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Hematology Manual Procedure - Sop 2022

The document outlines the standard operating procedures for conducting a white blood cell (WBC) count and platelet count in a hematology laboratory. It includes details on specimen collection, handling, safety precautions, equipment, quality control, and the methodology for performing the tests. The document also provides normal value ranges for WBC and platelet counts, as well as limitations and references for further reading.

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0% found this document useful (0 votes)
44 views26 pages

Hematology Manual Procedure - Sop 2022

The document outlines the standard operating procedures for conducting a white blood cell (WBC) count and platelet count in a hematology laboratory. It includes details on specimen collection, handling, safety precautions, equipment, quality control, and the methodology for performing the tests. The document also provides normal value ranges for WBC and platelet counts, as well as limitations and references for further reading.

Uploaded by

rachellabelong00
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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You are on page 1/ 26

ST.

CECILIA
DIAGNOS
TIC CLINIC
STANDARD OPERATING
PROCEDURE

HEMAT
OLOGY
LABORATORY PROCEDURE MANUAL for WHITE BLOOD CELL OCUNT (WBC)

WHITE BLOOD CELL


- A type of blood cell that is made in the bone marrow and found in the blood and
lymph tissue. White blood cells
- White blood cells are part of the body’s immune system. They help the body fight
infection and other diseases
- Types of white blood cells are granulocytes (neutrophils, eosinophils and basophils),
monocytes and lymphocytes.
- Also called leukocyte

1.0 INTRODUCTION
- The objective of this procedure is to check the number of white blood cells in the
blood and is usually part of a complete blood cell (CBC) test, to accurately count
WBC in chamber, to perform reliable dilution of blood cells and to calculate the
number of cells/ µL
- It may be used to look for conditions such as infection, inflammation, allergies and
leukemia

2.0 TEST PRINCIPLE


* Whole blood collected in EDTA is diluted according to the type of cell count obtained
* The diluted blood suspension is then placed in a chamber and the cell counted
* The count is multiplied by dilution factor and reported as number of cells per
Microliter (µL) of the whole blood

3.0 SAFETY AND ENVIRONMENTAL PRECAUTIONS


- all human blood and other potentially infectious material (OPIM) are to be treated as
infectious and handled according to “standard precautions” Therefore, all personnel handling
specimens or other materials should wear appropriate Personal Protective Equipment’s (PPEs)
Which include but not limited to: Gloves, Gowns, Masks.

4.0 SPECIMEN
4.1 PATIENT PREPARATION: If room temperature EDTA blood is being used the blood
should be place on the blood rocker for 10-15 minutes to allow adequate mixing of the
blood cells. If the blood was refrigerated, mix the tube for at least 20 minutes. Clean the
microscope lens cleaner and paper
4.2 COLLECTION

* Specimen type: Free flowing capillary or thoroughly mixed anticoagulated venous blood
* Preferred collection container: 2 mL Lavender-top (K2 EDTA) tube
* Alternate collection container: EDTA microcollection tubes
* Specimen Required: 2 mL whole blood or two (2) full microcollection tubes

NOTE: Draw a sufficient amount of blood with the required anticoagulant tube.
To achieve an optimum ratio of blood to anticoagulant, the volume of blood should fill
to the line indicated on the tube label.

4.3 HANDLING CONDITIONS


For Whole blood or EDTA

* Mix immediately after collection, before clotting can occur, gently mix the blood
collection tube by inverting 8 to 10 times
* Analyze the sample within 4 hours
* Ensure that the sample are at ambient temperature (20-25°C)
* If leukocyte count cannot be performed immediately after blood is diluted, store reservoir
at room temperature. Perform count within three (3) hours of making dilution

4.4 SAMPLE STORAGE


* Room temperature

5.0 EQUIPMENT AND MATERIALS

5.1 EQUIPMENT
- Microscope

5.2 MATERIALS
- Hemocytometer with Neubauer grid
- Cover glass
- Diluents

6.0 QUALITY CONTROL


* Test is performed in duplicate and results must agree within 10%. If not
repeat test
* At least one cell count control specimen must be analyzed, or a procedural
control employed for each 8 hours of patient testing
* If infrequently performed, perform prior to all patient testing
*This can be performed with a previously assayed patient sample or by
comparison with a visual blood film concentration estimate

7.0 PROCEDURE

7.1 METHODOLOGY
* Put the cover slip or glass slip on the top of grid area in the Chamber
(use air tight technique)
* Dilute your sample:
-1:20 for WBC count
-1:200 for RBC counts and Platelets
* Load your sample in to the loading area in the chamber
* Count the cells in the 4 Large squares for WBC
* Calculate the number of cells counted/µL

7.2 SAMPLE DILUTION


* Dilution of whole blood sample:

-Diluents:
* Acetic acid
* or Distilled water

-Purpose:
* Dilute the amount of WBC, RBC to be able to count it (NRBC: M
4.3-6.2 x 106 /µL) (F: 3.8-5.5 x 106 /µL)
(NR WBC: 4.3-10.8 x 103/µL)
*To lyses RBC and Platelets (the diluents lyses also the WBC but takes
longer time) (time factor is critical)

-Dilution:
* 1:20 dilution or 1:50 (ex: chronic leukemia)
* (1+19=20)
* (50 µL of blood + 950 µL diluent)

Loading the sample:


-WBC COUNT:
*The hemocytometer contains 2 Neubauer counting chamber

*Each chamber contains:


- 4 WBC counting squares
- Each contains 16 squares
100 RBC= 10 Platelets= 1 WBC

*Choose 90 ° lines, count only the cells that on those lines


(ex: L-shape)
 Apply it to all squares for maximum accuracy

8.0 CALCULATIONS

Cells/ µL =

Number of cells in 1 large square x Dilution factor


_________________________________________
Volume Factor (0.1)

 Dilution factor= reciprocal of dilution (20)


 Volume factor = (width x length x height)= 0.1

9.0 REPORTING OF RESULT

* ADULT:
The normal number of WBCs in the blood is 4,500 to 11,000 WBCs per microliter
(4.5 to 11.0 x 109 /L)
* 1 YEAR OLD:
The normal number of WBCs in the blood is 6,000 to 17,500 WBCs per microliter
(6.0 to 17.5 x 109 /L)
*NEWBORN:
The normal number of WBCs in the blood is 9,000 to 30,000 WBCs per microliter
(9.0 to 30.0 x 109 /L)

10.0 LIMITATIONS OF THE PROCEDURE


- a highly elevated leukocyte count may make accurate counting difficult. In this instance
a secondary dilution should be made. When calculating the total count, adjust the formula to
allow for secondary dilution

-In order to avoid increasing the imprecision of particle counting, an increased number
of hemocytometer squares must be enumerated when there is leukopenia or thrombocytopenia

11.0 REFERENCES

1. https://www.slideshare.net/Tshering_Namgyal_Wangdi/manual-total-leucocyte-
count-sop
2. https://kau.edu.sa/Files/0007058/Files/60503_WBC%20manual%20count%20using
%20hemocytometer.pdf
PROCEDURES:
Hemoglobin Determination ( Sahli’s Method )
Materials Needed:

 Hemometer
 Hemoglobin Tube
 Hemoglobin Pipette
 N/10 HCL

Procedure:

1. Place N/10 HCL into Hb tube upto 2 grams.


2. Blood sample in Sahli’s HB pipette upto 20 um litre.
3. Add blood sample to acid solution.
4. Mix with a stirrer.
5. Allow blood sample to acid solution
6. Mix with a stirrer
7. Allow to stand for 10 minutes.
8. Add distilled water drop by drop til
the color of the solution matches to brown glass standard
9. Take the reading of the lower meniscus from the graduated tube in grams.

Normal Value:

Male: 140-180 g/L

Female: 120-160 g/L

Hematocrit Determination (Packed Cell Volume)


Materials:

 Capillary tube
 Clay sealer
 Microhematocrit reader
 Microhematocrit centrifuge
 Sterile lancet

Procedure:

1. Clean the finger tips with 70% alcohol and let it dry.
2. Prick the finger with lancet, near the tip but not too close to the nail.
3. Place the tip of a capillary tube onto a drop of blood on your finger. The tubes should be filled ¾
full.
4. Seal the end of each tube with small amount of clay sealer at a 90 degrees angle.
5. Record the position number of each specimen.
6. Securely fasten the flat lid or cover plate from the centrifuge.
7. Centrifuge for 5 minutes at a set speed ( speed is about 10,000 g). This separates the RBC from
plasma and leaves a band of buffy coat consisting of WBC and platelets.
8. Read the result using the microhematocrit reader.

Normal Value:

Male: 0.41 – 0.53

Female: 0.37 – 0.47

Newborn: 0.48 – 0.60

Differential Count
Reagents:

Hema Quick Solution 1: Fixative

Hema Quick Solution 2: Eosin

Hema Quick Solution 3: Methylene blue

Hema Quick Solution 4: Wash Buffer

Procedure:

1. Transfer an amount of each solution in a coplin jar.


2. Prepare slides in the same way as preparing slides for differential cout.
3. Dip air dried smear five (5) times for about one second each dip into Solution 1, Solution 2, and
Solution 3.
4. Rinse the slide into solution 4.
5. Allow to dry.
6. Examine under oil immersion lens.

Results:

Erythrocytes…………….. Pinkish Color

Platelets…………………….Purple Granules

Polymorphonuclear Neutrophils:

Nucleus……………..dark blue

Cytoplasm…………pale pink

Granules……………reddish purple
Normal Value:

Neutrophils: 55-72%

Lymphocytes: 25-35%

Monocytes: 2-8%

Eosinophils: 1-3%

Basophils: 0-0.5 %

RBC count
Materials and Reagents:
 Anticoagulated whole blood ( using EDTA or heparin or capillary blood)
 Hayem’s solution
 Distilled water
 RBC micropipette
 Neubauer counting chamber
 Cell counter
Procedure:
1. Aspirate blood from collected sample into RBC pipette up to 0.5 mark.
2. Aspirate diluting Hayem’s solution to the 101 marks.
3. Hold the pipette horizontally and role it with both hands between finger and thumb.
4. Touch the tip of the pipette on the surface of the counting chamber 45 degrees angle.
5. Place the Neubauer counting chamber on the stage of the microscope and allow 2
minutes for the cell to settle.
6. Scan the counting area with the 10x objective lens.
7. Use 40x objective, including all cells lying on the upper and left lines of any square, omit
the cells on the lower and right-hand lines.
8. Count the cells in five groups of 16 small squares. Ie. 80 small squares.
Calculations:
The total number of red cells/ c.mm= NX 10 000.
N is the number of red cells found in 80 squares.
LABORATORY PROCEDURE MANUAL for PLATELET COUNT

1.0 INTRODUCTION
- The objective of this procedure is to measure the number of platelets in the blood.
Platelets are cells that help the blood clot. Too few platelets can be a sign of cancer,
infections or other health problems. Too many platelets put at risk for blood clots or
strokes. There are tens of thousands of platelets in a single drop of blood.

2.0 TEST PRINCIPLE


-Whole blood is diluted with a 1% ammonium oxalate solution.
-The isotonic balance of the diluent is such that all erythrocytes are lysed while the
leukocytes, platelets, and reticulocytes remain intact.
-The standard dilution for platelet counts is 1:100.
- This dilution is prepared using the leukocyte/platelet Unopette system.
- The dilution is mixed well and incubated to permit lysis of the erythrocytes.
- Following the incubation period, the dilution is mounted on a hemacytometer.
-The cells are allowed to settle and then are counted in a specific area of the
Hemacytometer chamber under the microscope.
- The number of platelets is calculated per µL (x 109 /L) of blood.

3.0 SAFETY AND ENVIRONMENTAL PRECAUTIONS


- All human blood and other potentially infectious material (OPIM) are to be treated as
infectious and handled according to “standard precautions” Therefore, all personnel handling
specimens or other materials should wear appropriate Personal Protective Equipment’s (PPEs)
which include but not limited to: Gloves, Gowns, Masks

4.0 SPECIMEN

4.1 PATIENT PREPARATION: If room temperature EDTA blood is being used the blood
should be place on the blood rocker for 10-15 minutes to allow adequate mixing of the
blood cells. If the blood was refrigerated, mix the tube for at least 20 minutes. Clean the
microscope lens cleaner and paper

4.2 COLLECTION

* Specimen type: Free flowing capillary or thoroughly mixed anticoagulated venous blood
* Preferred collection container: 2 mL Lavender-top (K2 EDTA) tube
* Alternate collection container: EDTA microcollection tubes
* Specimen Required: 2 mL whole blood or two (2) full microcollection tubes

NOTE: Draw a sufficient amount of blood with the required anticoagulant tube.
To achieve an optimum ratio of blood to anticoagulant, the volume of blood should fill
to the line indicated on the tube label.

4.3 HANDLING CONDITIONS


For Whole blood or EDTA

* Mix immediately after collection, before clotting can occur, gently mix the blood
collection tube by inverting 8 to 10 times
* Analyze the sample within 4 hours
* Ensure that the sample are at ambient temperature (20-25°C)
* If leukocyte count cannot be performed immediately after blood is diluted, store reservoir
at room temperature. Perform count within three (3) hours of making dilution

4.4 SAMPLE STORAGE


* Room temperature

5.0 EQUIPMENT AND MATERIALS

5.1 EQUIPMENT
- Microscope

5.2 MATERIALS
- Hemocytometer with Neubauer grid
- Cover glass
- Diluents
- 2 leukocyte/platelet Unopette reservoirs; each containing 1.98mL of the
following diluent:

Ammonium oxalate 11.45 g


Sorensen’s phosphate buffer 1.0 g
Thimerosal 0.1 g
QS with distilled water to 1 liter

- 2 Unopette capillary pipets, 20 µL


- Petri dish with filter paper
- hand counter
6.0 QUALITY CONTROL
- Commercial quality control materials with established control limits should be run
periodically. The frequency is determined by each laboratory’s workload. For instance, quality
control material may be run at the beginning of each eight hour shift.

7.0 PROCEDURE

7.1 Prepare two leukocyte/platelet Unopettes as follows:


a. Using the protective shield on the capillary pipet, puncture the diaphragm as
follows:

1) Place reservoir on a flat surface. Grasping the reservoir in one hand,


take the pipet assembly in the other hand and push the tip of the pipet
shield firmly through the diaphragm in the neck of the reservoir, then
remove.

b. Remove the shield from the pipet assembly with a twist and fill the capillary
pipet with whole blood. Transfer the whole blood to reservoir as follows:

1) Wipe excess blood from the outside of the capillary pipet, making
certain that no blood is removed from the capillary bore.

2) Squeeze the reservoir slightly to force out some air. Maintain pressure
on the reservoir.

3) Cover opening of overflow chamber of the pipet with your index finger

and seat the pipet securely in the reservoir neck.


4) Release pressure on the reservoir. Then remove your finger from the
pipet opening. Negative pressure will draw the blood into the diluent.

5) Squeeze the reservoir gently two or three times to rinse the capillary
bore, forcing diluent into, but not out of, the overflow chamber, releasing
pressure each time to return the mixture of the reservoir.

6) Place your index finger over the pipet opening and gently invert
several times to thoroughly mix the blood with diluent.
7) Let stand for 10 minutes to allow erythrocytes to hemolyze.

7.2 Clean the hemacytometer and cover glass by flooding them with 70% alcohol. Dry
thoroughly with gauze or tissue; do not allow the alcohol to dry on the hemacytometer.
Be sure to remove all lint. Place the cover glass in position over the ruled area.

7.3 Following incubation, mix diluted blood thoroughly by inverting reservoir to


resuspend cells. Charge hemacytometer as follows:
a. Convert to dropper assembly by withdrawing the pipet from the reservoir and
reseating it securely in its reverse position.

b. Clean the capillary bore by inverting the reservoir, gently squeeze the sides,
and discard the first three or four drops.

c. Place the pipet tip on the edge of the ruled area of the counting chamber.
Carefully charge the hemacytometer with diluted blood by gently squeezing the
sides of the reservoir to expel the contents until the chamber is properly filled.

d. Repeat procedure to charge the other side of the hemacytometer with the
first Unopette reservoir.

e. Place the hemacytometer on moistened filter paper in a Petri dish, and allow
to stand 10 minutes to permit the cells to settle

f. Using this same procedure, charge a second hemacytometer with the second
Unopette reservoir.

7.4 Carefully place the hemacytometer on the microscope stage. Perform cell count as follows:

a. With the low-power (10x) objective, locate the ruled area and the center large
square (1 mm2). Examine the entire center square for even distribution of platelets,
then carefully switch to the high-dry-power (40x) phase objective for counting
platelets. With phase microscopy, platelets appear as round or oval bodies.

b. Platelets are counted in the entire center large square (1 mm2) (Figure 7-7) as
follows:

1) Count the platelets in the first row of squares going from left to right,
then from right to left in the second row; follow this pattern until all rows
are counted.

2) Within each square, count all platelets touching the top and left-hand
borders. Do not count any cells touching the bottom or right-hand
borders.
3) Use the fine adjustment knob to focus up and down to identify the
platelets.

c. Repeat this counting procedure for the other side of the hemacytometer.

d. Record the counts for each center square. The difference between these two counts
should not exceed 10%.

e. Count the platelets on the second hemacytometer following the above counting
procedure.

Loading the sample:

Platelet Counting chamber:

8.0 CALCULATIONS

8.1 The calculation formula for hemacytometer cell counts determines the number of
cells within 1 µL (1 mm3) of blood (Figure 7-9). To make this determination, the total
number of cells counted must be corrected for the initial dilution of blood and the
volume of diluted blood used. The standard dilution of blood for platelet counts is
1:100; therefor the dilution factor is 100. The volume of diluted blood used is based on
the area and depth of the counting area. The area counted is 2 mm2 and the depth is
0.1 mm; therefore the volume factor is 0.2 mm3.

Total number of cells counted • dilution factor • 1/volume factor = cells/mm3


Cells/mm3 = cells/µL or cells/µL • 103 µL /L = cells x 109 /L

Example: 100 x 103 platelets/µL • 103 µL/L = 100 x 109 platelets/L

8.2 Average the platelet counts from the duplicate pipets and report result (x 109/L
or /mm3).

9.0 REPORTING OF RESULTS

10.0 LIMITATIONS OF THE PROCEDURE

 Unmixed blood sample


 Blood collection from capillaries (heal/finger prick) – results falsely low
platelet count however if we remove first blood drop we can minimize the
error
 If clumps or platelets present in the hemocytometer, the procedure should
be repeat
 Pipetting errors
 Improper mixing after dilution
 Uneven distribution of platelets
 Dirty counting chamber or cover glass
 Over filling or under filling of counting chamber
 Wrong counting techniques
 Intense illumination
 Wrong calculation

11.0 REFERENCES

1. https://aladdincreations.com/manual-platelet-count-test/

2. https://wps.prenhall.com/wps/media/objects/684/700987/
ch07MNLP.pdf#:~:text=MANUAL%20PLATELET%20COUNT%20Principle%20Whole
%20blood%20is%20diluted,The%20standard%20dilution%20for%20platelet
%20counts%20is%201%3A100.
MANUAL PROCEDURE for ABO BLOOD GROUPING

1.0 INTRODUCTION
The objective of this test is to apply blood typing method to determine the blood type for a
sample of synthetic human blood.

Blood grouping is the classification of blood based on the presence or absence of two inherited
antigenic substances on the surface of red blood cells (RBCs). The ABO and Rh are the major,
clinically significant and the most important of all the blood group systems.

The human ABO blood group system is divided into the following four major groups depending
on the antigen present on the surface of their red blood cells.

1.1 “A” group


1.2 “B” group
1.3 “AB” group
1.4 “O” group

2.0 TEST PRINCIPLE


The ABO and Rh blood grouping system is based on agglutination reaction. When red
blood cells carrying one or both the antigens are exposed to the corresponding antibodies they
interact with each other to form visible agglutination or clumping.

3.0 SAFETY AND ENVIRONMENTAL PRECAUTIONS


All human blood and other potentially infectious material (OPIM) are to be treated as
infectious and handled according to “standard precautions”. Therefore, all personnel handling
specimens or other materials should wear appropriate Personal Protective Equipment’s (PPEs)
which include but not limited to:
1. Gloves
2. Gowns
3. Masks

4.0 SPECIMEN

4.1 PATIENT PREPARATION: N/A

4.2 COLLECTION
* Specimen type: Whole blood
* Preferred collection container: 2 mL Lavender-top (K2 EDTA) tube
* Alternate collection container: EDTA microcollection tubes
* Specimen Required: 2 mL whole blood or two (2) full microcollection tubes

NOTE: Draw a sufficient amount of blood with the required anticoagulant tube.
To achieve an optimum ratio of blood to anticoagulant, the volume of blood should fill
to the line indicated on the tube label.

4.3 HANDLING CONDITIONS


For Whole blood or EDTA

* Mix immediately after collection, before clotting can occur, gently mix the blood
collection tube by inverting 8 to 10 times
* Analyze the sample within 4 hours
* Ensure that the sample are at ambient temperature (20-25°C)

4.4 SAMPLE STORAGE


* Samples stored 7 days at 2-8°C

5.0 EQUIPMENT AND MATERIALS

5.1 EQUIPMENT
* Serological centrifuge

5.2 REAGENTS
* Anti-A antisera
* Anti-B antisera
* A1 reagent red cells
* B reagent red cells
* Isotonic saline

5.3 SUPPLIES
* Test tubes and test tube rack
* Transfer pipettes
6.0 QUALITY CONTROL

6.1 All reagents shall be used and controlled according to the manufacturer’s
written instructions
6.2 All antisera must be visually inspected for contamination such as
discoloration, cloudiness, turbidity and/or particulate matter
6.3 All reagent red cells must be visually inspected for hemolysis and/or
discoloration
6.4 The results of the visual inspection, reagent lot number, expiry date,
date of the inspection and the individual performing the inspection
must be documented
6.5 The expiry date should be checked on each reagent used. Do not use
reagents beyond expiry date
6.6 The reactivity of blood grouping reagents shall be confirmed each day of
use by control test with known antigen positive and negative red cells.
Positive control cells should be selected to represent weak expression
of the specific antigen

7.0 PROCEDURE

7.1 Centrifuge specimen according to facility policy


7.2 Determine specimen acceptability
7.3 Perform a patient history check
7.4 Ensure patient information on the sample corresponds with the patient
information on the worksheet

FORWARD GROUP
a. Place 1 drop of Anti-A in a clean, labeled test tube
b. Place 1 drop of Anti-B in a separate, clean, labeled test tube
c. Add 1 drop of a 3-5% patient cell suspension to each tube
d. Mix the contents of the tube gently
e. Centrifuge for the calibrated spin time
f. Gently resuspend the cell buttons and examine them for agglutination
g. Read, interpret and record the test results

REVERSE GROUP
a. Add 2 or 3 drops each of serum or plasma to two clean, labeled test tubes
b. Add 1 drop of A1 reagent red cells to the tube labeled A1
c. Add 1 drop of B reagent red cells to the tube labeled B
d. Gently mix the contents of the tubes, then centrifuge for the calibrated
spin time
e. Examine the serum overlying the red cell buttons for evidence of hemolysis.
Gently resuspend the cell buttons and examine them for agglutination
f. Read, interpret and record the test results

8.0 REPORTING RESULTS


Agglutination
The strength of agglutination reactions should be recorded as follows:

8.1 Grading System for Agglutination Reaction

4+ (++++) One complete mass of agglutination, readily visible on the tube


3+ (+++) Large separated masses of agglutination, readily visible on tube
2+ (++) Smaller agglutinates, still readily visible on the tube
1+ (+) A granular appearance, just visible on the tube
NEGATIVE All cells are free and evenly distributed

8.2 DETERMINATION OF BLOOD GROUPS


9.0 REFERENCES

1. https://apps.who.int/iris/bitstream/handle/10665/119501/dsa24.pdf?sequence=1&isAllowed=y
2. https://www.gov.nl.ca/hcs/files/bloodservices-pdf-abo-grouping.pdf
3. http://www.labflorida.com/internal/COLA/guides/elf27.pdf

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