It means patient now has new antibody which
does not cause detectable hemolysis. The patient must have antigen
negative red cells transfusion.
A Gram stain or cultures on the donor unit can be done any time
in case the clinical symptoms suggest bacterial contamination exposure
(Febrile reaction > 20, and other signs of shock).
The sulfosalicylic acid test helps to differentiate between hemo­
globin and non-protein pigment, probably porphyrin in the urine.
The ammonium sulfate precipitation test is based on the fact that
hemoglobin and myoglobin are precipitated in urine at different degrees
of ammonium sulfate saturation.
204 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Tests on post-transfusion urine sample

Red color urine indicates hematuria or hemoglobinuria

If the filter retains color, it is due to myoglobin.

In case filter is clear and the precipitate is colored, it is due to Hb.

D. Phase IV
If any findings in Phases 1, II and III reveal a transfusion error or if
transfusion-related hemolysis is strongly suspected, the following tests
are carried out to support the diagnosis of immune hemolysis.
• Serum bilirubin (Unconjugated bilirubin 5–7 hours post-
transfusion) to determine the severity of hemolysis.
• Serum creatinine.
• Serum urea.
• Quantitative serum hemoglobin using ortho-tolidine method to
det­er­mine the severity of hemolysis.
• Serum haptoglobin, pre and post-transfusion.
• Peripheral blood film for spherocytes in hemolytic transfusion reac­
tions.
• Serial hemoglobin, hematocrit and platelet count for monitoring
course of hemolysis.
• Blood coagulation studies for detection and monitoring of DIC.
• Coagulation and renal output study.
• Urine hemosiderin.
 Investigation of Transfusion Reactions 205

• Anti-IgA Ab and quantitative IgA testing in anaphylactic (non-

hemo­lytic) reaction.
• WBC antibody screen in donor and recipient and chest X-ray
for infil­trates in transfusion-related acute lung injury—TRALI
(hypoxia/respiratory failure/pulmonary edema).
• ECG for evidence of hyper-kalemia.
• Quantitative measurement of electrolytes.

Limitations
The non-serologic possibilities of hemoglobinemia and hemoglobinuria
are:
1. Hemolysis of blood before transfusion.
2. Poor technique of collecting post-transfusion sample.
3. Myoglobinuria following major surgery.
4. Infusion of distilled water during prostatectomy.
5. Hemolysis due to artificial valve.
6. Patient’s clinical condition; autoimmune hemolytic anemia or
paroxysmal nocturnal hemoglobinuria.
7. Use of glucose or dextrose through the same line before starting blood.
8. Addition of certain drugs to blood such as ethacrynic acid,
hydrocortisone or diphenyl hydantoin.
When serological testing is complete, the Medical Officer of
Blood Bank evaluates the workup and may write recommendations to
clinicians for future hemotherapy and treatment, perhaps including the
use of leukocyte-depleted or antigen-negative red cells, premedication
with antipyretics or antihistamines before transfusion, the use of
crossmatch-compatible or apheresis platelets instead of random platelet
concentrates, or obtaining the patient’s tissue type in preparation for
supplying HLA-matched components.
Whenever the laboratory investigation rules out the primary causes,
the Medical Officer of Blood Bank shall report as: “No evidence of
clerical error, hemolysis, or serologic incompatibility found.”
206 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Acute Reactions < 24 hrs

S. Reaction Frequency Usual Prevention
No. cause
1. Hemolytic Uncommon ABO Proper identification of the patient
transfusion incompati­ from sample collection to blood
reaction bility administration, proper labelling of
samples and products is essential.
Prevention of non-immune hemoly-
sis requires adherence to proper
handling, storage and administra-
tion of blood products.
2. Febrile non- Common Cytokines, A proportion of patients who
hemolytic anti- have febrile reactions will have
reaction leukocyte similar reactions to subsequent
antibody transfusions. Many are prevented
by leukocyte filtration (either
bedside or pre-storage).
3. Allergic Uncommon Antibodies In case of history of experiencing
transfusion to plasma repeated urticarial reactions,
reaction proteins give an antihistamine such as
chlorpheniramine 0.1 mg/kg IM or
IV 30 minutes before commencing
the transfusion, where possible.
4. Anaphylactic Uncommon Antibodies Patients with anti-IgA antibodies
transfusion to IgA require special blood products such
reaction as washed red blood cells and
plasma products prepared from IgA
deficient donors. Manage further
transfusion in consultation with the
hematologist-on-call.
5. Transfusion- Rare Antibodies Firstly, there should be a clear indi-
related to leuko- cation for the transfusion. Screen-
lung injury cytes ing of the donors for the presence
(TRALI) of HLA antibodies is expensive and
time consuming. Earlier detection
may lead to early diagnosis, treat-
ment, and prevention.
*Recently method for prevention
of transfusion-related acute lung
injury using riboflavin and light has
been patented. This prevents the
formation of bioactive substances
in a pathogen inactivated blood
component. The steps include
illuminating the blood component
with light at a sufficient energy so
that an alloxazine photosensitizer
Contd...
 Investigation of Transfusion Reactions 207

Contd...

present in the blood component

may be photolyzed to inactivate any
pathogens which may be present;
preventing the formation of bioactive
substances in the pathogen
inactivated blood component; and
storing the pathogen inactivated
blood component.
*http://www.freshpatents.com/-
dt20090122ptan 20090023130.php
6. Marked Rare Bacterial Inspect blood products prior to
fever with contamina- transfusion. Bacterial contamination
septic shock tion and other abnormalities can be
detected by visual inspection in
some of the units (clots, clumps,
or abnormal color). Maintaining
appropriate cold storage of red cells
in a monitored blood bank refrigerator
is important. Transfusions should not
proceed beyond the recommended
infusion time (4 hours).
7. Congestive Common Volume Packed cells should be given
heart failure overload slowly over 4 hours. The usual rate
of transfusion is about 200 ml per
hour. In patient at risk, rate of 100
ml per hour or less are appropriate.
Diuretics should be given at the
start of the transfusion and only one
or two units of concentrated red
cells should be transfused in any
24-hour period.
Blood transfusion should be given
during the daytime, overnight
transfusion should be avoided.
Wherever possible, packed cell
should be used instead of whole
blood.
8. Hypocalce- Uncommon Citrate The anticoagulant citrate is usually
mia toxicity metabolized to bicarbonate
following blood transfusion.
Therefore the prophylactic use of
calcium salts, such calcium chloride
is not recommended. Their use
should only be restricted in cases of
clinical or biochemical evidence of
reduced ionized calcium.
Contd...
208 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Contd...
9. Hypothermia Uncommon Rapid infu- Appropriately maintained blood
sion of cold warmers should be used during
blood massive or exchange transfusion.
Additional measures include warm-
ing of other intravenous fluids and
the use of devices to maintain pa-
tient body temperature.
10. Hyper Uncommon Red cell Fresh blood less than 7 days old is
kalemia storage used.

Delayed Reactions> 24 hrs

S. Reaction Frequency Usual cause Prevention
No.
1 Hemolytic Uncommon Development An antibody screen is
of a red cell performed as part of
antibody pre-transfusion testing.
When an antibody is
detected, it is identified
and appropriate antigen
negative blood is provided.
Sometimes antibodies fall
below detectable limits
and may not be detected
by pretransfusion testing.
However, some reactions
are due to rare antigens
(i.e. anti-Jka blood group
antibodies that are very
difficult to detect during
pretransfusion testing.
2 Graft- Rare Engraftment It is prevented by gamma
versus- of transfused irradiation of cellular blood
host functional components given to
disease lymphocytes patients at risk to stop the
(GvHD) proliferation of transfused
lymphocytes. Alternately
leukocyte depletion of
blood products is done.
3 Post- Rare Anti-platelet Leukocyte reduction of
transfu- antibodies blood products to levels
sion less than 106/unit reduces
purpura the likelihood of allo-
immunization. This can be
achieved through the use
of pre-storage or bedside
leukocyte reducing blood
products.
Contd...
 Investigation of Transfusion Reactions 209

Contd...

4 Iron The transfusion Multiple Iron binding agents, such

overload dependent transfusions as desferrioxamine, are
patients widely used to minimize
accumulate iron the accumulation of iron
over a period in these patients. The aim
of time since is to keep serum ferritin
there is no levels at <2000 ug/litre.
physiological
mechanism to
eliminate iron
5 Transfu- 1 in 10,000 e.g. HIV, The single most effective
sion trans- transfused units Hepatitis, way of protecting patients
mitted Variant against both known and
diseases Creutzfeldt- unrecognized blood-
Jackob borne infections is to
Disease avoid the use of blood
(vCJD), etc. products or tissues unless
there is a well-founded
reason. Donor selection
criteria and subsequent
screening of all donations
are designed to prevent
disease transmission, but
these do not completely
eliminate the hazards.
Blood or blood product
should be released for
transfusion until these and
all other nationally required
tests are shown to be
negative.

HEMOVIGILANCE PROGRAM
A hemovigilance program as an integral part of pharmacovigilance
program of India (PvPI) at national level has been launched on
December 10, 2012. The objectives of this program are:
1. Monitor transfusion reactions.
2. Create awareness among healthcare professionals.
3. Generate evidence-based recommendations.
4. Advise central drugs standard control organization (CDSCO) for
safety related regulatory decisions.
5. Communicate findings to all stakeholders.
6. Create national and international linkages.
210 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

National Institute of Biologicals (NIB) is the National Coordinating

Centre for Hemovigilance.
Hemovigilance is a continuous process of data collection and
analysis of transfusion-related adverse reactions in order to investigate
their causes and outcomes, and prevent their occurrence or recurrence.
It includes the identification, reporting, investigation and analysis of
adverse reactions and events in recipients and blood donors as well
as incidents in manufacturing processes and, eventually errors and
“near-misses”. A hemovigilance system is also an integral part of quality
management in a blood system, triggering corrective and preventive
actions, and for the continual improvement of the quality and safety of
blood products and the transfusion process.
Initially, 60 medical colleges were enrolled under this program.
This number has increased to 117. These institutions collect data in
respect of adverse reactions associated with blood transfusion and
blood product administration in transfusion reaction reporting form
(TRRF) (Annexure 54.1) developed by NIB and forward this information
to the Coordinating Centre NIB through software. This data will be
collated and analyzed to identify trends and recommend best practices
and interventions required to improve patient care and safety. These
recommendations will be forwarded to national coordinating center
PvPI for onward transmission to Drugs Controller General (India),
Central Drugs Standard Control Organization for formulating safety
related regulatory decisions on blood and blood products transfusion.
 Investigation of Transfusion Reactions 211

Annexure 54.1: Transfusion Reaction Reporting Form

(TRRF) for Blood & Blood Products
Indian Pharmacopoeia Commission–National Institute of Biologicals
Ministry of Health & Family Welfare–Govt. of India
Hemovigilance
(Pharmacovigilance Programme of India)
Transfusion Reactions Reporting Form for Blood & Blood Products
For Reporting of Transfusion Reactions by Healthcare Professionals
A. Patient Information *Mandatory Field
Patient initials* ...................... DoB/Age in years* ................. Blood Group* ....................
Diagnosis ................................ Hospital Code No* .............................................................
Hospital Admission No.* ........................................................ Sex.* F M
Date & Time of Transfusion* ............................ Date & Time of Reaction* .......................
Date & Time of Recovery ..................................
B. Transfusion Product Details*
Components Select Unit Expiry Manufacture Batch Indications 1st time/
components number date blood bags number Repeat
(transfused) Transfusion
(No. of
repeats)

Whole Blood

Red Blood Cells

Platelets
Apheresis

Platelets Pooled/
RDP

Solvent
detergent (SD)
Plasma

FFP

Cryoprecipitate

Any other

Blood Products Manufacturer Batch Number Expiry Date

(Please Specify)

C. Nature of Adverse Reactions*

S. No. Reactions Please Tick ()

1. Immunological Haemolysis due to ABO Incompatibility

2. Immunological Haemolysis due to other allo-antibodies

3. Non-immunological Heamolysis

4. Transfusion Transmitted Bacterial Infection

5. Anaphylaxis/Hypersensitivity

6. Transfusion Related Acute Lung Injury (TRALI)

Contd...
212 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Contd...
7. Transfusion Transmitted Viral Infection (HBV)

8. Transfusion Transmitted Viral Infection (HCV)

9. Transfusion Transmitted Viral Infection (HIV-1/2)

10. Transfusion Transmitted Viral Infection, other (Specify)

11. Transfusion Transmitted Parasitic Infection (Malaria)

12. Transfusion Transmitted Parasitic Infection, other (Specify)

13. Post-transfusion Purpura

14. Transfusion-associated Graft versus Host Disease (TAGv HD)

15. Febrile Non-hemolytic Reactions (FNHTR)

16. Transfusion-associated Dyspnea (TAD)

17. Transfusion-associated Circulatory Overload (TACO)

18. Other Reaction(s)

D. Outcomes of the Adverse Reactions* E. Reporter*

• Death following the adverse reactions Name and professional Address: __________________
• Recovered Pin Code: ______________ Email: __________________
• Recovered with sequelae Tel No. (with STD code) __________________________
• Permanently disabled
• Unknown

Any other information _____________________ F. Casualty Date of this report (DD/MM/YYYY)

Assessment*
 C H A P T E R 55
Quality Control/Assurance of the
Blood/Blood Components

SCOPE AND APPLICATION

Quality control (QC) is a procedure or set of procedures intended to
ensure that a manufactured product or performed service adheres to
a defined set of quality criteria or meets the requirements of the client
or customer. QC is similar to, but not identical with, quality assurance
(QA). QA is defined as a procedure or set of procedures intended to
ensure that a product or service under development (before work is
complete, as opposed to afterwards) meets specified requirements. QA
is sometimes expressed together with QC as a single expression, quality
assurance and control (QA/QC).
In blood transfusion service, the primary goal of quality is trans­
fusion of safe unit of blood. The objective is to ensure availability of
a sufficient supply of blood, blood components of high quality with
maximum efficacy and minimum risk to both donors and patients.
The quality assurance in blood transfusion service is multifaceted.
This requires a correctly organized scheme of management with
adequate emphasis on the following areas:
• Personnel and premises.
• Standard operating procedures.
• Laboratory testing (Techniques).
• Reagents.
• Equipment.
• Documentation.
It is important to develop an elaborate quality assurance program
and procedures as defined by Drugs and Cosmetics Act 1940 and Rules
1945, Indian Pharmacopoeia and National Blood Policy, keeping in mind
that the available resources are used in most efficient, cost-effective and
ethical manner.

RESPONSIBILITY
It is the responsibility of the administration/management to see that the
staff in position and the design and construction of blood transfusion
214 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

premises meets the requirements as laid down in the Drugs and

Cosmetics Act 1940 and Rules 1945.
The technical staff of the Blood Bank is responsible for providing a
high level of assurance of safe blood and transfusion practices to blood
donors, physicians, patients and patients’ families.
Assurance of a quality product includes issues of safety of patients
and blood donors, reagent quality control, monitoring of equipment
repair and maintenance, competence of personnel, and testing of a
defined number of units of each product for the appropriate parameters
and hazardous waste management. Inherent in this goal is the provision
of a safe work environment. Adequate record keeping is essential to
the process of quality and safety assurance. Besides, appropriate data
collection, retrieval and analysis are also essential to the process of
monitoring quality.
It is the responsibility of the blood bank to develop its own SOPs for
meeting the specified standards uniformly, monitoring the performance
of the task in more effective way, standardizing the training of the
staff, reducing the adverse effects on performance in case of change/
absence of staff, etc. The technical staff is also responsible for blood/
blood component collection, processing, labeling, storage, issue and
documentation.
It is also for the technical staff to ensure that the each equipment
in the blood bank is in perfect order and that the reagents are stored
and used as per guidelines of the manufacturers as well as statutory
requirements.

SCHEME FOR QUALITY CONTROL/ ASSURANCE OF VARI-

OUS BLOOD COMPONENTS
A. Whole Blood
Quality control
• Frequency of control: 1% of all units with minimum of 4 units per
month.
• Storage: 2°C to 6°C, for CPDA-1 the storage time is 35 days.
• On expiry date: Measure HCT, pH, total Hb, K+ and perform sterility
assays.

Quality assurance
• Volume: 350 ml/450 ml ± 10 percent excluding anticoagulant which
is 49/63 ml for 350/450 ml blood collection bag respectively.
 Quality Control/Assurance of the Blood/Blood Components 215

• HCT: 40 ± 5 percent.
• pH > 6.5.
• K < 27 mmol/L.
• Hb: minimum 45 g/unit for 450 ml blood collection bag.
• Sterility: No growth.

B. Packed Red Cells (PRC)

Quality control
• Frequency of control: 1 percent of all units with minimum of 4 units
per month.
• Storage: 2°C to 6°C, for CPDA-1, the storage time is 35 days.
• On expiry date: Measure HCT, pH, total Hb, K+ and perform sterility
assays.

Quality assurance
• Volume: 280 ml ± 50 ml, frequency of control 1% of all units.
• HCT: 55–65%.
• pH > 6.5.
• K < 78 mmol/L.
• Hb: minimum 45 g/unit.
• Sterility: no growth.

C. Platelet Concentrates
Quality control
• Prepared within 6 hours of blood collection.
• Must evaluate at least 4 platelet preparations monthly for platelet
count, pH and plasma volume.
• Platelets should be selected from each centrifuge in use.
• The temperature at which pH is measured should be the same as
stored.
• Label the volume, the actual volume by measurement must be 10
percent of the stated volume.
• Storage: 20–24°C.
• Temp. should be recorded at least every 4 hours during storage.

Quality assurance
• Volume > 50–60 ml from 1 unit of whole blood (450 ml). By apheresis
volume: 200–300 ml.
• pH: > 6.0 (at the end of permissible storage).
216 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Platelet count: > 4.5 × 1010/Unit. By apheresis: > 3–5 × 1011.

• WBC contamination: < 1.50 × 109/litre. By apheresis: < 5.0 × 106/
litre.
• RBC contamination: < 0.1 × 1012/litre. By apheresis: traces to 0.5 ml.
• Macroscopic appearance no visible platelets aggregates.
• Sterility: No growth.

D. Fresh Frozen Plasma

Quality control
• 4 units per month.
• Storage:
■ 24 months at below –30°C.
■ 12 months at –25 to –30°C.
■ 3 months at –18 to –25°C.
• Thawed at temp. between 30–37°C and transfused within 24 hours
after thawing.

Quality assurance
• Volume: 220–250 ml.
• Factor VIIIc: > 0.7 IU/ml-every 2 months.
• Stable coagulation factors: 200 units of each factor
• Fibrinogen: 200–400 mg
• No leakage after pressure in plasma extractor, before freezing and
after thawing.
• Macroscopic: no abnormal color or visible clots.
• Residual cell:
■ Red cell: < 6.0 × 109/l.
■ Leukocyte: < 0.1 × 109/l.
■ Platelets : < 50 × 109/l.

E. Cryoprecipitate
Quality control
• Assayed on at least 4 bags/month—for factor VIII.
• Storage:
■ 24 months at below –30°C.
■ 12 months at –25 to –30°C.
■ 3 months at –18 to –25°C.
• Must be thawed at 37°C and used within 6 hours.
 Quality Control/Assurance of the Blood/Blood Components 217

Quality assurance
• Volume: 10–20 ml.
• Factor VIII: > 70 IU/unit.
• Fibrinogen: > 140 mg per unit.
• Macroscopic: Homogeneous.
• Sterility: No growth.

Note
• Since it is difficult to measure the volume of the blood collected in a blood
bag, the volume is indirectly inferred by weighing the blood bag using blood
collection scale. The factor to convert blood volume to weight is 1.05, i.e.
1 ml of blood = 1.05 gm of blood. Accordingly following formula is used to
calculate the amount of blood collected in 350 ml/450 ml capacity blood
bag.

Total weight of blood bag = (vol. of blood x 1.05) + wt. of empty bag
• The hemoglobin is determined by Cyanmethemoglobin method
and is expressed as gms per unit.
• pH of the blood and components is measured after the expiry date
labeled on the unit, using a pH meter.
• Packed cell volume is determined using micro-hematocrit centri­
fuge after mixing the sample thoroughly.
• Total leukocytes, red cells and platelet counts are done by recognized
methods.
• Factor VIII coagulation activity is determined only by the laboratory
involved in its regular testing.
• Sterility testing is done as per IP.
• Macroscopic inspection of all blood components is done by
the blood bank staff before processing and before issue for any
abnormal appearance, e.g. hemolysis.
• All tests are done on units which are expired in the blood bank.
Proper record keeping of each and every aspect of blood transfusion
service forms an important part of quality assurance system. SOPs
constitute one of the significant elements of documentation.
 C H A P T E R 56
Storage of the Consumables,
Reagents and Kits

SCOPE AND APPLICATION

The quality assurance system requires that all the reagents used for
various test procedures are stored according to the manufacturer’s
instructions. Any lacunae in the storage conditions, reduce the effectivity
of the rea­gents.

RESPONSIBILITY
It is the responsibility of Laboratory Technicians to store all the reagents
and kits as per manufacturer’s instructions.

MATERIALS REQUIRED
• Blood bank refrigerator.
• Refrigerator.
• All reagents and kits.
• Stock register.

PROCEDURE
a. Donor Room
i. Store disinfectants for preparation of phlebotomy sites at room
temperature (22°C–25°C) in the donor room.
ii. Store blood collection bags in air-conditioned room (22°C–25°C).
b. Red Cell Serology Laboratory (RCS):
Store ABO reagents anti-A, anti-B, anti-D, ID Diluent (LISS), pooled
A, B, and O red blood cells, reagents in the refrigerator maintained
at 2°–8°C or as per manufacturer’s instructions. The Diclon cards are
stored at room temperature.
c. Transfusion of Transmissible Infections Testing (TTI):
Store HBsAg, HIV, HCV and VDRL kits at 4°–6°C in the designated
refrigerator in the TTI laboratory or as per manufacturer’s instru­cti­
ons.
 Storage of the Consumables, Reagents and Kits 219

d. Quality Control (QC):

i. Store Copper sulfate stock and working solutions, 0.9% normal
saline, and distilled water at RT (22°–25°C).
ii. Store chemicals like copper sulfate, sodium chloride and cal­cium
chloride powders at RT (22°–25°C).
It is important to carry out the following activities for quality assu­
rance:
• Maintain a stock register for all reagents.
• On receipt, make entries of number of vials/kits received, name of
manufacturer, batch number and expiry date in this register.
• Issue the reagents for use, only after a QC check is performed.
• Enter all issue records in the stock register.
• Order all reagents/kits; no sooner the critical level is reached.

Note
Critical level for all reagents/kits is normally maintained as per the requirement
of reagents, as well as the time taken by the procurement procedure to ensure
that reagents are received before the stock in use is exhausted. The new batch
received should be tested against the batch in use.
 C H A P T E R 57
Equipment Maintenance

SCOPE AND APPLICATION

This procedure applies to all the instruments and equipments used
within the blood bank.

RESPONSIBILITY
It is the responsibility of Laboratory Technicians to ensure that the equ­
ip­ment is in perfect order. Equipment used in the collection, proce­
ssing, testing, storage and sale/distribution of blood and its components
shall be maintained in a clean and proper manner and so placed as to
facilitate cleaning and maintenance. The equipment shall be observed,
standardized and calibrated on a regularly scheduled basis as described
in the Standard Operating Procedures Manual based on Manufacturer’s
Instruction manual of the equipment and provisions of Drugs and
Cosmetics Act 1940 and Rules 1945 as amended from time to time and
shall operate in the manner for which it was designed so as to ensure
compliance with the official requirements (the equipments).
All equipments need preventive maintenance including cleaning,
regular calibration and periodic verification of performance accuracy
which is either carried out by the hospital maintenance section or by
the manufacturer under maintenance contract. The maintenance plan
as given by the manufacturer of the equipment should be adhered to.

PROCEDURES
The maintenance work described in the maintenance plans of the major
equipment in the blood bank is carried out according to the instructions
in the User Manual as well as the provisions of the Drugs and Cosmetics
Act,1940 and Rules 1945.
A. Centrifuge for Gel Cards
No internal part of the centrifuge is to be lubricated or oiled
 Equipment Maintenance 221

Sr. Time Activity To be Method

No. carried
out by
a. As Cleaning LT Clean the tub, lid, outer housing and
required surface of centrifuge head with a
lint-free cloth moistened with 70%
ethyl alcohol after switching off and
removing the power cord. Clean inside
of the card holder in the same way.
Allow the machine to dry.
b. As Decontamina- LT/Tech In case of contamination, the
required tion Support machine should be decontaminated
immediately. Never use cleaning agent
containing chloride or ammonium.
c. Weekly Checking the LT/Tech The ID card holders are checked for
mobility of Support ease of movement and the full 90
the ID card degree swivel angle. If it does not fall
holder back into its original position or the
movement is stiff, contact bio-rad.
d. Weekly Inspecting the LT/Tech Seal and lid are safety components.
seal and lid Support Check for any damage, e.g. nicks,
tears and pressure marks.
e. Yearly Checking the Tech Contact manufacturer
target speed Support
f. Yearly Checking the It is compared with the stop watch. If
centrifugation the test is negative, contact Bio-rad
time

B. Incubator for Gel Cards

No internal part of the centrifuge is to be lubricated or oiled
Sr. Time Activity To be Method
No carried out
by
a. As required Cleaning LT Clean the tub, lid, outer
and housing with a
lint-free cloth moistened
with 70% ethyl alcohol
after switching off and
removing the power cord.
Remove the hot plate to
clean the entire tub. Allow
the machine to dry.
b. As required Decontamination LT/Tech In case of contamination,
support the incubator should
be decontaminated
immediately. Never
use cleaning agent
containing chloride or
ammonium.
Contd...
222 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Contd...

c. Weekly Checking /replace LT/Tech Check the O ring for

O ring support damage, e.g. nicks tears
and pressure marks. In
case damaged, it is to be
replaced.
d. Every 6 Check/ Replace Tech support Manufacturer
months fan filter
e. Yearly Checking Tech support Manufacturer
incubation temp.
f. Yearly Oil the springs of Tech support Manufacturer
the hinges
C. Hemo Control
Sr. Time Activity To be Method
No carried
out by
a. As required Cleaning LT Clean the housing and the touch screen
with a lint-free cloth lightly moistened
with clear waterpower cord.
Clean the cuvette holder with mild soap
solution.
Cleaning the optical unit is very delicate.
It is done with a special cleaner and no
cleaning agent is used.
b. As required Charging LT To maintain the full capacity, batteries
and care should always be discharged as far as
of the possible before being recharged.
battery
c. As required Repairs Tech In case the instrument is not
support functioning even after resetting,
contact bio-rad
• VDRL Shaker RS-12R
Sr. Time ActivityTo be carried Method
No out by
a. As required Cleaning LT Clean the instrument with a lint-
free cloth lightly moistened with
mild soap water and then dried
with a soft absorbent cloth
b. As required Electrician Rotating tracks under the platform
in lubrication be lubricated with a deep or two
or light machine oil
c. As required Repairs Tech support In case the instrument is not
functioning, contact manufacturer
 Equipment Maintenance 223

D. Microplate ELISA Reader

Sr. Time Activity To be Method

No carried out
by
a. As required Cleaning LT Clean the instrument with a lint-
free cloth lightly moistened with
clear. Decontamination is done
using 70% isopropanol
Clean LCD using soft cloth
b. As required LT Disconnect the power cord and
Chang-
then change the fuse.
ing the Open the rear cover of the
lamp instrument by pressing the release
latch located on the top left portion
of the cover. Remove the two
screws on the black plastic cover
and carefully remove the plastic
cover. Remove the screws on the
metal cover. Lift the cover up and
away from the instrument. This will
allow the lamp to be pulled free
from the lamp housing. Carefully
replace the lamp and place the
metal cover. Tighten the screws
and replace the plastic cover.
c. As required Chang- LT Replace with the same type of
ing the fuse on the rear panel of the
fuse instrument. If instrument continues
to blow fuses, discontinue the use
and contact the manufacturer.
d. As required Repairs Tech support In case the instrument is not
functioning, contact manufacturer.

E. Incubator

Sr. Time Activity To be Method

No carried
out by
a. As required Cleaning LT Clean the chamber and outer
surface at regular intervals with a
lint-free cloth lightly moistened with
mild soap water
b. Daily Accuracy of LT Compared against thermometer
thermometer
224 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

F. ELISA Washer

Sr. Time Activity To be Method

No carried
out by
a. As required Cleaning LT Clean the instrument with a lint-free
cloth lightly moistened with clear
water. Decontamination is done
using 70% isopropanol.
Clean LCD using soft cloth.
b. As required Periodic LT Periodic check up to determine
check whether the microwells are correctly
filled and emptied
c. As required Repairs Tech In case the instrument is not
Support functioning, contact manufacturer

G. Bench Centrifuge R 8C

Sr. Time Activity To be Method

No carried out
by
a. As required Cleaning LT Clean all the buckets at
regular intervals
b. As required Greasing the Electrician Greasing and replacement
ball bearings of carbon brushes is done
and checking after 5000 and 100 hours of
the carbon run respectively if required
brushes
c. Every 3 Checking of Tech Accuracy of speed is
months speed Support checked with rpm meter and
stop watch

H. Blood Collection Monitor (BCM)

Sr. Time Activity To be Method

No carried
out by
a. As required Cleaning LT Clean the BCM using delicate
detergent. Take out the tray and
clean it separately. Dry the BCM
before connecting it to mains
b. As required Repairs Tech In case the instrument is not
Support functioning, check for the
fuse/power supply. Contact
manufacturer
c. Every 6 Calibration Tech To be done by the manufacturer
months Support
 Equipment Maintenance 225

I. Tube Sealer
Sr. Time Activity To be Method
No carried
out by
a. As required Cleaning LT Clean the tube sealer with soft
cloth dipped in delicate detergent.
In case of blood spills, disinfest
the electrodes with spirit. Allow
the electrodes to dry up before
connecting to mains.
b. As required Repairs Tech In case the instrument is not
Support functioning, check for the
fuse/power supply. Contact
manufacturer

J. Donor Station
Sr. Time Activity To be Method
No carried
out by
a. As required Cleaning LT Before cleaning, be sure to
disconnect the AC power cord. Clean
the rexine with clean cloth dipped in
mild detergent solution. Blood stains
are to be removed by using isopropyl
alcohol. For disinfection, the
surface cis wiped with cloth dipped
in hospital spirit or any antiseptic
germicide solution.
b. As required Repairs Tech In case the instrument is not
Support functioning, check for the fuse/
power supply. Contact manufacturer

K. Blood Storage Cabinets (Blood Bank Refrigerator/Deep Freezers)

Sr. Time Activity To be Method
No carried
out by
a. As required Cleaning LT Clean the Blood Storage Cabinets
with clean soft cloth Alcohol/Spirit/
Ether/Any other organic solvents
are not to be used.
Finned condenser to be cleaned
with vacuum cleaner.
b. Daily Checking of LT Observation
temperature
chart daily
c. Weekly Checking of LT By immersing the sensor in ice
alarm system and in water at 15–20°C
Contd...
226 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Contd...
d. Weekly Changing of LT Chart change switch is pressed
temperature and needle moves to rest
chart position. Pull the chart holding
knob out. Replace the chart and
put the knob back. Then press
chart set switch to adjust the
starting time of recording. When
you hold the switch down the
chart rotates. Press chart change
again to restart the recording.
e. Every 6 Calibration Tech Done by the manufacturer
months Support

L. Platelet Agitator and Incubator

Sr. Time Activity To be Method
No carried
out by
a. As required Cleaning LT Clean the Platelet Agitator/Incubator
using a clean dry cloth. Do not clean
with wet cloth, cloth dipped in ether,
or other organic solvents.
Cleaning of trays is to be done with
soft detergent solution after removing
the trays from tray stand and rinse the
trays with clean water to remove the
detergent.
The openings between the fins of the
condenser should be cleaned with the
vacuum cleaner whenever there is
clogging with dust.
Do not lubricate any internal parts.
b. As required Repairs Tech In case the instrument is not
Support functioning, check for the fuse/power
supply. Contact manufacturer

M. Cryobath and Plasma Bath

Sr. Time Activity To be Method
No carried
out by
a. As required Cleaning LT Clean the external surface regularly
by using clean dry cloth.
Empty the water in the tank and
clean with soap solution at regular
intervals. Wash the coating/scaling
of the inner tank surface and
evaporator coil.
b. As required Repairs Tech In case the instrument is not
Support functioning, check for the fuse/ power
supply. Contact manufacturer
 Equipment Maintenance 227

List of all equipment and instruments used in all sections in the

blood bank is maintained and has been assigned asset number.

Service contracts
All the major equipments purchased are under warranty for one year
and under annual maintenance contract for 4 years.

Repair and breakdown

a. Operating instructions for each item of equipment. Identify the steps
required to be taken in the event of a fault or breakdown, and shall
identify who is responsible for organizing service or replacement.
b. A log book of errors and corrective actions will be maintained for
all equipment items. In the event of equipment breakdown, it is
essential that it be clearly labeled and identified as being “OUT OF
SERVICE”.

Note
• Maintain individual files of service reports of all equipments.
• Enter the details of all routine as well as trouble-shooting service calls by
the manufacturer’s engineer in the equipment maintenance register.
• Maintain a file of all manufacturer’s instructions and where required display
them close to the equipment.
• Record the name, address and telephone number of the service engineer
to be contacted in case of need.
 C H A P T E R 58
Quality Control of the Reagents

SCOPE AND APPLICATION

The primary objective of quality control of reagents is to ensure their
desired quality at the time of their use for various diagnostic tests. The
quality standards of anti-sera have been included in the Indian Phar­ma­
copoeia which are mandatory for the manufacturers. Therefore, manu­
facturer’s instructions are also of importance while performing the
quality checks on these reagents.

RESPONSIBILITY
It is the responsibility of Laboratory Technicians to ensure that blood
bank reagents including anti-sera react as expected on each day of use
and that these are stored as per manufacturer’s instructions.

QUALITY CONTROL OF THE REAGENTS

Antisera
A. General Principles for Quality Control
It is essential that blood grouping reagents are prepared using reliable
manufacturing procedures that are consistently capable of producing
safe and efficacious products. The products must comply with
requirements of Indian Pharmacopoeia.
Examine entire stocks of anti-sera/reagents on arrival for their
acceptability. Quality control on receipt is typically limited to inspection
of the reagents for turbidity, discoloration or hemolysis.
Ensure that the reagents have minimum shelf-life of one year and
are stored as per manufacturers’ instructions.
Ensure that the reagents are clearly labeled with lot/batch number,
date of manufacture, date of expiry and storage conditions.
All reagents are used according to the manufacturer’s instructions.
 Quality Control of the Reagents 229

Positive and negative controls are put up with each batch in order to
see that the reagents are specific and potent.
The blood bank should maintain uniformity in use of the reagents/
kits which must be of the good quality and of reputed manufacturer. In
case of change over, these should be properly assessed and tested for
their quality parameters before switch over.
Polyclonal antibody reagents (human source) must carry the
label that the product is negative for HBsAg and non-reactive for HIV
antibodies.

B. Frequency of Quality Control Tests

The Drugs and Cosmetics Act 1940 and Rules 1945 require that
representative samples of the following reagents and/or solutions shall
be tested regularly on a scheduled basis by methods described in the
Standard Operating Procedures Manual to determine their capacity to
perform as given below:
Reagents and solutions Frequency of testing along with controls
Anti-human serum Each day of use
Blood grouping serums Each day of use
Lectin Each day of use
Antibody screening and reverse Each day of use
grouping cells
Hepatitis test reagents Each run
Syphilis serology reagents Each run
Enzymes Each day of use
HIV I and II reagents Each run
Normal saline (LISS and PBS) Each day of use
Bovine albumin Each day of use

C. Parameters for Quality Control of Anti-sera

The parameters to be tested for quality control of the anti-sera are:
1. Specificity: It means that the sera reacts with the corresponding
antigen as designed by the manufacturer and fail to react with other
antigens. Similarly, reagent RBCs which are designed to express
a specific blood group antigen are shown to react with anti-sera
of the corresponding specificity. This circularity invites creation
of a simple QC system in which demonstration of reactivity of
a reagent RBC is linked to demonstration of the specificity of the
corresponding antiserum.
230 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

2. Potency
a. Avidity (Reactivity): It is a measure of the speed with which the
anti-serum agglutinates the red cells.
According to IP, the following are the minimum requirements
for the time taken by anti-A and anti-B sera to show naked eye
agglutination when mixed on a slide with an equal volume of a
5.0 to 10.0 per cent v/v suspension of A1, A2, , A2B cells and B cells
respectively.

Serum Test red cells Titer

Anti-A A1 15 seconds
A2 30 seconds
A2B 30 seconds
Anti-B B 15 seconds

b. Intensity: Strength of reaction which is scored as follows:

+ + + + = Complete agglutination into one or two large clumps.
+ + + = Numerous clumps clearly visible to the naked eye.
+ + = Granularity just visible to the naked eye; very big
clumps and unagglutinated corpuscles seen under the
microscope.
+ = Not quite such big clumps; numerous unagglutinated
corpuscles.
(+) = Clumps of 8 to 12 corpuscles.
w = A definite but weak reaction in which there is a uniform
distribution of very small clumps of 4 to 6 corpuscles.
? = Uneven distribution of corpuscles with no definite
clumps.
– = All corpuscles separate and evenly distributed.
c. Titer: It is reciprocal of the highest dilution showed weak
agglutination. Denotes the strength of the reagent. According to
IP, the following are the minimum requirements for titer of anti-A
serum with A1, A2 and A2B corpuscles and of anti-B serum with
B red cells.

Serum Test red cells Titer

Anti-A A1 256
A2 128
A2B 64
Anti-B B 256
 Quality Control of the Reagents 231

3. Stability: The reagents and anti-sera are stable for the period of
storage and retain their activity as expected on each day of use.
d. Testing for avidity of anti-sera
1. Anti-A
Material Required
• 20 percent A cells.
• Glass slide
• Anti-A serum.
• Tooth picks.
Proecdure
• Put 1 drop of (20%) A cells on a slide.
• Add 1 drop anti-A and start mixing.
• Simultaneously start a stop watch.
• Stop the watch as soon as agglutination becomes visible.
Limit
• 15 seconds.
• Appearance should be clear.
2. Anti-B
Materials Required
• 20 percent B cells.
• Glass slide.
• Anti-B serum.
• Tooth picks.
Procedure
• Put 1 drop of (20%) B cells on a slide.
• Add 1 drop anti-B and start mixing.
• Simultaneously start a stop watch.
• Stop the watch as soon as agglutination becomes visible.
Limit
• 15 seconds - Appearance should be clear.
3. Anti-D
Material Required
• 20 percent Rh positive cells.
• Glass slide.
• Anti-D serum.
• Tooth picks.
Procedure
• Put 1 drop of (20%) Rh positive cells on a slide.
• Add 1 drop anti-D and start mixing.
• Simultaneously start a stop watch.
• Stop the watch as soon as agglutination becomes visible.
Limit
• 60 seconds appearance should be clear.
232 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

E. Testing for potency of Anti-sera

Materials Required
• Anti sera.
• Test cells.
• Test tube.
• Pasteur pipette.
• Centrifuge.
• Normal saline.
Procedure
• Label a row of test tubes, according to anti-sera dilution (1:1
through 1:512).
• Put 1 drop saline into all tubes except the first tube.
• Add 1 drop anti-sera to tube 1 and 2 (dilution 1:1).
• Mix the contents of tube 2 with a clear Pasteur pipette and
then transfer 1 drop of the mixture to tube 3.
• Continue the same technique, through all the tubes and
remove 1 drop from the dilution tube of 1:512 and discard.
• Add 1 drop of 5 percent saline suspension of appropriate red
cells to each tube.
• Incubate for 5–10 minutes.
■ For anti-A and anti-B at room temperature.
■ For anti-D at 37°C.
• Centrifuge at 1000 rpm for 1 minute.
• Gently resuspend the red cells and look for agglutination
macroscopically.

Result
The agglutination titer is recorded as the reciprocal of the highest
dilution showing weak agglutination.
Anti-serum Test cells Limits
Anti-A AB 64
Anti-B AB 256
Anti-D Rh (Positive) cells 32

Quality Control of Red Cell Reagents

Parameter Quality requirements Frequency
Appearance No hemolysis or turbidity in the Daily
supernatant
Reactivity and Clear reaction with known antisera Daily
specificity
 Quality Control of the Reagents 233

Wash the red cells if there is slightest hemolysis. If the supernatant

becomes clear. The cells are suitable for use.

Quality control of antihuman globulin reagent (AHG) quality of anti-globulin

reagent
Parameter Quality requirement Frequency of control
Appearance No precipitate, particles or gel formation Each day
by visual inspection
Reactivity • No prozone phenomenon. Each new lot
and • No hemolysis or agglutination of Each new lot
Specificity unsensitized red cells.
• Agglutination of red cells sensitized Each day and each
with anti-D serum containing not new lot
more than 0.2 mg/ml antibody
activity. Each new lot
• Agglutination of red cells sensitized
with a complement binding antibody
(e.g. anti-Lea). Each new lot
• Agglutination of red cells coated
with C3b and C3d, and no/ weak
agglutination with C4 coated red
cells.

Minimum requirements for quality product of AHG are:

Anti-IgG 1:64
Anti-C3/C4 1:4

Quality control of gel cards and reagents

• Gel cards must be carefully examined for decreased fluid levels,
drying, contamination, and discoloration, opened or damaged
seals, bubbles or artifacts. Discard any card showing unacceptable
visual appearance.
• Visually check the foil seal over the gel wells. If the foil is punctured,
the gels may dry up and be unusable. If there is no fluid above the
gel, do not use the well and discard.
• Record the lot numbers and expiration dates of the reagents in use
on the QC log.
• Follow the test procedures as described in the specific package
inserts of the gel cards and the reagents.
• Controls are included as per guidelines of quality assurance.
• The gel cards are stored at room temperature (18–25°C).
• The test cells are stored at 2–8°C and are used before expiry date for
their stability.
234 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Quality control of kits for testing transfusion transmitted infections (TTI)

• These kits are strictly used as per the manufacturers’ instructions.
• Both positive and negative controls provided with the kits are run
along with the tests.
• Each blood bank should have internal and external quality control
mechanism.

Note
Documentation of all observations of all tests is an important part of QC/QA.
 C H A P T E R 59
Assessing Suitability of the Donor
for Platelet Apheresis

SCOPE AND APPLICATION

This SOP describes the criteria for a donor to be accepted for platelet-
apheresis, for ensuring safety of donor as well as recipient. The purpose
of doing the platelet apheresis procedure is to transfuse it to patients
who are thrombocytopenic and it also minimizes the risk of multiple
donor exposure.

RESPONSIBILITY
The Medical Officer is responsible for determining the suitability of
donor for platelet-apheresis. He/She should confirm that the criteria
are fulfilled after evaluation of health history questionnaire and medical
examination including the results of pre-donation screening tests.

MATERIALS REQUIRED
• Donor Questionnaire and Informed Consent Form (Annexure 59.1).

PROCEDURE
Healthy individuals have a surplus of platelets, so removal of platelets
by apheresis has no adverse affects as body is constantly replacing
platelets. Properly screened healthy donor is selected for donation.
Donor selection follows the standard blood donation criteria as given in
SOP for “Criteria for Donor Selection “and the following:
• Platelet count is not required before the first platelet-apheresis or if
4 weeks or more have elapsed since the last procedure.
• If the donation interval is less than 4 weeks, the donor platelet count
should be above 150,000/µl before subsequent platelet-apheresis.
• Interval between donations should be at least 2 days and donors
should not undergo platelet-apheresis more than twice in a week or
more than 24 times in a year.
236 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• If the donor donates a unit of whole blood or if it becomes impossible

to return the donor’s red cells during previous platelet apheresis,
at least three months should have elapsed before a subsequent
platelet-apheresis procedure unless the extracorporeal volume is
less than 100 ml.
• Donors who have taken anti-platelet medications within specific
intervals before donation (aspirin/aspirin containing medications,
48 hours; Feldene, 48 hours; Plavix/Clopidogrel, 14 days; Ticlid/
Ticlopidine, 14 days) are deferred because these medicines affect
platelets’ ability to function properly.
• The donor is screened prior to platelet-apheresis procedure for
all the legally mandatory infectious disease markers, i.e. HIV,
HBsAg, HCV, VDRL and MP preferably by ELISA a day prior to the
procedure as for whole blood donation. The infectious markers
are taken as valid for 30 days to facilitate repeat directed platelet/
plasma donation.
• For urgent apheresis, a signed consent form treating doctor and
patient for rapid screening must be taken.
• Other donor parameters such as hematocrit, height, and weight are
required to be entered into the machine.
• The donor should not be fasting prior to the procedure, however,
should refrain from oily/fatty food.
• Informed written consent is taken from the donor.
• A prominent and easily accessible anti-cubital vein on one of the
arms is selected for the apheresis.

DOCUMENTATION
Enter all the details in the platelet-apheresis donor selection, registration
and Informed consent form, donor register and the computer.
 Assessing Suitability of the Donor for Platelet Apheresis 237

Annexure 59.1
Platelet Apheresis Donor Selection, Registration and Informed
Consent Form
Blood Bank .................. {Licence No. ......................}
Routine Apheresis Emergency Apheresis
Donor’s Name:..................................
BB No: ............................................ Father’s Name:..................................
Apheresis No:................................. Age/DOB:....................Sex:..............
Blood Donor Questionnaire
CONFIDENTIAL
(√ )Tick wherever applicable
Please answer the following questions correctly. This will help to protect
you and the patient who receives your blood.
Occupation: .................... Address: ............................. Tel No: .......................
Mobile No:.....................Email:......................................
Type of Donor:
Voluntary Replacement, if Replacement, Patient Name:......................
MR No.: ................................. Indoor Patient (IP) No: .............................
Would you like us to call you on your mobile: Yes No
Have you denoted previously : Yes No. If yes,
Whole Blood Platelets
Date/period when last donated:........................................................
Did you have any discomfort during/after donation? Yes No
Your Blood Group:............................... Time of last meal:..............................

[√] the appropriate answer:

1. Do you feel well today? 7. Are you taking or have taken any
Yes No of these in the past 72 hours?
2. Did you have something to Antibiotics
eat in the last 4 hours? Aspirin
Yes No Tlclopidine
Alcohol
3. Did you sleep well last night? Steroids
Yes No Clopidogrel
Vaccinations
Ibuprofen/Diclofen Sodium
Dog bite/Rabies vaccine (1 yr)
Contd...
238 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Contd...

4. Have you any reason to 7. Is there any history of surgery

believe that you may be or blood transfusion in the
infected by either Hepatitis, past 6 months?
Malaria, HIV/AIDS and/or Major
venereal disease? Minor
Yes No Blood transfusion
5. In the last 6 months have 8. Do you suffer from or have
you had any history of the suffered from any of the
following? following diseases?
Unexplained weight loss Heart Disease
Repeated diarrhea Lung Disease
Swollen glands Kidney Disease
Continuous low-grade Cancer Malignant Disease
fever Epilepsy
6. In the last 6 months have you Diabetes
had any? Tuberculosis
Tattooing Abnormal bleeding tendency
Ear piercing Hepatitis B/C
Dental extraction Allergic Disease
Jaundice (last 1 yr)
Sexually trans. diseases
Malaria (6 months)
Typhoid (last 1 yr)
Fainting spells
9. For women donors
a. Are you pregnant?
Yes No
b. Have you had an abortion in the last 3 months?
Yes No
c. Do you have a child less than one year old?
Yes No
10. Would you like to be informed about any abnormal test result at the
address furnished by you?
Yes No
I have read and understood all the information presented and answered
all the questions truthfully, as any incorrect statement or concealment
may affect my health or may harm the recipient.
Yes No
 Assessing Suitability of the Donor for Platelet Apheresis 239

INFORMED CONSENT FOR BLOOD DONATION

I understand that:
a. Blood donation is a totally voluntary act and no inducement or
remuneration has been offered.
b. During apheresis, blood is withdrawn through a needle and mixed
with an anticoagulant as it is drawn. The blood is pumped through
the cell-separator and the desired components are collected in a
sterile plastic container. Most of the blood in the cell-separator is
then returned to the donor. All equipment used are commercially
available, and all materials coming in contact with the donor's blood
are sterile, only used once and then disposed as biomedical waste.
c. Apheresis is a medical procedure and that by undergoing the
procedure voluntarily, I accept the risks associated with the procedure
which is safe and most people tolerate very well. Reactions are rare
but can happen, especially if a person has not eaten or is tired or
nervous. Reactions that may occur include bruising around the vein,
pain, infection (caused by needle slick); dizziness, fainting, nausea,
vomiting focused by low blood volume, pain or the sight of blood;
muscle spasm (caused by anxiety and rapid breathing or fainting;
or an allergic reaction (caused by arm scrub or tape) or temporary
symptoms of bingling of the lips and/or fingers and increased muscle
tension during the return of blood due to possible anticoogulant
discomfort).
d. My blood will be tested for Hepatitis B, Hepatitis C, Malaria parasite,
HIV/AIDS and venereal diseases in addition to any other screening
tests required to ensure blood safely. The medical and personal
information and results of testing will be held by the Blood Bank
in strict confidence and will not be disclosed to anyone unless
specifically authorized by me in writing or as required by the Govt.
authority or legal process.
I acknowledge that I have read and understood the information provided
on this form about Blood Donation. I have truthfully, completely and
accurately answered all the questions on this form.
I hereby voluntarily consent to one of the following procedures checked
below:
• Platelet pheresis
• Plasma pheresis
I hereby authorize Blood Bank Services personnel to perform the
withdrawal of my blood by either a continuous or intermittent flow
cell-separator, the extraction of the appropriate blood component, the
reinfusion of my own anticoagulated blood and replacement fluids.

Contd...
240 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Contd...

The procedure and risks have been explained to me. I have been given
ample opportunity to ask questions about the procedures and about
the risks, hazards and possible complications involved. All questions
have been answered to my satisfaction. In the event of a reaction or
complication, the Medical Staff will provide immediate emergency
medical care as per protocol.
I hereby authorize the blood bank that the platelets/plasma removed
from me may be either utilized for my/any other patient or discarded as
necessary as per policy of the Govt. for the blood safety.
Date and Time: .....................
Donor's Sign: .....................
I (attendant name) ...............................
Related to (patient's name) ........................................................................
have been explained that my patient needs an urgent transfusion of
....................................... (Blood product) which has to be prepared from
one of my known directed donor Mr/Mrs (Donor Name) ..................... I
understand that since this product is needed for my patient's treatment
urgently, it has to be tested for infectious diseases like HIV, HCV, VDRL,
MP, by Rapid method which have slightly lower sensitivity and may miss
such infection, if present in the donor even after testing. Keeping in view
of the urgency, I am willing to take the risk.
I also give my consent that in case the procedure is aborted or unsuccessful
for any reason, the cost of the kit and consumables shall be borne by us as
per the hospital policy.

........................................................
Signature of the patient's guardian
............................
Date
Physical/Medical Examination
Hemoglobin: _____________ gm/dl Weight: ______________ kgs
Hematocrit: _____________% Height ______________ cms
Platelet count:_____ thousands/ BP ______________ mm of Hg
cmm Pulse ___________ /minute
Blood Group: _________Rh_______ Temperature ___________ ºF
Antibody screening______________
 Assessing Suitability of the Donor for Platelet Apheresis 241

Screening for TTI

Test Method Result Date/Time Signature
HIV I and II
HBsAg
HCV
VDRL
MP
ABS

Accepted Rejected   Deferred Sign of BTO/Consultant

Apheresis Procedure Detail

Name of the patient: ………………………………………. MR No: ……………
IPD No: …….......Age: ……….Sex: …………….
Ward/Bed No:………………………………………………
Consultant in charge: ……………………………………….....................
Clinical Diagnosis :………………....................................................................
Blood group: …………………………..Platelet count: ……………………………
Yield………………………………………………
Set used: ………………………… Batch used: …………………………
Batch No. & Expiry (ACD)…………………………………………….
Venepuncture performed by (Name): ……………………………………………
Date and time of commencement of apheresis: …………………………………
Total volume of blood processed: ……………………… Plt. conc. volume
collected……………………….ml
Total volume of ACD used: In Plt. Conc.……………………ml and to
donor………………………………….ml
Total volume of replacement fluid used: ……………………………………….
Date and time of completion of apheresis procedure: …………………………
Expiry date of component: …………………………………………………………
Any complaint by the donor during the procedure: ...................................
Any medicine given during the procedure: ……………………………………..
Remarks: ........................................................................................................
Signature of BTO/Consultant: ...................................................................
242 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Space for Sticker Space for Sticker

Bill No & Date: ...............................................................................................

Spillovers no. Blood process volume Time IDO

 C H A P T E R 60
Plateletpheresis Using Blood Cell
Separator (Dual Needle)
SCOPE AND APPLICATION
This SOP describes the Dual Needle PLT-5d program of platelet-
apheresis using blood cell Separator (COM. TEC version 4.02xx only).
SOP may be amended accordingly by the blood banks using other cell
separators according to the instruction manual of the manufacturers.
With this program, platelets are separated in continuous mode via
two venous donor accesses. The C5L set is a closed system per­mitting
the storage of platelets for a maximum of up to 5 days. For this reason
not only the sterile filters in the saline and ACD lines but also the inlet
needle are pre-connected in this set.
This SOP provides instructions for installation of apheresis set C5L,
priming, separation, reinfusion and removal of the apheresis set.

RESPONSIBILITY
The Medical Officer is responsible for determining the suitability of
donor for platelet apheresis. He/She should confirm that the criteria are
fulfilled after evaluation of health history questionnaire and medical
examination including the results of pre-donation screening tests.

MATERIALS REQUIRED
• COM.TEC blood cell separator version 4.02 xx.
• Double needle closed system Com Tec kit ( C5L).
• Material for phlebotomy.
• EDTA (Lavender top) vacutainer for product sampling.
• Tube sealer.
• Oral calcium tablets.
• Emergency medicine tray.
244 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 60.1: Pressing power on

PROCEDURE
Selection of the Platelet Program
1. Connect the COM.TEC blood cell separator to the power supply,
2. Press the I (Power: On) key in the front panel (Fig. 60.1).
3. Screen will display, showing software version. Press continue
(Fig. 60.2).

Fig. 60.2: Software version display

 Plateletpheresis Using Blood Cell Separator (Dual Needle) 245

Fig. 60.3: Screen display for selection of program

4. Following screen will display (Fig. 60.3).

5. Use the ↑ and ↓ keys to select the program Group Platelet Donation.
Press the OK key (Fig. 60.4).
6. Use the ↑ and ↓ keys to select the PLT-5d program. Press the OK key.

Fig. 60.4: Selection of program for platelet donation

246 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 60.5: Display for install set

Installation of the Apheresis Set (C5L)

7. The message “Install set C5L” will display (Fig. 60.5). Follow the help
instructions on the screen on total 5 screen pages as below:
1. Open all 4 pump lids. Press the turn pumps key. The threading
pins automatically stop at the threading position (12 O’clock/
ACD pump: 5 O’clock).
2. Hang up blood inlet/return lines on right side IV rack hooks of
cell separator.
3. Close RED inlet clamp just beyond Y piece to the pre-sampling
bag.
4. Close WHITE needle clamp.
5. Hang up storage platelet bags at left side of device, close clamp
between 2 platelet concentrate storage bags.
6. Close clamp at platelet concentrate sample bag.
7. Hang empty bag at hook above return drip chamber.
8. Hang Plasma bag at hook above clamp 4.
9. Install all 4 pump segments by inserting the top pump adapters
to color coded pumps, hold with fingers and Press Turn Pumps.
Pumps will be loaded automatically. Close Pump Lids to all
4 Pumps.
10. Install the Return Drip Chamber at the Air Detector in such a
way that Drip Chamber is equally outside the Detector on both
Top/Bottom side of Detector.
 Plateletpheresis Using Blood Cell Separator (Dual Needle) 247

11. Install the plasma line in clamp 4 between the Y-piece and drip
cham­­ber.
12. Install the plasma line section marked by the yellow tabs into
the Hb/Hct detector.
13. Insert the line leading to the empty bag in clamp 5.
14. Insert the return line in clamp 1.
15. Insert the inlet line of the cell pump into the cell detector.
16. Install the drip chamber of the ACD line, which is identified by
a green clamp, in the ACD detector and pull the drip chamber
completely down.
17. Install the pump adapter for the ACD pump. The threading pin
for the ACD pump must be in the 5 o’clock position. If this is not
the case prior to installing the adapter, press the Turn Pumps
key. Pump will be automatically loaded.
18. Install the two saline lines in clamp 2 and clamp 3. Make sure
that the lines are installed so that their color matches the color
coding of the covering foil and the color of the roller clamp (red
line clamp 2, blue line clamp 3).
19. Screw the filters of the red-coded pressure measuring lines for
the inlet pressure onto the red-coded pressure measurement
port for the inlet pressure.
20. Screw the blue-coded pressure measuring line for the return
pressure onto the blue-coded pressure measurement port for
the return pressure.
21. Hold the separation chamber at the upper centrifuge adapter
and let it hang down freely beside the device to avoid twisting
of the line.
22. Open the hinged door of the chamber holder. Push the circular
adapter of the separation chamber into the groove in the
central funnel. Close the hinged door until it locks into place.
The locking pin on the flat side of the chamber holder must no
longer be visible.
23. Remove the empty packaging from the centrifuge door. Press
the Open Door key. Slide the centrifuge door open. If Open
Door is not displayed, the door cannot be opened.
24. Turn the centrifuge rotor until one of the two line guides is
on the right of the rotor. Push the chamber holder with the
installed separation chamber under the rotor with the lines
pointing down.
25. Push the chamber holder onto the guide rail until it is felt to
be locked. Swing the locking flap down until it clicks into
place. Pass the centrifuge tubing through the line guide. Push
248 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

the upper centrifuge adapter narrow side first into the upper
adapter holder.
Should it be necessary to slightly turn the adapter, and then turn
it in the direction in which the individual lines are twisted around one
another? After a test revolution and another visual check, close the
centrifuge door. The door is properly locked, when the Open Door key is
displayed. Place the Air Protect in the holder.

Caution
Ensure that the centrifuge tubing is not trapped between chamber
holder and rotor.

Note
Manually turn the rotor one full revolution counter-clockwise to verify correct
installation. After a test revolution and another visual check, close the centrifuge
door. The door is properly locked, when the Open Door key is displayed. Place
the Air Protect in the holder.

Checks after Apheresis Set and Separation Chamber Install­ation

(Fig. 60.6)
Visually verify the following:
• The red clamp on the inlet line below the branch to the pre-sampling
bag, the white needle clamp, the clamp between the concentrate
bags and the clamp on the concentrate sampling bag must be
closed. All other stop clamps on the set must be open.
• The separation chamber must be correctly installed.
• The drip chamber in the air detector and the ACD-A drip chamber
must be correctly installed in their holders.
• The ACD-A pump adapter and the ACD-A line must be correctly
installed in the ACD-A pump below the drip counters.
• The return line is installed in clamp 1. The saline lines must be
inserted in clamp 2 and clamp 3.
• The plasma line must be installed in clamp 4.
• The line leading to the empty bag must be installed in clamp 5.
• The inlet line of the cell pump must be inserted in the spillover
detector.

Preparing for priming

8. Press the “Continue” key; following screen will display (Fig. 60.7).
 Plateletpheresis Using Blood Cell Separator (Dual Needle) 249

Fig. 60.6: Checks after apheresis set and separation chamber installation

Fig. 60.7: Screen display after continuing on installation of set

250 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Prior to priming the set, a user guide is available to assist with

the connection of the saline and ACD-A solutions. Connect the
ACD-A bag to the green connector and hang it from the upper left
hook on the front of the device. Break the cone of the ACD-A bag.
Deaerate the ACD-A drip chamber by pressing it and set the level
to approximately 1 cm, so that the fluid level is approximately 1 cm
below the optical sensor.
Connect the saline bag to the two transparent connectors. Hang the
saline bag from the front left hook. Break the cones and set the fluid
level in the two-drip chambers.

Priming
During priming, saline and ACD-A will be pumped into the set to
displace the air. The program step:
Prime is divided into the following phases:
Detection of set and procedure: The COM.TEC automatically detects
which procedure has been selected and which set matches the
procedure. The additional ACD pump has a miniature contact which is
actuated by the pump segment when a C5 set is installed. When a C4 or
a PL1 set is used, this contact will not be actuated. If the position of the
contact is identical with the position expected by the software (actuated
for the C5 procedure, not actuated for C4 and PL1 procedures), the
ACD drip test will be performed. If the actual position and the expected
position are not identical, a failure message will be displayed (Failure:
Wrong set).
Pressure test/test clamp 1: With clamp 1 and clamp 2 closed, the whole
blood pump delivers until a negative pressure has developed at the inlet
pressure monitor to check whether the lines are installed in the clamps
and whether the clamps close tightly.
Priming phase V1: The priming phase V1 is usually terminated as soon
as the air detector is in a permanent alarm-free state and an increased
volume of 100 ml has been delivered into the empty bag. If the air
detector is not or not permanently alarm-free, the error message Drip
chamber will be displayed.
Priming phase V2/V3: The centrifuge is accelerated or decelerated for a
certain number of cycles to remove residual air in the chamber.
Priming phase V4: The speed of the centrifuge is increased to operating
speed. As soon as this speed has been reached, the interface detection
is tested.
 Plateletpheresis Using Blood Cell Separator (Dual Needle) 251

Fig. 60.8: Sreen display during alarm test

Skip priming
If an inserted set is already primed and the device was turned off or a
program was aborted, the priming program can be skipped.

Start Prime by Pressing Prime Key

1. The alarm test screen will be displayed (Fig. 60.8).
A bargraph indicates that the test is in progress. Start and end of the
test are indicated by an audible alarm.
The alarm test is performed automatically.

Note
If an alarm system fails to correctly detect an alarm, the alarm message which
failed to occur will be displayed as an error. In case of several test errors, the
first error which occurred is displayed. After correcting the test error, the alarm
test must be repeated. The alarm test can be repeated by pressing the prime
key again.
After completion of the alarm test, priming is started automatically. The
following screen is displayed while priming. (Fig. 60.9).
Prime takes around 6 minutes to complete

2. Second Priming can be done by selecting the Prime key again after
the completion of Automatic Prime. The option 2nd priming is only
available in the Set primed screen. (Fig. 60.10)
252 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 60.9: Screen display during priming

3. At the end of Prime, Prime Saline Diversion can be selected by using

Up/Down keys to Donor or to Waste Bag.

Fig. 60.10: Screen display after priming of set

 Plateletpheresis Using Blood Cell Separator (Dual Needle) 253

Fig. 60.11: Screen display for “Prepare separation”

Preparing for Separation

After selection of saline diversion, the following screen (Fig. 60.11) is
displayed:

Fig. 60.12: Screen display for various checks

254 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

10. Visually check the apheresis set. Check and, if necessary, adjust the
fluid level of the connected solutions in all drip chambers. Check
that the white needle clamp is closed and close, if necessary. Close
the two blue return clamps. Set the roller clamps to 50%.
Press the continue key (Fig. 60.12).

Connecting the Donor and Collecting a Pre-donation Sample

10. Insert the needle after vein compression. Open the white needle
clamp and collect a pre-donation sample of at least 20 ml. Close
the clamp on the pre-sampling bag and seal off the pre-sampling
bag. Open the red clamp on the inlet line and check that the two red
clamps are open. Connect the return needle to the blue Luer lock
connector. Deaerate and place the return needle. Open the blue
return clamp.
Press the continue key and the screen for donor values appears.
(Fig. 60.13)

Menus
The donor values (height, weight, etc.) must be entered with the up,
down, +and- keys. After entering the data, press the OK key. The software
calculates the maximum possible blood flow based on the ACD rate and
the donor values set in the Config.sys.

Fig. 60.13: Screen display for adding donor values

 Plateletpheresis Using Blood Cell Separator (Dual Needle) 255

Fig. 60.14: Menu screen for calculated values

The calculated values will be displayed in the procedure Menu
screen (Fig. 60.14).

Procedure Menu
11. Select the value to be altered with the up and the down keys. Use
the + and – keys to alter the selected value. All values affected by this
change will be automatically adapted. A change of the yield will, for

Fig. 60.15: Screen display for starting separation

256 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 60.16: Screen display during platelet separation

example, result in an automatic recalculation of the blood volume
to be processed.
Press OK; All Values will be Saved.

Separation
All separation parameter changes should be made before starting the
program, but can also be made at a later stage.
12. Press Start: Press the Start key (Fig. 60.15). The device automati­cally
performs a system pressure test to check the pressure measuring
lines for tight connection and to verify correct position of all clamps
at the return drip chamber.

Automatic Operation
The following display screen appears during the separation program
(Fig. 60.16).
Process will be fully automatic till the target volume is processed.
The plasma pump is automatically controlled to maintain the interface
which has developed. In the C5 separation chamber the blood is
separated into red blood cells, buffy coat and platelet-rich plasma.
The platelet-rich plasma is separated off behind a dam which holds
back the red blood cells, and the platelet concentrate at the cell port is
delivered into the collection bag. At the end of the chamber channel the
platelet-poor plasma is returned to the donor by the plasma pump.
 Plateletpheresis Using Blood Cell Separator (Dual Needle) 257

During the entire separation procedure the device is monitored by

a complex alarm system. This ensures donor safety. In the event of an
alarm or a malfunction the device will automatically be switched into the
stop mode. The help menu assists with troubleshooting and remedies to
correct the problems.

Decomposition phase
20 ml before the PC target volume has been achieved, a chime will sound
and the centrifuge speed will be reduced by 100 rpm. The plasma and
the cell flow are automatically controlled to decompose the interface
until the end of the separation.

Pausing and Continuing the Separation Program

Press the STOP key. Separation will be stopped; the centrifuge speed
will be reduced to1000 rpm, if the COM.TEC is stopped for more than 30
seconds. If the device stops for another 3 minutes, the centrifuge speed
will be reduced to 600 rpm.
Pressing the STOP key will cause the pumps to stop, clamp 1 to close
and clamp 2 and clamp 3 to open.
Press the Start key. The separation will be continued.

Additional Plasma Collection

Additional plasma can be collected, if desired by selecting program
Menu and Config. sys. Enter the desired PLS volume to be collected.

Aborting the Separation with Premature Reinfusion

Procedure can be terminated any time in between, by going to Option
and selecting Direct Reinfusion followed by OK at any time.

Completing the Separation Program

At the end of the separation program, the following screen is displayed.
When the target yield has been achieved, the separation will be
terminated automatically (Fig. 60.17).

Reinfusion
During this phase the blood components still left in the line will be
returned to the donor.
Disconnect the inlet line and add saline and ACD (120 ml of saline
at a collection flow of 35 ml/min) to displace the blood from the line set
258 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 60.17: Screen display after completion of platelet separation

and to return the blood to the donor via the return line. The cell pump
simultaneously delivers the platelets still present in the separation
chamber into the platelet concentrate bag.
Press Continue, following Reinfusion start menu (Fig. 60.18) will be
displayed:

Fig. 60.18: Screen display guiding for the start of reinfusion

 Plateletpheresis Using Blood Cell Separator (Dual Needle) 259

Fig. 60.19: Screen display during reinfusion

Follow the instructions on display as follows:

1. Close the lower red inlet clamp.
2. Completely open the red roller clamp of the saline line.
3. Close the yellow clamp on the plasma collection bag.
4. Disconnect the donor from the inlet line.
5. Press the Start key.
The following screen (Fig. 60.19) is displayed during reinfusion
Reinfusion can be terminated prematurely, if desired by selecting
Option and Exit Reinfusion followed by OK key.

Terminating the Reinfusion

1. Display at the end of reinfusion.
2. Reinfusion can be continued for another 30 ml by pressing the
START key.
3. The reinfusion will be terminated by pressing the STOP key.
4. Close the stop clamp of the return line.
5. Close the yellow stop clamp of the plasma bag.
6. Press the Continue key.
7. Disconnect the donor from the return line.
8. Press the Continue key.
260 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Deaeration of Concentrate Bags and Collection of Samples

The deaerating option is only available if the menu option FPC deaerate
or PC deaerate (Vol) in the configuration program has not been
deactivated.

Display
Use forceps to clamp both the red blood cell line leading to the drip
chamber and the whole blood line below the air separator.
• Remove the cell pump line segment from the pump.
• Remove the concentrate bag, holding its connector up.
• Press the Start key.

Display
• The concentrate bag is being deaerated.
• As soon as the concentrate bag is completely deaerated, press the
STOP key.
• If the concentrate bag has not yet been completely deaerated, press
the START key to remove another 5 ml of air. On completion of the
deaeration, press the STOP key.

Removing Platelet Concentrate and Set

• Seal off the concentrate bag and remove the set.
• Press the Continue key.
• After the report has been printed, the device can be started for
another separation by pressing the RESET key.
• Press the O key.
• The device switches off.

Collection of Concentrate Samples

• On completion of reinfusion thoroughly mix the concentrate in the
open concentrate bag.
• Allow the concentrate bag to rest for 1 h.
• Then agitate the bags on an agitator for a minimum of 30 minutes.
Collect the sample (Make sure to obtain a representative sample!).
 C H A P T E R 61
Plateletpheresis Using Blood Cell
Separator (Single Needle)

SCOPE AND APPLICATION

This SOP describes the Single Needle PLT-5d-SN program of Platelet
apheresis using blood cell separator (COM.TEC version 4.02xx only)
SOP may be amended accordingly by the blood banks using other cell
separators according to the instruction manual of the manufacturers.
With this program, platelets are separated in intermittent mode via one
venous donor access using single needle method. The intermittent flow
centrifugation is a one arm procedure, i.e. blood is drawn and re-infused
after separating desired component, through the same vein. The S5L set
is a closed system permitting the storage of platelets for a maximum of
up to 5 days. For this reason not only the sterile filters in the saline and
ACD lines but also the inlet needler are pre-connected in this set.
This SOP provides instructions for installation of apheresis set S5L,
Priming, Separation, Reinfusion and removal of the apheresis set.

RESPONSIBILITY
The Medical Officer is responsible for determining the suitability of
donor for platelet apheresis. He/She should confirm that the criteria are
fulfilled after evaluation of health history questionnaire and medical
examination including the results of pre-donation screening tests.

MATERIALS REQUIRED
• COM.TEC blood cell separator version 4.02xx.
• Single needle closed system Com Tec kit (S5L).
• Material for phlebotomy.
• EDTA (Lavender top) vacutainer for product sampling.
• Tube sealer.
262 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 61.1: Pressing power on

• Oral calcium tablets.

• Emergency medicine tray.

PROCEDURE
A. Selection of the Platelet Program
1. Connect the COM.TEC Blood Cell Separator to the power supply.
2. Press the I (Power: On) key in the front panel (Fig. 61.1).
3. Screen will display, showing software version. Press Continue
(Fig. 61.2).

Fig. 61.2: Software version display

 Plateletpheresis Using Blood Cell Separator (Single Needle) 263

Fig. 61.3: Screen display for selection of program

4. Following screen (Fig. 61.3) will display.

5. Use the ↑ and ↓ keys to select the program. Group “Platelet Don­
ation” (Fig. 61.4). Press the OK key.
6. Use the ↑ and ↓ keys to select the PLT-5d-SN program. Press the OK
key.
a. Installation of the Apheresis Set (S5L).

Fig. 61.4: Selection of program for platelet donation

264 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 61.5: Display for install set

7. The message “Install set C5L” will display (Fig. 61.5). Follow the help
instructions on the screen on total 5 screen pages as below:
1. Open all 4 pump lids. Press the Turn Pumps key. The threading
pins automatically stop at the threading position (12 o’clock/
ACD pump: 5 o’clock).
2. Deposit the packing on the centrifuge door. Ensure that each
pump line segment is located under the matching color-coded
pump.
3. Take the rolled-up donor connecting lines out of the packaging,
close the red inlet clamp below the branch to the pre-sampling
bag, close the white needle clamp and suspend the connecting
line laterally at the upper right of the device. Suspend the
concentrate bag from the rear hook on the left of the device.
4. Close the clamps between the concentrate bags and at the PC
sampling bag.
5. Suspend the empty bag above clamp 5
6. Suspend the plasma bag above clamp 4.
7. Suspend the single needle bag at the left side of the plasma bag.
8. Install all pump line segments so that the colors of pump
adapter and the pump coding match. Make sure to push the
pump adapter fully to the rear.
 Plateletpheresis Using Blood Cell Separator (Single Needle) 265

9. Press the turn pumps key. The pump lines will automatically be
threaded into place.
10. Install the plasma line section leading to the bag behind the
Y-piece in the upper part of clamp 4.
11. Install the section leading to the air detector drip chamber in
the lower part of clamp 4.
12. Install the plasma line section marked by the yellow tabs into
the Hb/Hct detector.
13. Insert the line leading to the empty bag in clamp 5.
14. Insert the return line in clamp 1.
15. Insert the inlet line of the cell pump into the spillover detector.
16. Install the ACD drip chamber, which is identified by a green
clamp, in the ACD detector and pull the drip chamber
completely down.
17. Install the pump adapter for the ACD pump. One of the three
threading pins for the ACD pump must be in the 6 O’clock posi­
tion, if this is not the case prior to installing the adapter.
18. Press the Turn Pumps key. The pump lines will automatically
be threaded into place.
19. Install the saline line in clamp 2 (The S5L set is provided with
one saline line only).
20. Install the SN collecting/return line in clamp 3 between the
blue marks.
21. Install the line segment between the red-coded Y-piece and the
uncoded Y-piece on the left in clamp 6.
22. Install the other line segment above the red-coded Y-piece on
the right in clamp 6.
23. Screw the filter of the blue-coded pressure measuring line for
the return pressure onto the blue-coded pressure measurement
port for the return pressure.
24. Screw the filter of the red-coded pressure measuring line for
the inlet pressure onto the red-coded pressure measurement
port for the inlet pressure.
25. Hold the separation chamber at the upper centrifuge adapter
and let it hang down freely beside the device to avoid twisting
of the line. Open the hinged door of the chamber holder. Push
the circular adapter of the separation chamber into the groove
in the central funnel. Close the hinged door until it locks into
place. The locking pin on the flat side of the chamber holder
must no longer be visible. Remove the empty packaging
from the centrifuge door. Press the Open Door key. Slide the
266 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

centrifuge door open. If Open Door is not displayed, the door

cannot be opened.
26. Turn the centrifuge rotor until one of the two line guides is on
the right of the rotor. Push the chamber holder with the installed
separation chamber under the rotor with the lines pointing down.
Push the chamber holder onto the guide rail until it is felt to be
locked. Swing the locking flap down until it clicks into place. Pass
the centrifuge tubing through the line guide. Push the upper
centrifuge adapter narrow side first into the upper adapter holder.
Should it be necessary to slightly turn the adapter, then turn it in
the direction in which the individual lines are twisted around one
another.
27. Manually turn the rotor one full revolution counter-clockwise
to verify correct installation.
28. After a test revolution and another visual check, close the
centrifuge door. The door is properly locked, when the Open
Door key is displayed. Place the air protect in the holder.

Checks after Apheresis Set and Separation Chamber Installation

(Fig. 61.6)
Visually verify the following:
• The red clamp on the inlet line below the branch to the pre-sampling
bag, the white needle clamp, the clamp between the concentrate
bags and the clamp on the sampling bag must be closed. All other
stop clamps on the set must be open.
• The air detector drip chamber and the ACD drip chamber must be
correctly installed in their holders.
• The return line is installed in clamp 1.
• The saline line must be installed in clamp 2.
• The plasma line must be installed in clamp 4.
• The line leading to the empty bag must be installed in clamp 5.
• For SN control the inlet line must be installed in clamp 6.
• The SN collecting/return line must be installed in clamp 3.

B. Preparing for Priming

8. Press the “Continue” key; following screen (Fig. 61.7) will dis­play.
Prior to priming the set, a user guide is available to assist with the
connection of the saline and ACD-A solutions. Connect the ACD-A bag
to the green connector and hang it from the upper left hook on the front
 Plateletpheresis Using Blood Cell Separator (Single Needle) 267

Fig. 61.6: Checks after apheresis set and separation chamber installation

Fig. 61.7: Screen display after continuing on installation of set

268 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

of the device. Break the cone of the ACD-A bag. Deaerate the ACD-A drip
chamber by pressing it and set the level to approximately 1 cm, so that
the fluid level is approximately 1 cm below the optical sensor. Connect
the saline bag to the two transparent connectors. Hang the saline bag
from the front left hook. Break the cones and set the fluid level in the two
drip chambers.

C. Priming
During priming saline and ACD-A will be pumped into the set to displace
the air. The program step Prime is divided into the following phases:
Detection of set and procedure: The COM.TEC automatically detects
which procedure has been selected and which set matches the
procedure. The additional ACD pump has a miniature contact which is
actuated by the pump segment when a C5 set is installed. When a C4 or
a PL1 set is used, this contact will not be actuated. If the position of the
contact is identical with the position expected by the software (actuated
for the C5 procedure, not actuated for C4 and PL1 procedures), the
ACD drip test will be performed. If the actual position and the expected
position are not identical, a failure message will be displayed (Failure:
Wrong set).
Pressure test/test clamp 1: With clamp 1 and clamp 2 closed, the whole
blood pump delivers until a negative pressure has developed at the inlet
pressure monitor to check whether the lines are installed in the clamps
and whether the clamps close tightly.
Priming phase V1: The priming phase V1 is usually terminated as soon
as the air detector is in a permanent alarm-free state and an increased
volume of 100 ml has been delivered into the empty bag. If the air
detector is not or not permanently alarm-free, the error message Drip
chamber will be displayed.
Priming phase V2/V3: The centrifuge is accelerated or decelerated for a
certain number of cycles to remove residual air in the chamber.
Priming phase V4: The speed of the centrifuge is increased to oper­ating
speed. As soon as this speed has been reached, the interface detection
is tested.

Skip priming
If an inserted set is already primed and the device was turned off or a
program was aborted, the priming program can be skipped.
9. Start prime by pressing prime key
 Plateletpheresis Using Blood Cell Separator (Single Needle) 269

Fig. 61.8: Screen display during alarm test

The alarm test screen (Fig. 61.8) will be displayed.

1. A bargraph indicates that the test is in progress. Start and end of
the test are indicated by an audible alarm.
The alarm test is performed automatically.

Note
If an alarm system fails to correctly detect an alarm, the alarm message which
failed to occur will be displayed as an error. In case of several test errors, the
first error which occurred is displayed. After correcting the test error, the alarm
test must be repeated. The alarm test can be repeated by pressing the Prime
key again.

After completion of the alarm test, priming is started

automatically. The following screen is (Fig. 61.9) displayed
while priming.
PRIME takes around 6 minutes to complete.
2. Second PRIMING can be done by selecting the PRIME key
again after the completion of Automatic Prime. The option 2nd
priming is only available in the Set primed screen (Fig. 61.10).
3. At the end of Prime, Prime Saline Diversion can be sele­cted by
using Up/Down keys to Donor or to Waste Bag.
270 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 61.9: Screen display during priming

Fig. 61.10: Screen display after priming of set

 Plateletpheresis Using Blood Cell Separator (Single Needle) 271

Fig. 61.11: Screen display for “Prepare separation”

Preparing for Separation

After selection of saline diversion, the following screen (Fig. 61.11) is
displayed:

Fig. 61.12: Screen display for various checks

272 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 61.13: Inflate cuff display

4. Visually check the apheresis set. Check and, if necessary, adjust the
fluid level of the connected solutions in all drip chambers. Check
that the white needle clamp is closed and close, if necessary. Close
the two blue return clamps. Set the roller clamps to 50%.
Press the Continue key (Fig. 61.12).
Connecting the Donor and Collecting a Pre-donation Sample

Applying the Cuff

Apply the cuff to the fully extended and relaxed upper arm of the donor.
It must be ensured that the cuff contains no air; this is done by opening
the red deflation valve on the air-pressure pump and by pressing the cuff
with your hand to remove all air. It is then imperative to close the red
valve again to prevent malfunctions in the separation program. With all
air removed, the pressure gauge on the cuff should indicate 0 mmHg. A
subsequent adjustment can be made by turning the scale.
Connect the cuff to the cuff port on the rear connector panel and
ensure that the connecting line is not squeezed or kinked.
By pressing the Start key, the cuff is inflated up to a pressure of
approx. 50 mmHg to facilitate puncturing of the donor. Due to its design,
the pump has a maximum pressure of 100 mmHg. The inflate cuff display
can be skipped by pressing the Continue key (Fig. 61.13).
Insert the needle after vein compression. Open the white needle
clamp and collect a pre-Donation sample of at least 20 ml. Close the
 Plateletpheresis Using Blood Cell Separator (Single Needle) 273

Fig. 61.14: Screen display for adding donor values

clamp on the pre-sampling bag and seal off the pre-sampling bag.
Open the red clamp on the inlet line and check that the two red clamps
are open. Connect the return needle to the blue Luer lock connector.
Deaerate and place the return needle. Open the blue return clamp.
Press the Continue key and the Screen for Donor Values appears
(Fig. 61.14).

Menus
11. The donor values (height, weight, etc.) must be entered with the up,
down +and – keys. After entering the data, press the OK key. The
software calculates the maximum possible blood flow based on the
ACD rate and the donor values set in the Config.sys.
The calculated values will be displayed in the Procedure Menu
screen (Fig. 61.15).

Procedure Menu
12. Select the value to be altered with the up and the down keys. Use
the + and – keys to alter the selected value. All values affected by this
change will be automatically adapted.
A change of the yield will, for example, result in an automatic
recalculation of the blood volume to be processed.
PRESS OK; All Values will be Saved.
274 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 61.15: Menu screen for calculated values

Separation
All separation parameter changes should be made before starting the
program, but can also be made at a later stage.
Press Start: Press the Start key (Fig. 61.16). The device autom­
atically performs a system pressure test to check the pressure measuring

Fig. 61.16: Screen display for starting separation

 Plateletpheresis Using Blood Cell Separator (Single Needle) 275

Fig. 61.17: Screen display during platelet separation

lines for tight connection and to verify correct position of all clamps at
the return drip chamber.
Automatic Operation: The following display Screen (Fig. 61.17)
appears during the separation program.
Process will be fully automatic till the target volume is processed.
The plasma pump is automatically controlled to maintain the inter­
face which has developed. In the C5 separation chamber the blood is
separated into red blood cells, buffy coat and platelet-rich plasma.
The platelet-rich plasma is separated off behind a dam which holds
back the red blood cells, and the platelet concentrate at the cell port is
delivered into the collection bag. At the end of the chamber channel the
platelet-poor plasma is returned to the donor by the plasma pump.
During the entire separation procedure the device is monitored by
a complex alarm system. This ensures donor safety. In the event of an
alarm or a malfunction the device will automatically be switched into the
stop mode. The help menu assists with troubleshooting and remedies to
correct the problems.

Cycle Control
Collection cycle
During the collection phase, the processed blood is delivered into the
SN bag.
276 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

At the beginning of the collection phase, the cuff is inflated to a

pressure of approx. 50 mmHg. This slightly compresses the donor’s vein
to avoid possible flow problems. While the cuff is being inflated, the
blood flow is reduced by 20%.
At the end of the last collection phase of a standard separation
procedure, the total extracorporeal volume amounts to 230 ml (set) +
150 ml (SN bag) + 250 ml (platelet concentrate bag) = 630 ml (incl.ACD).
It is at the discretion of the attending physician to decide whether
this volume is tolerated by the donor. The extracorporeal volume can be
altered in by reducing the SN cycle volume Config.sys.
In the first collection phase an approx. 80 ml buffer will be
delivered into the SN bag in addition to the preset collection volume
(volume buffer). This buffer prevents blood with a low hematocrit from
entering the separation chamber which would cause a reduction of the
interface (the hematocrit in the upper part of the SN bag drops due to
sedimentation of the platelets during collection).
If the last collection phase of the COM.TEC is less than 50 ml, the
collection phase preceding the last collection will be increased up to a
maximum of 50 ml and automatically becomes the last collection.

Return Cycle
The volume of blood delivered to the SN transfer bag during collection
(= cycle volume) is returned to the donor by means of the whole blood
pump by reversing clamp 3 and clamp 6. The blood thus passes once
again through the separation chamber.
The final return is automatically terminated when the volume of
blood collected in the SN bag during the collection has been completely
returned to the donor. The last return can, however, also be terminated
with the option Exit last return.

Return Blood Flow

Return blood flow can be set in the procedure menu. The separation time
can be reduced by increasing the return blood flow. The ACD infusion
rate during this phase will then increase. It is at the discretion of the
attending physician to select an adequate return blood flow. The return
blood flow is coupled to the collection flow. The maximum difference
which can be set is 10 ml/min. In the default setting the return blood
flow is 5 ml/min higher than the collection flow. Depending on other
settings the program will only allow for certain maximum flow rates. If
the alarm limit of the permitted maximum value is reached, this flow
will be reduced by the menu by 2 ml/min to prevent that the keys for
changing the parameters are suppressed.
 Plateletpheresis Using Blood Cell Separator (Single Needle) 277

Fig. 61.18: Screen display after completion of platelet separation

Changing Options
Use the Up and the Down keys to select the parameter to be changed.
Use the + and the – keys to change the selected parameter. Press the OK
key to confirm the change.

Options
• Print parameters prints the parameter list.
• Direct reinfusion–Controls the saline diversion at the start of the
separation.
• Cuff SN–Controls the automatic cuff.
• Chime SN–Controls the audible signal when switching from
collection to return and vice versa.
• Exit program–Terminates the program prematurely when the stand­
ard program is used, e.g. with a donation time of approximately
80 minutes, the ACD load to the donor is approx. 375 ml ACD. This
corresponds to a mean ACD rate of 4.7 ml/min. Since no fluid is
infused to the donor during the collection phase, the maximum
ACD load is approx 9.4 ml/min.

Completing the Separation Program

When the target yield has been achieved the separation will be
terminated automatically (Fig. 61.18).
278 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 61.19: Screen display for stopping SN-bag

Empty SN-Bag
If the SN bag is not empty, SN return can be continued for another 20
ml each time the Start key is pressed. Emptying of the SN bag can be
stopped at any time by pressing the Stop key. If the SN bag is not empty,
SN return can be continued for another 20 ml each time the Start key is
pressed. Emptying of the SN bag can be stopped at any time by pressing
the STOP key. (Fig. 61.19).

Reinfusion
At the end of processing Target volume Display will show Separation
Finished. Press Continue, Following Reinfusion start menu
(Fig. 61.20) will be displayed as following:
• Follow the instructions on display as follows:
• Close the lower red inlet clamp.
• Open the roller clamp at the saline line.
• Press the Start key.
You can terminate Reinfusion premature, if desired by selecting
Option and Exit Reinfusion followed by OK key.

END OF REINFUSION
• Close the stop clamp of the return line.
• Close the yellow stop clamp of the plasma bag.
 Plateletpheresis Using Blood Cell Separator (Single Needle) 279

Fig. 61.20: Screen display guiding for start of reinfusion

• Press the Continue key.

• Disconnect the donor.
• Press the Continue key.
• Deaeration of Concentrate Bags and Collection of Samples.
The “Prepare deaerating” option is only available if the menu option
F PC deaerate or PC deaerate (Vol.) in the configuration program has
not been deactivated. Use forceps to clamp both the red blood cell line
leading to the drip chamber and the whole blood line below the air
separator. Remove the cell pump line segment from the pump. Remove
the concentrate bag, holding its connector up. Press the Start key. The
concentrate bag is being deaerated. As soon as the concentrate bag is
completely deaerated, press the STOP key. If the concentrate bag has not
yet been completely deaerated, press the START key to remove another 5
ml of air. On completion of the deaeration process, press the STOP key.

Removing platelet concentrate and set

• Seal off the concentrate bag and remove the set.
• Press the Continue key.
• After the report has been printed, the device can be started for
another separation by pressing the Reset key (Fig. 61.21).
• Report will be printed as above on the Printer.
• Turn the device Off by O key.
• Collection of Concentrate Samples.
280 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Fig. 61.21: Display at the end of procedure for print out

• On completion of reinfusion thoroughly mix the concentrate in the

open concentrate bag.
• Allow the concentrate bag to rest for 1 h.
• Then agitate the bags on a suitable agitator for a minimum of 30
minutes.
• Collect the sample. To obtain a representative sample, the lines must
be sufficiently rinsed with platelet concentrate before collecting the
sample.
 C H A P T E R 62
Donor Care during and after
Apheresis

SCOPE AND APPLICATION

Although apheresis is a relatively safe procedure, it is not without
poten­tial complications to donors. These include problems related to
antico­agulant use, replacement fluids, fluid and electrolyte imbalance,
vascular access, hemolysis, air embolus and infection. Staff, therefore,
must be trained to highest standards of proficiency in the operation
of apheresis equipment and the care of donors during and after the
procedure.

RESPONSIBILITY
The blood bank staff under direct supervision of the Medical Officer is
responsible for the care of donors during and after the procedure and
managing any adverse reaction if any.

MATERIALS REQUIRED
Following materials are required to attend to any emergency arising in
the post-donation period.

Oral Medication
• Analgesic tablets, e.g. Paracetamol tablet IP 500 mg.
• Calcium and Vitamin C tablets.

Injection
• Epinephrine (Adrenaline).
• Atropine sulfate.
• Heparin.
• Calcium gluconate.
• Pheniramine maleate.
282 Standard Operating Procedures and Regulatory Guidelines-Blood Banking
282

• Diazepam.
• Corticosteroid.
• Glucose (Dextrose 25%).
• Metoclopramide.
• Prochlorperazine maleate.
• Sodium bicarbonate.
• Glucose saline (Sodium chloride and Dextrose 500 ml).
• Deriphyllin.
• Lasix.

Antiseptics
• Savlon solution.

Miscellaneous
• Bandages/Dressings.
• Band-aids.
• Heparin and benzyl nicotinate ointment (Thrombophob).
• Spirit of ammonia.
• Tongue depressor.
• Disposable syringes (2/5 ml) and needles 22 g.
• Clinical thermometer.
• Oxygen cylinder.
• Infusion set.
• Paper bag.

GENERAL CARE DURING PROCEDURE

a. Perform the thorough assessment of the donor including:
• Diagnosis and medical history.
• Medications.
• Vital signs and laboratory, investigations.
• Changes since last apheresis, if any.
b. Many apheresis complications may develop as a result of the proce­
dure itself or alternatively from the donor’s primary or secondary
medical conditions.
c. Identifying any pre-existing medical conditions and determining
the possible impact on the management of the donor’s during an
apheresis procedure, aids in minimizing or preventing any adverse
events.
d. The donors should never be left in the room without the attendance
of trained staff member.
 Donor Care during and after Apheresis 283

e. Good vascular access is essential for a successful apheresis proce­

dure. At least one (1) good cubital fossa vein is required to draw
whole blood from the donor. Steel needle of 16 to17 gauge is used
for the inlet/draw side to maintain blood flow rate at least 40–50 ml/
min. Lower arm or hand veins may be used for the return side. Steel
needle of 17 to 18 gauge is used for the return side to support rapid
flow of whole blood during apheresis procedure.
f. Knowledge of signs, symptoms and treatment of adverse reactions
will prevent a mild reaction from becoming serious.
g. The donor should be under constant supervision till he/she leaves
the premises.

COMPLICATIONS
The donor complications associated with the use of cell separators for
platelet/plasma-apheresis can be due to:
A. Delivery of anti-coagulant
B. Vasovagal
C. Allergy
D. Vascular access
E. Machine malfunction

Delivery of Anti-coagulant

Hypocalcemia (Citrate toxicity)

During apheresis procedure anticoagulant must be used to prevent
clott­ing in the extracorporeal circuit. The most commonly used antico­
agulant is Acid Citrate Dextrose Solution Formula A (ACD-A) at an
anticoagulant: whole blood ratio of 1:12 to 1:15. The citrate anticoagulant
which binds calcium is used to prevent the blood from clotting in the
apheresis machine tubing.
The healthy adult liver will metabolize 3g citrate every 5 minutes.
Transfusion at rates higher than one unit every 5 minutes/150 mL/70
kg/minute, impaired liver function and if the anticoagulant line of
an apheresis instrument is not properly housed in its rotary pump or
becomes disengaged during the procedure may thus lead to citrate
toxicity and hypocalcemia.
It is most commonly seen in the donor setting and is related to the
infusion of citrate anticoagulant and has been recorded up to 15% of the
procedures.
284 Standard Operating Procedures and Regulatory Guidelines-Blood Banking
284

The Symptoms of Citrate Toxicity

Mild Moderate Severe
Paresthesias Lightheadedness Tetany
– Perioral (Lips) Muscle cramps Laryngeal spasm
– Peripheral (Fingertips, legs) Weakness Seizures
Chills Nausea Arrhythmia
Shivering Vomiting Prolonged QT interval
Crawling Feeling Chest pain Bradycardia

Prevention: The donor should be warned about the symptoms and

asked to report any immediately. Oral calcium supplements during the
procedure may prevent the development of hypocalcemia.

Treatment of Citrate Toxicity

Mild
• Slow the procedure (inlet rate) to decrease the citrate infusion rate.
• Give calcium replacement orally or IV.
• Cover with a warm blanket.
• Reassure the donors but observe carefully.

Moderate
• Stop the inlet blood pump (pause tx).
• Begin or increase IV calcium infusion.
• Consider risk-benefit of reinfusion.
Severe
• Stop the procedure.
• Do not rinse back.
• Begin or continue IV calcium replacement. Calcium gluconate
5 ml of 10% given slowly can be used for the treatment of serious
citrate reactions where clinical and electrocardiographic evidence
of hypocalcemia exists.
• Notify the physician and the support team.
• Begin life support measures.

Inadequate citration
This is because of kinks in the tubing of the apheresis kit. Inadequate
levels of citrate may lead to clotting in the extracorporeal cell separator
 Donor Care during and after Apheresis 285

circuit and may result in either reinfusion of material with pro-coagulant

activity and potentially precipitate disseminated intravascular
coagulation (DIC) or cause hemolysis in the cell separator leading to
reinfusion of the hemolyzed blood.

Prevention
• Use manufacturer’s recommended anticoagulant at the correct
ratio.
• Monitor the anticoagulant pump, the rate of delivery via the drip
chamber and the volume of anticoagulant used throughout the pro­
ced­ure to ensure constant correct delivery of anticoagulant.
• Monitor the separation chamber of the return line filter for evidence
of clotting. Also monitor the return line for evidence of negative pre­
ssure which can be an early indicator of clotting within the cir­cuit.
• Monitor the color of the separated plasma for evidence of he­
molysis.

Treatment
• Check tubing for kinks.
• Increase anticoagulant inlet ratio.
• In event of hemolysis, stop the procedure.

Vasovagal
There is sudden fainting due to hypotension induced by the response
of the nervous system to abrupt emotional stress, pain, or trauma. It is
common in whole blood donation and also seen with apheresis pro­
cedures but with less frequency.

Symptoms
• Apprehension.
• Lightheadedness.
• Nausea.
• Decreased pulse rate.
• Hypotension.
• Perspiration.
• Can progress to cardiac arrest.

Treatment
• Stop the procedure.
• Raise feet and lower head end.
286 Standard Operating Procedures and Regulatory Guidelines-Blood Banking
286

• Loosen tight clothing (belt, tie, etc.).

• Ensure adequate airway.
• Check pulse and blood pressure.
• Apply cold compresses to forehead and back.
• Administer inhalation of spirit of ammonia if needed. The donor
should respond by coughing which will elevate the blood pressure.
• If there is bradycardia and hypotension—
■ Administer atropine injection 1 ml IM, if bradycardia continues
for more than 20 minutes.
■ Administer IV normal saline or dextrose saline infusions if
hypotension is prolonged.

Allergic Reactions
Donors may be hypersensitive to—
• Iodine used in cleansing of skin.
• Ethylene oxide used in sterilization of the disposable sets.
• Plastic of the disposable set and replacement fluids.
• Latex.

Symptoms

Mild Moderate Severe

Itching Intense itching Shortness of breath
Urticaria (rash) Widespread urticaria Hypotension
Rhinitis Hives or welts Diarrhea
Cough Rhinitis Laryngeal Edema
Tearing Wheezing Cardiopulmonary arrest
Tongue or Facial swelling

Treatment
Mild
• Determine cause.
• Consider hydrocortisone as per order of physician.

Moderate
• Stop the procedure.
• Maintain access for IV medication, i.e. Hydrocortisone, Epinephrine
as per order of physician.
 Donor Care during and after Apheresis 287

Severe
• Stop the procedure.
• Medication as ordered as per order of physician.

Complications of Vascular Access

The safest venous access is by venepuncture, most commonly of ante-
cubital fossa veins. It may be associated with hematoma formation,
bruising and occasionally nerve damage. High Return Pressure may also
produce hematoma. Poor vascular access may require re-sitting of the
vene-puncture or abandonment of a procedure. Careful explanations
must be given to the donor/patient when these complications occur and
appropriate medical management must be undertaken.
Complications of access can be:
• Infection.
• Occlusion: Mechanical or thrombotic.
• Mechanical displacement.
• Bleeding.
• Air embolus.
• Pneumo- or hemothorax.

Prevention
• Careful selection of the vascular site is very important. Steel needle
of 16 to 17 gauge is used for the inlet/draw side to maintain blood
flow rate at least 40–50 ml/min. Lower arm or hand veins may be
used for the return side. Steel needle of 17 to 18 gauge is used for the
return side.
• Check for the kinks in the tubing.

Complications due to Machine Malfunction

Machine malfunction leading to hemolysis, Thrombus formation, air-
embolism, or leakage occurs in less than 0.5% of the procedures, but the
effects can be severe. It is usually secondary to improper manufacturing
of the disposable set or improper mounting of the set. Newer devices
have improved safety features to protect the donor and mitigate these
complications.

Hemolysis
Forcing blood by pump through a narrow orifice particularly when,
blood is concentrated to a high hematocrit, may result in hemolysis.
Inadequate anticoagulation is also associated with hemolysis.
288 Standard Operating Procedures and Regulatory Guidelines-Blood Banking
288

Prevention
• Cell separator machines should be serviced in accordance with
manufacturers’ instructions. A planned maintenance scheme
should be followed.
• In the event of a mechanical failure of the machine, a service
engineer should be able to be contacted by telephone during
normal working hours.
• All the software must be carefully examined prior to setting up the
machine to ensure there are no kinks or twists in the tubing.
• Constant observation of the color of the plasma to detect for the
presence of hemolysis.
• When using filtration machines, constant monitoring of the trans-
membrane pressure is essential and particular care taken if frequent
episodes of low flow occur, as in this situation hemolysis is more
likely to occur.
• If hemolysis is suspected the procedure must be terminated as the
return of damaged red cells to the patient/donor could precipitate
DIC and mimic a hemolytic transfusion reaction.

Air embolus
Most cell separators incorporate air detector devices in the reinfusion
line. However, with the use of blood warmers and other software beyond
the machine’s air detectors, there is a risk of air embolism if all the lines
are not fully primed.
Never rely totally on ‘fail/safe’ alarm systems. Occasionally, they
can fail and constant monitoring of all reinfusion lines is necessary to
prevent air embolism from occurring.

Infection
i. Equipment Contamination: Do not leave cell separators and
associated equipment primed for longer than necessary and not for
more than one hour prior to use.
Apheresis machines should be routinely cleaned with a suitable
decontaminating agent. A standard procedure for dealing with
blood spillage must be in operation.
ii. Bacterial Infection: If bacterial contamination has occurred during
the set-up and priming procedure, there is a risk of causing a severe
bacteremia, which could be fatal in an immunosuppressed patient.
Plasma exchange using crystalloid, colloid or albumen as replacement
fluid depletes the patient’s immunoglobulin level. The combination
of low immunoglobulins and immunosuppressive therapy pre­dis­
 Donor Care during and after Apheresis 289

poses the patient to infection. Prophylactic administration of intra­

venous immune globulin to patients particularly at risk should only
be considered under special circumstances.

POST-DONATION CARE
It is important to ensure as far as possible that all donors/patients take
the required amount of rest and drink at least one cup of fluid before
leaving the apheresis venue and if no adverse reactions have occurred,
this information is noted in the relevant notes.
Any adverse reaction must be dealt with promptly, appropriately
and sympathetically and must be documented. The donor must have
recovered as fully as possible before being allowed to leave the venue.
The nurse/doctor in-charge must remain on the unit until the donor has
left the premises.
 C H A P T E R 63
Bio-medical Waste
Management

SCOPE AND APPLICATION

The blood banks as well as the pathological laboratories are governed by
the regulations of Bio-medical Waste Management (BMW) Rules, 1998.
Audit of biomedical waste is required to understand the type and quantity
of waste generated during various procedures undertaken therein. It
helps in formulation of the plan for segregation, waste handling and
mana­gement to prevent common health hazard of infection.

RESPONSIBILITY
It is the responsibility of all the Laboratory Technicians and other staff in
the Blood Bank to dispose of the waste generated as per requirements of
Bio-medical Waste Management (BMW) Rules, 1998.

MATERIALS REQUIRED
• 1% Hypochlorite (HOCl) Solution.
• Color Coded Plastic Buckets.
• Red-capped punctures proof containers.
• Red, yellow, blue and black plastic bags.

TYPES OF WASTE GENERATED, SEGREGATION AND ON-SITE

STORAGE
Segregation at source is the most critical step towards a well-functioning
waste management system. Separation of infectious and non-infectious
waste becomes impossible once mixed, resulting in greater risk to all
concerned. If mixed; non-infectious also becomes infectious.
The Bio-medical Rules provide color coding for waste segregation
as listed in Table 63.1.
The different type of waste material generated in various sections of
the blood bank and their segregation in various color coded plastic bags
is given in Table 63.2.
 Bio-medical Waste Management 291

Table 63.1: Color coding for segregation of bio-medical waste

291

The blood bank ensures that there are designated segregation points,
as close to the generation points as possible. Appropriate consumables,
such as good quality and adequately sized containers, non-chlorinated
plastic bags, needle cutters and safety boxes are used for segregation. The
specifications and color-coding provided in the Bio-medical Rules are
strictly followed. The disinfectant solution used for disinfection of glass-
wares and plastic tips must be changed daily. The containers are kept
closed all the time. The waste storage containers are not allowed to be more
than 3/4th full at any point of time and are emptied at least once everyday.

IMPORTANT NOTES
a. The blood/blood product units which need disposal are
• Units which have tested positive for infectious agents.
• Unsuitable products, such as under- and overweight blood packs,
or
• Those where the storage temperature has not been maintained.
• Discarded products, e.g. expired units.
• Blood returned unused but unsuitable for reissue.
• Blood packs in which leaks have been detected.
Secure and exclusive quarantine storage must be done for blood
units awaiting disposal. Discarded blood components must not be
left at room temperature, but should be stored in a designated com­
292 Standard Operating Procedures and Regulatory Guidelines-Blood Banking
292

Table 63.2: Waste segregation and color coding in blood bank

Sr Room Waste inventory Category Color code for bag
No. No.
1. Donor room Cotton swabs 6 Yellow plastic bag
Needles 4 Red-capped container
Lancets 4 Red-capped container
Syringe without 7 Red plastic bags
292 needle
Plastic tubing 7 Red plastic bags
2. Plasma- Cotton swabs 7 Yellow plastic bag
pheresis
room
Needles 4 Red capped container
Surgical gloves 7 Red plastic bags
Disposable plastic 7 Red plastic bag
kit
3. Component Plastic tubing of 7 Red plastic bags
lab blood bags
Cotton and gauze 7 Yellow plastic bags
Surgical gloves 7 Red plastic bag
Broken/Leaking 7 Red plastic bag
blood bags
4. Serology lab Vacutainers 7 Red plastic bag
Yellow and blue tips 7 Red plastic bag
Diamed gel cards 7 Red plastic bag
Blood bags 7 Red plastic bag
Test tubes (RIA) 7 Blue plastic bag
Surgical gloves 7 Red plastic bag
Glass slides 4 Blue plastic bag
5. TTI lab Vacutainers 7 Red plastic bag
Yellow and blue tips 7 Red plastic bag
Microplates 7 Red plastic bag
Rapid test devices 7 Red plastic bag
Plastic droppers 7 Red plastic bag
Blood bags 7 Red plastic bag
Test tubes RIA 7 Blue plastic bag
Surgical gloves 7 Red plastic bag
Glass slides 4 Blue plastic bag
Waste wash 8 To be discharged into
Solutions drain
6. Stationery and Other gen.waste Black plastic bag
 Bio-medical Waste Management 293

partment refrigerator to minimize bacterial growth. It is also impor­

tant to keep discarded products under a security lock to reduce the
risk of misuse and prevent access by unauthorized persons.
The treatment of PVC blood bags forms an integral part of BMW
management. Discarded Blood and Blood Products packs need to
be safely treated and disposed of in an environment-friendly way.
These are autoclaved at +121°C for 20 minutes as autoclaving of PVC
blood bags is a safer and reliable method as compared to chemical
293
disinfection. Thereafter, blood bags are discarded in Red plastic
bags.
b. Disposal of sharps is an important BMW issue in blood banks. Lan­
cets used for rapid blood group screens/Hb testing and the needles
of donor sets form the major sharps. The needles containing por­tion
of cut tubing and lancets are discarded in red-capped con­tainers.
c. Post analysis plasma/serum/blood from various containers are
decan­ted carefully into stainless steel kettle/container which is
auto­claved before disposing it to manure pit.
d. The glass tubes, slides and blue/yellow tips are discarded in trough
containing 1% hypochlorite solution. After 1 hour immersion in
hypochlorite solution glass tubes and slides are washed and reused.
Blue/yellow tips are discarded in red bags. The residual hypochlorite
solution used for disinfection is drained into sink.
e. The blood donor sets, after cutting out the needle parts and gel
cards are discarded in red bags.
f. Spills on the floor, of infected or potentially infected material,
are covered with paper towel/blotting paper/newspaper. 1%
hypochlorite solution is poured on and around the spill area and
covered with paper and kept covered for 10 minutes. Then the paper
is removed with gloved hands and discarded as infectious waste in
yellow bags.
Color-coded plastic bags and red-capped containers containing
waste are kept in a central place till these are handed over to the
government approved bio-medical waste contractor for further disposal.
 C H A P T E R 64
The Management of Sharps/
Needle Stick Incidents and
Other Exposure Incidents

SCOPE AND APPLICATION

The purpose of this SOP is to:
• To define the process and policy of management for needle stick
injuries in the hospital.
• Implement the use of devices with safety features and evaluate their
use to determine which are most effective and acceptable.
• Analyze needle stick and sharps-related injuries in the hospital to
identify hazards and injury trends.
• Set priorities and strategies for prevention by examining local and
national information about risk factors for needle stick injuries and
successful intervention efforts.
• Ensure that health care workers are properly trained in the safe use
and disposal of needles and sharps.
• Modify work practices that pose a needle stick injury hazard to
make them safer.
• Establish procedures for and encourage the reporting and timely
follow-up of all needle stick and other sharps-related injuries.
• Encourage health care workers to report any hazards from needles
they observe in their work environment and to participate in
blood. Borne pathogen training and follow recommended injury
prevention practices, incl uding getting a hepatitis B vaccination.
• To prevent the likelihood of transmission this policy must be
followed in the event of a sharps/splash incident.
The policy is applicable to all health care workers that all health care
workers adopt a responsible attitude in preventing and reporting such
who come into contact with patients’ blood or body substances. This
necessitates incidents. Accurate documentation is crucial to the success
of this policy.
 The Management of Sharps/Needle Stick Incidents ... 295

RESPONSIBILITY
Hospital Infection Control Committee (HICC) has the responsibility to
safely manage needle stick injuries that are produced while delivering
care. Staff has the responsibility to report any exposure incidents that
occur related to contaminated needle and to take appropriate measures
to avoid these in the first instance. HICC has the responsibility to
ensure local risk assessments are carried out where necessary, e.g. to
identify the use of appropriate personal protective equipment (PPE),
adherence to safe practices, including the provision of resources for this,
immunization programs are offered appropriately and any incidents that
occur are reviewed and subsequent actions taken where appropriate.
Training should be provided on all aspects of the management of needle
stick injury. The infection control team has the responsibility to ensure
training is available and it is mandatory for staff to attend such training
sessions.

Infection Control Officer (ICO)

• Conducts audit every month.
• Maintains records (Incidents and actions taken).
• Ensures that PEP drugs are available in the hospital.
• Ensures provision of PPE at all concerned locations.
• All NSI injuries should be reported to the state AIDS control societies
giving the exposure code and the HIV status code.
• Ensures that the code of conduct for infection control is followed in
the hospital.

Infection Control Nurse (ICN)

• Conducts daily rounds in all departments.
• Checks that puncture proof containers are available in all locations.
• Checks that all needles are being discarded in puncture proof
containers.
• Checks that PPC are being disposed as per BMW policy.
• All NSI injuries should be reported to the infection control team/
committee.
• Informs all nonconformities to Infection Control Officer/Chairperson.
• Maintains records of incidents in the hospital.

PROCEDURE AND GUIDELINES

Management of needle stick/exposure incidents requires the following:
• Reporting.
• Administration of first aid.
296 Standard Operating Procedures and Regulatory Guidelines-Blood Banking
296

• Risk assessment and evaluation of exposure.

• Evaluation of the source.
• Management of the exposed person.
• Follow up of the health care worker or injured person.

Reporting
Report the exposure to the appropriate authority (Infection Control
Officer/Medical Officer) and condition must be treated as an emergency.
Prompt reporting is essential because in some cases, HIV post-
exposure prophylaxis (PEP) may be commended and it should be started
as soon as possible, preferably within a few hours. Initiating treatment
after 72 hours of exposure is not recommended.

Administration of First Aid

Needle stick, sharps injury or cut

• Needle-sticks and cuts should be washed with soap and water.
• Cover with wate rproof dressing if necessary.
• Pricked finger should not be put into mouth reflexly.
• Dispose of sharp carefully into an approved sharps container.

Splash to mucous membrane, conjunctiva or non-intact skin

• Flush the exposed site immediately with water.
• Identify the source person if possible.
There is no need of local antiseptic cream or disinfectant.

Risk Assessment and Evaluation of Exposure

Risk assessment requires knowledge of:
i. The infection status and/or risk factors for infection in the source
patient where identified. Identification of potential risk factors in
the source patient, for example:
• Transfusion/injection with blood products.
• Birth in an area of high endemicity for HIV, hepatitis B or hepatitis
C.
• Close contacts infected with HIV, hepatitis B or hepatitis C.
• People with identified risk factors for HIV, hepatitis B or hepatitis
C, e.g. intravenous drug users.
ii. The susceptibility of the health care worker to blood-borne viruses
e.g. previous vaccination history and immune status.
iii. The nature of the exposure and the type of body fluid. The risk
of transmission of blood-borne viral infection post-exposure is
increased with per-cutaneous exposure involving:
 The Management of Sharps/Needle Stick Incidents ... 297

• Deep injury to health care worker.

• Visible blood on the device causing the injury.
• Device previously in the source patient’s vein or artery (e.g.
phlebotomy needle rather than a suture needle).
• Device previously in a patient with a high viral load (e.g. advanced
disease).
• Volume of blood injected, or with mucous membrane or skin
exposure involving:
■ Prolonged contact with blood.
■ An extensive area of skin.
■ Where skin integrity is compromised.
The HCW must be evaluated for potential to acquire HIV based on
the type of biological material involved, route of injury and severity of
exposure as shown in Algorithm (Fig 64.1).

Fig. 64.1: Algorithm to determine exposure code (EC) of an injury

(OPIM-Other potentially infectious material; PEP-Post exposure prophylaxis;
MM-Mucous membrane; Vol.-Volume)
298 Standard Operating Procedures and Regulatory Guidelines-Blood Banking
298

Susceptibility to Hepatitis B will be determined by vaccination

status against HBV and antibody response.
The baseline status of the HCW in terms of HBsAg, anti-HCV and
anti-HIV should be carried out within 72 hours of injury.

Cost (staff)
Hospital will bear all expenses that include all lab tests and treatment
recommended by the HICC.
Recommendation of the same would be given by the HICC.

Evaluation of the Source

Once the source patient is known, the clinical team is notified. The
sample of clotted blood is collected after the written consent and pretest
counseling. Urgent testing for hepatitis B surface antigen, hepatitis C
antibody and HIV antibody is got done. Testing is confidential.
The HIV test is done routinely in many circumstances such as
ante-natal patients, patients referred for surgery. Patients having the
test are not discriminated against and the information is handled in a
confidential sympathetic manner.
Having a HIV test does not affect current and future life insurance
or any other insurance policy as long as the person is HIV negative. The
source status of source for HIV is determined by the algorithm given
below (Fig 64.2):

Fig. 64.2: Determining HIV status code

 The Management of Sharps/Needle Stick Incidents ... 299

If HIV antibody is positive, then absolute lymphocyte count/CD4

count, viral load, history of antiretroviral therapy (ART) and clinical
stage of the disease is assessed. In individuals where outcome of therapy
is poor, drug resistance should be looked for, if possible.

Confidentiality
Patients having the test are not discriminated against and the information
is handled in a confidential sympathetic manner. Testing is confidential.
If HIV test is positive, refer the case to expert for further management.
Strict confidentiality of the HIV status of source patient and injured
person must be observed. When the source is HBsAg positive, then
HBeAg is required, if anti-HCV positive, then HCV viral load and HCV
genotype are required.
At times the source individual may not be available for testing
or may refuse to be tested. In such circumstances, details of medical
diagnosis, clinical symptoms and history of high-risk behavior will
determine administration of PEP to the exposed HCW.

Management of the Exposed Person

For optimal efficacy, PEP for HIV and HBV should be commenced as
soon as possible after the incident and ideally within one to two hours.

HIV
PEP for exposure to HIV should be started preferably within the next
one hour. If delay is more than 36 hours, expert consultation is advised.
PEP, when started, should continue for 28 days. The recommendations
of NACO for post-exposure prophylaxis (PEP) are followed and are as
under (Table 64.1):
Typical schedules are basic two-drug regimen appropriate for low
risk exposures and expanded three-drug regimens for exposures with
increased risk of transmission. If source case is found to be negative
for anti-HIV and does not belong to the “high-risk” category, PEP is
discontinued. When PEP is initiated, baseline serum creatinine, liver
function tests with enzymes and complete blood counts must be done.

Basic Regimen
1. Zidovudine (AZT) 600 mg in divided doses (300 mg/twice a day or
200 mg/thrice a day for 4 weeks)
2. Lamivudine (3TC) 150 mg twice a day for 4 weeks.
300 Standard Operating Procedures and Regulatory Guidelines-Blood Banking
300

Table 64.1: Recommendations of NACO for post-exposure prophylaxis (PEP)

EC HIV SC PEP Recommendation
1 1 PEP may not be warranted
1 2 Consider basic regimen (Negligible risk)
2 1 Recommend Basic Regimen (most exposures are in this
category)
2 2 Recommended expanded regimen
3 1 or 2 Recommended expanded regimen
2/3 Unknown If setting suggests a possible risk
(epidemiological risk factors) and EC is 2 or 3, consider
Basic Regimen

Expanded Regimen
1. Basic regimen (4 weeks therapy) +
2. Indinavir 800 mg/thrice a day or any other protease inhibitor.

HBV
PEP for HBV should be instituted immediately, preferably within 24
hours but definitely within 7 days following guidelines as given in the
table 64.2 below:
Children: 32–48 IU/Kg body wt.
Individuals who lack HBsAg and have not previously developed
satisfactory immune response to the virus may be susceptible. They
could be offered HBIg for immediate protection of significant exposure
to HBV.
An individualized approach based on risk assessment is
recommended for the management of a health care worker with
unknown response to hepatitis B vaccination, one who has been
exposed to an unknown source or a source with unknown hepatitis
status. In such circumstances, the HBV status of the source and/or the
exposed should be determined where appropriate and available. The
exposed person may be managed as in the case of an injury involving
an HBsAg positive source person if the HBV status of the latter cannot
be ascertained.
A comprehensive strategy for eliminating transmission of HBsAg is
through universal vaccination of the HCW.
 The Management of Sharps/Needle Stick Incidents ... 301

Table 64.2: Hepatitis B prophylaxis for reported sharps injuries and

mucocutaneous exposure to blood or other body fluids

Hepatitis B Status Hepatitis B Status of Source

of the HCW
Negative Unknowm/ Positive
Untested
One dose or less Initiate/continue Accelerated *Accelerated
of Hep B vaccine course of Hep. course of Hep. B course of Hep. B
prior to exposure B vaccine vaccine vaccine and one
dose of Hep B
immunoglobulin
Two or more doses Check HbsAb Check HbsAb Check HbsAb
of Hep B vaccine titer. titer. titer. One dose
prior to exposure Complete Complete course of Hep B vaccine
but antibody titer course of Hep of Hep B vaccine immediately
not known B vaccine if if indicated and complete
indicated the course as
indicated
Responder to Hep No action Booster dose of A booster dose of
B vaccine (anti- required Hep B vaccine Hep B vaccine is
HBs>10 IU/L) may be required required
depending on risk
assessment
Known non- Consider re- Following risk Re-immunization
responder to immunization if assessment if not previously
Hep B vaccine not previously consider attempted
(Titer <10 IU/L, attempted two doses of
two months after HBIG
course or booster) ** One month
apart and
consider re-
immunization if
not previously
attempted

* An accelerated course of vaccine consists of vaccine doses spaced at 0,

1 and 2 month intervals. HCW undergoing accelerated vaccination may
require a 4th dose at 12 months. A 1.0 ml dose of Hep vaccine contains
20 mcg of HBsAg protein and is given intramuscularly in deltoid region for
adults and children 10 years and older.
** Hepatitis B Globulin (HBIg) should be offered as soon as possible and
within 48 hours post-exposure–if considered appropriate following risk
assessment. Dose of Hepatitis B immunoglobulin (HBIg) is 1000–2000 IU
intramuscularly for adults. It is available as 0.5 ml ampoule of 1000 IU and
1 ml vial of 2000 IU.
302 Standard Operating Procedures and Regulatory Guidelines-Blood Banking
302

HCV
At this time, there are no recommendations of PEP for HCV
Immunoglobulin and ribavirin are ineffective. For the source, baseline
testing for anti-HCV antibodies is warranted. For the exposed HCW,
carry out baseline anti-HCV and ALT. Efforts should be made to
determine the genotype of source HCV for management.

Management of the Source Person

Once the source patient is known, the clinical team is notified. The
sample of clotted blood is collected after the written consent. Urgent
testing for hepatitis B surface antigen, hepatitis C antibody and HIV
antibody is got done.
When the source is HBsAg positive, then HBcAg is required, if anti-
HCV positive, then HCV viral load and HCV genotype are required. If
HIV antibody is positive, then absolute lymphocyte count/CD4 count,
viral load, history of antiretroviral therapy (ART) and clinical stage of
the disease is assessed. In individuals where outcome of therapy is poor,
drug resistance should be looked for, if possible.
At times, the source individual may not be available for testing
or may refuse to be tested. In such circumstances, details of medical
diagnosis, clinical symptoms and history of high-risk behavior will
determine administration of PEP to the exposed HCW.

CONSENT AND COST (SOURCE PERSON)

If viral markers are not conducted for any patient, then consent of
conducting the same would be taken by the consultant/doctor on duty.
Consent would be signed by the patient and if due to some reason
patient is unable to give the consent, then patient’s relative would give
the consent. Patient would bear the cost of lab tests.

Follow up of the Healthcare Worker or Injured Person

HBV
Hepatitis exposed HCW should be tested for HBsAg at 6 weeks,
three months and again at 6 months. If vaccinated test for anti-HBs
antibodies, two months after the last dose of the vaccine. However,
testing for antibodies for hepatitis surface antigen cannot be undertaken
if hepatitis B immunoglogulin (HBIG) has been given within the last 6–8
weeks. The HCW is advised to use barrier precautions (condom) and
refrain from donating blood/plasma/organs/tissue or semen during the
follow-up period.
 The Management of Sharps/Needle Stick Incidents ... 303

HCV
HCV-exposed HCW should be tested for anti-HCV and ALT at 4–6 weeks
and at least 4–6 months post-exposure; confirm repeatedly positive anti-
HCV ELISA results with supplemental tests (Recombinant Immunoblot
assay RIBA or HCV RNA). The test for HCV RNA, where facilities exist, may
be done at 4 weeks for an earlier diagnosis. HCV sero-conversion occurs
silently; hence tests should be carried out periodically. Genotyping is
helpful in planning therapy should sero-conversion occur. Genotype 2
and 3 are more likely to respond to therapy with pegylated interferon
along with ribavirin as compared to genotype 1.

HIV
The exposed person should be followed up for at least 6 months and be
asked to report signs/symptoms of acute HIV sero-conversion. Blood
sampling should be performed soon after injury, at 6 weeks, at 12 weeks
after exposure and when there is suggestion of sero-conversion. On
all three occasions, HCW must be provided with a pre-test and post-
test counseling if the HIV test is found to be positive at anytime within
12 weeks, the HCW should be referred to a physician for treatment.
Expanded follow-up of 12 months is recommended for a HCW exposed
to a HIV-HCV co-infected source. Complete blood counts, serum
creatinine, LFT including enzymes should be repeated two weekly. Those
receiving protease inhibitor, should have blood sugar levels monitored.
If a patient on indinavir (IDV) or tenofovir (TDF), then urine analysis
should be included. He should be advised to refrain from donating
blood, semen or organ/tissues and abstain from sexual intercourse. In
addition, women HCW should not breast-feed their infants during the
follow-up period. Individual started on chemoprophylaxis should also
be monitored for drug toxicity and tolerance.
The protocol for the needle stick/sharp injury/body fluid exposure
is summarized as:

NEEDLE STICK INJURY/SHARP OBJECT/BODY FLUID

EXPOSURE
1. Do not squeeze the area, allow free bleeding. Wash the injured site
with soap and water (antiseptics may be used).
2. Check patient’s status for HIV, HBsAg, and HCV, if not known, get it
done.
304 Standard Operating Procedures and Regulatory Guidelines-Blood Banking
304

3. Immediately go to Casualty/Triage or any designated area in the

health care setup.
4. The needle stick injury protocol form should be filled and signed
with the help of the MO/ICO and provide pre-test counseling to the
employee.
5. Inform infection control nurse during working hours and thereafter
on next working day.
6. Inform matron or night supervisor on duty.
7. Give your blood sample for HIV, HBsAg, HCV in the main laboratory
(needle stick injury protocol form should be shown).
8. Follow up treatment regimen within two hours of the injury.
a. If source patient is HIV (+), start anti-retroviral treatment as
soon as possible as per the hospital protocol.
b. If the patient is HBsAg + start treatment as per protocol.
9. a. If source patient is HIV negative, then the healthcare worker
must repeat the blood test after 6 wks and 12 wks.
b. If source patient is HBsAg –ve, then:

10. If source is unknown, then follow the protocol point no. 8a and b.

TRAINING AND PREVENTION

Most health care workers (HCW) working around patients or biological
samples stand the risk of accidental exposure to blood and blood-borne
pathogens. The risk of transmission of infection is higher with exposure
to blood especially in advanced disease; prolonged exposure of even
non-intact skin/mucous membrane to blood or other infectious fluid;
cut with a contaminated device drawing blood and injury with a hollow-
bore, blood-filled needle.
Prevention of exposure in a workplace setting needs to be inculcated
into every health care provider right from the time of recruitment
through regular trainings in safe work practices and management of
sharp instruments for all staff members by the HICC.
 The Management of Sharps/Needle Stick Incidents ... 305

An important element of a needle stick injury prevention program

is the education and training of health care personnel in sharps injury
prevention as a part of continuing medical education. Health care
workers need to know how to use, assemble, disassemble, and dispose
of needles properly. An effective program should address all the aspects
of needle stick injuries including risk of injury, potential hazards,
recommended precautions, etc.
All health care providers must be vaccinated against hepatitis
B (Three doses) and the antibody levels checked after a month of
completion of three doses (anti-HBs). The protective antibody level is
10 IU/L or more.

RECORD AND DOCUMENTATION

Infection control nurse will be responsible for maintaining record of:
• All needle stick injuries.
• Reason of injury.
• Treatment in emergency department.
• Follow-up treatment of staff member.
• All other documentations.

SAFE WORK PRACTICES

Infection Control and Occupational Health Measures to Prevent

Transmission of Blood-borne Viruses
• Apply good basic hygiene practices with regular handwashing.
• Cover existing wounds or skin lesions with waterproof dressings.
• Appropriate disposable gloves should always be worn whenever
there is known or anticipated contact with blood, saliva or other
body fluids.
• Avoid invasive procedures if suffering from chronic skin lesions on
hands.
• Avoid contamination by appropriate use of personal protective
clothing.
• Protect mucous membranes of eyes, mouth and nose from blood
splashes.
• Care must be taken when opening ampoules to avoid minor cuts.
• Non-disposable clothing contaminated with blood should be
placed separately for washing.
• Institute safe procedures for handling and disposal of needles and
other sharps.
306 Standard Operating Procedures and Regulatory Guidelines-Blood Banking
306

• Never re-sheath a needle by hand.

• All needles, syringes and sharps must be disposed of into the
puncture proof sharps disposal bins.
• Sharps disposal bins must not be overfilled above the level indicated.
They should be carefully sealed prior to disposal.
• Remember when you use a sharp; you are responsible for its
immediate disposal.
• Institute procedures for sterilizing and disinfecting instruments
and equipment.
• Apply 1% Sodium Hypochlorite before wiping up. If the spillage is
on the floor, granules should be used.
• Institute procedures for the safe disposal of contaminated waste.
• Vaccinate all clinical and laboratory workers against hepatitis B
There is no vaccine for bad practice.

Management of Sharp Instruments

Seven points to remember
1. Extra care must be taken during the use and disposal of sharp
instruments.
2. The person who uses sharps must dispose of them safely.
3. Always dispose of used sharps directly into an approved sharps
disposal container.
4. Do not re-sheath needles by hand.
5. Do not overfill sharps boxes–Change when three-quarters full.
6. Whenever possible, take a sharps disposal box to the point of use.
7. Report all sharps injuries immediately to ICO/MO.

Know the Rules

Be a Sharps Safety Stickler
Exposure Incident Record Form for Health Care Workers (HCWS)
Incident Details

Section A: Details of Person Exposed

Name: _____________________ Age and Sex ___________________
Home Address:__________________________________________________
________________________________________________________________
Telephone No: _____________________________
Date of Birth: ____/____/_______ Sex: Male:     Female:
Specify Category (e.g. Nurse/Lab. Tech): ____________________________
 The Management of Sharps/Needle Stick Incidents ... 307

Section B: Details of Exposure

Date and time when incident occurred: ___/___/____ Time: _____am/pm
Date and time when HCW/client reported the incident: ___/___/___
Time:______am/pm
Give a brief description of the incident (what happened and why):
________________________________________________________________
________________________________________________________________
Describe or specify the procedure in which the HCW was involved at the
time of the exposure (e.g. venipuncture, removal of sutures, IM injection,
fixing the patient’s bed etc.)
________________________________________________________________
________________________________________________________________
Was the procedure an elective procedure?
Or an emergency? _______________________________________________
To what material was the HCW/client exposed?
Blood/other body/laboratory fluid (specify): ________________________
What type of exposure occurred?
• Percutaneous (the skin of the HCW was cut or penetrated by a
needle or other sharp object e.g., scalpel blade, trochar, tooth, bone
spicule):
• Mucocutaneous (contamination of the eye(s), nose, mouth or non-
intact skin:
• Both per-cutaneous and mucocutaneous:
• Not Known:
If percutaneous, please specify the sharp object which caused the injury:
______________ And give the size/gauge of object: ______________ Not
Known: ______________
If percutaneous, was the injured HCW/client the original user of the
sharp object? Yes No Not Known
Where did it happen? ICU Theatre Other Ward Other
Specify: _________________________________________________________
(A) Was the HCW/client wearing gloves at the time of the incident?
Yes No
Was the HCW/client wearing protective goggles at the time of the
incident? Yes No
308 Standard Operating Procedures and Regulatory Guidelines-Blood Banking
308

Section C: Immediate Post-exposure Care

Has an initial post-exposure blood specimen been obtained from HCW/
client?   Yes    No Date: ____/____/______
Has HCW/client had Hepatitis B vaccine in the past?
No 1 dose 2 doses 3 or more doses not sure
Was HBIG (Hepatitis B immunoglobulin) given post exposure?
Yes No Date:____/_____/______
Was HBIG (Hepatitis B immunoglobulin) offered to HCW/client?
Yes No Date: ____/____/______
Was serological (blood) test carried out post vaccination? Yes No
Do you know the result? Yes No
If yes: Immune Non-immune Not Known
Was follow-up arranged for the HCW/client, e.g., appointment for
further vaccination? Yes No

Signed: Date:
 S E C T I O N 2

Regulatory Guidelines
 C H A P T E R 65
Regulatory Requirements of
Blood and/or Its Components
Including Blood Products

INTRODUCTION
Blood Transfusion Service is a vital part of the National Health Service and
there is no substitute for human blood and its components. Increasing
advancement in the field of transfusion technology has necessitated
enforcing stricter control over the quality of blood and its components.
In most of the developed countries, the blood banking system has
advanced in all facets of donor management, storage of blood, grouping
and cross-matching, testing for transmissible diseases, rationale use of
blood and distribution. The Government has full responsibility for the
blood program, even though, in some countries, the managements of
blood transfusion services are delegated fully or partly to appropriate
non-governmental organizations (NGOs) working on non-profit basis,
e.g. Red Cross Society. When a NGO is assigned this responsibility, the
Government should formally recognize it and give a clear mandate
formulating the national blood policy. It is important to consider policy
decisions enforcing appropriate regulations or necessary functions of
health services to ensure high-quality blood transfusion services and
safe blood.
The Government of India has taken necessary steps from time to
time in order to improve the standards of Whole Human Blood and
its components by making necessary amendments in the statutory
provisions as well as by creating additional regulatory functionaries.
The Central Government through Drugs Controller General of India has
formulated a comprehensive legislation to ensure better quality control
system on collection, storage, testing and distribution of blood and its
components. The Government of India made numerous amendments
in the Drugs and Cosmetics Act, 1940 and Rules thereunder to meet the
latest standards.
In order to provide abundant availability of blood and blood
transfusion facilities at first referral units the Drugs and Cosmetics
Act 1940 and Rules 1945 was amended with an objective of setting up
312 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

blood storage facilities at the FRUs/CHCs/PHCs. The main aim of this

notification was to provide human blood as well as components to these
hospitals without taking license under the Drugs and Cosmetics Act
1940.
Further in order to check growth and mushrooming of stand-alone
blood banks in the country, the Government of India further amended
the Rules 1945 by prohibiting such stand-alone blood banks to apply for
grant/renewal of blood bank.

NATIONAL BLOOD POLICY

Government of India notified the National Blood Policy on blood safety
in the year 2002. The objective of the policy was to provide safe, adequate
blood and its components to the masses in distress. The main aim of
the policy was to procure blood from non-remunerated regular blood
donors by the blood banks. The policy also addresses various issues
with regard to technical personnel, research and development and to
eliminate profiteering by the blood banks. The policy also envisages
that no fresh licences to stand-alone blood banks in private sector
shall be granted and renewal of such blood banks shall be subjected to
thorough scrutiny.

SCENARIO OF LEGAL FRAMEWORK

Whole Human Blood has been included in the definition of ‘drug’
under section 3(b) of The Drugs and Cosmetics Act 1940. Therefore, the
health care institutions/organizations engaged in collection, storage,
processing, distribution, etc. of Whole Human Blood drawn from donors
and/or preparation, storage and distribution of blood components need
to be covered under the purview of the provisions made under the Drugs
and Cosmetics Act 1940 and Rules 1945.
With the onset of prevalence of AIDS virus in the country, testing
of every blood unit for HIV was made mandatory before issue for
transfusion by the Ministry of Health and Family Welfare, Government
of India through notification issued in the year 1989 under the Drugs
and Cosmetics Rules 1945. For this purpose the Government of India
notified National Institute of Communicable Disease, Department of
Microbiology, Delhi; National Institute of Virology, Pune; and Centre
for Advanced Research in Virology, Christian Medical College, Vellore
under Rule 3-A(6) of Drugs and Cosmetics Rules 1945 to test HIV
antibodies in respect of human blood and its components.
Following M/s Ferguson’s report highlighting various deficiencies
with regard to quality control of blood and blood components, etc. in
 Regulatory Requirements of Blood and/or Its ... 313

the year 1990 as well as the concerns expressed in different forum and
Parliament, the Drugs and Cosmetics Rules 1945 were again amended
(Rules 68A, Part- XB and Part- XIIB of Schedule F) in the year 1992–93.
These amendments provided for a Central license Approving Authority
to achieve uniformity in blood bank licensing throughout the country
and some other amendments were made. The Government of India
notified Drugs Controller General (India) as Central License Approving
Authority (CLAA) to approve the licenses for Blood, Blood Components
and Blood Products, IV Fluids and Vaccines and Sera, etc. to achieve
uniformity in the licensing of such products throughout the country.
The Government of India vide notification GSR 28(E), dated
22.1.1993 has further amended the existing provisions for proper and
safe functioning and operation of a blood bank and/or for preparation of
blood components by inserting Part XII B under Schedule F of the Drugs
and Cosmetics Act 1940 and Rules 1945. Various requirements such as
accommodation, technical staff, equipments and instruments, etc. for
operation of blood bank as well as for blood components are included
in this Part. Licensing Authorities as per provisions under the Drugs
and Cosmetics Act 1940 and Rules 1945 are authorized to issue licenses
for operation of blood banks in the country. For the first time, statutory
provisions were incorporated about organizing blood donation camps. In
view of provisions contained in Part XII- B of Schedule F of the Drugs and
Cosmetics Rules 1945, only Licensed Designated Regional Transfusion
Centre, Licensed Government blood bank and Indian Red Cross Society
are allowed to organize blood donation camps. The standards for ‘Whole
Human Blood’ have been prescribed in Indian Pharmacopoeia.
The Government of India further in the year 2002 put regulatory
control on “In vitro diagnostic devices” which were being used for
testing of human blood and its components for HIV, HbsAg and HCV
by notifying these devices as “drug” under section 3(b) of the Drugs and
Cosmetics Act 1940. National Institute of Biologicals, Noida, was notified
Central Testing Laboratory to carry out testing of these notified drugs
including blood grouping sera under Rule 3-A(8) of Drugs and Cosmetics
Rules 1945. The procedure of making applications by a blood bank, fees
to be paid for grant/renewal of license and conditions of license to be
followed after grant/renewal of licenses were inserted under the added
rules. Good manufacturing practices, standard operating procedures,
validation of equipments, etc. were also made mandatory for the
blood banks. The Government of India further amended Rule 122-G by
inserting Rule 122-G (2) vide notification GSR 733(E) dated 21.12.2005
in order to check the growth and mushrooming of stand-alone blood
banks in the country.
 C H A P T E R 66
Drugs and Cosmetics Rules,
1945 (Part X-B)

Requirements for the Collection, Storage, Processing and Dis­

tri­bution of Whole Human Blood, Human Blood Compo­nents
by Blood Banks and Manufacture of Blood Products

RULE 122-EA OF THE DRUGS AND COSMETICS RULES, 1945

Definitions
1. In this part and in the forms contained in Schedule A and in
Part XII-B and Part XII-C of Schedule F, unless there is anything
repugnant in the subject or context—
a. “apheresis” means the process by which blood drawn from a
donor, after separating plasma or platelets or leukocytes, is re-
transfused simultaneously into said donor;
b. “autologous blood” means the blood drawn from the patient
for re-transfusion into himself later on;
c. “blood” means and includes whole human blood, drawn from
a donor and mixed with an anticoagulant;
d. “blood bank” means a place or organization or unit or
institution or other arrangements made by such organization,
unit or institution for carrying out all or any of the operations
for collection, apheresis, storage, processing and distribution
of blood of blood components drawn from donors and/or for
preparation, storage and distribution of blood components;
e. “blood component” means a drug prepared, obtained, derived
or separated from a unit of blood drawn from a donor;
f. “blood product” means a drug manufactured or obtained from
pooled plasma of blood by fractionation, drawn from donors;
g. “donor” means a person who voluntarily donates blood after he
has been declared fit after a medical examination, for donating
blood, on fulfilling the criteria given hereinafter, without
 Drugs and Cosmetics Rules, 1945 (Part X-B) 315

accepting in return any consideration in cash or kind from any

source, but does not include a professional or a paid donor;
Explanation: For the purpose of this clause, benefits or incentives
like pins, plaques, badges, medals, commendation certificates,
time-off from work, membership of blood assurance program,
gifts of little or intrinsic monetary value shall be construed as
consideration.
h. “Leucapheresis” means the process by which the blood drawn
from a donor, after leukocyte concentrates have been separated
is re-transfused simultaneously into the said donor;
i. “Plasmapheresis: means the process by which the blood drawn
from a donor, after plasma has been separated, is re-transfused
during the same sitting into the said donors;
j. “Plateletpheresis” means the process by which the blood drawn
from a donor, after platelet concentrates have been separated,
is re-transfused simultaneously into the said donor;
k. “Professional donor” means a person who donates blood for
a valuable consideration, in cash or kind, from any source, on
behalf of the recipient-patient and includes a paid donor or a
commercial donor.
l. “Replacement donor” means a donor who is a family friend or
a relative of the patient-recipient.

122-F. Form of Application for Licence for Operation of Blood

Bank/Processing of Whole Human Blood for Components/
Manufacture of Blood Products for Sale or Distribution
1. Application for grant and/or renewal of licence for the operation
of a blood bank/processing of human blood for components/
manufacture of blood products shall be made to the Licensing
Authority appointed under Part VII in Form 27-C [or Form 27-E],
shall be accompanied by licence fee of [rupees six thousand and an
inspection fee of rupees one thousand and five hundred for every
inspection thereof or for the purposes of renewal of licence].
Provided that if the applicant applied for renewal after its expiry
but within six months of such expiry the fee payable for the renewal
of licence shall be six thousand and inspection fee of rupees one
thousand and five hundred plus an additional fee at the rate of
rupees one thousand per month or a part thereof in addition to the
inspection fee:
316 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Provided further that a licensee holding a licence in Form 28-C

[or Form 28-E, as the case may be] for operation of Blood Bank/
processing of whole human blood for components/manufacture of
blood products shall apply for renewal of licence under sub-rule (1)
before the expiry of the said licence on Form 27-C [or Form 28-E, as
the case may be], and he shall continue to operate the same till the
orders on his application are communicated to him.
2. A fee of rupees one thousand shall be paid for a duplicate copy of a
license issued under thus rule, if the original is defaced, damaged or
lost.
3. Application by a licensee to manufacture additional drugs listed
in the application shall be accompanied by a fee of rupees three
hundred for each drug listed in the application.
4. On receipt of the application for the grant or renewal of such license,
the Licensing Authority shall–
i. verify the statements made in the application form.
ii. cause the manufacturing and testing establishment to be
inspected in accordance with the provision of Rule 122-I; and
iii. in the case the application is for renewal of license, call for
informations of past performance of the licensee.
5. If the Licensing Authority is satisfied that the applicant is in a position
to fulfill the requirements laid down in the rules, he shall prepare a
report to that effect and forward it along with the application [and
the licence (in triplicate) to be granted or renewed, duly completed]
to the Central License Approving Authority:
Provided that if the Licensing Authority is of the opinion that the
applicant is not in a position to fulfill the requirements laid down in
these rules, he may, by order, for reasons to be recorded in writing,
refuse to grant or renew the license, as the case may be.
6. If, on receipt of the application and the report of the licensing
authority referred to in sub-rule (5) and after taking such measures
including inspection of the premises, by the Inspector, appointed by
the Central Government under Section 21 of the Act, and/or along
with the expert in the field concerned if deemed necessary, the
Central License Approving Authority is satisfied that the applicant
is in a position to fulfill the requirements laid down in these rules,
he may grant or renew the licence, as the case may be:
Provided that if the Central License Approving Authority is of
the opinion that the applicant is not in a position to fulfill the
requirements laid down in these rules he may, notwithstanding the
report of the Licensing Authority, by order, for reasons to be recorded
in writing, reject the application for grant or renewal of license, as
 Drugs and Cosmetics Rules, 1945 (Part X-B) 317

the case may be, and shall supply the applicant with a copy of the
inspection report.

122-G. Form of License for the Operation of a Blood Bank/Process­ing

of Whole Human Blood for Components and Manufacture of
Blood Products and the Conditions for the Grant or Renewal
of Such License
(1) A license for the operation of a Blood Bank or for processing whole
human blood for components and manufacture of blood products
shall be issued in Form 28-C [or Form 28-E or Form 26-G or Form
26-I, as the case may be]. Before a license in form 28-C [or Form
28-E or Form 26-G or Form 26-I, as the case may be] is granted or
renewed, the following conditions shall be complied with by the
applicant:
i. The operation of Blood Bank and/or processing of whole
human blood for components shall be conducted under
the active direction and personal supervision of competent
technical staff consisting of at least one person who is whole
time employee and who is Medical Officer, and possessing:
a. Postgraduate degree in Medicine–MD (Pathology/Trans­
fusion Medicines); or
b. Degree in Medicine (MBBS) with Diploma in Pathology
or Transfusion Medicines having adequate knowledge
in blood group serology, blood group methodology and
medical principles involved in the procurement of blood
and/or preparation of its components; or
c. Degree in Medicine (MBBS) having experience in Blood
Bank for one year during regular service and also has
adequate knowledge and experience in blood group
serology, blood group methodology and medical principles
involved in the procurement of blood and/or preparation
of its components, the degree or diploma being from a
University recognized by the Central Government.
Explanation: For the purposes of this condition, the experience in
Blood Bank for one year shall not apply in the case of persons who
are approved by the Licensing Authority and/or Central License
Approving Authority prior to the commencement of the Drugs and
Cosmetics (Second Amendment) Rules, 1999]
ii. The applicant shall provide adequate space, plant and
equipment for any or all the operations of blood collection or
blood processing. The space, plant and equipment required for
318 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

various operations is given in Schedule ‘F’, Part XII-B and/or

XII-C.
iii. The applicant shall provide and maintain adequate technical
staff as specified in Schedule ‘F’, Part XII-B and/or XII-C.
iv. The applicant shall provide adequate arrangements for storage
of whole human blood, human blood components and blood
products.
v. The applicant shall furnish to the Licensing Authority, if
required to do so, data on the stability of whole human blood, its
components or blood products which are likely to deteriorate,
for fixing the date of expiry which shall be printed on the labels
of such products on the basis of the data so furnished.
(2) Application for grant or renewal of license for operation of Blood
Bank or processing of human blood components shall be made by
the Blood Bank run by the Government, Indian Red Cross Society,
hospital, charitable trust or voluntary organization approved by a
State/Union Territory Blood Transfusion Council only.
Explanation: For the purpose of this subrule, “renewal” shall
include renewal of any license issued after the commencement of the
Drugs and Cosmetics (Sixth Amendment) Rules 2005.

122-H. Duration of License

An original license in Form 28-C [or Form 28-E] or a renewed license in
Form 26-G [or Form 26-I] unless sooner suspended or cancelled shall be
valid for a period of five years on and from the date on which it is granted
or renewed.

122-I. Inspection Before Grant of Renewal of License for Operation

of Blood Bank, Processing of Whole Human Blood for
Components and Manufacture of Blood Products
Before a license in [Form 28-C or Form 28-E is granted or a renewal
of license in Form 26-G or Form 26-I is made, as the case may be] the
Licensing Authority or the Central License Approving Authority, as
the case may be, shall cause the establishment in which Blood Bank
is proposed to be operated/whole human blood for components is
processed/blood products are manufactured to be inspected by one or
more Inspectors, appointed under the Act and/or along with the Expert
in the field concerned. The Inspector or Inspectors shall examine all
portions of the premises and appliances/equipments and inspect the
process of manufacture intended to be employed or being employed
along with the means to be employed or being employed for operation
 Drugs and Cosmetics Rules, 1945 (Part X-B) 319

of blood bank/processing of whole human blood for components/

manufacture of blood products together with their [testing] facilities and
also enquire into the professional qualification of the expert staff and
other technical staff to be employed.

122-J. Report by Inspector

The Inspector or Inspectors shall forward a detailed descriptive
report giving his findings on each aspect of inspection along with his
recommendation in accordance with the provisions of Rule 122-I to the
Licensing Authority or to the Central License Approving Authority.

122-K. Further Application after Rejection

If within a period of six months from the rejection of application for a
license the applicant informs the Licensing Authority that the conditions
laid down have been satisfied and deposits an inspection fee of rupees
two hundred and fifty, the Licensing Authority if after causing further
inspection to be made, is satisfied that the conditions for the grant or
renewal of a license have been complied with, shall grant or renew the
license in Form 28-C or Form 28-E:
Provided that in the case of a drug notified by the Central Government
under Rule 68-A, the application, together with the inspection report
and the form of license (in triplicate to be granted or renewed), duly
completed shall be sent, to the Central License Approving Authority,
who may approve the same and return it to the Licensing Authority for
issue of the license].

122-L. Delegation of Powers by the Central License Approving

Authority
The Central License Authority may, with the approval of the Central
Government, by notification delegate his powers of signing licenses and
any other power under rules to persons under his control having same
qualifications as prescribed for Controlling Authority under Rule 50-A,
for such areas and for such period as may be specified.

122-M. Provision for Appeal to the State Government by a Party

whose License has not been Granted or Rene­wed
Any person who is aggrieved by the order passed by the Licensing
Authority or Central License Approving Authority, as the case may be,
may within thirty days from the date of receipt of such order, appeal to
the State Government or Central Government, as the case may be and
the State Government or Central Government may after such enquiry
320 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

into the matter as it considers necessary and after giving the said person
an opportunity for representing his view in the matter may pass such
order in relation thereto as it thinks fit.

122-N. Additional Information to be Furnished by an [App­li­cant]

for License or by a Licensee to the Licensing Authority
The applicant for the grant of license or any person granted a license
under the Part shall, on demand, furnish to the Licensing Authority,
before the grant of the license or during the period the license is in force,
as the case may be, documentary evidence in respect of the ownership
or occupation, rental or other basis of the premises, specified in the
application for license or in the license granted, constitution of the firm
or any other relevant matter, which may be required for the purpose of
verifying the correctness of the statement made by the applicant or the
licensee, while applying for or after obtaining the license, as the case may
be.

122-O. Cancellation and Suspension of Licenses

1. The Licensing Authority or Central License Approving Authority
may for such licenses granted or renewed by him after giving the
licensee an opportunity to show cause why such an order should
not be passed by an order in writing stating the reason thereof,
cancel a license issued under this part or suspend it for such period
as he thinks fit, either wholly or in respect of some of the substances
to which it relates [or direct the licensee to stop collection, storage,
processing, manufacture and distribution of the said substances and
thereupon order the destruction of substances and stocks thereof
in the presence of an Inspector], if in his opinion, the licensee has
failed to comply with any of the conditions of the license or with any
provision of the Act or Rules thereunder.
2. A licensee whose license has been suspended or cancelled may,
within three months of the date of the order under sub-rule (1)
prefer an appeal against that order to the State Government or
Central Government, which shall decide the same.

122-P. Conditions of License

A license in Form 28-C, Form 28-E, Form 26-G or Form 26-I shall be
subject to the special conditions set out in Schedule F, Part XII-B and
Part XII-C, as the case may be, which relate to the substance in respect
of which the license is granted or renewed and to the following general
conditions, namely:
 Drugs and Cosmetics Rules, 1945 (Part X-B) 321

i. (a) The licensee shall provide and maintain adequate staff, plant
and premises for the proper operation of a Blood Bank for
processing whole human blood, its components and/or
manufacture of blood products.
(b) The licensee shall maintain staff, premises and equipment as
specified in Rule 122-G. The licensee shall maintain necessary
records and registers as specified in Schedule F, Parts XII-B and
XII-C.
(c) The licensee shall test in his own laboratory whole human
blood, its components and blood products and [maintain
records and] registers in respect of such tests as specified
in Schedule F, Part XII-B and XII-C. The records and register
shall be maintained for a period of five years from the date of
manufacture.
(d) The licensee shall maintain/preserve reference [sample and]
supply to the Inspector the reference sample of the whole
human blood collected by him in an adequate quantity to
conduct all the prescribed tests. The licensee shall supply to the
Inspector the reference sample for purpose of testing.
ii. The licensee shall allow an Inspector appointed under the Act
to enter, with or [without] prior notice, any premises where the
activities of the Blood Bank are being carried out for the processing
of Whole Human Blood and/or Blood Products, to inspect the
premises and plant and the process of manufacture and the means
employed for standardizing and testing the substance.
iii. The licensee shall allow an Inspector appointed under the Act to
inspect all registers and records maintained under these rules and
to take samples of the manufactured product and shall supply to
the Inspector such information as he may require for the purpose of
ascertaining whether the provisions of the Act and rules thereunder
have been observed.
iv. The licensee shall from time to time report to the Licensing Authority
any changes in the expert staff responsible for the operation of a
Blood Bank/processing of whole human blood for components and/
or manufacture of blood products and any material alterations in
the premises or plant used for that purpose which have been made
since the date of last inspection made on behalf of the Licensing
Authority after the grant of the license.
v. The licensee shall on request furnish to the Licensing Authority,
or Central License Approving Authority or to such Authority as the
Licensing Authority, or the Central License Approving Authority
may direct, from any batch unit of drugs as the Licensing Authority
322 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

or Central License Approving Authority may from time to time

specify, sample of such quantity as may be considered adequate by
such Authority for any examination and, if so required, also furnish
full protocols of the test which have been applied.
vi. If the Licensing Authority or the Central License Approving Authority
so directs, the licensee shall not sell or offer for sale any batch/unit
in respect of which a sample is, or protocols are furnished under
the last preceding sub-paragraph until a certificate authorizing the
sales of batch/unit has been issued to him by or on behalf of the
Licensing Authority or the Central License Approving Authority.
vii. The Licensee shall on being informed by the Licensing Authority
or the Controlling Authority that any part of any batch/unit of the
substance has been found by the Licensing Authority or the Central
License Approving Authority not to conform with the standards
of strength, quality or purity specified in these rules and on being
directed so to do, withdraw, from sales and so far as may in the
particular circumstances of the case be practicable recall all issues
already made from that batch/unit.
viii. No drug manufactured under the license shall be sold unless the
precautions necessary for preserving its properties have been
observed throughout the period after manufacture. Further no
batch/unit manufactured under this license shall be supplied/
distributed to any person without prescription of a Registered
Medical Practitioner.
ix. The licensee shall comply with the provisions of the Act and of
these Rules and with such further requirements, if any, as may be
specified in any Rules subsequently made under Chapter IV of the
Act, provided that where such further requirements are specified in
the Rules, these would come in force four months after publication
in the Official Gazette.
x. The licensee shall maintain an Inspector Book in Form 35 to enable
an Inspector to record his impression and defects noticed.
xi. The licensee shall destroy the stocks of batch/unit, which does not
comply with standard tests in such a way that it would not spread
any disease/infection by way of proper disinfection method.
xii. All biomedical waste shall be treated, disposed of or destroyed as
per the provisions of the Bio-Medical Wastes (Management and
Handling) Rules, 1996.
xiii. The licensee shall neither collect blood from any professional
donor or paid donor nor shall he prepare blood components and/
or manufacture blood products from the blood drawn from such a
donor.
 C H A P T E R 67
Schedule F (Part XII B)
Under the Drugs and Cosmetics
Rules, 1945

Requirements for the Functioning and Operation of a Blood

Bank and/or for Preparation of Blood Components

1. BLOOD BANKS/BLOOD COMPONENTS

A. GENERAL
1. Location and Surroundings: The blood bank shall be located at a
place which shall be away from open sewage, drain, public lavatory
or similar unhygienic surroundings.
2. Building: The building(s) used for operation of a blood bank and/
or preparation of blood components shall be constructed in such
a manner so as to permit the operation of the blood bank and
preparation of blood components under hygienic conditions and
shall avoid the entry of insects, rodents and flies. It shall be well
lighted, ventilated and screened (mesh), wherever necessary.
The walls and floors of the rooms, where collection of blood or
preparation of blood components or blood products is carried out
shall be smooth, washable and capable of being kept clean. Drains
shall be of adequate size and where connected directly to a sewer,
shall be equipped with traps to prevent back siphon age.
3. Health, Clothing and Sanitation of Staff: The employees shall be
free from contagious or infectious diseases. They shall be provided
with clean overalls, head-gears, foot-wears and gloves, wherever
required. There shall be adequate, clean and convenient hand-
washing and toilet facilities.
324 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

B. ACCOMMODATION FOR A BLOOD BANK

A blood bank shall have an area of 100 square meter for its operations
and an additional area of 50 square meters for preparation of blood
components. It shall be consisting of a room each for—
1. Registration and medical examination with adequate furniture and
facilities for registration and selection of donors;
2. Blood collection (air-conditioned);
3. Blood component preparation (this shall be air-conditioned to
maintain temperature between 20°C to 25°C);
4. Laboratory for blood group serology (air-conditioned);
5. Laboratory for blood transmissible diseases like hepatitis, syphilis,
malaria, HIV-antibodies (air-conditioned);
6. Sterilization-cum-washing;
7. Refreshment-cum-rest room (air-conditioned);
8. Store-cum-records.

Notes
1. The above requirements as to accommodation and area may be relaxed,
in respect of testing laboratories and sterilization-cum-washing room, for
reasons to be recorded in writing by the Licensing Authority and/or the
Central License Approving Authority, in respect of blood banks operating
in hospitals, provided the hospital concerned has a pathological laboratory
and a sterilization-cum-washing room common with other departments in
the said hospital.
2. Refreshments to the donor after phlebotomy shall be served so that he is
kept under observation in the Blood Bank.

C. PERSONNEL
Every blood bank shall have following categories of whole time com­
petent technical staff:
a. Medical Officer, possessing the qualifications specified in condition
(i) of Rule 122-G.
b. Blood Bank Technician(s), possessing –
i. Degree in Medical Laboratory Technology (MLT) with
six months’ experience in the testing of blood and/or its
components; or
ii. Diploma in Medical Laboratory Technology (MLT) with one
year’s experience in the testing of blood and/or its components,
the degree or diploma being from a University/Institution
recognized by the Central Government or State Government.
c. Registered Nurse(s)
 Schedule F (Part XII B) Under the Drugs and... 325

d. Technical Supervisor (where blood components are manufactured),

possessing–
i. Degree in Medical Laboratory Technology (MLT) with six
months’ experience in the preparation of blood components; or
ii. Diploma in Medical Laboratory Technology (MLT) with one
year’s experience in the preparation of blood components,
the degree or diploma being from a University/Institution
recognized by the Central Government or State Government.

Notes
1. The requirements of qualification and experience in respect of Technical
Supervisor and Blood Bank Technician shall apply in the cases of persons
who are approved by the Licensing Authority and/or Central License
Approving Authority after the commencement of the Drugs and Cosmetics
(Amendment) Rules, 1999.
2. As regards the number of whole time competent technical personnel, the
blood bank shall comply with the requirements laid down in the Directorate
General of Health Services Manual.
3. It shall be responsibility of the licensee to ensure through maintenance of
records and other latest techniques used in blood banking system that the
personnel involved in blood banking activities for collection, storage, testing
and distribution are adequately trained in the current Good Manufacturing
Practices/Standard Operating Procedures for the tasks undertaken by each
personnel. The personnel shall be made aware of the principles of Good
Manufacturing Practices/Standard Operating Procedures that affect them
and receive initial and continuing training relevant to their needs.

D. MAINTENANCE
The premises shall be maintained in a clean and proper manner to
ensure adequate cleaning and maintenance of proper operations. The
facilities shall include:
1. Privacy and thorough examination of individuals to determine their
suitability as donors.
2. Collection of blood from donors with minimal risk of contamination
or exposure to activities and equipment unrelated to blood
collection.
3. Storage of blood or blood components pending completion of tests.
4. Provision for quarantine, storage of blood and blood components in
a designated location, pending repetition of those tests that initially
give questionable serological results.
5. Provision for quarantine, storage, handling and disposal of products
and reagents not suitable for use.
326 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

6. Storage of finished products prior to distribution or issue.

7. Proper collection, processing, compatibility testing, storage and
dis­tri­bution of blood and blood components to prevent contamin­
ation.
8. Adequate and proper performance of all procedures relating to
plasmapheresis, plateletpheresis and leukapheresis.
9. Proper conduct of all packaging, labeling and other finishing oper­
ations.
10. Provision for safe and sanitary disposal of–
i. Blood and/or blood components not suitable for use, distribution
or sale.
ii. Trash and items used during the collection, processing and com­
patibility testing of blood and/or blood components.

E. EQUIPMENT
Equipment used in the collection, processing, testing, storage and sale/
distribution of blood and its components shall be maintained in a clean
and proper manner and so placed as to facilitate cleaning and main­
tenance. The equipment shall be observed, standardized and calibrated
on a regularly scheduled basis as described in the Standard Operating
Procedures Manual and shall operate in the manner for which it was
designed so as to ensure compliance with the official requirements (the
equipments) as stated below for blood and its components.
Equipment that shall be observed, standardized and calibrated
with at least the following frequencies:

Equipment Performance Frequency Frequency of

Calibration
1. Temperature Compare against Daily As often as
thermometer necessary recorder
2. Refrigerated Observe speed Each day of As often as
centrifuge and temperature use necessary
3. Hematocrit – – Standardize before
centrifuge
4. General lab. – – Tachnometer, every
centrifuge 6 months
5. Automated Observe controls Each day of –
blood typing for correct result use
Contd...
 Schedule F (Part XII B) Under the Drugs and... 327

Contd...
6. Hemoglo- Standardize Each day of –
binometer against cyname- use
themoglobulin
standard
7. Refractometer Standardize -ditto- –
or Urinometer against distilled
water
8. Blood Standardize -ditto- As often as
container against container necessary
weighing of known weight
device
9. Water bath Observe -ditto- -ditto-
temperature
10. Rh view box -ditto- -ditto- -ditto-
(Wherever
necessary)
11. Autoclave -ditto- Each time -ditto-
of use
12. Serologic Observe controls Each day of Speed as often as
rotators for correct results use necessary
13. Laboratory – – Before initial use
thermometers
14. Electronic ther- – Monthly –
mometers
15. Blood agitator Observe weight of Each day of Standardize with
the first container use container of known
of blood filled for mass or volume
correct results before
initial use, and
after repairs or
adjustments

F. SUPPLIES AND REAGENTS

All supplies and reagents used in the collection, processing, compatibility
testing, storage and distribution of blood and blood components shall
be stored at proper temperature in a safe and hygienic place, in a proper
manner and in particular:
i. All supplies coming in contact with blood and blood components
intended for transfusion shall be sterile, pyrogen-free, and shall not
interact with the product in such a manner as to have an adverse
effect upon the safety, purity, potency or effectiveness of the
product.
328 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

ii. Supplies and reagents that do not bear an expiry date shall be stored
in a manner that the oldest is used first.
iii. Supplies and reagents shall be used in a manner consistent with
instructions provided by the manufacturer.
iv. All final containers and closures for blood and blood components
not intended for transfusion shall be clean and free of surface solids
and other contaminants.
v. Each blood collecting container and its satellite container(s), if any,
shall be examined visually for damage or evidence of contamination
prior to its use and immediately after filling. Such examination
shall include inspection for breakage of seals, when indicated, and
abnormal discoloration. Where any defect is observed, the container
shall not be used or, if detected after filling, shall be properly discar­ded.
vi. Representative samples of each lot of the following reagents and/
or solutions shall be tested regularly on a scheduled basis by
methods described in the Standard Operating Procedures Manual
to determine their capacity to perform as required:

Reagents and solutions Frequency of testing along with

controls
Anti-human serum Each day of use
Blood grouping serums Each day of use
Lectin Each day of use
Antibody screening and reverse grouping Each day of use
cells
Hepatitis test reagents Each run
Syphilis serology reagents Each run
Enzymes Each day of use
HIV I and II reagents Each run
Normal saline (LISS and PBS) Each day of use
Bovine albumin Each day of use

G. GOOD MANUFACTURING PRACTICES (GMPS)/

STANDARD OPERATING PROCEDURES (SOPS)
Written Standard Operating Procedures shall be maintained and shall
include all steps to be followed in the collection, processing, compatibility
testing, storage and sale or distribution of blood and/or preparation of
blood components for homologous transfusion, autologous transfusion
and further manufacturing purposes. Such procedures shall be available
 Schedule F (Part XII B) Under the Drugs and... 329

to the personnel for use in the areas concerned. The Standard Operating
Procedures shall inter alia include:
1. a. Criteria used to determine donor suitability.
b. Methods of performing donor qualifying tests and
measurements including minimum and maximum values for a
test or procedure, when a factor in determining acceptability;
c. Solutions and methods used to prepare the site of phlebotomy
so as to give maximum assurance of a sterile container of blood;
d. Method of accurately relating the product(s) to the donor;
e. Blood collection procedure, including in-process precautions
taken to measure accurately the quantity of blood drawn from
the donor;
f. Methods of component preparation, including any time
restrictions for specific steps in processing.
g. All tests and repeat tests performed on blood and blood
components during processing;
h. Pre-transfusion testing, wherever applicable, including precau­
tions to be taken to identify accurately the recipient blood com­
ponents during processing;
i. Procedures of managing adverse reactions in donor and
recipient reactions;
j. Storage temperatures and methods of controlling storage tem­
peratures for blood and its components and reagents;
k. Length of expiry dates, if any, assigned for all final products;
l. Criteria for determining whether returned blood is suitable for
reissue;
m. Procedures used for relating a unit of blood or blood component
from the donor to its final disposal;
n. Quality control procedures for supplies and reagents employed
in blood collection, processing and re-transfusion testing;
o. Schedules and procedures for equipment maintenance and
calibr­ation;
p. Labeling procedures to safeguard its mix-ups, receipt, issues,
rejected and in-hand;
q. Procedures for plasmapheresis, plateletpheresis and leuka-
pheresis if performed, including precautions to be taken to en-
sure re-infusion of donor’s own cells.
r. Procedures for preparing recovered (salvaged) plasma if per­
for­
med, including details of separation, pooling, labeling,
storage and distribution,
s. All records pertinent to the lot or unit maintained pursuant
to these regulations shall be reviewed before the release
330 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

or distribution of a lot or unit of final product. The review

or portions of the review may be performed at appropriate
periods during or after blood collection processing, testing and
storage. A thorough investigations, including the conclusions
and follow-up, of any unexplained discrepancy or the failure of
a lot or unit to meet any of its specification shall be made and
recorded;
2. A licensee may utilize current Standard Operating Procedures,
such as the Manuals of the following organizations, so long as such
specific procedures are consistent with, and at least as stringent as,
the requirements contained in this Part, namely:
i. Directorate General of Health Services Manual.
ii. Other Organizations’ or individual blood bank’s manuals, sub­
ject to the approval of State Licensing Authority and Cen­tral
License Approving Authority.

H. CRITERIA FOR BLOOD DONATION

Conditions for donation of blood:
1. General: No person shall donate blood and no blood bank shall
draw blood from a person, more than once in three months. The
donor shall be in good health, mentally alert and physically fit and
shall not be intimate of jail, persons having multiple sex partners and
drug-addicts. The donors shall fulfill the following requirements,
namely:
a. The donor shall be in the age group of 18 to 65 years;
b. The donor shall not be less than 45 kilograms;
c. Temperature and pulse of the donor shall be normal;
d. The systolic and diastolic blood pressures are within normal
limits without medication;
e. Hemoglobin which shall not be less than 12.5 grams;
f. The donor shall be free from acute respiratory diseases;
g. The donor shall be free from any skin diseases at the site of
phlebotomy;
h. The donor shall be free from any disease transmissible by
blood transfusion, insofar as can be determined by history and
examination indicated above;
i. The arms and forearms of the donor shall be free from skin
punctures or scars indicative of professional blood donors or
addiction of self-injected narcotics.
2. Additional qualifications of a donor–No person shall donate blood,
and no blood bank shall draw blood from a donor, in the conditions
mentioned in column (1) of the Table given below (Table 67.1)
 Schedule F (Part XII B) Under the Drugs and... 331

before the expiry of the period of deferment mentioned in the

column (2) of the said Table.
3. No person shall donate blood and no blood bank shall draw blood
from a person suffering from any of the diseases mentioned below
namely:
a. Cancer
b. Heart disease
c. Abnormal bleeding tendencies
d. Unexplained weight loss
e. Diabetes controlled on insulin
f. Hepatitis infection
g. Chronic nephritis
h. Signs and symptoms suggestive of AIDS
i. Liver disease
j. Tuberculosis
k. Polycythemia vera
l. Asthma
m. Epilepsy
n. Leprosy
o. Schizophrenia
p. Endocrine disorders
Table 67.1: Deferment of blood donation
Conditions Period of deferment
(1) (2)
a. Abortions 6 months
b. History of blood transfusion 6 months
c. Surgery 12 months
d. Typhoid 12 months after recovery
e. History of malaria and duly treated 3 months (endemic) 3 years
(non-endemic area)
f. Tattoo 6 months
g. Breastfeeding 12 months after delivery
h. Immunization (Cholera, Typhoid- 15 days
Diphtheria, Tetanus, Plague, Gamma
globulin
i. Rabies vaccination 1 year after vaccination
j. History of Hepatitis in family or close 12 months
contact
k. Immunoglobulin 12 months
332 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

I. GENERAL EQUIPMENT AND INSTRUMENTS

For Blood Collection Room
i. Donor beds, chairs and tables: These shall be suitably and com­for­
tably cushioned and shall be of appropriate size.
ii. Bed side table
iii. Sphygmomanometer and Stethoscope
iv. Recovery beds for donors
v. Refrigerators, for storing separately tested and untested blood,
main­taining temperature between 2 to 6 degree centigrade with
digital dial thermometer, recording thermograph and alarm device,
with provision for continuous power supply
vi. Weighing devices for donor and blood containers.

For Hemoglobin Determination

i. Copper sulfate solution (specific gravity 1.053)
ii. Sterile lancet and impregnated alcohol swabs
iii. Capillary tube (1.3 × 1.4 × 96 mm for Pasteur pipettes)
vi. Rubber bulbs for capillary tubings
vii. Sahli’s hemoglobinometer/Colorimeteric method.

For Temperature and Pulse Determination

i. Clinical thermometers
ii. Watch (fitted with a seconds-hand) and a stop-watch.

For Blood Containers

a. Only disposable PVC blood bags shall be used (closed system) as
per the specifications of IP/USP/BP.
b. Anticoagulants: The anticoagulant solution shall be sterile, py­ro­gen-
free and of the following composition that will ensure satisfactory
safety and efficacy of the whole blood and/or for all the separated
blood components.
i. Citrate Phosphate Dextrose Adenine solution (CPDA) or cit­
rate Phosphate Dextrose Adenine-1(CPDA-1) – 14 ml. Solu­tion
shall be required for 100 ml of blood.

Note 1
i. In case of single/double/triple/quadruple blood collection bags used for
blood component preparations, CPDA blood collection bags may be used.
ii. Acid Citrate Dextrose solution (ACD with Formula-A). IP- 15 ml solution
shall be required for 100 ml of blood.
 Schedule F (Part XII B) Under the Drugs and... 333

iii. Additive solutions such as SAGM, ADSOL, NUTRICEL may be used for
storing and retaining Red Blood Corpuscles up to 42 days.

Note 2
The licensee shall ensure that the anticoagulant solutions are of a licensed
manufacturer and the blood bags in which the said solutions are contained have
a certificate of analysis of the said manufacturer.

Emergency Equipments/Items
i.Oxygen cylinder with mask, gauge and pressure regulator.
ii.5 per cent glucose or normal saline.
iii.Disposable sterile syringes and needles of various sizes.
iv.Disposable sterile IV infusion set.
v.Ampoules of adrenaline, noradrenaline, mephentin, betam­etha­
sone or dexamethasone, metoclopramide injections.
vi. Aspirin.

Accessories
i. Such as blankets, emesis basins, hemostats, set clamps, sponge for­
ceps, gauze, dressing jars, solution jars, waste cans.
ii. Medium cotton balls, 1.25 cm. adhesive tapes.
iii. Denatured spirit, tincture iodine, green soap or liquid soap.
iv. Paper napkins or towels.
v. Autoclave with temperature and pressure indicator.
vi. Incinerator.
vii. Stand-by generator.

Laboratory Equipment
i. Refrigerators for storing diagnostic kits and reagents, main­taining a
temperature between 4 to 6°C (± 2°C) with digital dial thermometer
having provision for continuous power supply.
ii. Compound microscope with low and high power objectives.
iii. Centrifuge table model.
iv. Water bath: having range between 37°C to 56°C.
v. Rh viewing box in case of slide technique.
vi. Incubator with thermostatic control.
vii. Mechanical shakers for serological tests for syphilis.
viii. Hand lens for observing tests conducted in tubes.
ix. Serological graduated pipettes of various sizes.
x. Pipettes (Pasteur).
334 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

xi. Glass slides.

xii. Test tubes of various sizes/micrometer plates (U or V type)
xiii. Precipitating tubes 6 mm × 50 mm of different sizes and glass
beakers of different sizes.
xiv. Test tube racks of different specifications.
xv. Interval timer electric or spring wound.
xvi. Equipment and materials for cleaning glass wares adequately.
xvii. Insulated containers for transporting blood, between 2°C to 10°C
temperatures, to wards and hospitals.
xviii. Wash bottles.
xix. Filter papers.
xx. Dielectric tube sealer.
xxi. Plain and EDTA vials.
xxii. Chemical balance (wherever necessary).
xxiii. ELISA reader with printer, washer and micropipettes.

J. SPECIAL REAGENTS
i. Standard blood grouping sera Anti-A, Anti-B and Anti-D with
known controls. Rh typing sera shall be in double quantity and each
of different brand or if from the same supplier each supply shall be
of different lot numbers.
ii. Reagents for serological tests for syphilis and positive sera for
controls.
iii. Anti-human globulin serum (Coomb’s serum)
iv. Bovine albumin 22 per cent enzyme reagents for incomplete anti­
bodies.
v. ELISA or Rapid or RPHA test kits for hepatitis and HIV I and II.
vi. Detergent and other agents for cleaning laboratory glass wares.

K. TESTING OF WHOLE BLOOD

i. It shall be responsibility of the licensee to ensure that the whole
blood collected, processed and supplied conforms to the standards
laid down in the Indian Pharmacopoeia and other tests published,
if any, by the Government.
ii. Freedom from HIV antibodies (AIDS) test. – Every licensee shall get
samples of every blood unit tested before use, for freedom from HIV
I and HIV II antibodies either from laboratories specified for the
purpose by the Central Government or in his own laboratory. The
results of such testing shall be recorded on the label of the container.
iii. Each blood unit shall also be tested for freedom from hepatitis B
surface antigen and hepatitis C virus antibody, VDRL and malarial
 Schedule F (Part XII B) Under the Drugs and... 335

parasite and results of such testing shall be recorded on the label of

the container.

Note
a. Blood samples of donors in plot tube and the blood samples of the recipient
shall be preserved for 7 days after issue.
b. The blood intended for transfusion shall not be frozen at any stage.
c. Blood containers shall not come directly in contact with ice at any stage.

L. RECORDS
The records which the licensee is required to maintain shall include
inter alia the following particulars, namely:
1. Blood Donor Record: It shall indicate serial number, date of
bleeding, name, address and signature of donor with other
particulars of age, weight, hemoglobin, blood grouping, blood
pressure, medical examination, bag number and patient’s detail
for whom donated in case of replacement donation, category
of donation (voluntary/replacement) and deferral records and
signature of Medical Officer Incharge.
2. Master Records for Blood and its Components: It shall indicate
bag serial number, date of collection, date of expiry, quantity in ml.
ABO/Rh Group, results for testing of HIV I and HIV II antibodies,
Malaria, VDRL, Hepatitis B surface antigen and Hepatitis C virus
antibody and irregular antibodies (if any), name and address of
the donor with particulars, utilization issue number, components
prepared or discarded and signature of Medical Officer Incharge.
3. Issue Register: It shall indicate serial number, date and time of
issue, bag serial number, ABO/Rh Group, total quantity in ml, name
and address of the recipient, group of recipient, unit/institution,
details of cross-matching report, indication for transfusion.
4. Records of Components Supplied: Quantity supplied;
compatibility report, details of recipient and signature of issuing
person.
5. Records of ACD/CPD/CPD-A/SAGM bags giving details of
manufacturer, batch number, date of supply, and results of testing.
6. Register for Diagnostic Kits and Reagents Used: Name of the kits/
reagents, details of batch number, date of expiry and date of use.
7. Blood Bank must issue the cross-matching report of the blood to the
patient together with the blood unit.
8. Transfusion adverse reaction records.
9. Records of purchase, use and stock in hand of disposable needles,
syringes, blood bags, shall be maintained.
336 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Note
The above said records shall be kept by the licensee for a period of five years.

M. LABELS
The labels on every bag containing blood and/or component shall
contain the following particulars, namely:
1. The proper name of the product in a prominent place and in bold
letters on the bag.
2. Name and address of the blood bank.
3. License number.
4. Serial number.
5. The date on which the blood is drawn and the date of expiry as
prescribed under Schedule P to these rules.
6. A colored label shall be put on every bag containing blood. The
following color scheme for the said labels shall be used for different
groups of blood:

Blood group Color of the label

O Blue
A Yellow
B Pink
AB White

7. The results of the tests for hepatitis B surface antigen and hepatitis
C virus antibody, syphilis, freedom from HIV I and HIV II antibodies
and malarial parasite.
8. The Rh group.
9. Total volume of the blood, the preparation of blood, nature and
percentage of anticoagulant.
10. Keep continuously temperature at 2 degree centigrade to 6
degree centigrade for whole human blood and/or components as
contained under III of Part XII B.
11. Disposable transfusion sets with filter shall be used in administration
equipment.
12. Appropriate compatible cross-matched blood without a typical
antibody in recipient shall be used.
13. The contents of the bag shall not be used if there is any visible
evidence of deterioration like hemolysis, clotting or discoloration.
14. The label shall indicate the appropriate donor classification like
“Voluntary Donor” or “Replacement Donor” in no less prominence
than the proper name.
 Schedule F (Part XII B) Under the Drugs and... 337

Note
1. In the case of blood components, particulars of the blood from which such
components have been prepared shall be given against item numbers (5),
(7), (8), (9) and (14).
2. The blood and/or its components shall be distributed on the prescription of
a Registered Medical Practitioner.

2. BLOOD DONATION CAMPS

A blood donation camp may be organized by–
a. A licensed designated regional blood transfusion center; or
b. A licensed Government blood bank; or
c. The Indian Red Cross Society; or
d. A licensed blood bank run by registered voluntary or charitable
organizations recognized by State or Union Territory Blood
Transfusion Council.

Note
i. “Designated Regional Blood Transfusion Center” shall be a centre approved
and designated by a Blood Transfusion Council constituted by a State
Government to collect, process and distribute blood and its components
to cater to the needs of the region and that center has also been licensed
and approved by the Licensing Authority and Central License Approving
Authority for the purpose.
ii. The designated Regional Blood Transfusion Center, Government blood
bank and Indian Red Cross Society shall intimate within a period of seven
days, the venue where blood camp was held and details of group-wise
blood units collected in the said camp to the Licensing Authority and
Central License Approving Authority.
For holding a blood donation camp, the following requirements shall be
fulfilled/complied with, namely:

(A) Premises, Personnel, etc.

a. Premises under the blood donation camp shall have sufficient area
and the location shall be hygienic so as to allow proper operation,
maintenance and cleaning.
b. All information regarding the personnel working, equipment used
and facilities available at such a camp shall be well-documented
and made available for inspection, if required, and ensuring:
i. Continuous and uninterrupted electrical supply for equipment
used in the camp;
ii. Adequate lighting for all the required activities;
iii. Handwashing facilities for staff;
338 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

iv. Reliable communication system to the central office of the con­

troller/organizer of the camp;
v. Furniture and equipment arranged within the available place;
vi. Refreshment facilities for donors and staff;
vii. Facilities for medical examination of the donors;
viii. Proper disposal of waste.

(B) Personnel for Outdoor Blood Donation Camp

To collect blood from 50 to 70 donors in about 3 hours or from 100 to
120 donors in 5 hours, the following requirements shall be fulfilled/
complied with:
i. One Medical Officer and two nurses or phlebotomists for mana­ging
6–8 donor tables;
ii. Two medico-social workers;
iii. Three blood bank technicians;
iv. Two attendants;
v. Vehicle having a capacity to seat 8–10 persons, with provision for
carriage of donation goods including facilities to conduct a blood
donation camp.

(C) Equipments
1. BP apparatus
2. Stethoscope
3. Blood bags (single, double, triple, quadruple)
4. Donor questionnaire
5. Weighing device for donors
6. Weighing device for blood bags
7. Artery forceps, scissors
8. Stripper for blood tubing
9. Bed sheets, blankets/mattress
10. Lancets, swap stick/tooth picks
11. Glass slides
12. Portable Hb meter/copper sulfate
13. Test tube (big) and 12 x 100 mm (small)
14. Test tube stand
15. Anti-A, Anti-B and Anti-AB Anti-sera and Anti-D.
16. Test tube sealer film
17. Medicated adhesive tape
18. Plastic waste basket
19. Donor cards and refreshment for donors
20. Emergency medical kit
 Schedule F (Part XII B) Under the Drugs and... 339

21. Insulated blood bag containers with provisions for storing between
2 degree centigrade to 10 degree centigrade
22. Dielectric sealer or portable tube sealer
23. Needle destroyer (wherever necessary).

3. PROCESSING OF BLOOD COMPONENTS FROM WHOLE

BLOOD BY A BLOOD BANK
The blood components shall be prepared by blood banks as a part of the
blood bank services. The conditions for grant or renewal of license to
prepare blood components shall be as follows:

(A) Accommodation
i. Rooms with adequate area and other specifications, for preparing
blood components depending on quantum of workload shall be
as specified in item B under the heading “BLOOD BANKS/BLOOD
COMPONENTS” of this Part.
ii. Preparation of Blood components shall be carried out only under
closed system using single, double, triple or quadruple plastic bags
except for preparation of Red Blood Cells, Concentrates, where
single bags may be used with transfer bags.

(B) Equipment
i. Air conditioner
ii. Laminar air flow bench
iii. Suitable refrigerated centrifuge
iv. Plasma expresser
v. Clipper and clips and or dielectric sealer
vi. Weighing device
vii. Dry rubber balancing material
viii. Artery forceps, scissors
ix. Refrigerator maintaining a temperature between 2°C to 6°C, a
digital dial thermometer with recording thermograph and alarm
device, with provision for continuous power supply
x. Platelet agitator with incubator (wherever necessary).
xi. Deep freezers maintaining a temperature between minus 30°C to
minus 40°C degree centigrade and minus 75°C to minus 80°C;
xii. Refrigerated water bath for plasma thawing
xiii. Insulated blood bag containers with provisions for storing at
appropriate temperature for transport purposes
340 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

(C) Personnel
The whole time competent technical staff meant for processing of blood
components (that is Medical Officer, Technical Supervisor, Blood Bank
Technician and Registered Nurse) shall be as specified in item C, under
the heading “I. BLOOD BANKS/BLOOD COMPONENTS” of this part.

(D) Testing Facilities

General: Facilities for A, B, AB and O groups and Rh (D) grouping.
Hepatitis B surface antigen and hepatitis C virus antibody, VDRL, HIV I
and HIV II antibodies and malarial parasites shall be mandatory for every
blood unit before it is used for the preparation of blood components.
The results of such testing shall be indicated on the label.

(E) Categories of Blood Components

1. Concentrated Human Red Blood Corpuscles: The product shall
be known as “Packed Red Blood Cells” that is Packed Red Blood
Cells remaining after separating plasma from human blood.

General requirements
a. Storage: Immediately after processing, the packed red blood Cells
shall be kept at a temperature maintained between 2º to 6ºC.
b. Inspection: The component shall be inspected immediately after
separation of the plasma, during storage and again at the time of
issue. The product shall not be issued if there is any abnormality
in color or physical appearance or any indication of microbial
contamination.
c. Suitability of Donor: The source blood for packed red blood cells
shall be obtained from a donor who meets the criteria for blood
donation as specified in item H under the heading “I. BLOOD
BANKS/BLOOD COMPONENTS” of this part.
d. Testing of whole blood: Blood from which packed red blood cells
are prepared shall be tested as specified in item K relating to Testing
of Whole Blood under the heading “I. BLOOD BANKS/BLOOD
COMPONENTS” of this part.
e. Pilot Samples: Pilot samples collected in integral tubing or in
separate pilot tubes shall meet the following specifications:
i. One or more pilot samples of either the original blood or of the
packed red blood cells being processed shall be preserved with
each unit of packed red blood cells which is issued.
 Schedule F (Part XII B) Under the Drugs and... 341

ii. Before they are filled, all pilot sample tubes shall be marked
or identified so as to relate them to the donor of that unit or
packed red blood cells.
iii. Before the final container is filled or at the time the final
product is prepared, the pilot sample tubes accompanying a
unit of packed red blood cells shall be attached in a tamper-
proof manner that shall conspicuously identify removal and re-
attachment.
iv. All pilot sample tubes, accompanying a unit of packed red blood
cells, shall be filled immediately after the blood is collected or
at the time the final product is prepared, in each case, by the
person who performs the collection of preparation.

Processing
i. Separation: Packed red blood cells shall be separated from the
whole blood:
a. If the whole blood is stored in ACD solution within 21 days, and
b. If the whole blood is stored in CPDA-I solution, within 35 days,
from the date of collection. Packed red blood cells may be
prepared either by centrifugation done in a manner that shall
not tend to increase the temperature of the blood or by normal
undisturbed sedimentation method. A portion of the plasma,
sufficient to ensure optimal cell preservation, shall be left with
the packed red blood cells.
ii. Packed Red Blood Cells Frozen: Cytophylactic substance may be
added to the packed red blood cells for extended manufacturer’s
storage not warmer than minus 65 degree centigrade provided
the manufacturer submits data to the satisfaction of the Licensing
Authority and Central License Approving Authority, as adequately
demonstrating through in vivo cells survival and other appropriate
tests that the addition of the substance, the material used and the
processing methods result in a final product meets the required
standards of safety, purity and potency for packed red blood cells,
and that the frozen product shall maintain those properties for the
specified expiry period.
iii. Testing: Packed red blood cells shall conform to the standards as
laid down in the Indian pharmacopoeia.
2. Platelets Concentrates: The product shall be known as “Platelets
Concentrates” that is platelets collected from one unit of blood and
re-suspended in an appropriate volume of original plasma.
342 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

General requirements
i. Source: The source material for platelets shall be platelet-rich
plasma or buffy coat which may be obtained from the whole blood
or by plateletpheresis.
ii. Processing:
a. Separation of buffy-coat or platelet-rich plasma and platelets
and re-suspension of the platelets shall be in a closed system
by centrifugal method with appropriate speed, force and time.
b. Immediately after collection, the whole blood or plasma shall be
held in storage between 20 to 24ºC. When it is to be transported
from the venue of blood collection to the processing laboratory,
during such transport action, the temperature as close as
possible to a range between 20 to 24ºC shall be ensured. The
platelet concentrates shall be separated within 6 hours after the
time of collection of the unit of whole blood or plasma.
c. The time and speed of centrifugation shall be demonstrated
to produce an unclamped product, without visible hemolysis,
that yields a count of not less than 3.5 × 1010 (3.5 × 10 raised to
the power of 10) and 4.5 × 1010 (4.5 × 10 raised to the power ten),
i.e. platelets per unit from a unit of 350 ml. and 450 ml. blood
respectively. One per cent of total platelets prepared shall be
tested, of which, 75 percent of the units shall conform to the
above said platelets count.
d. The volume of original plasma used for re-suspension of the
platelets shall be determined by the maintenance of the pH
of not less than 6 during the storage period. The pH shall be
measured on a sample of platelets which has been stored for
the permissible maximum expiry period at 20 to 24ºC.
e. Final containers used for platelets shall be colorless and
transparent to permit visual inspection of the contents.
The caps selected shall maintain a hermetic seal to prevent
contamination of the contents. The container material shall not
interact with the contents, under the normal conditions of the
storage and use, in such a manner as to have an adverse effect
upon the safety, purity, potency, or efficacy of the product.
At the time of filling, the final containers shall be marked or
identified by number so as to relate it to the donor.
iii. Storage: Immediately after re-suspension, platelets shall be placed
in storage not exceeding a period of 5 days, between 20°C to 24°C,
 Schedule F (Part XII B) Under the Drugs and... 343

with continuous gentle agitation of the platelet concentrates

maintained throughout such storage.
iv. Testing: The units prepared from different donors shall be tested at
the end of the storage period for—
a. Platelet count;
b. pH of not less than 6 measured at the storage temperature of
the unit;
c. measurement of actual plasma volume;
d. one per cent of the total platelets shall be tested for sterility;
e. the tests for functional viability of the platelets shall be done by
swirling movement before issue;
f. if the results of the testing indicate that the product does not
meet the specified requirements, immediate corrective action
shall be taken and records maintained.
v. Compatibility Test: Compatible transfusion for the purpose of
variable number of red blood cells, A, B, AB and O grouping shall
be done if the platelets concentrate is contaminated with red blood
cells.
3. Granulocyte Concentrates
i. Storage: It shall be kept between 20°C to 24°C for a maximum
period of 24 hours.
ii. Unit of granulocytes shall not be less than 1 × 1010 (i.e. 1 × 10
raised to the power of 10) when prepared on cell separator.
iii. Group specific tests/HLA test wherever required shall be
carried out.
4. Fresh Frozen Plasma: Plasma frozen within 6 hours after blood
collection and stored at a temperature not warmer than minus
30°C, shall be preserved for a period of not more than one year.
5. Cryoprecipitate: Concentrate of anti-hemophiliac factor shall be
prepared by thawing of the fresh plasma frozen stored at minus
30ºC.
a. Storage: Cryoprecipitate shall be preserved at a temperature
not higher than minus 30ºC and may be preserved for a period
of not more than one year from the date of collection.
b. Activity: Anti-hemophiliac factor activity in the final product
shall be not less than 80 units per bag. One per cent of the total
cryo­precipitate prepared shall be tested of which seventy five
percent of the unit shall conform to the said specification.

(F) Plasmapheresis, Platepheresis and Leukapheresis Using a Cell

Separator
An area of 10 square meters shall be provided for apheresis in the blood
bank. The blood banks specifically permitted to undertake the said
344 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

apheresis on the donor shall observe the criteria as specified in item

H relating to CRITERIA FOR BLOOD DONATION under the heading
“I. BLOOD BANKS/BLOOD COMPONENTS” of this part. The written
consent of the donor shall be taken and the donor must be explained
the hazards of apheresis. The Medical Officer shall certify that donor is
fit for apheresis and it shall be carried out by a trained person under
supervision of the Medical Officer.

(A) Plasmapheresis, plateletpheresis and leukapheresis

The donors subjected to plasmapheresis, plateletpheresis and leuka-
pheresis shall, in addition to the criteria specified in item H relating to
the CRITERIA FOR BLOOD DONATION, under the heading “I. BLOOD
BANKS/BLOOD COMPONENTS” of this part being observed, be also
subjected to protein estimation on post-pheresis/first sitting whose
results shall be taken as a reference for subsequent pheresis/sitting. It
shall also be necessary that the total plasma obtained from such donor
and periodicity of plasmapheresis shall be according to the standards
described under validated standard operating procedures.

Note
i. At least 48 hours must elapse between successive apheresis and not more
than twice in a week.
ii. Extra-coporeal blood volume shall not exceed 15% of donor’s estimated
blood volume.
iii. Platelet pheresis shall not be carried out on donors who have taken
medication containing aspirin within 3 days prior to donation.
iv. If during plateletpheresis or leukapheresis, RBCs cannot be re-transfused
then at least 12 weeks shall elapse before a second cytapheresis procedure
is conducted.

(B) Monitoring for apheresis

Before starting apheresis procedures, hemoglobin or hematocrit shall
be done. Platelet count, WBC counts, differential count may be carried
out. In repeated plasmapheresis, the serum protein shall be 6 gm/100
ml.

(C) Collection of plasma

The quantity of plasma separated from the blood of a donor shall not
exceed 500 ml. per sitting and once in a fortnight or shall not exceed
1000 ml. per month.
 C H A P T E R 68
Storage Conditions, Expiry of
Blood, Blood Components and
Blood Products as Per Schedule P
of the Drugs and Cosmetics Act,
1940, and Rules, 1945

S. No. Name of drug Expiry Storage conditions

(Months)
1. Anti-hemophilic human 12 In a cool place
globulin
2. Dried plasma 60 At temperature not
exceeding 25°C
3. Dried normal human serum 60 At temperature not
albumin exceeding 25°C
4. Frozen plasma 60 In deep freeze
5. Liquid plasma 24 In cold place
6. Liquid normal human serum 60 In cold place
albumin
7. Whole human blood –
a. Collected in ACD solution 21 days At temperature between
4°C and 6°C
b. Collection in CPDA solution 35 days At temperature between
4°C and 6°C

Note
1. The term “cool place” means ‘place having a temperature between 10°C
and 25°C.
2. The term “cold place” means ‘place having a temperature not excee­ding
8°C.
346 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Storage conditions, expiry of blood, blood components and blood

products as per WHO guidelines

S. No. Name of drug Expiry (Months) Storage conditions

1. Concentrated RBC 35 days 2° to 6°C
2. Platelet concentrate Max. 5 days 20°–24°C in agitator
3. FFP 7 years –65°C or below
4. FFP/Cryoprecipitate 24 months –40° to –64°C
5. -do- 12 months –30° to –39°C
6. -do- 6 months –25° to –29°C
7. -do- 3 months –2° to –24°C
 C H A P T E R 69
Extract of Schedule K Under the
Drugs and
Cosmetics Rules, 1945
Class of drugs Extent and conditions of exemption
“5B Whole Human The provisions of Chapter IV of the Act and the
Blood IP and/or its Rules made thereunder which require obtaining of a
components stored for licence for operation of a blood bank or processing
transfusion by a First Whole Human Blood and/or its components, subject
Referral Unit. Comm­ to the following conditions, namely:
unity Health Cen­ter,
Primary Health Cen­ter
and a Hospital.
1. The First Referral Unit, Community Health Centre,
Primary Health Center and/or any Hospital
shall be approved by the State/Union Territory
Licensing Authority after satisfying the conditions
and facilities through inspection.
2. The captive consumption of Whole Human Blood
IP or its components in the First Referral Unit,
Community Health Center, Primary Health Center
and/or any Hospital shall not be more than 2000
units annually.
3. The Whole Human Blood and/or its components
shall be procured only from Government Blood
Bank and/or Indian Red Cross Society Blood Bank
and/or Regional Blood Transfusion Center duly
licensed.
4. The approval shall be valid for a period of two
years from the date of issue unless sooner
suspended or cancelled and First Referral Unit,
Community Health Center, Primary Health Center
or the Hospital shall apply for renewal to the State
Licensing Authority three months prior to the date
of expiry of the approval.
5. The First Referral Unit, Community Health Center,
Primary Health Center and/or any Hospital shall
have the following technical staff for storage of
blood or its components.
348 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

a. A trained Medical Officer for proper pro­

curement, storage and cross-matching
of blood and/or its components. He/She
shall also be responsible for identifying
hemolyzed blood and ensure non-supply of
expired blood or its components.
b. A blood bank technician with the qualifi­
cation and experience as specified in Part
XII B of Schedule F or an experience labor­
atory technician trained in blood grouping
and cross-matching.
6. The First Referral Unit, Community Health Center
Primary Health Center and Hospital shall have
an area of 10 sq. metres. It shall be well lighted,
clean and preferably air-conditioned. Blood bank
refrigerator or appropriate capacity fitted with
alarm device and temperature indicator with
regular temperature monitoring shall be provided
to store blood units between 2°C to 8°C and
if the components are proposed to be stored,
specialized equipments as specified in Part XII B
of Schedule F shall also be provided.
7. The First Referral Unit, Community Health Center,
Primary Health Center and Hospital shall main­
tain records and registers including details of
procurements of Whole Human Blood IP and/or
blood components, as required under Part XII B of
Schedule F.
8. The First Referral Unit, Community Health Center,
Primary Health Center and Hospital shall store
samples of donor’s blood as well as patient’s sera
for a period of seven days after transfusion.”
30. Whole Human Blood All the provisions of Chapter IV of the Act and rules
collected and transfused made thereunder which require them to be covered
by Centers run by by a licence to operate a Blood Bank for collection,
Armed Forces Medi­ storage and processing of whole human blood for
cal Services in border sale or distribution subject to the following conditions:
areas, small mid-zonal
i. These Centers shall collect, process and transfuse
hospitals including
blood in emergent situations, which require life-
peripheral hospitals,
sav­ing emergency surgeries/or transfusion.
field ambulances, mobile
medical units and other ii. These Centers shall be under the active direction
field medical units and personal supervision of a qualified Medical
including blood supply Officer, processing the qualifications and experi­
units in border, sensitive ences specified in condition (i) of Rule 122-G.
and field areas.
 Extract of Schedule K Under the Drugs ... 349

iii. Each blood unit shall be tested before use for

freedom from HIV I and II antibodies, hepatitis
B surface antigen, malarial parasites and other
tests specified under the monograph “Whole
Human Blood” in current edition of Indian Phar­
ma­copoeia.
iv. These Centers shall have adequate infrastruc­
ture facilities for storage and transportation of
blood.
v. The blood collected and tested by such Centers
shall be transfused by the Center itself and may
be made available for use of other peripheral
Armed Forces hospitals or Centers during oper­
ational circumstances.
 C H A P T E R 70
Blood Storage Centers

With an objective of providing safe and quality blood supply to all in

need wherever and whenever required, it was felt necessary to establish
blood storage centers which can receive tested and processed blood
and blood components from regional centers for use by patients in the
hospitals in the area where storage center is located. The circumstances
leading up to establishing such centers were:
1. Many doctors working in the first referral units and other hospitals
in the rural areas specially those working in the vicinity of the
highways constantly complained of unavailability of blood.
2. In large cities and towns, the number of blood banks has been
increasing, as all hospitals small and big required establishing their
own center.
3. For supplying blood to many private nursing homes in the
areas, private blood banks working on small scales have started
mushrooming.
4. For proper regulation of the system it is necessary to reduce the
number of blood banks.
With these factors in mind the blood storage centers have taken
birth.

REQUIREMENTS
• The storage center can be established at any first referral unit,
community health center, primary health center and/or any
hospital. It may be in rural or urban area.
• Any blood bank presently collecting up to 2000 units of blood can
be converted into a storage center provided it can get affiliated to a
blood bank for its regular supply of blood.
• The storage center can get affiliated to any government or regional
blood bank; which is approved by SBTC and licenced for the
purpose. Private or commercial blood banks should not be given
permission to supply blood to storage centers by the SBTC.
 Blood Storage Centers 351

• The area required is only 10 sq. mts. well-lighted, clean and pre­
ferably air-conditioned and should have equipments for storage as
prescribed by Drugs and Cosmetics Rules 1945.
• The storage center should have following equipment:
■ Blood bank refrigerator.
■ Insulated boxes for transport.
■ Microscope.
■ Centrifuge.
■ Incubator.
■ Pipettes.
■ Glassware.
• The storage center should have adequate provision for blood group­
ing reagents.
• The center will have to maintain records of procurement, cross-
matching and issue of blood and blood components and archive
these for at least 5 years.
• The license issued to the storage center will require renewal every 2
years. In case if the license of the affiliated parent center is cancelled,
the license of the storage center will be automatically cancelled.
• The storage center can procure blood or components from more
than one blood bank to ensure availability but an approval will be
required for each case from SBTC and Drug Controller.
• It is necessary to adhere to biosafety guidelines.

STAFF
• The staff, i.e Medical Officer and technician is not required to be full
time employees for the storage center. They may have other duties
in the hospital.
• The staff should preferably undergo an orientation training of
approxi­mately 1–2 weeks at the regional center to which the storage
center is affiliated.
• The storage center should work round the clock.

STORAGE
• It is necessary to maintain the cold chain at all times during trans­
port, storage and issue. Proper insulated carry boxes should be used
during transportation of blood. The ice in use should be clean and
should not come in direct contact with blood bags.
• Whole blood and packed cells are kept in blood bank refrigerator
at 4–6ºC ± 2°C up to 35 days. Red cells in additive solutions can be
stored up to 42 days.
352 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• FFP (Fresh Frozen Plasma)/cryoprecipitates can be stored up to

one year at –30ºC or lower.
• Platelet concentrates can be stored at 20–22ºC for 3–5 days
depending on the bags in which these are prepared.
• Due care should be taken to maintain sterility of blood and blood
components by keeping all storage areas clean.
• The temperature of all storage areas should be monitored
continuously preferably using a graphic recorder. The alarm system
should indicate any deflection in temperature and this should be
checked every month.
• All storage equipment should be connected to a generator to ensure
continuous electrical supply.
• The storage centers should check the condition of the blood on
receipt from the regional center and also during the period of
storage, as the responsibility of problems arising from storage,
cross-matching, issue and transfusion will be of the storage center.
Any unit showing hemolysis, turbidity or change in color should not
be taken on stock for transfusion.

ISSUE OF BLOOD/COMPONENTS
• Blood may be issued to any hospital in the area against the
prescription of a registered medical practitioner.
• Patient’s blood grouping and cross-matching should be carried out
before issue.
• First in first out (FIFO) policy whereby older blood, closer to expiry
date is used first, should be followed to ensure use of all available
blood and prevent outdating. Any blood that remains unused
should be sent back to Regional Center after its expiry date, which
originally supplied the unit.
• Medical Officer should also review the requirement of blood before
it is issued.

BLOOD GROUPING
• ABO/Rh grouping should be done by tube technique.
• Cell and serum grouping should be done and cross-checked.
• Blood grouping reagents in use should be approved by regional
center and should undergo QC test on receipt and daily.
 Blood Storage Centers 353

CELL GROUPING
• Add 1 drop of anti-A, anti-B, anti-AB and anti-D in 4 different tubes.
• Add 1 drop of 2–5% cell suspension of patient’s blood in each tube.
• Mix the contents and incubate at room temperature for 15 minutes.
• Centrifuge at 1000 rpm for 1 minute.
• Look for agglutination, record all the negative results and should be
confirmed under microscope.

SERUM GROUPING
• Add 2 drops of patient’s serum in each of the 4 different tubes.
• Add 1 drop of 2% pooled A cells in first tube.
• Add 1 drop of 2% pooled B cells in second tube.
• Add 1 drop of 2% pooled O cells in third tube.
• Add 1 drop of 22% albumin and O cells in fourth tube.
• Incubate first 3 tubes at room temperature for 15 minutes.
• Incubate fourth tube at 37ºC for 15 minutes.
• Centrifuge at 1000 rpm for 1 minutes.
• Look for agglutination and record.
• Proceed by washing cells in the 4th tube 3 times with saline and add
2 of AHG.
• Incubate for 15 minutes at room temperature.
• Centrifuge, read and record.
• While carrying out grouping always record results before documen­
ting interpretations.
• If there is discrepancy between cell and serum grouping, repeat the
test.

CROSS-MATCHING
For cross-matching, routinely use saline and albumin or enzyme
method; when patient requires regular or massive transfusion, use IAT
method.

Saline Method
• Add 2 drops patient’s serum in the test tube
• Add 1 drop 5% donor’s cell in the same tube.
• Mix, incubate at room temperature for 15 minutes.
• Centrifuge, read and record.
354 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Albumin/Enzyme Method
• Add 2 drops patient’s serum.
• Add 2 drops 22% bovine albumin or 1 drop papain crysteine.
• Add 1 drop 5% donor’s red cells.
• Incubate at 37°C for 15 minutes.
• Centrifuge, read and record.

IAT Method
• Add 2 drops patient’s serum.
• Add 1 drop 5% donor’s red cells.
• Add 2 drops 22% bovine albumin.
• Incubate at 37°C for 15 minutes.
• Wash cells with isotonic saline three times.
• Decant after last wash.
• Add 2 drops AHG.
• Mix well, centrifuge.
• Read and record.

POINTS TO REMEMBER
• All tests performed should be recorded and signed by technician.
• Medical Officer should supervise the technician’s work of grouping,
cross-matching and storage.
• Sample of patient and donor should be stored for 7 days after issue.
When transfusion is required 48 hours after one transfusion, fresh
sample should be asked for cross-matching.
• Standard operating procedures for storage, transport, issue, equip­
ment maintenance, grouping and cross-matching should be written
and made available for use.
• The storage center should maintain adequate stocks of colloids and
crystalloids for initial volume replacement in emergency.
• Medical Officer will be responsible for the overall working of the
storage center and hence, he/she should ensure that the work is
carried out systematically to avoid any errors leading to adverse
transfusion reactions.
 Blood Storage Centers 355

GUIDELINES BEFORE GRANT OF APPROVAL FOR OPERA-

TION OF WHOLE HUMAN BLOOD AND/OR ITS COMPO-
NENTS STORAGE CENTERS RUN BY FIRST REFERRAL UNIT,
COMM­UNITY HEALTH CENTER, PRIMARY HEALTH CENTER
OR ANY HOSPITAL
The following guidelines may be followed before exempting the said
institutions for obtaining of a license for operation of a blood bank or
processing whole human blood/or its components:
1. The applicant shall be first referral unit, community health center,
primary health center or any hospital.
2. The applicant shall furnish an undertaking to the licensing authority
that the captive consumption of whole human blood or components
shall not be more than 2000 units annually.
3. The applicant shall enclose list of equipment needed for storage
viz. blood bank refrigerator with alarm system and temperature
indicator. A separate list of blood components would be enclosed if
proposed to be stored.
4. The applicant shall furnish the following:
a. Name of the medical officer responsible for conducting
operation of blood storage center
b. Attested certified copies of MBBS or MD qualification
c. Name, certified copies of qualification and experience of the
blood bank technician
d. Name, attested certified copies of qualification and experience
of the blood bank technician having non-DMLT qualification
5. The applicant shall furnish the source of procurement of whole
human blood/blood components namely the name and address of
the blood banks.
a. The source of procurement of blood/components shall be from
licensed blood banks run by Govt. hospitals/Indian Red Cross
Society/regional blood transfusion centers only.
b. A letter of consent from the above blood banks who intend to
supply whole human blood/blood components to the blood
storage centers shall be furnished along with the application.
6. The applicant shall submit the plan of the premises. A minimum
area of 10 sq. meters is essential for the blood storage center.
7. In order to satisfy the conditions and facilities, an inspection of the
proposed blood storage center may be carried out by the respective
State Drug Control Department.
356 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

8. The inspection team shall also inspect the blood banks who have
given consent letters for supply of whole human blood/components.
The inspection team may verify whether the blood banks have
sufficient quantity of blood units to be supplied to the blood storage
centers and also verify the mode of shipper or containers used for
supply of blood units/components to ensure that the proper storage
condition is maintained as per the pharmacopeia. The blood bank
shall label the blood units/components as per the Drugs and
Cosmetics Rules, 1945.
9. The blood banks who intend to supply the blood units/components
shall test the following mandatory tests before supplying to blood
storage centers.
a. Blood grouping
b. Antibody testing
c. Hemoglobin contents
d. HIV I and II antibodies
e. Hepatitis B surface antigen
f. Hepatitis C antibody
g. Malarial parasite
h. Syphilis or VDRL
The label of the tested blood unit shall contain the above particulars
with date of testing before supplying to blood storage centers.
The blood bank shall maintain a separate register for supply of blood
units/components to blood storage centers with all necessary details.
10. The validity of approval shall be for a period of 2 years from the date
of issue of the approval.
11. The state licensing authority shall forward the approved blood
storage centers to the concerned zonal officer immediately.
12. A format of the approval performa is enclosed.
 Blood Storage Centers 357

SPECIMEN OF CERTIFICATE OF APPROVAL TO BLOOD STORAGE

CENTER FOR STORAGE OF WHOLE HUMAN BLOOD AND/OR ITS
COMPONENTS

No. _____________ Date of issue _____________

M/s __________________________ is hereby approved to store the
following items on the premises situated at _______________________
under the supervision of the following technical staff:
1. Names of the approved Medical Officer:
2. Names of the items:
3. Names of the qualified blood bank technician:
4. Name and address of the licensed blood.
bank from whom the blood units would be procured:
5. The approval shall be in force from __________________ to
_______________

Dated: Signature
Designation
Licensing Authority

CONDITIONS
The blood storage center shall comply with the conditions as stipulated
under item 5B of Schedule K of the Drugs and Cosmetics Rules which
also includes as under:
1. The captive conception of whole human blood or its components in
the above said center shall not be more than 2000 units annually.
2. In the event of any change in the technical staff it shall be forthwith
reported to the licensing authority.
3. In the event of any change in the name of the licensed blood
bank from whom the blood units are procured, the same shall be
intimated to the licensing authority for approval.
4. The center shall apply for renewal of the approval to the licensing
authority three months prior to the date of expiry of the approval.
5. The center shall maintain records and registers including the details
of procurement of blood/its components.
6. The center shall store samples of donor’s blood as well as patient’s
sera for a period of 7 days after transfusion.
 C H A P T E R 71
Recent Amendments in the Drugs
and Cosmetics Rules, 1945, and
Guidelines
I. AMENDMENTS

DRUGS AND COSMETICS (2nd AMENDMENT) RULES, 2011

MINISTRY OF HEALTH AND FAMILY WELFARE
(Department of Health)
NOTIFICATION
New Delhi, the 18th February, 2011
*G.S.R. 101(E).— Whereas a draft of certain rules further to amend
the Drugs and Cosmetics Rules, 1945, was published, as required by
Sections 12 and 33 of the Drugs and Cosmetics Act, 1940 (23 of 1940),
vide notification of the Government of India, Ministry of Health and
Family Welfare (Department of Health), number G.S.R. 303(E), dated 9th
April, 2010, in the Gazette of India, Extraordinary, Part II, Section 3, Sub-
section (i), dated the 9th April, 2010, inviting objections and suggestions
from all persons likely to be affected thereby before the expiry of a period
of forty five days from the date on which the copies of the Official Gazette
in which this notification is published are made available to the public;
And whereas copies of the Gazette were made available to the
public on the 13th April, 2010;
And whereas, no objections and suggestions were received from the
public on the said draft rules;
Now, therefore, in exercise of the powers conferred by Sections 12
and 33 of the Drugs and Cosmetics Act, 1940 (23 of 1940), the Central
Government, after consultation with the Drugs Technical Advisory
Board, hereby makes the following rules further to amend the Drugs and
Cosmetics Rules, 1945, namely:
1. i. These rules may be called the Drugs and Cosmetics (2nd
Amendment) Rules, 2011.
ii. They shall come into force on the date of their publication in the
Official Gazette.
 Recent Amendments in the Drugs and Cosmetics Rules, 1945... 359

2. In the Drugs and Cosmetics Rules, 1945,

(i), (ii), (iii), (v)
x x x x
iv. In Schedule F, under heading “I. Blood Banks/Blood
Components,” under sub-heading “H. Criteria for Blood
Donation,” in condition (1), for item (a), and entry relating
thereto, the following shall be substituted, namely:
a. The donor shall be in the age group of 18 to 65 years
[F. No. X-11014/6/2009-DFQC]
ARUN PANDA, Jt. Secy.
Foot Note: The principal rules were published in the Gazette of
India vide notification No. F.28-10/45–1–1(1), dated 21st December,
1945 and last amended vide notification number G.S.R. 45(E), dated
24–1–2011. The Drugs and Cosmetics Rules, 1945, as amended up to
1–5–1979 is contained in the publication of the Ministry of Health and
Family Welfare (Department of Health) containing the—Published in
the Gazette of India (extraordinary) Part-II, section 3, sub-section (i), vide
G.S.R. 101(E) dated 18th February, 2011.

DRUGS AND COSMETICS (3rd AMENDMENT) RULES, 2011

MINISTRY OF HEALTH AND FAMILY WELFARE
(Department of Health)
NOTIFICATION
NEW DELHI, 27thDecember 2011
G.S.R 899 (E): Whereas a draft of certain rules further to amend the
Drugs and Cosmetics Rules, 1945, was published, as required by
Sections 12 and 33 of the Drugs and Cosmetics Act. 1940 (23 of 1940),
vide notification of Government of India, Ministry of Health and Family
Welfare (Department of Health), number GSR 304 (E), dated the 9th
April, 2010, in the Gazette of India, Extraordinary, Part II, section 3, Sub-
section (i) dated the 9th April, 2010, inviting objections and suggestions
from all persons likely to be affected thereby before the expiry of a period
of forty five days from the date on which the copies of the Official Gazette
in which this notification is published are made available to public.
And whereas copies of the Gazette were made available to public on
the 13th May, 2010.
And whereas the objections and suggestions received from
the public on the said rules have been considered by the Central
Government.
Now, therefore, in exercise of the powers conferred by Sections 12
and 33 of the Drugs and Cosmetics Act 1940 (23 of 1940), the Central
360 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Government, after consultation with the Drugs Technical; Advisory

Board, hereby makes the following rules further to amend the Drugs and
Cosmetics rules, 1945, namely:
1. 1. These rules may be called the Drugs and Cosmetics (3rd
Amendment) Rules, 2011.
2. They shall come into force on the date of their publication in the
Official Gazette.
2. In the Drugs and Cosmetics rules, 1945, (herein after referred to as
said rules):
1. Under the heading to Part X B, for the words, “And Manufacture
of Blood Products”, the words, “Manufacture of Blood Products
and Collection, Processing, Testing, Storage, Banking and
Release of Umbilical Cord Stem Cells”, shall be substituted.
2. In rule 122 EA of the said rule, in sub-rule 1:
i. For the words, figures and letter “and Part 12 C”, the words,
figures and letter “Part XII C and PART XII D”, shall be
substituted;
ii. After clause (f), the following shall be substituted, namely:
“(fa) ‘cord blood bank’ means a place or organization
or unit for carrying out and responsible for operations of
collection, processing, testing, banking, selection and
release of blood cord units.”
iii. After clause (l), the following shall be substituted, namely:
“(m) ‘umbilical cord blood’ is the whole blood including
Hematopoietic progenitor cells collected from placental
and or umbilical cord blood vessels after the umbilical cord
have been clamped”.
3. In rule 122 F:
i. In the heading after the words, “ for sale or distribution” the
words “collection, processing, testing, storage, banking,
selection and release of umbilical cord blood stem cells”, shall
be inserted;
ii. In the subrule 1:
a. After the words, “manufacture of blood products”, the
words “collection, processing, testing, storage, banking,
selection and release of umbilical cord blood stem cells”,
shall be inserted;
b. For the words figures and letter, “or Form 27E”, the words
figures and letters, “Form 27E or Form 27F”, shall be
substituted;
c. For the second proviso, the following proviso shall be
substituted, namely:
 Recent Amendments in the Drugs and Cosmetics Rules, 1945... 361

“provided further that a licensee holding a licence in

Form-28C, Form-28E or Form-28F as the case may be,
for operation of blood bank/processing of whole human
blood for components/manufacture of blood products/
collection, processing, testing, storage, banking and release
of umbilical cord blood stem cells shall apply for grant of
licence under sub-rule (1) before the expiry of the said
licence on Form-27C, Form 27E or Form 27F as the case
may be, and he shall continue to operate the same till the
orders on his application are communicated to him.”
4. In rule 122 G:
i. In the heading and for words, “manufacture of blood products”
the words “manufacture of blood products/collection,
processing, testing, storage, banking, and release of umbilical
cord blood stem cells “shall be inserted;
ii. For the words, figures and letters, “Form 26-G or Form 26-l,
as the case may be, before a licence in “Form 28-C, or Form
28-E or Form 26-G or Form 26- l” the words “Form-28F or Form
26-G or Form 26-l or Form 26-J, as the case may be, before a
licence in Form 28-C or Form 28-E or Form 28-F or Form 26-G
or Form 26-l or Form 26-J’, shall be substituted.
iii. In clause (ii), after the figures and letter, “XII C”, the words,
figures and letter, “or Part XII D”, shall be inserted;
iv. In clause (iii), after the figures and letter, “XII C”, the words,
figures and letter, “or Part XII D”, shall be inserted.
5. In rule 122-H:
i. After the figures and letter, “Form 28-E”, the words, figures and
letter, “or Form 28-F”, shall be inserted;
ii. After the figures and letter, “Form 28-I”, the words, figures and
letter, “or Form 28-J”, shall be inserted;
6. In rule 122-I:
i. After the figures and letter, “Form 28-E”, the words, figures and
letter, “or Form 28-F”, shall be inserted;
ii. After the figures and letter, “Form 28-I”, the words, figures and
letter,“or Form 28-J”, shall be inserted.
7. In rule 122-K, after the figures and letter, “Form 28-E”, the words,
figures and letter, “or Form 28-F”, shall be inserted.
8. In rule 122-P:
i. For the words, figures and letters, “Form 26-G or Form 26-l,”
shall be subject to the special conditions set out in “Schedule F,
XII B and Part XII C” the words, figures and letters “Form 28-F,
Form 26-G, Form 26-l or Form 26-J shall be subject to special
362 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

conditions set out in Schedule F, Part XII B and Part XII C, Part
XII D”, shall be substituted.
ii. In clause (i), in sub-clause (c) after the figures and letters,
“XII C”, the words, figures and letter “or Part XII D”, shall be
inserted.
9. In Schedule A
i. After the Form 26-I, the following Form shall be inserted,
namely:

“Form 26-J”
[See rules 122-G, 122-H, 122-I, 122P]
Certificate of renewal of licence for collection, processing, testing,
storage, banking and release of umbilical cord blood stem cells
Certified that licence number _______________________ granted on
____________________to M/s____________________________________for
collection, processing, testing, storage, banking and release of umbilical
cord blood stem cells at the premises situated at___________________
is hereby renewed with effect from ___________________________ to
________________________
1. Name(s) of competent Technical Staff:
1.
2.
3.
Signature___________________
Designation_________________
Licensing authority___________
Date: _____________________________
Central Approving Authority
ii. After the Form 27-E, the following Form shall be inserted,
namely:

“Form 27-F”
[See rule 122-F]
Application for grant/renewal* of licence for collection, processing,
testing, storage, banking and release of umbilical cord blood stem
cells
I/We________________________ of M/S____________________________
Hereby apply for the grant/renewal* of license number _______________
dated ______________________ for collection, processing, testing,
storage, banking and release of umbilical cord blood stem cells on the
premises situated at______________________________________________
 Recent Amendments in the Drugs and Cosmetics Rules, 1945... 363

2. Name(s), qualification and experience of competent Technical Staff

are as under:
1. Medical Director
2. Laboratory In-charge
3. Technical Supervisor
4. Cord Blood Bank Technician(s)
3. The premise and plant are ready for inspection/will be ready for
inspection on ___________________
4. A licence fee of rupees ____________________ and inspection fee of
rupees__________________ has been credited to the Government
under Head of Accounts _____________________ (Receipt enclosed)
Signature__________________
Dated: _____________ Name & Designation____________
*Delete whichever is not applicable.

Note
1. The application shall be accompanied by a plan of the premises, list of
machinery and equipment for collection, processing, testing, storage,
banking and release of umbilical cord blood stem cells, memorandum
of association/constitution of the firm, copies of certificate relating to
educational qualification and experience of the competent technical staff
and documents relating to ownership of tenancy of the premises.
2. A copy of the application together with the relevant enclosure shall also be
sent to the Central Licence Approving Authority and to the Zonal/ Sub-Zonal
Officers concerned of the Central Drugs Standard Control Organization

iii. After the Form 28-E, the following Form shall be inserted,
namely:

“Form 28-F”
[See rules 122-F, 122-G, 122-H, 122-I, 122-K, 122-P]
License to collect, process, test, store, banking and release of umbilical
cord blood stem cells
1. Number of licence_____________________ date of issue _________ at
the Premises situated at ______________________________________
2. M/s __________________ is hereby licensed to collect, process, test,
store, banking and release of umbilical cord blood stem cells.
3. Name(s) of competent Technical Staff:
1.
2.
3.
4.
5.
364 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

4. The license authorizes licensee to collect, process, test, store,

banking and release of umbilical cord blood stem cells subject
to the conditions applicable to this licence.
5. The license shall be in force from ______________________ to
______________________.
6. The license shall be subject to the conditions stated below and
to such other conditions as may be specified from time to time
in the Rules made under the Drugs and Cosmetics Act, 1940.
Signature__________________________
Name & Designation_________________
Licencing authority___________________
Dated: ______________________
Central Approving Authority

Conditions of License
1. Umbilical cord blood specific for an individual will be collected after
signing an agreement with the parent(s), whose child’s umbilical
cord blood is to be collected, and the cord blood bank.
2. Umbilical cord blood shall be collected from hospitals, nursing
homes, birthing centers and from any other place where a
consenting mother delivers, under supervision of the qualified
Registered Medical Practitioner responsible for the delivery.
3. The license and any certificate of renewal in force shall be displayed
on the approved premises and the original shall be produced at the
request of an inspector appointed under the Drugs and Cosmetics
Act, 1940.
4. Any change in the technical staff shall be forthwith reported to the
Licensing Authority and/or Central Approving Authority.
5. The lincesee shall inform the Licensing Authority and/or Central
Approving Authority in writing in event of any change in the
constitution of the firm operating under the license. Where any
change in the constitution of the firm takes place, the current
licence shall be deemed to be valid for a maximum period of three
months from the date on which the change has taken place unless,
in the meantime, a fresh licence has been taken from the Licensing
Authority and/or Central Approving Authority in the name of the
firm with the changed constitution.”
 Recent Amendments in the Drugs and Cosmetics Rules, 1945... 365

Note
In Schedule F, after Part XII C, the following shall be inserted, namely:

PART XII-D of Schedule-F of the Drugs and Cosmetics Rules

1945

REQUIREMENTS FOR COLLECTION, PROCESSING, TESTING,

STORAGE, BANKING AND RELEASE OF UMBILICAL CORD BLOOD
DERIVED STEM CELLS

A. General Requirements
1. Location, Surroundings and Building: The building(s) for storage
of umbilical cord blood shall be so situated and shall have such
measures to avoid risk of contamination from external environment,
including open sewage, drain, public lavatory or any factory which
produces disagreeable or obnoxious odor or fumes, excessive soot,
smoke, chemical or biological emissions.
2. Building and Premises: (1) The premises used for processing and
storage shall be designed, constructed and adapted and maintained
to ensure that the above operations and other ancillary functions
are performed smoothly under hygienic conditions and in sterile
areas wherever required. They shall also conform to the conditions
laid down in the Factories Act, 1948 (63 of 1948). The premises shall
be:
a. Adequately provided with working space to allow orderly and
logical placement of equipment, material and movement
of personnel so as to maintain safe operations and prevent
contamination;
b. Designed/constructed/maintained to prevent entry of insects,
pests, birds, vermins and rodents. Interior surfaces (walls,
floors, ceilings and doors) shall be smooth and free from cracks,
and permit easy cleaning, painting and disinfection, and in
aseptic areas the surfaces shall be impervious, non-shredding,
non-flaking and non-cracking.
c. Flooring shall be unbroken and provided with a cove both at
the junction between the walls and the floor as well as the wall
and the ceiling.
d. Provided with light fittings and grills which shall flush
with the walls and not hanging from the ceiling to prevent
contamination.
366 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

e. If provided with fire escapes, these shall be suitably installed in

the walls without any gaps.
f. Provided with the furniture in aseptic areas which is smooth,
washable and made of stainless steel or any other appropriate
non-shedding material other than wood.
g. Provided with separate areas for processing and storage of
products to prevent mix-ups, product contaminations and
cross contamination;
h. Provided with defined environmental conditions for
temperature, humidity, ventilation and air filtration.
Classification shall be defined and, if appropriate, monitored.
2. A periodical record of cleaning and renovating of the premises shall
be maintained.
3. Disposal of waste and infectious material:
a. Waste materials awaiting disposal shall be stored safely;
b. The disposal of sewage and effluents from the facility shall be
in conformity with the requirements of the Pollution Control
Board;
c. All biomedical waste shall be dealt with in accordance with
the provisions of the Bio-medical Waste (Management and
Handling Rules, 1996)
4. Health, Clothing and Sanitation of personnel
a. All personnel shall undergo medical examination prior to
employment and shall be free from infectious and contagious
diseases and thereafter they should be medically examined
periodically at least once a year and for this purpose records
shall be maintained thereof;
b. All personnel, prior to and during employment, shall be trained
in practices which ensure personal hygiene and a high level of
personal hygiene shall be observed by all those engaged in the
collection, processing, banking of umbilical cord blood;
c. All persons shall wear clean body coverings appropriate for
their duties before entering the Processing Zone and the
Change Rooms with adequate facilities shall be provided prior
to entry into any specific zone;
d. Smoking, eating, drinking , is prohibited inside the Laboratory;
e. All personnel working in the Laboratory shall be protected
against virus infections.
5. Requirements for Processing, Testing, and storage areas for
umbilical cord blood stem cells:
a. Separate dedicated areas specifically designed for the purpose
and the workload shall be provided
 Recent Amendments in the Drugs and Cosmetics Rules, 1945... 367

b. There shall be separate areas for designated work purposes,

namely:
i. Cord Blood Reception: Cord blood reception area
with space for transient storage of units and physical
examination shall have adequate facilities for registration,
data entry and generation of bar-coded labels. Air
condition area of at least 10.00 sq. meters shall be provided.
ii. Cord Blood Processing Area: The room shall be clean
and have an air handling system to provide a class 10,000
environment. Entry to this area shall be through air lock.
The vroom will house Class 100 biological safety cabinets
for umbilical cord blood processing. The temperature of
the clean room shall be maintained 20 to 25°C and with
a positive differential pressure of 10 to 15 pascals and
relative humidity of 50 to 60%. Minimum area shall be
10.00 sq. meters for the activity.
iii. Hematology and Serology Laboratory: The laboratory
shall be equipped and utilized for the purpose of testing
of umbilical cord blood for ABO grouping and Rh typing,
total nucleated cell count, progenitor cell count and
viability test. The room shall be air-conditioned and area
of at least 10.00 sq. meters shall be provided.
iv. Transfusion Transmissible Disease Screening Labora­
tory: The laboratory shall be equipped and utilized for
screening tests on maternal blood for infectious diseases
viz. HIV I and II; Hepatitis B and C virus, syphilis, malaria,
CMV and HTLV. The room shall be air-conditioned and
area of at least 10.00 sq. meters shall be provided.
v. Sterility Testing Laboratory: The laboratory shall be
used for performing sterility tests on umbilical cord blood
unit. The premises may be classified depending on the
testing method used. The room shall be air-conditioned
with adequate and ancillary area for media preparation,
sterilization, incubation and decontamination. Area of at
least 10.00 sq. meters shall be provided.
vi. HLA Typing Laboratory: The umbilical cord blood unit
shall have arrangements for HLA typing and genetic
disease testing. In-house testing can be done by providing
a well-demarcated laboratory from the processing area
for evaluation of possible genetic disease and HLA typing.
The area shall have class 100,000 environments and air-
conditioned and area of at least 10.00 sq. meters shall be
provided.
368 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

vii. Sterilization-cum Washing: Appropriate facility shall

be provided within the premises for proper washing and
sterilization. This facility would be optional for laboratories
using entirely disposable items.
viii. Records and Store Room: There shall be designated record
room(s) and store room(s) of at least 10.00 sq. meters
each. The access to record room shall be permitted only
to the authorized persons. The room will have adequate
protective facilities as the documents and records are to be
preserved for long years.
ix. Cryogenic Storage Unit: A minimum space of 20.00 sq.
meters shall be provided by the Licensee. The Cryogenic
storage room shall have provision for temperature
monitoring of storage vessels, liquid nitrogen level in
storage vessels and oxygen vessel, supply cylinders and
connecting hose should be minimum 1.00 sq. meters.
Separate storage space for other accessories required shall
be provided. The room shall be air-conditioned.
x. General Storage Area: General storage area shall be
provided to store all consumables, under conditions
deemed optimum for storage by manufacturers.

B. Collection and Storage of Processed Umbilical Cord Blood

Component
1. Collection:
a. Umbilical cord blood specific for an individual will be collected
after signing an agreement with the parents, whose child’s
umbilical cord blood is to be collected, and the cord blood
bank. Private and Public umbilical Cord Blood Banking to have
different agreements;
b. Umbilical cord blood shall be collected from hospitals, nursing
homes, birthing centers and from any other place where a
consenting mother delivers, under supervision of the qualified
Registered Medical practitioner responsible for the delivery;
c. The cord blood shall be collected aseptically in a disposable
PVC bag, containing adequate quantity of sterile, pyrogen free
anti-coagulant and sealed effectively and such PVC bag shall
be procured from licensed manufacturer;
d. The umbilical cord blood would be collected from a premises
operating in hygienic conditions to allow proper operation,
maintenance and cleaning.
 Recent Amendments in the Drugs and Cosmetics Rules, 1945... 369

2. Transportation:
a. Umbilical cord blood shall be transported from birthing center
to the designated laboratory under and as per procedure
prescribed by the cord blood bank;
b. The transportation procedure shall be validated to ensure
optimum survival of stem cells;
c. The transportation temperature should be between 18 to 28°C;
d. The time period between collection and processing shall not
exceed 72 hours.
3. Storage:
a. The umbilical cord blood shall be stored at room temperature
between 20 to 25°C in the reception area prior to processing;
b. Samples pending tests for specific transfusion transmissible
infectious diseases shall be stored in a segregated manner.

Note
Temperature range between 4 to 37°C, for the whole time period of transit
may be allowed beyond the 18°C to 28°C in exceptional cases. The effects
of deviation of transit temperature from the optimum, on the product shall be
adequately explained by the licensee in the client education booklet.

C. Personnel
Cord blood bank shall have the following categories of whole time
competent technical staff, namely:
1. Medical Director: The operation of cord blood bank shall be
conducted under the active directions and supervision of a Medical
Director who is whole time employee and is possessing a post-
graduate degree in Medicine-MD (Pathology/training in cord blood
processing and cryogenic storage.
2. Laboratory In-charge: The laboratory in-charge shall have post-
graduate qualification in Physiology or Botany or Zoology or
Cell Biology or Microbiology or Biochemistry or Life Sciences,
or Graduate in Pharmacy and one year working experience in
Pathological laboratory licensed by the local health authority or
any microbiology laboratory of a licensed drug manufacturing/
testing unit and experience/training in cord blood processing and
cryogenic storage.
3. Technical Supervisor (Cord Blood Processing): The technical
supervisor shall have a.
a. Degree in Physiology or Botany or Zoology, Pharmacy or Cell
Biology or Bio-sciences or Microbiology or Biochemistry or
Medical Laboratory Technology (MLT) with minimum of three
370 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

years of experience in the preparation of blood components

and/or experience or training in cord blood processing and
cryogenic storage; or
b. Diploma in Medical Laboratory Technology (MLT) with five
years experience in preparation of blood components and
experience or training in cord blood processing and cryogenic
storage shall be essential.
4. Cord Blood Bank Technician (s): The technicians employed shall
have a:
a. A Degree in Physiology or Botany or Zoology or Pharmacy or
Cell Biology or Bio-science or Microbiology or Biochemistry
or Medical Laboratory Technology (MLT) with six months
experience and or training in cord blood processing and
cryogenic storage; or
b. Diploma in Medical Laboratory Technology (MLT) with one
year’s experience in the testing of blood and/or its components
and/or experience or training in cord blood processing and
cryogenic storage shall be essential.

D. Air Handling Systems

1. Air handling for sterile areas shall be different from those for other
areas. The filter configuration in the air handling system shall be
suitably designed to achieve the grade of air as given in the Table
71.1. The environmental microbiological monitoring of clean areas
shall be in accordance to the recommended limits given in Table
71.2.
2. The Processing area shall have HVAC system and fitted with HEPA
Filters having Grade C (Class 10,000) environment as given in
Table 71.1.
3. The entire processing shall be done conforming to Grade A (Class
100) standard of air quality.
Table 71.1: Air-borne particulate classifications for manufacture
of sterile products
Grade Maximum number of permitted particles per cubic meter equal to
or above
At rest (b) In operation (a)
0.5 um 5 um 0.5 um 5 um
A 3,500 0 3,500 0
B (a) 3,500 0 3,50,000 2000
C (a) 3,50,000 2,000 35,00,000 20,000
D (a) 35,00,000 20,000 Not defined Not defined
 Recent Amendments in the Drugs and Cosmetics Rules, 1945... 371

Notes
a. In order to reach the B, C and D air grades, the number of air changes shall
be related to the size of the room and the equipment and personnel present
in the room. The air system shall be provided with the appropriate filters
such as HEPA for grades A, B and C. The maximum permitted number of
particles in the “at rest” condition shall approximately be as under:
[Grade A and B corresponds with class 100 or M 3.5 or class 5];
Grade C with class 10,000 or M5.5 or ISO class 7; Grade D with Class
1,00,000 or M 6.5 or ISO Class 8.
b. The requirement and limit for the area shall depend on the nature of
operation carried out.

Table 71.2: Recommended limits for microbiological monitoring of clean

areas “in operation”
Grade Air Sample Settle Plates Contact Plates Glove Points
cfu/m3 (dia 90 mm) (dia. 55 mm) ( five fingers)
cfu/2 hrs. cfu per plate cfu per glove
A <1 <1 <1 <1
B 10 5 5 5
C 100 50 25 –
D 500 100 50 –

Notes
a. These are average values.
b. Individual settle plates may be exposed for not less than two hours in
Grade B, C and D areas for not less than thirty minutes in Grade A area.

E. Quality Control
1. Facilities shall be provided for quality control such as hematologi-
cal, microbiological and Instrumental testing.
2. Following duties shall be performed under the function of quality
control:
a. To prepare detailed instructions for carrying out such tests and
analysis;
b. To approve or reject raw materials and consumables, used in
any step, on the basis of approved specifications;
c. Hematological tests like Total Nucleated cell counts,
Mononuclear cell count, enumeration of the population of
stem cells, stem cell viability;
d. Microbiological tests shall be done on maternal blood sample
for freedom from hepatitis B surface antigen, hepatitis C virus
antibody, HIV I and II antibodies, syphilis, malaria, CMV
372 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

and HTLV. Bacterial and Fungal Culture shall be done on the

umbilical cord blood samples;
e. Instruments which would be used to processes and store
the UCB unit would be validated before commissioning and
calibrated from time to time to check their conformity to
specific standards according to an approved and valid protocol;
f. The environmental monitoring of the clean rooms would
be done at periodic intervals according to an accepted and
validated protocol;
g. All tests mentioned above shall be done in house except tests
under item number (e), (f) and test for enumeration of stem
cell population, HLA typing and genetic disease testing which
may be outsourced to a competent third party approved by the
licensing authority.

F. Screening Tests
1. The maternal Blood Sample shall be tested for—
a. Hepatitis B;
b. Hepatitis C;
c. HIV I and II;
d. Syphilis;
e. Malaria;
f. CMV;
g. HTLV.
2. The umbilical cord blood shall be tested for—
a. Total nucleated cell count;
b. Total mononuclear cell count;
c. Progenitor cells (CD34+) enumeration;
d. Cell viability;
e. ABO group and Rh type;
f. Sterility as regards bacterial and fungal contamination status;
g. HLA matching (only for allogeneic cord blood units)

G. Storage
1. The umbilical cord blood shall be cryo-preserved using a controlled
rate freezing or equivalent validated procedures. The frozen storage
shall be at minus 196°C and shall not be warmer than minus 150°C.
2. There will be no shelf-life for this class of product.
 Recent Amendments in the Drugs and Cosmetics Rules, 1945... 373

H. Reference Samples
1. At least two reference samples shall be collected from cord blood
unit product prior to cryopreservation and stored at –196°C and
shall not be warmer than minus 150°C.
2. At least one additional reference sample shall be stored at –78°C or
colder for the purposes other than viability analysis.

I. Labeling
1. Initial label placed during collection shall specify:
a. Human umbilical cord blood;
b. Approximate volume or weight of contents in the collection bag
(UCB + Anticoagulant);
c. Mother’s name;
d. Place of collection;
e. Date and time of collection;
f. Collected by;
g. To be labeled in bold “Room Temperature Only. Do Not
Refrigerate, Donot Irradiate.”
h. Manufacturing licence number.
2. Label at completion of processing and before issue-Cryogenic
Storage Label (statutory label) shall indicate the following:
a. Name of Product-human Progenitor Cells (HPC)-Cord Blood;
b. Volume or weight of contents;
c. Percentage of cryo-precipitant (DMSO);
d. Percentage of any other additive/preservant;
e. Date of collection…………….
f. Date of Processing ………….
g. Name of manufacturer ………………
h. Manufacturing licence number …………….
i. Storage temperature not less than minus 196°C and shall not be
warmer than minus 150°C.
j. Unique traceability number and/or BAR code
3. Issue label at the time of release of cord blood unit shall indicate the
following, namely:
a. Name of manufacturer ……………
b. Licence number …………….
c. All details of the cryogenic storage label
d. The results of total nucleated cells, progenitor cell percentage
(CD34+), viability;
e. Results of transfusion transmissible diseases testing on
maternal blood;
374 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

f. ABO group and Rh type;

g. Date of processing;
h. Result of HLA typing (allogeneic);
i. Statement “Properly identify recipient and product”;
j. A statement indicating that leuko-reduction filters should not
be used;
k. Statement “Do not irradiate”;
l. Name and address of receiving hospital.

J. Records and Documentation

1. The licensee shall maintain the following records:
a. Client/Donor enrolment/agreement record;
b. Collection of unit and transportation record;
c. Master record of stored unit;
d. HLA matching record;
e. Unit release register;
f. Stock register for blood collection bag cryoprotectant and
preservant, RBC sedimentation enhancer;
g. Stock register for diagnostic kits, reagents and consumables;
h. Record on feedback after use of cord blood/adverse reaction
record
2. The following Standard Operating Procedures shall be maintained
by the licensee, namely:
a. Umbilical cord blood collection;
b. Transportation of umbilical cord blood unit;
c. Processing of umbilical cord blood unit;
d. Cryogenic storage of processed umbilical cord blood;
e. Testing of maternal blood for transfusion transmissible
infections;
f. Testing of umbilical cord blood for ABO grouping and Rh
typing;
g. Testing of umbilical cord blood unit for total nucleated cell
count, mononuclear cell count, progenitor cell (CD34+)
enumeration and viability;
h. Testing of umbilical cord blood stem cell unit for sterility;
i. Disposal of bio-medical waste;
j. Dispensation of umbilical cord blood unit;
k. Preventive maintenance protocol for all instruments;
l. Acceptance/Rejection procedure of consumables;
m. Environment monitoring of classified areas;
n. Any other standard operating procedure as requirements.
 Recent Amendments in the Drugs and Cosmetics Rules, 1945... 375

K. Cord Blood Release

1. There shall be designated area with adequate space for procedures
and records related to cord blood unit selection and release.
2. The cord blood bank shall obtain written or electronic request from
transplant physician or designee for shipment of the cord blood
unit.
3. Accompanying documents at the time of issue from cord blood bank
shall include indications, contraindications, caution, instructions
for handling and use of cord blood unit including short-term
storage and preparation for transplantation.
4. Procedure for transportation of cryo-preserved cord blood unit
within the facility shall be designed to protect the integrity of the
unit and the health and safety of the personnel.
5. Cryopreserved cord blood unit stored at –150°C or colder shall be
transported in a liquid nitrogen cooled dry shipper that contains
adequate absorbed liquid nitrogen and has been validated to
maintain temperature below –150°C for at least 48 hours beyond
the expected time of arrival at the receiving facility.
[F.No.X-11014/2/2008-DFQC]
ARUN K PANDA, Jt. Secy.
Footnote: The principal rules were published in the Gazette of India
vide notification No F.28-10/45-H (1), dated 21st December, 1945 and
last amended vide Notification no. GSR 101 (E), dated 18-2-2011.
The Drugs and Cosmetics Rules 1945 as amended up to 1-5-1979
is contained in the publication of the Ministry of Health and Family
Welfare (Department of Health) containing the Drugs and Cosmetics
Act, 1940 (PDGHS-61)

II. GUIDELINES
I. Copy of the letter no. CLAA/B&BP/DCG (I)/10/2002-D dated
8.6.2006 of the Drugs Controller General (India), Nirman Bhawan,
New Delhi, adressed to all state drugs controllers
Sub: Gazette notification GSR 733 (E) dated 21/12/2005
Sir,

Consequent upon the subject notification, blood banks run by the

Government, Indian Red Cross Society, Hospitals, Charitable trusts
or Voluntary organizations approved by State/UT Blood Transfusion
Council only can apply for grant of license for operations of blood bank
or processing of blood components. It was, however, felt that there is
376 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

a lack of clarity regarding the criteria for accepting a blood bank as a

charitable trust blood bank.
The issue was taken up with the National Blood Transfusion Council,
New Delhi, and accordingly the issue was discussed by the Technical
Resource Group (TRG) of NBTC. The suggestions received from them in
this regard for identification and acceptance of blood bank application
run by a charitable trusts are quoted below:
1. Be registered with Charity Commissioner for at least last five years.
2. The trusts should not be a family trust.
3. The trust deed and the activities undertaken by the trust should
showcase social accountability.
You are, therefore, requested to henceforth follow this guideline
while considering application for grant of license to run a blood bank by
charitable trust.
This guideline may also be brought to the notice of State Blood
Transfusion Council of your state for their information and necessary
action.
II. Copy of the letter No. S.J2015/81/2006-dated 5 January 2009 of
NACO (BS), Government of India, Ministry of Health and Family
Welfare (National AIDS Control Organization) addressed to the
Director/Member Secretary of all SBTCs.
Subject: Guidelines for SBTC to grant “No Objection Certificate” for
approval /renewal of Blood Bank license: Forwarding of
Sir/Madam,
This is in reference to Govt. of India Gazette Notification No
R.S.R.733 (E) dated 21st December 2005, (copy enclosed) on the above
mentioned subject.
2. Para 2(ii) of above Gazette Notification denotes that “Application
for grant or renewal of a license for operation of Blood Bank or
processing of human blood components shall be made by the Blood
Bank run by the Government, Indian Red Cross Society, Hospital,
Charitable Trust or Voluntary Organization approved by a state/
Union Territory Blood Transfusion Council only.
3. Accordingly guidelines for SBTC to grant “No Objection Certificate’
for approval/renewal of blood bank license has been formulated by
TRG (BS), NACO and the same have been approved by the governing
Body of National Blood Transfusion Council in its meeting held on
5th February, 2007. A copy of the guidelines is enclosed.
4. You are requested to adhere the guidelines strictly while issuing No
objection certificates for issue of license/renewal of license of blood
banks.
 Recent Amendments in the Drugs and Cosmetics Rules, 1945... 377

III. Guidelines for SBTC to grant “No Objection Certificate” for

approval/renewal of blood bank license:
1. While considering the application for approval of license for blood
bank, SBTC should satisfy on assessment regarding the following
points.
a. Reasons and justification.
b. The geographical area of blood bank and the population of that
area, and the medical facility available in the area, any facility
for handling accident victim, any trauma center.
c. Whether they have the capability of fulfilling the criteria laid
down by NACO.
d. Any blood bank having collection less than 2000 units to be
given the status of blood storage centers to maintain quality in
the blood bank and this will reduce number of blood banks in
the area.
e. Applicant should supply details of demand of blood unit in that
area.
f. If it is a charitable trust then whether proper memorandum of
association is being prepared. They should also show the proof
of their social work related to health.
g. No license will be given to stand alone blood bank for private
commercial purpose.
2. While considering the application for renewal of license, the
following points should be clearly investigated:
a. If any malpractice had been reported about the blood bank, no
renewal of license should be given.
b. 400 bedded hospital with the capacity of more than 2000 units
of voluntary blood collection.
c. Voluntary blood collection should be close (within 10%) of
national average.
d. Whether the component preparation has been started by the
blood bank within the stipulated time period allotted to them
(2–5 years).
 C H A P T E R 72
Guidelines for the Preparation of
Application for Grant/Renewal of
License to Operate Blood Bank

A. Blood Bank
The operation, functional activities and regulatory matters of Blood
Banks are being governed by Central Legislation. As per the statutory
provisions, the above matter is to be regulated in accordance with the
provisions of Drugs and Cosmetics Act, 1940 and Rules thereunder
(Whereas here in after referred as “Act”).
As per the provisions laid down under the Act, the collection,
distribution, storage, etc. of Whole Human Blood IP and its components
shall be carried out only in accordance with the condition of the license
issued under the Act. Normally the procedures and provisions made
under the Act followed strictly for submitting an application to obtain
a fresh license for starting a new blood bank, but in some of the States/
UT’s the applicant is required to get the plan approved in advance for the
proposed premises from the Licensing Authority but it is not universally
applicable for all the cases. The proposed premises must have at least
100 sq.mt. built-up area. The premises are to be suitably divided into at
least 7 separate sections. The 7 separate sections are listed as follows:
1. Registration and medical examination section with adequate
furniture and facilities for registration and selection of donors.
2. Blood collection section (Air-conditioned).
3. Laboratory for blood group serology (Air-conditioned).
4. Laboratory for blood transmissible disease like hepatitis, syphilis,
malaria, HIV-antibodies (Air-conditioned).
5. Sterilization-cum-washing area.
6. Refreshment-cum-rest room (Air-conditioned).
7. Store-cum-records room.
Blood Components: The manufacturing of blood components
requires additional space of 50 square meter area which shall be fully
air-conditioned. Plan is required to be approved in some of the States in
advance. The whole area of 50 sq. meters shall be suitably divided into
three portions as follows:
 Guidelines for the Preparation of Application for Grant/Renewal... 379

1. Component manufacturing area with air lock

2. Component storage area.
3. Component testing area.
At present, standalone blood bank in private sector is not permitted.
While charitable organization/voluntary organizations approved by
State/UT Blood Transfusion Council only can apply for grant of license
for operation of blood bank or processing of blood components. 379
The applicant is supposed to file his application in 3 copies with
the documents and other relevant enclosures as per the check-list given
below:
1. Application on Form 27-C Specimen Form enclosed as
Annexure 72.1
2. Fees Rs. 7500/– deposited through
treasury challan–original challan to be
submitted
3. Constitution of Applicant form– Proprie- Specimen format enclosed as
tor/Partnership/Ltd/Pvt. Ltd./Voluntary/ Annexure 72.2
Charitable organization/NGOs, etc. (at-
tested copy of partnership deed/trust
deed, etc.required)
4. Affidavits of Proprietor/Partner/Direc- Specimen format enclosed as
tor/Trustee and Responsible person/ Annexure 72.3
authorized signatory
5. Details of technical staff (whole-time) a. Name, address, qualifi-
cation and experience of
technical staff. Specimen
format enclosed as Annex-
ure 72.4
b. Attested photocopies of
educational qualification of
the technical staff.
c. Original/attested copies of
their experience certificate.
d. Copies of their appoint-
ment letters and their ac-
ceptance with date of join-
ing.
6. Affidavits of all technical staff, i.e. Medi- Specimen format enclosed as
cal Officer/BTO/Consultant, Technical annexure 72.5
Supervisor, Laboratory technician and
Registered nurse
Contd...
380 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Contd...

7. Blue-print of the premises indicating Specimen site plan enclosed as

area of each section/room (total area Annexure 72.6
should not be less than 100 sq. mt. (for
whole human blood IP) additional area
of 50 sq. mt. for manufacture of blood
components in the same premises. For
apheresis, additional 10 sq. mt.
8. Documents relating to ownership or Specimen enclosed as Annex-
tenancy of the premises ure 72.7
9. List of prospective voluntary donors
with their address and telephone/Mo-
bile numbers and email address
10. List of products with undertaking
11. Copy of draft label for each blood group
12. Undertaking of the owner for not to col-
lect blood from professional donors
13. Standard Operating Procedure/Blood
Bank Manual
14. List of equipment [as per Schedule F
Part XII-B]
15. Purchase invoice (Photocopies) of pur-
chase bills of above (14)
16. Specimen donor card, blood bank Specimen enclosed as Annex-
stock, issue register/records (perfor- ures 72.8 to 72.14
mas), etc
17. Registration from respective State/UT
Registrar of co-operative Societies in
case of Voluntary/Charitable registered
societies
18. Recognition of registered voluntary or
charitable organization by State Blood
Transfusion Council
19. Provision for disposal of biomedical
waste—documents to be submitted

Note
Any change in the technical staff must be intimated forthwith to the concerned
State. Licensing Authority along with the attested copies of testimonials/
experience of the technical staff, copies of their appointment letter/joining letter
and non-conviction affidavit of the new person(s) joining blood bank in original.
 Guidelines for the Preparation of Application for Grant/Renewal... 381

Upon receipt of application and other documents from the firm, the
SLA (State Licensing Authority) shall forward one set of application and
documents to the Dy Drugs Controller (I) CDSCO, of the respective zone,
requesting him to arrange for joint inspection of the blood bank (Along
with Blood Bank Expert as per the Procedure followed by the Concerned
State) under intimation to the applicant. The performa for the inspection
is provided as Annexure 72.15. After completion of satisfactory381 joint
inspection and favorable recommendation by the team of inspecting
officer, the SLA will examine the suitability and eligibility of the firm in
respect of the facilities provided by the firm. If he is satisfied that the
applicant is able to fulfill the minimum requirements, conditions of
license, rules, regulations and provisions of the Act, he shall prepare the
license in FORM-28C, (3 copies) and will send these to CLAA (Central
Licensing Approving Authority), New Delhi, for his approval and counter
signature. The power of CLAA has been designated to Drugs Controller
General (I), DGHS, New Delhi. Once the license has been duly approved
and countersigned by CLAA, it shall be handed-over to the applicant
through SLA to allow him blood bank activities.

B. Blood Storage Center

Applicant is required to submit application on simple white paper to the
State Licensing Authority with the following enclosures:
1. Consent letter (preferably in affidavit shape) from mother blood
bank.
2. Name, qualification and experience of Medical Officer and his
undertaking to supervise the functioning of Blood Storage Center
(He may not be whole time).
3. Name, qualification, experience, etc. (as per Part XII B of Schedule
F of Drugs and Cosmetics Rules, 1945) of Lab Technician.
4. Site plan preferably blue print with an area not less than 10 sq mt.
duly signed by authorized signatory.
5. List of equipments installed as well as the facilities provided for
ensuring uninterrupted supply of electricity.
6. Undertaking that captive consumption of Whole Human Blood IP
and its components shall not exceed 2000 units annually.
A copy of the application after receipt in the office of State
Licensing Authority is sent to the Senior Drug Control Officer/Drug
Control Officer of the area for the inspection and verification of the
applicant’s site, facilities, technical staff, etc. On receipt of inspection
report, recommendation, etc. from the inspecting Drug Control Officer,
approval is granted to the applicant, which will be valid for a period of
two years from date of its issue unless sooner suspended or cancelled.
382 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Annexure 72.1
Form 27-C
[See Rule 122-F]
Application for grant/renewal of license for the operation of Blood
Bank for processing of whole blood and/or preparation of Blood
components
1. I/We ________________________ of M/s ________________________
____________________________________________________________
hereby apply for the grant of license/renewal of license number
_______________ dated __________________ to operate a Blood
Bank, for processing of whole blood and/or* for preparation of its
components on the premises situated at ________________________
___________________________________________________________ .
2. Name(s) of the item(s):
i.
ii.
iii.
3. The name(s), qualification and experience of competent technical
staff are as under:
a. Name(s) of Medical Officer
b. Name(s) of Technical Supervisor
c. Name(s) of Registered Nurse
d. Name(s) of Blood Bank Technician
4. The Premises and Plant are ready for inspection/will be ready for
inspection on _______________________
5. A license fee of rupees six thousand and an inspection fee of rupees
one thousand five hundred has been credited to the Government
under the Head of Account 0210-Medical and Public Health (receipt
enclosed).
Dated: ____________________ Signature of Applicant
(Name and Designation)
*Delete, whichever is not applicable.

Notes
1. The application shall be accompanied by a plan of the premises, list of
machinery and equipment for collection, processing, storage and testing of
whole blood and its components, memorandum of association/constitution
of the firm, copies of certificates relating to educational qualifications and
experience of the competent technical staff and documents related to
ownership or tenancy of the premises.
2. A copy of the application together with the relevant enclosures shall also be
sent to the Central License Approving Authority and to the Zonal/Sub-zonal
officers concerned of the Central Drugs Standard Control Organization]
 Guidelines for the Preparation of Application for Grant/Renewal... 383

Annexure 72.2
Name and Address of Blood Bank ___________
Constitution: Proprietorship/Partnership/Pvt. Ltd./Ltd. Voluntary or
Charitable Organization/NGO/Any other
Sr. Name of Prop/Partner/ Complete Permanent & Designation
No. Director/Member/Trustee Correspondence Address 383

Date: _____________
Signature of Proprietor/Partner/
Place: _____________ Director/Managing Director/Chairman
384 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Annexure 72.3
Specimen of Affidavit of Proprietor/Partner/Director
(on Rs. 3/- stamp paper duly attested by 1st class/ Executive Magistrate)
I ___________ S/o ___________ aged ___________ R/o ___________ do
hereby solemnly affirm as under:
1. That I have never been convicted under Drugs and Cosmetics Act
1940 and Rules 1945.
2. That I am Proprietor/ Partner/ Director of M/s __________________.
3. That Dr. ________________________ S/o ________________________
qualification ____________________ will work as whole time Medical
Officer in this Blood Bank. He will be responsible for running the
Blood Bank.
4. That Sh. ________________________ S/o ________________________
qualification will work as whole time
Blood Bank Technician in this Blood Bank.
5. That Ms. ____________________ W/o/D/o ____________________
qualification ________________________________________________
will work as whole time Registered Nurse in this Blood Bank.
6. That the blood will not be drawn from any paid or professional
donor in this blood bank.
7. That the blood will not be issued to any patient without prescription/
requisition duly signed by the registered medical practitioner.
8. That the blood will be issued only after getting it tested for the tests
mentioned in the Part XII-B of the Drugs and Cosmetics Rules, 1945.

Deponent

Verification: Verified that the contents as stated above are true and
correct to the best of my knowledge and belief and nothing has been
concealed therein.

Deponent
 Annexure 72.4
Name, Address, Qualification and Experience of Technical Staff
Sr. No. Designation Name Date of Address Qualification Year of Experience
Appointment Passing,
& Joining University/
Institute
1 Medical
Officer

2 Tech.
Supervisor

3 Lab. Tech.

4 Regd. Nurse

Date ___________
Place: ___________
Guidelines for the Preparation of Application for Grant/Renewal...

Signature of Proprietor/Partner/
385
385

Director/Managing Director/Chairman
386 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Annexure 72.5
Specimen of Affidavit of Technical Staff (MO/LT/Nurse)
(On Rs. 3/- stamp paper duly attested by 1st class/Executive Magistrate)
I __________________ S/o __________________ aged __________________
R/o __________________ do hereby solemnly affirm as under:
1. That I have never been convicted under Drugs and Cosmetics Act
1940 and Rules 1945.
2. That I have accepted the appointment offer of M/s _____________
to work as Medical Officer/Lab Tech./Regd. Nurse at a salary of
Rs. ______________ pm on whole time basis, w.e.f. ______________.
3. That I will be responsible for running this Blood Bank as per
provisions under the Drugs and Cosmetics Act 1940 and Rules 1945.
4. That I will not work at any other place during my services with this
blood bank.
5. That the blood will not be drawn from any paid or professional
donor in this blood bank.
6. That the blood will not be supplied to any patient without
prescription/requisition duly signed by the registered medical
practitioner.
7. That the blood will be issued only after getting it tested for the tests
specified under Part XII-B of the Drugs and Cosmetics Rules, 1945.

Deponent

Verification: Verified that the contents as stated above are true and
correct to the best of my knowledge and belief and nothing has been
concealed therein.

Deponent
 Guidelines for the Preparation of Application for Grant/Renewal... 387

Annexure 72.6
Suggested specimen site plan of blood bank for manufacture of whole
human blood

387
388 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Annexure 72.7
Specimen of Rent Receipt of the premises

I, __________________ S/o __________________ R/o __________________

(Tel No. __________________) have rented out my premises situated at
_______________________________________________________________.
Total covered area approx _____________________________________ to
Sh. __________________ S/o __________________ R/o __________________
for running blood bank under the name and style _____________
______________________________________________ at a monthly/
yearly rent of Rs. _____________________ month/year. I have received
Rs. ________________ as the advance rent payment for the month of
________________________ from Sh. ________________________. I have
also signed the site plan of my premises, which has been rented by me.

Date ______________
(Signature of the Landlord)

(Signature of Tenant)

Witnessed by ______________
 Guidelines for the Preparation of Application for Grant/Renewal... 389

Annexure 72.8
Name and Address of the Blood Bank
S.No. ___________ Manufacturing Licence No. ___________
Specimen
Whole Human Blood IP
(Voluntary/Replacement) 389
Date of Collection ___________ Date of Expiry ___________

Tested for Following:

Hepatitis B surface antigen and Hepatitis C virus antibody, syphilis,
freedom from HIV I and HIV II antibodies and malarial parasite Found
Negative.

Total quantity /volume of blood ___________________________________

Name and Percentage of anticoagulant _____________________________

Notes
1. Keep continuously at 2°C–6°C.
2. Disposable Transfusion sets with filter must be used in administration
equipment.
3. The content of the bag should not be used if there is any visible evidence
of deterioration like hemolysis, clotting or discoloration.
4. Appropriate compatible cross-matched blood without a typical antibody in
recipient shall be used.
A colored label shall be put on every bag containing blood. The following color
scheme for said labels shall be used for different groups of blood.

Blood Group Color of the Label

O Blue
A Yellow
B Pink
Ab White

Notes
The above colored labels should be pasted on the bags of blood units in such
a way that the original manufacturer’s label of the CPD-A bag is not concealed.
 Annexure 72.9 390

Blood Donor Register

Name & Address of Blood Bank_______________________
Sr Date of Name & Sig. of Age Weight Hb Blood B.P. Med. Bag Patient Category Referral Sig.
No Bleeding Address Donor Grouping Exam Sr. for Whom of Records Medical
of Donor No. Donated Donation Officer
(in Case of (V/R) Incharge
Replacement)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.

Note
It is advisable to maintain record of the blood units including the segment number as embossed on the blood bag tubing for better
traceability and safety in blood transfusion. Additional columns can be added as per the requirement in all the registers maintained for
Standard Operating Procedures and Regulatory Guidelines-Blood Banking

records purposes in the blood bank.

 Annexure 72.10
Master record for blood (Stock Register)
Name & Address of Blood Bank_______________________

Sr Bag Date Date Quantity ABO/ Result Malaria VDRL Hepatitis-B Hepatitis-C. Irregular Name & Utilization Components Sign. of
No Sr. of of in M.L. Rh of Antibodies Address Issue No. Prepared or Medical.
No. Collec- Expiry Group HIV-I (if any) of Donor Discarded Officer
tion & II I/C
Testing
1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.
Guidelines for the Preparation of Application for Grant/Renewal...

391
391
 Annexure 72.11 392

Issue Register
Name & Address of Blood Bank _______________________

Sr Date & Time Bag Sr. No. ABO/Rh Total Quantity Name & Blood Unit/ Institution Details of Cross- Indication for
No of Issue Group in ml Address of Group of the match Report Transfusion
Recipient Recipient
1.
2.
3.
4.
5.
6.
7.
Standard Operating Procedures and Regulatory Guidelines-Blood Banking
 Guidelines for the Preparation of Application for Grant/Renewal... 393

Annexure 72.12
Records of components supplied
Name of component _____________________
Name and Address of Blood Bank _____________________

Sr. Quantity Supplied Compatibility Detail of Signature of

No Report Recipient
393
Issuing Person

1.

2.

3.

4.

5.

6.

7.
 Annexure 72.13 394

Stock Register for Diagnostic Kits and Reagents

Name and Address of Blood Bank_______________________
Name of kit/reagent __________________

Sr. Date of Name of Supplier/ Invoice No. & Batch No. Date Quantity Date of Quantity Balance Sig. of MO
No Receipt Manufacturer Date of Expiry Received Issue Issued (I/C)

1.

2.

3.

4.

5.

6.

7.
Standard Operating Procedures and Regulatory Guidelines-Blood Banking
 Annexure 72.14
Stock Register for CPD-A bags/Sterile hypodermic needles/syringes
Name and Address of Blood Bank_______________________

Sr Date of Name of Invoice No. & Batch Date Quantity Result of Date of Quantity Balance Sig. of MO
No Receipt Supplier/ Date No. of Expiry Received Test Issue Issued (I/C)
Manufacturer

1.

2.

3.

4.

5.

6.

7.
Guidelines for the Preparation of Application for Grant/Renewal...

395
395
396 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Annexure 72.15
Blood Bank Inspection Checklist
(Use separate sheets, if necessary)
(Collect specimen forms, documents, labels,
record copies whenever necessary)
Name of Date of Inspection
Institution
Address of
the Institution
Telephone
No.:
Fax No.:
E-mail:
License
number and
date of issue
Inspected By CDSCO State Drugs Control
Expert, if any
Institution
represented
by
Purpose of
Inspection
Type of Government Charitable Red Cross Others
Institution (specify)
Constitution
Details
Products
Technical Qualification Experience
staff (Attach (check (check
sheet, if documents) testimonials)
reqd.)
Doctor
Registered
Nurse
Technician
Attendant
 Guidelines for the Preparation of Application for Grant/Renewal... 397

A Total Collection Year

(Last two calendar years) Voluntary
Replacement
Professional
Total
Distribution Used in own hospital
397
Issued to others
Discarded
B Premises Total area
Details of areas Comments
1. Registration and Medical Examination
2. Blood Collection
(A/C?)
3. Serology Lab.
(A/C?)
4. Transmissible diseases Lab. (A/C?)
5. Sterilization and washing

6. Refreshment and Rest Room (A/C?)

Comments on Area

C. Standard Books? Yes/No/NA

1. Obtain list
2. Blood Bank Manual Yes/No/NA
3. Standard operating procedures Yes/No/NA
a. Criteria to determine donor suitability Yes/No/NA
b. Method of donor selection Yes/No/NA
c. Preparation of phlebotomy site Yes/No/NA
d. Product to donor traceability Yes/No/NA
e. Collection procedures, precautions, Yes/No/NA
etc.
f. Method of components preparation Yes/No/NA
g. Test methods Yes/No/NA
h Pre-transfusion testing Yes/No/NA
i Adverse reaction management Yes/No/NA
j Storage temperature and its control Yes/No/NA
k Expiry date assignment Yes/No/NA
l Returned blood management Yes/No/NA
m QC for reagents and supplies Yes/No/NA
398 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

n Maintenance, calibration & validation Yes/No/NA

of equipment
o Labeling procedures Yes/No/NA
p Apheresis procedures Yes/No/NA
q Any other SOPs Yes/No/NA
D Procedure for disposal of blood Yes/No/NA
(expired, clotted, improperly collected,
HIV + etc.)
E Donor education/motivation material Yes/No/NA
F Donor selection Yes/No/NA
1 Donor record Yes/No/NA
2 Selection/rejection manual Yes/No/NA
3 Donor record details:
a. Age Yes/No/NA
b. Interval between donations Yes/No/NA
c. Last pregnancy/delivery/abortion Yes/No/NA
d. Immunization details Yes/No/NA
e. Recent drug intake Yes/No/NA
f. Major surgery Yes/No/NA
g. Malaria Yes/No/NA
h. Jaundice Yes/No/NA
i. Other viral infection Yes/No/NA
j. Fever and common cold Yes/No/NA
k. History-cancer, TB, Diabetes, Drug Yes/No/NA
addiction, etc.
l. Alcohol intake Yes/No/NA
m. Transfusion history Yes/No/NA
4. Donor Examination Yes/No/NA
a. Weight Yes/No/NA
b. Venipuncture site Yes/No/NA
c. Hemoglobin Yes/No/NA
d. Blood pressure Yes/No/NA
e. Pulse Yes/No/NA
f. Temperature Yes/No/NA
G. Collection of Blood
 Guidelines for the Preparation of Application for Grant/Renewal... 399

a. Preparation of phlebotomy site Yes/No/NA

b. Type and amount of anti-coagulant Yes/No/NA
used
c. Amount of blood collected (random Yes/No/NA
wt.)
d. Blood collected in bags/bottles Yes/No/NA
399
e. Pediatric Bags? Yes/No/NA
f. Is mixing done during collection? Yes/No/NA
How?
g. Is new bag used in case of 2nd punc- Yes/No/NA
ture?
h. How is sample tubes labelled? Yes/No/NA
i. Emergency kit available? Yes/No/NA
H. Storage of blood
a. Temperature recording graph pre- Yes/No/NA
served?
b. Alarm system checks done? Yes/No/NA
c. Physical verification done? Fre- Yes/No/NA
quency?
d. How is blood transported? Outside, to Yes/No/NA
wards?
I. Blood Testing
a. Sterility testing Yes/No/NA
b. Hemoglobin estimation-method Yes/No/NA
c. Method for ABO grouping Yes/No/NA Slide/Tube/
Others.
Describe
d. Procedure for grouping Yes/No/NA Cell/Serum/
Both. De-
scribe
e. Method of pooled cell preparation Yes/No/NA
f. Du test done on D-samples? Yes/No/NA
g. Test for unexpected antibodies done? Yes/No/NA
Hepatitis test done? Describe method Yes/No/NA
and Name of kit manufacturer
h. Syphilis test done? Yes/No/NA
Describe method and Name of kit
manufacturer
i. HIV test done? Describe method and Yes/No/NA
Name of kit manufacturer
400 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

j. HCV test done? Describe method and Yes/No/NA

Name of kit manufacturer
Malaria test done? Describe method Yes/No/NA
k. Donor informed in case of +ve Yes/No/NA
results?
l. In case of HbsAg/HIV +ve results Yes/No/NA
Donor debarred permanently
m. Are HbsAg/HIV +ve donors followed Yes/No/NA
up?
J. Testing of reagents, etc.
a. Antisera tested? Yes/No/NA
b. Method of antisera testing Yes/No/NA
c. CPDA solution testing Yes/No/NA
K. General equipment and instru-
ments
a. Refrigerators for blood storage Yes/No/NA
type, capacity and number
b. Temperature recorder in refrigerator Yes/No/NA
c. Audible alarm system in refrigerator Yes/No/NA
d. Balance for bag weighing Yes/No/NA
e. Autoclave with temp. and pressure Yes/No/NA
display
f. Incinerator Yes/No/NA
g. Emergency power supply (generator) Yes/No/NA
h. Donor beds, chairs, tables Yes/No/NA
i. Bedside table Yes/No/NA
j. Sphygmomanometer and Yes/No/NA
stethoscope
k. Recovery bed for donors Yes/No/NA
l. Donor weighing scale Yes/No/NA
L. Emergency equipment
a. Oxygen cylinder, mask, gauge and Yes/No/NA
pressure regulator
b. 5% Dextrose or Normal Saline inj. Yes/No/NA
c. Sterile Disposable syringes and Yes/No/NA
needles (various sizes)
d. Sterile disposable IV sets Yes/No/NA
 Guidelines for the Preparation of Application for Grant/Renewal... 401

e. Adrenaline, Noradrenaline, Mephen- Yes/No/NA

tin, Betamethasone (or dexametha-
sone), Metoclopramide injections
M Accessories
a. Blankets, emesis basins, haemostats, Yes/No/NA
set clamps, sponge forceps, gauze,
dressing jars, waste cans, etc. 401
b. Medium cotton balls, 1.25 cm adhe- Yes/No/NA
sive tapes
c. Denatured spirit, Tinc. Iodine, green Yes/No/NA
or liquid soap
d. Paper napkins or towels Yes/No/NA
N. Laboratory Equipment
a. Refrigerator for kits and reagents
storage
Refrigerator make and Capacity Yes/No/NA
Temperature display provided Yes/No/NA
b. Compound microscope with low & Yes/No/NA
high power objectives
c. Table centrifuge Yes/No/NA
d. Water bath- 37°–57° Yes/No/NA
e. Rh viewing box Yes/No/NA
f. Incubator with thermostat Yes/No/NA
g. Mechanical shakers for serological Yes/No/NA
test of syphilis test
h. Hand lens Yes/No/NA
i. Serological graduated pipettes of vari- Yes/No/NA
ous sizes
j. Pasteur pipettes Yes/No/NA
k. Glass slides Yes/No/NA
l. Test tube of various sizes/microplates Yes/No/NA
m. Precipitating tubes (6 × 50 mm) of Yes/No/NA
various sizes
n. Test tube racks Yes/No/NA
o. Interval timer Yes/No/NA
p. Material and equipment for glassware Yes/No/NA
cleaning
q. Blood transporting containers Yes/No/NA
r. Wash bottles Yes/No/NA
402 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

s. Filter papers Yes/No/NA

t. Dielectric tube sealer Yes/No/NA
u. Plain and EDTA vials Yes/No/NA
v. Chemical balance Yes/No/NA
w. ELISA reader with printer, washer and Yes/No/NA
micro-pipettes
x. Colorimeters/hemoglobinometer Yes/No/NA
(strike off which is not applicable) for
hemoglobin determination
O Records and Reports
Comments on
records, if any
a. Blood Stock Register Yes/No/NA
b. Blood donor record Yes/No/NA
c. Issue register Yes/No/NA
d. Record of blood bags Yes/No/NA
e. Cross-matching records Yes/No/NA
f. Register of diagnostic reagents and Yes/No/NA
kits
g. Adverse reaction records Yes/No/NA
h. Stock register of other consumable Yes/No/NA
articles
i. Are records destroyed? Yes/No/NA
j. Labels of blood containers as per Yes/No/NA
Schedule F of the D and C Act
P Outdoor camps
a. Eligible to hold outdoor camps Yes/No/NA
b. Average number of camps held per Yes/No/NA
month
c. Vehicle available? Yes/No/NA
d. How are blood bags transported Yes/No/NA
e. Proof sanitary conditions of camps Yes/No/NA
f. Detailed statement of blood collected Yes/No/NA
in camps
 Guidelines for the Preparation of Application for Grant/Renewal... 403

Processing of Blood Components from Whole Blood

Q Accommodation/Premises Comments
1. Area provided for component preparation.
2. Is an additional 10 sq. meter area provided Yes/No/NA
for apheresis procedures?
a. Is blood components room air-conditioned? Yes/No/NA
403
b. Is blood component room well-lighted? Yes/No/NA
c. Are walls and floors smooth and washable? Yes/No/NA
d. Are overall hygienic conditions maintained Yes/No/NA
in the premises?
e. Comments on area
R. Personnel Yes/No/NA
Whether technical supervisor with basic
qualification and experience is available
with the blood bank?
Name, Qualification and Experience

S1 Equipment Make/Model/
(as per GSR 245(E) dated 05.08.99) Capacity
i. Air-conditioner Yes/No/NA
ii. Laminar air flow bench Yes/No/NA
iii. Suitable refrigerated centrifuge Yes/No/NA
iv. Plasma expresser Yes/No/NA
v. Clipper and clips and/or dielectric sealer Yes/No/NA
vi. Weighing device Yes/No/NA
vii. Dry rubber balancing material Yes/No/NA
viii. Artery forceps, scissors Yes/No/NA
ix. Refrigerator maintaining a temperature Yes/No/NA
between 2°C to 6°C, a digital
dial thermometer with recording
thermograph and alarm device, with
provision for continuous power supply.
x. Platelet agitator with incubator Yes/No/NA
(wherever necessary)
xi. Deep freezers maintaining temperature Yes/No/NA
between –30° to –40°C and –75° to
–80°C
xii. Refrigerated water bath for plasma Yes/No/NA
thawing
404 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

xiii. Insulated blood bag containers with Yes/No/NA

provisions for storing at appropriate
temperature for transport purposes
xiv. Whether components are prepared only Yes/No/NA
in a closed system using single/double/
triple or quadruple plastic bags

S2 Equipment (GMP) Comments

1. Are equipments located in logical Yes/No/NA
sequence and permit effective cleaning?
2. Is equipment calibrated/validated Yes/No/NA
periodically?
T Preparation of Blood Components
Comments
1. Concentrated Human RBC’s (Packed
Red Blood Cells)
a. Whether SOP is available for Yes/No/NA
preparation of PRC?
Specify:
• Source material
• Method
• RCF
• Speed
• Time
b. Is blood collected from suitable donor? Yes/No/NA
(Check the donor record)
c. Are the packed red cells conforming to Yes/No/NA
the standard of I.P.1996?
d. How the pilot tubes/samples are Yes/No/NA
numbered?
e. Whether pilot tube is attached in a Yes/No/NA
tamper proof manner to the unit?
f. Who is responsible for filling of pilot Yes/No/NA
samples?
g. Whether pilot samples are filled Yes/No/NA
immediately after the blood is collected
or at the time the final product is
prepared?
h. Whether expiry is assigned as per Yes/No/NA
norms?
(Specify the period)
 Guidelines for the Preparation of Application for Grant/Renewal... 405

2. Platelets Concentrates Comments

a. Whether SOP is available for preparation Yes/No/NA
of platelets concentrates?
Specify:
• Source material
• Method
• RCF
• Speed
405
• Time
b. Whether the whole blood/source Yes/No/NA
material is stored at 20° to 24°C after
collection, before processing to platelet
concentrates?
c. Whether platelet concentrates are Yes/No/NA
separated within 6 hours after the time
of collection of whole blood/source
material?
d. Whether platelet concentrates are Yes/No/NA
tested:
• Platelet count
(Note the Count)
• Ph (not less than 6)
• Measurement of plasma volume
• Sterility
(1% of total platelets prepared shall be
tested for sterility, functional viability,
i.e. swirling movements)
e. Whether compatibility test prepared on Yes/No/NA
every unit before issue
e. Whether platelet yield is calculated Yes/No/NA
(1% of total platelets prepared shall be
tested of which 75% of the units shall
conform to the standards)?
3. Fresh Frozen Plasma (FFP) Comments
a. Whether SOP is available for Yes/No/NA
preparation of FFP?
Specify:
• Source material
• Method
• RCF
• Speed
• Time
b. Whether deep freezers capable of Yes/No/NA
maintaining temperature between
–75°to –80°C are available?
406 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

c. Whether the source material/human Yes/No/NA

blood stored at 4°C till processed?
d. Whether thawing facilities are provided Yes/No/NA
(Note the thawing temperature)?
e. Lag time between collecting of blood Yes/No/NA
and processing of FFP (Check records)
4. Cryoprecipitate Comments
a. Whether SOP is available for preparation Yes/No/NA
of cryoprecipitate?
Specify:
• Source material
• Method
• RCF
• Speed
• Time
b. Whether thawing facilities are available? Yes/No/NA
(Note the temperature.)
c. Whether anti-hemophiliac factor activity Yes/No/NA
is tested?
(NLT 80 units/bag),(1% of the total cryo
prepared shall be tested of which 75%
shall conform to specifications)
5. Apheresis Procedure
a. Whether cell separator facility provided? Yes/No/NA
b. Whether donor is certified fit for Yes/No/NA
apheresis (Check the record)
c. Time allowed between successive Yes/No/NA
apheresis on a single donor
d. Whether protein estimation of donor is Yes/No/NA
carried out if serial apheresis is to be
conducted?
e. Whether inquiries about aspirin intake Yes/No/NA
made before platelet apheresis?
f. Whether RBCs are retransfused during Yes/No/NA
platelet apheresis or leucopheresis? If
not, what precautions are taken?
g. Whether the following tests are carried
out before apheresis procedures:
 Guidelines for the Preparation of Application for Grant/Renewal... 407

Name of test Acceptance

criteria
i. Hemoglobin/Hematocrit
ii. Platelet count
iii. WBC count
iv. Differential count
v. Serum proteins 407
h. How much quantity of plasma is to be
collected (Plasmapheresis)
Duration Limit
Single sitting Not
exceeding
500 ml
Per month Not
exceeding
1000 ml
U. Storage of Blood Components
S. Blood Components Temperature Duration/
No. Expiry Period
1. FFP
2. Cryoprecipitate
3. Platelet concentrate
4. PBRC
5. Granulocyte concentrate
V. Records and Labels Comments
1. Whether details of quantity supplied, Yes/No/NA
compatibility report, details of receipts
and signatures of the issuing person
mentioned in the component record?
2. Whether master record for component
and issue register is mentioned as per
norms (GSR 245E dated 05-04-1999)?
3. Whether labels for components are
prepared as per norms (GSR 245E
dated 05-04-1999)?
4. Whether all details on labels are filled
by the responsible person on the final
container?
408 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Observations, irregularities and recommendations

Signature and Designations of Inspecting Officers

 Guidelines for the Preparation of Application for Grant/Renewal... 409

Names of Technical Personnel to be Endorsed on the License Copy:

S. Personnel Name Qualification Experience Testimonials
No
1. Medical Officer
Incharge(s)

409

2. Registered Nurse(s)

3. Blood Bank Technicians

4. Technical Supervisor(s)
 C H A P T E R 73
Judicial Pronouncements–
Blood Safety

1. P v. Union of India (2001)—Kolkata High Court (Negligence in

blood transfusion):
The petitioner, a pregnant lady was admitted for delivery of her child
at a hospital under the administrative control of the Indian Navy. After
delivery, the petitioner required blood transfusion. A sailor donated
fresh blood to hospital, which did not come from the blood bank of the
hospital as required under the provisions of the Drugs and Cosmetics
Act. The sailor’s blood was not tested for HIV at the time of donation. He
was later found to be HIV+. It was clear that the petitioner became HIV+
on account of the negligent transfusion of blood to her.
The court felt that since the hospital was under administrative
control of the Indian Navy, the Indian Navy had a duty to compensate
the petitioner. Correspondence was exchanged between the petitioner
and respondent Indian Navy during the pendency of the petition. Finally
the respondent made an offer of compensation which included:
a. A Government job at Kolkata or the place where she desired
b. Accommodation on her appointment on the usual terms and
conditions.
c. A sum of Rs. 10 lakhs from the date of filing of the writ petition
@ 18% interest.
d. Medical treatment at the cost of the Government
The petitioner was agreeable to the offer and the Court passed the
judgment in terms of the compromise arrived at by the parties.
2. M. Chinnaiyan v Sri Gokulam Hospital and Queen Mary’s
Clinical Laboratory (National Consumer Dispute Redressal
Commission, 2006):
The Appellant’s wife underwent a hysterectomy operation, at the
1st Respondent hospital in 1990 where she was transfused 2 units of
blood in the postoperative period which was procured from the 2nd
Respondent laboratory in 1990. In mid-1994, the, Appellant’s wife
developed recurrent loose motions, weight loss, respiratory infection
and difficulty in swallowing, etc. On being tested, she was found to
 Judicial Pronouncements–Blood Safety
411411

be HIV+ and showed symptoms of full-blown AIDS. In July 1995, she

developed left-sided hemiparesis, oral candidiasis and TB. Later she was
diagnosed with glioma of the brain and died in August 1995.
Her husband filed a complaint before the State Consumer Redressal
Forum suing the hospital and pathology laboratory for deficiency
of services under the Consumer Protection Act. His complaint was
rejected. Aggrieved by this order, he appealed to the National Consumer
Dispute Redressal Commission (National Commission). The National
Commission held:
a. The 1st respondent gave blood transfusion without obtaining
the consent of the patient and that the concerned doctor
negligently transfused blood, as he did not inform the
Petitioner’s wife about the benefits, risks or alternatives of
blood transfusion, which amounted to deficiency of service
under the Consumer Protection Act.
b. Furthermore, the Drugs and Cosmetics Rules, 1945, requires
that every licensee of a blood bank gets samples of every blood
unit tested for freedom from HIV antibodies, which the 2nd
respondent had failed to do.
c. As compensation, the Commission awarded Rs. 4,00,000 (Rs.
4 lakh) with interest at the rate of 6% pa from the date of filing
the complaint, which was to be paid jointly and severally by the
Respondents and Rs 10,000 as costs.

An appeal by one of the respondents to the Supreme Court was

dismissed.
412 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

I (2008) CPJ 205 (NC)

NATIONAL CONSUMER DISPUTES
REDRESSAL COMMISSION, NEW DELHI
Hon’ble Mr Justice MB Shah, President and Mrs. Rajyalakshmi Rao,
Member
Deo Kumar Singh—Appellant
versus
CBP Sinha (DR.)—Respondent
First Appeal No. 314 of 2004—Decided on 11.12.2007

Consumer Protection Act, 1986—Section 2(1)(g)—Medical Negli­

gence—Wrong blood report—Complainant’s wife having blood group
Rh Negative reported to be Rh Positive by OP—Pregnancies aborted
despite treatment given—Rh factor plays extremely important role
during pregnancy—Woman is at risk when she has Negative Rh factor
and husband has Positive Rh factor—Complications can be prevented
by appropriate medication only when Rh factor of mother is correctly
known—Appropriate treatment could not be given due to wrong blood
report about Rh factor—Giving report about Rh factors in casual manner
has to be condemned—Deficiency in service proved—Compensation
granted.
Testing blood type and Rh factor are basic and fundamental aspects
of blood test and Pathological Laboratories are required to be extremely
careful since the wrong report can make the difference between life and
death. Giving the report about these factors in a casual manner has to be
condemned since the Rh factor plays an extremely important role in the
case of the pregnant woman.
In view of the above discussion, this appeal is allowed. The order of
the State Commission is set aside. The respondent is directed to pay a sum
of Rs. 25,000 towards compensation to the complainant, within a period
of two months from that long, failing which the amount of Rs. 25,000 will
carry interest at the rate of 9% pa In the facts and circumstances of this
case, there shall be no order as to costs.
Appeal allowed
 Judicial Pronouncements–Blood Safety 413
413

II (2000) CPJ 439

UNION TERRITORY CONSUMER DISPUTES
REDRESSAL COMMISSION, CHANDIGARH
Hon’ble Mr Justice JB Garg, President;
Dr PK Vasudeva and Mrs Devinderjit Dhatt, Members
Jaspal Singh and ANR.—Complainants
versus
The Post Graduate Institute of Medical Education and
Research [PGI], Chandigarh and ANR.—Respondents
Complaint No. 12 of 1997—Decided on 1.2.2000

Consumer Protection Act, 1986—Sections 2(1)(g), 14(1)(d)—Medical

Negli­gence—Mismatching of blood—Deficiency in Service—Com­
pensation—Complainant’s wife suffered burn injuries to the extent
of 50%—Admitted to PGI Chandigarh—Patient’s blood group was A+,
transfused B+ group blood—Kidney damaged due to wrong transfusion
of blood—Mismatching of blood and wrong transfusion confirmed—
PGI Chandigarh liable to pay compensation.
After hearing the learned counsel for the parties and perusing
the relevant records with their assistance including evidence of the
parties and other documents on record, this Commission has come to
the conclusion that there has been serious deficiency and negligence
on the part of the PGI and its attending doctor (s)/staff for transfusing
wrong blood group to the patient which caused death of the wife of
complainant No. 1. Mis-matching of blood has been confirmed by the
Senior Resident in the Death Summary also. Once the patient is brought
to the PGI or any other Institute of Health Care, the background/history,
if any, for example that the patient was maltreated by the husband, does
not absolve the hospital from its professional obligations. The Post-
graduate Institute, (PGI), Chandigarh is, therefore, liable to pay a sum
of Rs. 2.00 lacs to the complainants out of which 3/4th shall be put in
the fixed deposit in favor of minor Amandeep Singh son of Jaspal Singh,
complainant No. 2, and ¼th be paid to the complainant No. 1 (husband)
a Government employee. The PGI shall also pay costs of Rs. 5,000/- to
the complainants. It is, however, upto the PGI Authorities to recover this
amount from the erring doctor(s)/staff.
Complaint allowed
414 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

I (2006) CPJ 136 (NC)

NATIONAL CONSUMER DISPUTES
REDRESSAL COMMISSION, NEW DELHI
Hon’ble Mr SN Kapoor, Presiding
Member and Mr BK Taimni, Member
Dr K Vidhyullatha—Appellant
versus
R Bhagawathy—Respondent
First Appeal No. 379 of 2001—Decided on 25.1.2005

i. Consumer Protection Act, 1986—Sections 2(1)(g) and 14(1)(d)—

Medical Negligence—Wrong blood transfusion after hysterec­tomy
operation resulted in intravascular hemolysis and renal failure—
Opposite party denied treatment to patient—Evidence that on
developing complications patient was referred to another hospital
where she had to undergo hemodialysis 8 times—Deficieny in
service proved on part of OP in transfusing wrong blood—Order of
Forums below awarding compensation upheld, amount reduced to
Rs. 1,00,000 from Rs. 1,50,000 and interest to 9 per cent from 12 per
cent.
(Paras 12, 13, 19)
ii. Res ipsa loquitur—Applicability—Complaint regarding wrong
blood transfusion after hysterectomy operation resulting intra-
vascular hemolysis and renal failure—Complainant not able to
pass urine after surgery—Principle of res ipsa loquitur attracted—
Deficiency in service proved.
In Harjot Ahluwalia (Minor through his parents) v. M/s. Spring
Meadows Hospital and Others, II (1997) CPJ 98 (NC), the Hon’ble
Supreme Court has observed:
“That the staff attending on the patient discharge their duties and
work on the patient under the control, guidance and supervision of the
consultant/doctor under whose treatment, care and supervision the
patient is admitted.”
While it is true as submitted by the learned Counsel for the appellant
that the compensation could not be awarded on the basis of mere
allegation. But amount of compensation in almost all cases is decided
on the basis of fair estimation. While we think that for the mental tension
and agony and acute renal failure, the learned State Commission might
have justifiably estimated the total compensation amounting to Rs.
1, 00,000, for we take the expenditure of Rs. 1,000 per day for hemodialysis
it would not exceed Rs. 15,000 plus overhead expenses ranging from 7,000
 Judicial Pronouncements–Blood Safety 415
415

to Rs. 7,500 in all. We feel that in the aforesaid circumstances, it would be

appropriate to award a sum of Rs. 1, 00,000 in all as compensation with
interest @ 9% p.a. from the date of complaint till the date of deposit of
a sum of Rs. 1, 50,000. The complainant shall be entitled to receive the
amount of Rs. 1,50,000 (sic) which is said to have been deposited with
the Commission along with interest which would have accrued thereon
and adjust the same towards the amount payable as aforesaid. With the
aforesaid modification, the appeal stands disposed of.
Appeal disposed of accordingly
416 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

II (2007)CPJ 395 (NC)

NATIONAL CONSUMER DISPUTES
REDRESSAL COMMISSION, NEW DELHI
Hon’ble Mr Justice KS Gupta,
Presiding Member and Dr PD Shenoy, Member
Susheel Kumar—Petitioner
versus
Dr Virendra Mahla and ANR.—Respondents

Revision Petition No. 798 of 2007—Decided on 11.7.2007

Consumer Protection Act, 1986—Sections 2(1)(g), 21(b)—Medical
Negli­gence—HIV reports—Patient operated for appendix—blood test
taken—Report showed patient HIV-Positive—Further investigations
done by other Hospitals—Patient found to be HIV-Negative—Complaint
filed for mental agony—Compensation awarded—Order reversed on
appeal—Hence revision—Test done by first Hospital (OP) from HIV
Niwa Kit of Cadila—Patient advised to co-relate result by Western Blot
Technique—Same not done—Harrison’s Book of Principles of Internal
Medi­cines referred—No deficiency in service on part of OP—Impugned
order upheld.
In the order under challenge the State Commission has quoted
two paras from Harrison’s Book of Principles of Internal Medicines.
Combined reading of these paras would show that the standard
screening test for HIV infection is the ELISA which is an extremely
good screening test with a sensitivity of 99.58%; Commercial use of EIA
kit by most of the diagnostic laboratories is not optimal with regard to
specificity and, therefore, it must be confirmed with a more specific
assay. It is not the case of petitioner that test through Western Blot
Technique was got done by him after 3.12.2002. Considering the contents
extracted above the aforesaid report dated 19.12.2003 does not help the
petitioner. To be noted that in aforementioned report dated 10.12.2003
the basis for reaching the conclusion in regard to petitioner being HIV-
Negative has not been disclosed. In this backdrop, we do not find any
merit in the contention advanced by Ms. Aishwarya Bhati, Advocate
about respondent being deficient in service for giving the wrong report.
There is no illegality or jurisdictional error in the order passed by State
Commission warranting interference in revisional jurisdiction under
Section 21(b) of the Consumer Protection Act, 1986. Revision petition is,
therefore, dismissed.
Revision Petition dismissed
 Judicial Pronouncements–Blood Safety 417
417

(Equivalent Citation:- 2005 (2) CPC 599 (NC))

IV (2005) CPJ 84 (NC)
NATIONAL CONSUMER DISPUTES
REDRESSAL COMMISSION, NEW DELHI
Hon’ble Mr Justice KS Gupta, Presiding
Member; Mrs. Rajyalakshmi Rao, Member
P Kallianikutty Amma and ORS—Complainant
versus
Unity Health Services (P) Ltd and ORS.—Opposite Parties
Original Petition No. 181 of 1996—Decided on 14.7.2005

Consumer Protection Act, 1986—Section 2(1)(g)—Medical Negli­

gence—blood transfusion—Alleged, contaminated blood trans­ fused
causing adverse reaction, which ultimately led to death—Allegation
not proved—No evidence produced to show that blood was improperly
stored from time of procurement till time of administration—blood
transfusion took place much before expiry date—Mild adverse reaction
properly attended to in time—Absence of expert evidence in support of
allegations—Medical negligence not proved—Complaint dismissed.
The complaint is not that the blood obtained from the blood bank was
not cross-matched with the patient’s blood. The complaint essentially
is that the blood obtained from the blood bank got contaminated at
the Unity Hospital and that the contaminated blood was administered
to the patient causing adverse reaction which ultimately led to death.
The crux of the argument of the opposite parties is that there was no
contamination in the blood and that though there was a mild adverse
reaction to the blood transfusion, it was properly attended to in time
and that the rest of the blood transfusion took place in an uneventful
manner. Their case is that the patient was suffering from dysfunctional
uterine bleeding for at least 14 months before she came to the Unity
Hospital and that septicemia took place because of the progression of
the disease leading to her death and that there was no negligence.
We also found that there is no substance in the argument of the
complainant that they had not given consent for blood transfusion. It
has come on record that the husband of the deceased enquired about
the availability of blood from the Royal Hospital from where it was
subsequently obtained. If there was no implied consent, he would not
have gone around making inquiries about the availability of blood for
transfusion.
In addition to all these, as mentioned above, after the death
of the patient, the Delhi based brother of the deceased; Shri P
Chandrashekharan filed a number of complaints before various
418 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

authorities alleging negligence and malafides against the opposite

parties. Firstly, on 23.5.1995 he filed a Criminal Complaint No. 246/1995
under Section 304-A of the IPC. This complaint was investigated by the
police and recorded by judicial order (filed as closed) as investigation
showed that there was no negligence and that the patient died
because of septicemia. Thereafter the Drug Controller, Govt. of Kerala,
Thiruvananthapuram was approached on 11.8.1995 alleging that the
blood obtained from the Royal Hospital was improperly stored and
hence got contaminated. The report of the Drug Controller dated
27.9.1995 clearly held that no evidence has been produced to show that
the blood was improperly stored from the time of procurement till the
time of administration.
As a result, the case of negligence against the opposite parties has
not been proved. The complaint stands dismissed.
Complaint dismissed
 Judicial Pronouncements–Blood Safety 419
419

IV (2005) CPJ 261(NC)

NATIONAL CONSUMER DISPUTES
REDRESSAL COMMISSION, NEW DELHI
Hon’ble Mr Justice MB Shah,
President and Mrs Rajyalakshmi Rao, Member
Supriya Gupta—Complainant
versus
Trustees of Beach Candy
Hospital and Research Centre—Opposite Party
Original Petition No. 7 of 1997—Decided on 27.10.2005

Consumer Protection Act, 1986—Section 2(1)(g), 21— Medical Negli­

gence—Surgery conducted—Patient contracted Hepatitis ‘B’—Alleged,
infection has arisen from OP’s hospital during blood transfusion at OP’s
Hospital or due to use of improperly sterilized medical equipment—
Absence of evidence in support of allegations—Fresh blood taken from
relatives and friends transfused—One unit given from hospital blood
bank which was not Hepatitis infected proved—Fresh and disposable
cardiac catheters were used on complainant—Infection caused from
OP’s hospital due to negligent procedure not proved—Complaint
dismissed.
420 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

IV 2007 CPJ 157(NC)

NATIONAL CONSUMER DISPUTES
REDRESSAL COMMISSION, NEW DELHI
Hon’ble Mr Justice SN Kapoor,
Presiding Member and Mr BK Taimni, Member
Chandigarh Clinical Laboratory—Petitioner
versus
Jagjeet Kaur—Respondent
Revision Petition No. 1377 of 2003—Decided on 30.8.2007

(i) Consumer Protection Atct, 1986—Section 2(1)(g)—Medical Negli­

gence—Clinical laboratory—Wrong blood test report given—Failure
on part of petitioner to take due care to return correct finding—
Whether harm caused to patient or not would not be a criteria—
Deficiency in service proved—Compensation awarded by Forum
affirmed by State Commission—No interference required in revision.
Very briefly stated the facts of the case are that the complainant Mrs.
Jagjeet Kaur was taken to the petitioner laboratory for getting her blood-
group checked up and the report was given of her having blood group
AB+. The blood-group report was required as she had been advised
blood-transfusion, for which she was transferred to GGS Medical
College and Hospital, where again blood sample was collected and it
gave a report of the complainant’s blood belonging to AB (—). It is in
these circumstances, a complaint was filed before the District Forum
alleging ‘medical-negligence’ who after hearing the parties and perusal
of material on record, directed the petitioner to pay a compensation of
Rs. 25,000 cost of Rs 2,000 to the complainant. Aggrieved by this order,
an appeal was filed before the State Commission, which was dismissed,
hence this revision petition before us.
We have no doubt that the petitioner is a qualified pathologist but
the ‘duty of care’, required in such case to give a correct finding, which
was not given in this case, is a clear instance of medical negligence on
the part of the petitioner.
The negligence stands proven in this case by the admitted fact
that the petitioner gave the report of blood group of the complainant
belonging to AB+ whereas in fact it was AB(—) which has not been
disputed by the petitioner. Whether harm came to the patient or not
would not be a criteria. It is the failure on the part of the petitioner to
 Judicial Pronouncements–Blood Safety 421
421

take due care to return a correct finding, that is at the heart of issue and
in which the petitioner completely failed.
In view of above, we do not find any ground to interfere with the
well-reasoned order passed by the District Forum and affirmed by the
State Commission. The revision petition has no merit, hence dismissed.
422 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Post Graduate Institute of Medical Education and Research,

Chandigarh v. Jaspal Singh (DK Jain and RM Lodha, JJ)

Post Graduate Institute of Medical Education and Research,

Chandigarh ____________________________ Appellant
versus
Jaspal Singh and ORS. ____________________ Respondent (s)

Civil Appeal No. 7950 of 2002, decided on May 29, 2009

The judgment of the court was delivered by RM Lodha, J.

1. In this appeal by special leave, the appellant, Post/Graduate
Institute of Medical Education and Research, Chandigarh (for short,
‘PGI’) has challenged the order dated September 29, 2000 passed by
the National Consumer Disputes Redressal Commission (for short,
“National Commission”). By its order, the National Commission
dismissed the appeal filed by PGI under Section 21 of the Consumer
Protection Act, 1986 (for short, `Act, 1986’) and affirmed the order
passed by the State Consumer Disputes Redressal Commission,
Chandigarh (for short, `State Commission’) whereby it directed
the PGI to pay compensation in the sum of Rupees two lacs to the
respondents 1 and 2 herein (for short, ‘the complainants’) and cost of
Rs. 5,000/-.
2. The brief facts of the case are thus:
On March 30, 1996, Smt. Harjit Kaur (wife of complainant No.
1 and mother of complainant No. 2) received accidental burns
while making tea on the stove. She sustained 50% TBSA III burns
involving upper limbs, part of trunk and most of both lower limbs.
Smt. Harjit Kaur was taken to Daya Nand Medical College and
Hospital, Ludhiana, immediately where she responded to the
treatment well. She remained admitted in Daya Nand Medical
College and Hospital up to April 19, 1996. Since the treatment
at Daya Nand Medical College and Hospital was expensive, the
complainant No. 1 decided to shift his wife to PGI for further
treatment. On April 19, 1996, Smt. Harjit Kaur was admitted in
PGI, Chandigarh. Dr Varun Kulshrestha, Senior Resident Doctor,
Department of Plastic Surgery attended to her. The condition of
Smt. Harjit Kaur started improving at PGI. On May 15, 1995, she
was transfused A+ blood which was her blood group. On May 20,
1996, the patient was transfused B+ blood group in the afternoon
although her blood group was A+. On the night of May 20, 1996,
 Judicial Pronouncements–Blood Safety 423
423

the urine of the patient was reddish like blood and the attendant
nurse was informed accordingly. As to the bad luck of Smt. Harjit
Kaur, on the next day, i.e., May 21, 1996 again one bottle of B+ blood
group was transfused although her blood group was A+. Because of
transfusion of mismatched blood, the condition of Smt. Harjit Kaur
became serious; her hemoglobin levels fell down to 5 mg. and urea
level went very high. Later on, it transpired that due to transfusion of
mismatched blood, the kidney and liver of the patient got deranged.
The complainant No. 1 made a written complaint to the Head of
the Department of Plastic Surgery for mismatched transfusion of
blood to the patient whereupon an inquiry was conducted through
senior doctor and wrong transfusion of the blood to the patient was
found. The condition of Smt. Harjit Kaur started deteriorating day
by day and she ultimately died on July 1, 1996. In the complaint
before the State Commission, the complainants alleged that the
death of Smt. Harjit Kaur was caused due to the negligence of
Dr Varun Kulshrestha and the medical staff at PGI; that there was
negligence in the discharge of service by the PGI and its doctors and
they claimed damages to the tune of Rupees nine lacs for the loss of
life of Smt. Harjit Kaur.
3. Dr. Varun Kulshrestha filed reply to the complaint. He principally set
up the plea that although the patient was transfused wrong blood
but it was not due to any negligence on his part. He stated that due
to the care exercised by him and the other nursing staff, the patient
became alright and her hematological and biochemical parameters
became almost normal and she recovered from mismatched
blood transfusion. It was stated in his reply that Smt. Harjit Kaur
died of septicemia and not by mismatched blood transfusion and,
therefore, the complaint was liable to be dismissed.
4. Insofar as PGI is concerned, no reply to the complaint was filed
separately but they adopted the reply filed by Dr Varun Kulshrestha.
The parties filed their respective affidavits and also produced before
the State Commission the summary report and the documents
concerning treatment of Smt Harjit Kaur.
5. The State Commission after hearing the parties and upon
consideration of the materials made available to it, came to the
conclusion that there was serious deficiency and negligence on the
part of PGI and its attending doctor (s)/staff in transfusion of wrong
blood group to the patient which resulted in death of Smt Harjit
Kaur. The State Commission in its order dated February 1, 2000 held
that PGI was liable to pay sum of Rupees two lac to the complainants
out of which 3/4th was to be put in the fixed deposit in favor of the
424 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

minor son Amandeep Singh (complainant no. 2) and 1/4th amount

to be paid to the complainant No. 1. The State Commission also
awarded the cost of Rs. 5000/-. 6. PGI chall­en­ged the order of the
State Commission in appeal before the National Commission but
without any success.
7. The learned counsel for PGI raised the same contentions before us
which were raised before the National Commission that the cause of
death of Smt Harjit Kaur was septicemia and not mismatched blood
transfusion. He would submit that Smt. Harjit Kaur recovered from
mismatched blood transfusion given to her on 20th and 21st May,
1996; her hemoglobin level was brought up and her vital organs
started functioning normal. The learned counsel would submit that
Smt Harjit Kaur died due to burn injuries and the other connected
reasons arising out of said injury and not due to mismatched blood
transfusion and, therefore, no negligence can be attributed to the
hospital and the attending doctor/s. He relied upon two decisions of
this Court namely (i) Jacob Mathew v. State of Punjab and Another 1
(2005) 6 SCC and (ii) Martin F D’Souza v. Mohd. Ishfaq.2 (2009) 3 SCC
8. The term negligence is often used in the sense of careless conduct.
Way back in 1866 in Grill vs. General Iron Screw Collier Co.3(1866)
L.R. 1 C.P. 600 at 612, Wills J. referred to negligence as “ ......... the
absence of such care as it was the duty of the defendant to use.”
9. Browen L.J. in Thomas v. Quatermaine 4 (1887)18 Q.B.D. 685 at 694
stated, “... idea of negligence and duty are strictly correlative, and
there is no such thing as negligence in the abstract; negligence is
simply neglect of some care which we are bound by law to exercise
towards somebody”.
10. In Donoghue v. Stevenson 5 (1932) A.C. 562 at 618-619, Lord
Macmillan with regard to negligence made the following classic
statement:
“The law takes no cognizance of carelessness in the abstract. It
concerns itself with carelessness only where there is a duty to take
care and where failure in that duty has caused damage. In such
circumstances carelessness assumes the legal quality of negligence
and entails the consequences in law of negligence. The cardinal
principle of liability is that the party complained of should owe
to the party complaining a duty to take care, and that the party
complaining should be able to prove that he has suffered damage in
consequence of a breach of that duty.”
11. In Jacob Mathew1 this Court while dealing with negligence as tort
referred to the Law of Torts, Ratanlal and Dhirajlal, (24th Edn., 2002
edited by Justice GP Singh) and noticed thus:
 Judicial Pronouncements–Blood Safety 425
425

“Negligence is the breach of a duty caused by the omission to do

something which a reasonable man, guided by those considerations
which ordinarily regulate the conduct of human affairs would do,
or doing something which a prudent and reasonable man would
not do. Actionable negligence consists in the neglect of the use of
ordinary care or skill towards a person to whom the defendant owes
the duty of observing ordinary care and skill, by which neglect,
the plaintiff has suffered injury to his person or property. ... the
definition involves three constituents of negligence: (1) A legal duty
to exercise due care on the part of the party complained of towards
the party complaining the former’s conduct within the scope of the
duty; (2) breach of the said duty; and (3) consequential damage.
Cause of action for negligence arises only when damage occurs; for,
damage is a necessary ingredient of this tort.”
12. Insofar as civil law is concerned, the term negligence is used for the
purpose of fastening the defendant with liability of the amount of
damages. To fasten liability in criminal law, the degree of negligence
has to be higher than that of negligence enough to fasten liability for
damages in civil law.
13. In Syed Akbar v. State of Karnataka 6 (1980) 1 SCC 30, this Court
dealt with in details the distinction between negligence in civil
law and in criminal law. It has been held that there is a marked
difference as to the effect of evidence, namely, the proof, in civil and
criminal proceedings. In civil proceedings, a mere preponderance
of probability is sufficient, and the defendant is not necessarily
entitled to the benefit of every reasonable doubt; but in criminal
proceedings, the persuasion of guilt must amount to such a moral
certainty as convinces the mind of the Court, as a reasonable man,
beyond all reasonable doubt.
14. In Bhalchandra Waman Pathe v. State of Maharashtra 7 1968 ACJ 38,
this Court held that while negligence is an omission to do something
which a reasonable man, guided upon those considerations which
ordinarily regulate the conduct of human affairs, would do, or doing
something which a prudent and reasonable man would not do.
15. With regard to the professional negligence, it is now well settled
that a professional may be held liable for negligence if he had
not possessed of the requisite skill which he professed to have
possessed or, he did not exercise, with reasonable competence in
the given case the skill which he did possess. It is equally well settled
that the standard to be applied for judging, whether the person
charged has been negligent or not; would be that of an ordinary
person exercising skill in that profession. It is not necessary for
426 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

every professional to possess the highest level of expertise in that

branch which he practises.
16. In Jacob Mathew1 as well as Martin F D’Souza2, this Court quoted
with the approval the opinion of MacNair, J in Bolam v. Friern
Hospital Management Committee 8 (1957) 2 All ER 118(QBD):
“[W]here you get a situation which involves the use of some
special skill or competence, then the test as to whether there has
been negligence or not is not the test of the man on the top of a
Clapham omnibus, because he has not got this special skill. The test
is the standard of the ordinary skilled man exercising and professing
to have that special skill. A man need not possess the highest expert
skill ... It is well-established law that it is sufficient if he exercises
the ordinary skill of an ordinary competent man exercising that
particular art.”
17. In Hucks v. Cole9 (1968) 118 New LJ 469, Lord Denning stated that
a medical practitioner would be liable only where his conduct fell
below that of the standards of a reasonably competent practitioner
in his field.
18. Lord President (Clyde) in Hunter v. Hanley10 1955 SLT 213
observed that the true test for establishing negligence in diagnosis
or treatment on the part of a doctor is whether he has been proved
to be guilty of such failure as no doctor of ordinary skill would be
guilty of, if acting with ordinary care.
19. In their classic work, `on professional negligence (fifth edition)’,
Jackson and Powell state that mistakes made in the course of
treatment may be purely physical; purely intellectual or they may
fall somewhere between the two. Whichever form the mistake
takes, there are two separate questions to consider: (i) whether the
defendant made a “mistake”; (ii) if so, whether the mistake was one
which a reasonably careful and skilful, medical practitioner would
not have made. The claimant must, of course, succeed on both
questions in order to establish negligence.
20. It needs no emphasis that in the medical negligence actions, the
burden is on the claimant to prove breach of duty, injury and
causation. The injury must be sufficiently proximate to the medical
practitioner’s breach of duty. In the absence of evidence to the
contrary adduced by the opposite party, an inference of causation
may be drawn even though positive or scientific proof is lacking.
21. `The Physiological Basis of Medical Practice (Eight Edition)’ by
charles H. Best and Norman B. Taylor in Chapter 26 deals with
transfusion; blood groups. In respect of incompatible transfusions,
while dealing with its effects, it is stated that if blood of the wrong
 Judicial Pronouncements–Blood Safety 427
427

(incompatible) ABO blood group is transfused, a hemolytic

transfusion reaction usually results red cells are destroyed and
there may be jaundice with hemoglobinemia and hemoglobinuria.
Chills, fever and shock may occur. Renal insufficiency may ensue
believed by some to be due to a reduced blood flow through the
glomeruli.
22. The patient, Harjit Kaur, got burn injuries to the extent of 50% on
March 30, 1996. She was initially treated at Daya Nand Medical
College and Hospital, Ludhiana, for about 20 days. Her condition
improved satisfactorily at Daya Nand Medical College and Hospital.
She was admitted to PGI, Chandigarh on April 19, 1996. The
available material placed before the State Commission shows that
at the time of her admission, Smt. Harjit Kaur was taking medicine
orally and passing urine; 75% of eschar was removed by May 1,
1996. Her condition had substantially improved at PGI before
May 20, 1996 and she had no signs of septicemia. It was only after
mismatched blood transfusion B+ on two consecutive days, i.e.,
20th and 21st May, 1996, that she became anemic (her hemoglobin
level was reduced to 5 per gram) and her kidney and liver were
deranged. It is true that her hemoglobin was brought up in a few
days but her condition otherwise got deteriorated. Although she
survived for about 40 days after mismatched blood transfusion but
from that it cannot be said that there was no causal link between
the mismatched transfusion of blood and her death. Wrong blood
transfusion is an error which no hospital/doctor exercising ordinary
care would have made. Such an error is not an error of professional
judgment but in the very nature of things a sure instance of medical
negligence. The hospital’s breach of duty in mismatched blood
transfusion contributed to her death, if not wholly, but surely
materially. Mismatched blood transfusion to a patient having
sustained 50% burns by itself speaks of negligence. Therefore, in the
facts and circumstances of the case, it cannot be said that the death
of Smt. Harjit Kaur was not caused by the breach of duty on the part
of the hospital.
23. The State Commission observed:
“..... that there has been serious deficiency and negligence on
the part of the PGI and its attending doctor(s)/staff for transfusing
wrong blood group to the patient which caused death of the wife
of complainant No. 1. Mismatching of blood has been confirmed
by the Senior Resident in the Death Summary also. Once the
patient is brought to the PGI or any other Institute of Health Care,
the background/history, if any, for example that the patient was
428 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

maltreated by the husband, does not absolve the Hospital from its
professional obligation... ...”
24. Affirming the aforesaid view of the State Commission, the National
Commission held thus:
“..... It is seen that the patient’s kidney was damaged and the
blood level reached to 100 gms. Percentage, hemoglobin came
down to 5 mg. after the mismatched blood transfusion was given
by the Doctor in the said Hospital. It was only after the complainant
gave the written complaint to the hospital regarding the wrong
transfusion of blood given to the patient, an inquiry was made and it
was found correct. The damage control treatment started only after
the written complaint was given by the complainant. Though it is
argued by the Counsel for the Appellant that the percentage levels
were brought down to normal, it is very clear to us that the internal
imbalances of liver and kidney functioning and deteriorating
hemoglobin levels started only after the mismatched blood
transfusion was given. Though septicemia has been written as the
ultimate cause of death, the patient’s health took a nose dive only
after wrong blood was given to her and this is clearly negligence on
the part of the Doctors of the Hospital which the appellants cannot
disown or absolve themselves....”
25. We concur with the view of the National Commission as it does not
suffer from any error of law.
26. In the result, the appeal fails and is dismissed with costs which we
quantify at Rs. 20,000/-.
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2. Anthony R Green, 6th edn. 2011.
3. A Victor Hoffbrand, Daniel Catovsky, Anthony R Green, Edward GD
Tuddenham. Postgraduate Haematology. Wiley Blacwell. 6th edn, 2010.
4. Chitnis V, Vaidya K, Chitnis DS. Biomedical waste in laboratory medicine:
Audit and management. Indian Journal of Medical Microbiology Vol.
23, No. 1, January-March, 2005; pp. 6–13.
5. Blood collection: Routine venepuncture and specimen handling:
http://library.med.utah.edu/WebPath/TUTORIAL/PHLEB/PHLEB05.
html 4.
6. Drugs and Cosmetic Act 1940 and Rules 1945.
7. Guidelines for setting up blood storage centers NACO Ministry of
Health and Family Welfare, Govt. of India, New Delhi 2007.
8. Guidelines for the clinical use of blood cell separators by Joint working
party of the transfusion and clinical hematology task forces of the British
Committee for standards in Haematology. Clin Lab Haem 1998;20:
265–78.
9. Hemovigilance Program-India, Asian Journal of Transfusion Science
Vol. 7, Issue 1 January-June 2013.
10. Manual on the management, maintenance and use of blood cold chain
equipment, WHO Geneva, 2005.
11. Model Standard Operating Procedures for Blood Transfusion Service-
WHO New Delhi.
12. Modern Blood Banking and Transfusion Practices by Denise M
Harmening, 5th edn. 2005.
13. National Guidebook on Blood Donor Motivation (MoH & FW, NACO,
GOI)-2nd edn. 2003.
14. Practice of safe Blood Transfusion-Compendium of Transfusion
Medicine by Dr RN Makroo, 2nd edn. 2009.
15. Quality Control: http://whatis.techtarget.com/definition/0,sid9_
gci112738200.html.
16. Screening donated blood for transfusion-transmissible infections-
Recommendations WHO, 2010.
17. Standards for blood banks and blood transfusion services, NACO
Ministry of Health and Family Welfare, Govt. of India, New Delhi, 2007.
434 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

18. Technical Manual of American Association of Blood Banks-16th edn.

2008.
19. Technical Resource Manual for Hospital Transfusion Services, BC
Provincial Blood Co-ordinating Office, 2nd edition, 2005.
20. Textbook of Medical Laboratory Technology by Praful B Godkar, 2nd
edn. 2003.
21. The Blood Cold Chain-Guide to selection and procurement of
equipment and accessories WHO Geneva 2002.
22. The Clinical Use of Blood (Handbook) WHO Geneva.
23. The User Manuals of Blood Bag Monitor and Tube Sealer.
24. Transfusion Medicine Technical Manual (DGHS) 1991.
25. Treatment of discarded blood units: Disinfection with hypochlorite/
formalin versus steam sterilization by V Chitinis, S Chitnis, S Patil,
D Chitnis-Department of Microbiology, Choithram Hospital and
Research Centre, Indore, India, Indian Journal of Medical Microbiology
Year: 2003, Volume: 21, Issue: 4, Page: 265–67.
26. Zarin Bharucha, Chauhan DM. Introduction to Transfusion Medicine.
1st edn. 1990.
 Glossary
• Absorption: The removal of antibodies from serum/plasma through
the addition of red blood cells having corresponding antigen.
• Adsorption: The uptake of antibody onto the specific receptors on
the red blood cell (RBC) surface under optimal conditions, therefore,
removing the antibody from the serum.
• Ambient Temperature: Means atmospheric temperature of the
immediate surroundings.
• Amplitude of the Agitation: Means side-to-side movement of the trays
in a platelet agitator. The amplitude is expected to be within the range
of 3.6 to 4.0 cm.
• Apheresis: Means the process by which blood drawn from a donor,
after separating plasma or platelets or leukocytes, is retransfused
simultaneously into the said donor.
• Atypical Antibody: An antibody which occurs as irregular a feature in
the serum of some individual, whose red cells lack the corresponding
antigen.
• Autoantibody: An atypical antibody that sensitizes or agglutinates own
red cells.
• Biomedical Waste: Waste generated during the diagnosis, treatment
or immunization of human beings or animals or in research activities
pertaining thereto or in the production and testing biological products.
• Blood Cold Chain: Means continuous maintenance of mandatory
storage conditions (e.g. 2 to 6°C for whole human blood) from the
point of collection/preparation to the point of use of blood and its
components.
• Blood: Means and includes whole human blood, drawn from a donor
and mixed with an anti-coagulant.
• Blood Bank: Means a place or organization or unit or institution or
other arrangements made by such organization, unit or institution for
carrying out all or any of the operations for collection, to be returned
for apheresis, storage, processing and distribution of blood drawn
from donors and/or for preparation, storage and distribution of blood
components.
• Blood Component: Means a drug prepared, obtained, derived or
separated from a unit of blood drawn from a donor, e.g. red cells,
platelet concentrates or fresh frozen plasma, etc.
• Blood Product: Means a drug manufactured or obtained from pooled
plasma of blood by fractionation, drawn from the donors.
• Cross-matched Blood: Donor’s whole blood or its components
matched with the blood of the recipient.
430 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

• Donor: Means a person who voluntarily donates blood after he has

been declared medically fit after medical examination, for donating
blood, on fulfilling the criteria given hereafter, without accepting in
return, any consideration in cash or kind from any source, but does not
include a professional or paid donor.
• Defrost Cycle: Process of removing of excess ice from plasma freezer
cabinets without change in the temperature of the freezer. Whenever,
such frost or ice builds up in the plasma freezer cabinets it requires
to be removed to avoid excessive running of the compressor. Modern
freezers have an automatic defrost cycle. The temperature of the cabinet
should not rise during the defrost cycle.
• Elution: The removal of antibody from the RBC surface. Total elution
removes the antibody coating the RBCs and destroys the antigens to
which they were attached. Partial elution removes the antibody, but
allows the antigen to remain intact.
• Eluate: A fluid medium containing the antibodies that have been
deliberately removed from RBCs, allowing for antibody identification.
• FIFO Policy: First-in-first-out policy.
• Incomplete Antibody: Any antibody which sensitizes red cells
suspended in saline but fails to agglutinate them. These antibodies
react in albumin/enzyme medium or with AHG serum.
• Major Cross-match: Test to determine compatibility between donor
serum and patient’s red cells.
• Minor Cross-match: Test to determine compatibility between patient’s
serum and donor red cells.
• Mixed Field Agglutination Reaction: A reaction wherein a few cells are
agglutinated while many cells are unagglutinated.
• Prozone Phenomenon: Negative reaction of antibody in low dilution
and positive reaction in higher dilution of the same antibody.
• Processed Blood: Blood which has been processed into components.
Generally refers to the red cell component. The essential tests may or
may not have been done.
• Professional Donor: Means a person who donates blood for a valuable
consideration, in cash or kind, from any source, on behalf of the
recipient-patient and includes a paid donor or a commercial donor.
• Quality Assurance: As part of the overall quality management program,
the range of activities and systems that provide confidence within the
organization and for the authorities that all quality requirements are
met.
• Quality Control: A component of quality management, these are tests
put in place to ensure that processes, procedures and products meet
the quality requirements.
 Glossary 431
431

• Quarantine: Temporary storage in isolation. For example, unprocessed/

untested blood is kept in isolation (not accessible for use) until all
essential processes/tests are completed.
• Rouleaux Formation: A form of pseudo-agglutination in which the red
cells look like pile of coins.
• Replacement Donor: Means a donor who is a family friend or a relative
of the patient/recipient.
• Standard Operating Procedure (SOP): Written instructions for the
performance of a specific procedure.
• Stroke: Number of times the tray of the platelet agitator moves from
side to side per minute; 65 to 75 strokes per minute are considered
adequate.
• Screening: Preliminary testing.
• Sensitivity: Probability that the test result will be reactive in an infected
individual.
• Serum: Fluid portion of clotted blood.
• Specificity: Special affinity between an antigen and its corresponding
antibody.
• Validation: Confirmation and provision of objective evidence that
the requirements for a specific intended use or application have been
fulfilled. It is a part of quality assurance system that evaluates in advance
the steps involved in operational procedures or product preparation to
ensure quality, effectiveness and reliability.
• Wharton’s Jelly: Mucoid connective tissues that make the matrix of the
umbilical cord.
• “30 Minute Rule”: General rule in the blood bank stating that a
maximum time of 30 minutes is allowed for a blood or its component
issued from the blood bank to a ward to be returned.
 435

Index

Page numbers followed by ‘f ’ refer to figure and ‘t’ refer to table

A test
ABO direct, 96
blood grouping, 57 indirect, 89, 133
confirmation of, 111 Anti-HIV testing, 158, 160
discrepancies Antihuman
and possible resolution, 74 globulin reagent, quality control
chart, resolving, 73 of, 233
resolving, 65 globulin serum, 96
group discrepancy, resolution of, 63 Apheresis set, installation of, 246
Acute reactions, 206 Apply cold compresses to forehead
Adhesive tapes, 26, 29 and back, 32
Adrenaline, 31 Applying cuff, 272
Agglutination, grading of, 61 Arterial puncture, 53f
AHG reagent, 97, 100 Artery forceps, 22, 26
Air Atropine sulfate, 31
embolus, 288 Autoclave, 327
handling systems, 370 Automatic operation, 256, 275
Air-borne particulate for manufacture of
sterile products, 370t B
Alarm test, screen display during, 251f Bacterial infection, 288
Albumin, 354 Bag after centrifuge, 42f
technique for Rh typing, 81 Bandages, 32
Allergic Band-aids, 32
balm, 32 Bench centrifuge, 224
reactions, 286 Betnovate ointment, 32
tablets, 31 Bevel of needle against vein wall, 52f
transfusion reaction, 206 Binometer, 327
Anaphylactic transfusion reaction, 206 Biomedical waste management, 290
Antibody screening, 87 Blood
antenatal mother, 123 and components, preservation
Anti-coagulant of, 174
blood samples of donors, 84 assurance of, 213
delivery of, 283 bag
Anti-globulin contains RBC, 39f
reagent, quality of, 233 for blood components, selection
technique, indirect, 94 of, 25
436 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

labeling of, 172 criteria for, 330

selection of, 24 deferment of, 331
total weight of, 217 donor
types of, 24 deferment of, 10t
banks, 323 questionnaire and informed
accommodation for, 324 consent form, 11
alternative technologies in, 104 record, 335
and release, 360 selection, criteria for, 8
of umbilical cord blood flow, return, 276
derived stem cells, 365 group, interpretation of, 61
deep freezers, 225 grouping, 352
operate, 378 license, conditions of, 364
refrigerator, 37, 225 pressure, checking of, 32
technician(s), 4 processing, 360
borne viruses, infection requirements for, 365
control of, 305 products, 186, 211
borne viruses, occupational health components including, 311
measures to prevent transmission manufacture of, 360
of, 305 transfusion of, 194
cells, packed, 36 quality
cold chain from collection to assurance, 214, 215-217
transfusion, 175 control of, 213
collecting CPD-A bags, 26 records for, 335
collection, 26, 360 requisition form for, 190
cycle, 275 return of whole, 181
monitor, 26, 224 safety and infection control, 50
requirements for, 365 sample
room, for, 332 collection, pre-requisites for, 46
compatibility test of, 343 for cross-matching, 46
components, 323, 335 for grouping, collection of, 46
and manufactured, 5 safegaurds/preventions
assurance of various, 214 during, 49
categories of, 340 stops flowing into tube– 52
from whole blood by blood bank, storage, 360
processing of, 339 cabinets, 225
issue of, 352 centers, 350, 381
requisition form for, 190 requirements for, 365
separation, 36 testing, 360
concentrate samples, 260 of whole, 334
containers for, 332 requirements for, 365
cross-matching, 353 transfusion of, 194
cycle don’ts for, 188
control, 275 reaction reporting form for, 211
return, 276 record/reaction form, 192
donation typing, 326
camps, 337 unit
consent for, 12 before transfusion, 185
 Index 437
437

checking, 184 Conjunctiva, 296

inventory of, 176 Control cuvette, 19
issue of, 178 Controls for Rh grouping, 79
physical checking of, 184f Convulsions, 32
traceability of, 34 Coomb’s
transport of, 178 cross-match, 127
Bovine albumin technique, 93 phase, antibody screening in, 121
BP apparatus, 22 serum, 97, 100
test
C direct, 96, 132, 134, 141
indirect, 100, 132, 136
Calcium tablets, 31
tubes
Cell
for patient pre-transfusion, 88
and serum results, discrepancies
prenatal testing, 88
between, 62, 116
Coplin jar, 15
control, 97, 100
Copper sulphate
grouping, 353
record book, 15
preparation of packed, 40
solution, 15
Centrifugation, 41f
method, 15
Centrifuge, 326
Cord blood
Chemiluminescent immunoassays, 143
bank technician, 370
Citrate toxicity, 283
processing, 369
symptoms of, 284
area, 367
treatment of, 284
reception, 367
Clean wooden sticks, 79
release, 375
Clinical thermometer, 32
stem cells
Clotted blood sample of
banking and release of
donors/patients, 92
umbilical, 363
patients, 84
process umbilical, 363
Code of injury, algorithm to determine
store umbilical, 363
exposure, 297
test umbilical, 363
Cold agglutinins, detection of, 142
umbilical, 360, 362
Collapsed vein, 52f
Cryobath, 226
Color coding
Cryogenic storage unit, 368
for components, 38f
Cryo-poor-plasma, 44
for segregation of bio-medical
Cryoprecipitate, 343
waste, 291
preparation of, 43
in blood bank, 292
thawing bath, 37
Community health center, 355
Crystalline CuSO4, 15
Compatibility testing, 91
Cysteine in frozen state, 92
Complete collection or no blood
obtained, 51
Component D
blood, 213 DCT/DAT, interpretation of, 98
laboratory, 292 Deaeration of concentrate bags and
preparation, 36f collection of samples, 260, 279
supplied, records of, 335 Deep freezer –40°C and –80°C, 37
438 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Demethylated spirit, 22 ELISA

Dextrose, 31 method, 155, 160, 166
Diazepam, 31 test for syphilis, 150
Direct antiglobulin test, 110, 132, 201 washer, 224
Disposal Emergency equipments/items, 333
box, 58, 76, 84, 88 Emergent/life-threatening
of used ID cards, 109 circumstances, 195
of waste and infectious material, 366 Enzyme
Distilled water, 15, 17 immunoassays, 143
Donor method, 354
additional qualifications of, 9 papaine, deep freezer to store, 92
and collecting pre-donation sample, phase, antibody screening in, 125
connecting, 254, 272 technique, 93
care during and after apheresis, 281 Epinephrine, 31
couch, 26 Equipment
for platelet apheresis, assessing contamination, 288
suitability of, 235 maintenance, 220
room, 218, 292
screening, 14
F
for hemoglobin, 15
station, 225 Febrile non-hemolytic reaction, 206
values, screen display for adding, Fever with septic shock, 207
254f, 273 First aid
Double bag for preparation of PRC administration of, 296
and FFP, 38f tray, 26
Dressings, 32 Fresh frozen plasma, 25, 36, 40, 343
Drugs and Cosmetics Rules Furesemide, 31
1945 (Part X-b), 314
1945, and guidelines, recent G
amendments in, 358
Gauze sponges, 46
1945, extract of schedule K
Gel cards
under, 347
centrifuge for, 220
2011, 2nd amendment, 358
quality control of, 233
2011, 3rd amendment, 359
Gel test, advantages of, 109
Dry
Getting blood from blood bank, 183
bath, 75, 84
Glass
rubber balancing material, 37
slide, 58, 76, 79, 84, 88, 92
Du weak antigen, testing for, 119
method, 58, 59
test tube, 58
E method, 60
E cryoprecipitate, 216 technique, 80
EDTA blood sample, 169 Glassware, 15
of hemoglobin concentration, 15 Glucocorticoid, 31
Electrolyte replacement fluid, 31 Glucose, 31
Electronic weighing scale, 37 saline, 31
 Index 439
439

Graft-versus-host disease, 208 I

Granulocyte concentrates, 343 IAT method, 354
ID microtyping system, 108
H ID-centrifuge, 106
H reference samples, 373 6S, 106f
HBV, 302 ID-incubator, 105
HCV, 302, 303 37 SI, 105f
antibodies, testing for, 163, 166 IGG coated cells, 97, 100
Healthcare worker or injured Inadequate citration, 284
Incompatibility, resolution of, 95
person, 302
Incubator, 57, 63, 75, 84, 223
Heart failure, congestive, 207
for gel cards, 221
Hemagglutination, 143
Infection control
Hematocrit, 326
nurse, 295
Hematology laboratory, 367 officer, 295
Hematoma, 33 Inflate cuff display, 272f
formation problem in older Informed consent, 10, 194
patients, 53 Infusion set, 32
prevention of, 49 Install set, display for, 264
under skin, 53f Installation of set, screen display after
Hemo-control photometer, 19 continuing on, 249f
Hemoglobin Iron overload, 209
by hemo-control, estimation of, 19
determination, for, 332 J
of donor, estimation of, 17
Judicial pronouncements–blood
Hemolysis, 287 safety, 410
Hemolytic transfusion reaction, 206
Hemovigilance program, 209
L
Heparin and benzyl nicotinate
ointment, 32 Laminar flow bench, 37
Hepatitis B Lamivudine, 299
prophylaxis for reported sharps Lancet, 17
Landsteiner’s law, 57
injuries, 301t
Leaflet for post-donation
surface antigen, testing for, 153, 155
instructions, 29
HIV, 303
Leukapheresis, 343, 344
status code, determining, 298
Licence for blood
HLA typing laboratory, 367 banking and release, 362
Hospital Infection Control collection, 362
Committee, 295 processing, 362
Human blood, operation of whole, 355 storage, 362
Human red blood corpuscles, testing, 362
concentrated, 340 License to collect umbilical cord blood
Hyperkalemia, 208 stem cells, 363
Hypocalcemia, 207, 283 Low-ionic strength saline
Hypothermia, 208 solution, 55, 64, 89, 97, 100
440 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

M O
Machine malfunction, complications Observation during transfusion, 192
due to, 287 Oxygen cylinder, 32
Management of sharps, 294 with accessories, 26
Menu screen for calculated
values, 255f, 274f P
Menus, 254 Pan malaria, 169
Metoclopramide, 31 Paracetamol, tablet 31
Microcuvettes, 19 Particle agglutination assays, 143
Microplate, 58 Pasteur pipettes, 17, 58, 76, 79, 84,
elisa reader, 223 88, 92
Microtubes, 58, 76, 84, 88 Pheniramine maleate, 31
Microwell plate, 58 Phlebotomy site, preparation of, 22
Minor cross-match, 128 Pilot
Monitoring transfused patient, 186 samples, 340
Monoclonal anti-D tubes, plain and edta, 26
reagents, 79 Plasma
saline agglutination test, using, 80 bath, 226
Mucocutaneous exposure to blood or collection of, 344
other body fluids, 301t additional, 257
expresser, 37
N fresh frozen, 216
or saline, 84
NACO for post-exposure prophylaxis, pheresis, 343, 344
recommendations of, 300 room, 292
National Blood Policy, 312 Plastic beakers, 80
Needle Platelet, 25
angle with surface of arm, 49f agitator
destroyer, 26 and incubator, 226
not in lumen of vein, 51f with incubator, 37
penetrated too far, 51f apheresis donor selection 237
stick concentrate, 40, 215, 341
incidents and exposure and set, removing 260, 279
incidents, 294 donation, selection of program
injury, 303 for, 245, 263
sharps injury or cut, 296 pheresis 344
Negative using blood cell separator
antibody screen, causes of dual needle 243
incompatible cross-match in single needle, 261
presence of, 94 poor plasma, 42f
test, 81 program, selection of, 244, 262
Newborn, ABO/Rh/DAT of, 140 rich plasma, 39, 42
Non-governmental organizations, 311 using double bags, 38
Non-intact skin, 296 separation, screen display
Nucleic acid amplification technology after completion of, 258f, 277
assays, 143 during, 256f, 275
 Index 441
441

Platepheresis 343 Records and store room, 368

Polyclonal human anti-D serum, 79 Red blood cells, 25
Positive test, 81 frozen, packed, 215, 341
Post-transfusion purpura, 208 Red cell
Post-donation care, 29, 289 preparation of packed, 38
Post-exposure prophylaxis, 299 reagents, quality control of, 232
Post-transfusion urine sample, serology laboratory, 218
tests on, 204 suspended in native serum, 84
Povidone iodine solution, 22 suspension, preparation of, 55
Prepare separation, screen display unit, packed, 181
for, 253f Red top vacutainer, 47f
Pressure test 250, 268 Redressal Commission, 412, 414, 416,
Pre-transfusion, 196 419, 420
testing, 91 Refrigerated centrifuge, 37
Prevent hemolysis, 50 Refrigerator, 63
Primary health center, 355 Register for diagnostic kits and reagents
Priming used, 335
of set, screen display after, 252f Registered
preparing for, 266 medical practitioner, 178
screen display during, 252f nurse(s), 5
Print out, display end of procedure Regulatory
for, 280 guidelines, 309
requirements of blood, 311
Prochlorperazine maleate, 31
Reinfusion, 257, 278
Program, screen display for selection
end of, 278
of, 263
screen display
Protect yourself, 50
during, 259f
PRP to bag 2, 41f
guiding for start of, 279
Pulse
Reverse grouping
checking of 32
of donor, 113
determination, for
with weak or missing reactions, 69
temperature and, 332 Rh blood grouping, 78
of donor, 111
Q Rh Du
Quality control tests, frequency of, 229 blood grouping, 83
grouping, interpretation of, 85
Rh view box, 80
R
Rubber teats, 58, 76, 80, 84, 88, 92
Rapid
antigen test for malaria 169
S
tests, 143, 153
SAGM
TP test, 148
solution, double bags or triple bags
tri-dot method, 158, 163
with, 37
RBCs in bag, packed, 39f
to bag 1, 42f
Reactions in blood donors, management
Sahli’s
of adverse, 31
hemoglobinometer, 17
Reagents, quality control of, 228, 233
method, 17
442 Standard Operating Procedures and Regulatory Guidelines-Blood Banking

Saline Sterile
addition and replacement connecting device, 37
technique, 102 cotton
method, 353 gauze/swabs, 22, 26
technique, 93 swabs, 17
Sample and materials required, 111 disposable
Savlon solution, 32 hypodermic needles, 26
Scenario of legal framework, 312 syringes, 26
Scheme for quality control, 214 gauze/cotton, spirit, 19
Scissors, 26 swabs, 29
Screening tests, 372 testing laboratory, 367
Separation Sterilization-cum washing, 368
preparing for, 253, 271 Sterilizing tray, 22
screen display for starting, 255f Suitability of donor, 340
Serology laboratory, 367 Syphilis
Serum
screening for, 146
and Coomb’s tubes, racks to
test for, 148
hold, 88, 92
grouping, 353
tubes, 58, 76, 84, 88, 92 T
racks to hold, 84 Table top centrifuge, 57, 63, 75, 84, 92
Sexually transmitted diseases, 148
machine, 55
Sharp instruments, management of, 306
Temperature, 326
Skin around venipuncture site,
Terminating reinfusion, 259
eczematous reactions of 33
Test
Skip priming, 268
kit, 169
Slide method for
tube, 15, 63, 79
blood grouping, 59f
rack for, 58, 63, 80
Rh blood grouping, 81f
stand, 15
SN-bag
empty, 278 Testing elute, 77
screen display for stopping, 278 Tetany/muscular spasm/twitching, 33
Sodium Thermometers, laboratory, 327
bicarbonate, 31 Thrombophob ointment, 29
chloride, 31 Tissue paper, 15
Software version display, 244f, 262f Tongue depressor, 32
Sops Tourniquet, 46
contents of, 6 Transfer bags, 37
use of, 7 Transfusion of
Spirit of ammonia, 32 reactions, investigation of, 198
Spirit wipes, 46 related lung injury, 206
Splash to mucous membrane, 296 right blood to right patient, 183
Standard operating procedure, 8 transmissible
Start of reinfusion, screen display disease screening laboratory, 367
guiding for, 258f infections testing, 218
Starting separation, screen display transmitted
for, 274 diseases, 209
 Index 443
443

infections, 143 Vein selection, 48

quality control of kits for Venipuncture performance of, 48
testing, 234 Venipuncture, 26
screening for, 143 site selection, 47
Treponema pallidum, 148, 150 Visual inspection, 201
hemagglutination, 148 Vitamin C tablets, 31
Triple bag system Vomiting, 32
for preparation of packed cells, 40f
with or without additive solution, 40
Troubleshooting guidelines, 51 W
Tube sealer, 26, 37, 225 Warming blood, 186
Washed packed red cell, 44
U Waste
Umbilical cord blood generated, types of, 290
component segregation, 292
collection of processed, 368 Water
storage of processed, 368 bath, 75, 327
stem cells, 362 distillation still, 37
Urinometer, 15 Weighing scale, 15
White tile method, 58
Whole blood, 214
V
Wooden blocks to hold
Vacutainers, 46 microtubes, 80, 84, 88
purple top, 47f
Vascular access, complications of, 287
Vasovagal, 285 Z
syndrome, 32 Zidovudine, 299