PROPERTY OF CHERRYANNE DOMINGO

PROPERTY OF CHERRYANNE DOMINGO

UNIT 1.1 LABORATORY SAFETY IN THE doesn’t have hema analyzers and if they
HEMATOLOGY SECTION OF THE CLINICAL only perform manual counting or CBC.
LABORATORY o Some laboratories derived hemoglobin
and RBCs from the hematocrit.
ANATOMY OF AN ACCIDENT  not a good practice
Hazard vs Risk  inaccurate results
 Hazard – a potential source of harm  only good for normal conditions
o Anything that can pose harm  Ex. If the patient has thalassemia
 Risk – the percentage of the hazard to pose (high RBC count, low
danger or accident to the individual hemoglobin)
 In the dictionary, hazard and risk are o Drabkin’s reagent is superior for manual
synonymous. hemoglobin determination
Danger – a state or condition in which personal injury  Physical Hazard
and/or asset damage is reasonably foreseeable
LABORATORY SAFETY
 Accidents happen anytime
o What can we do?
 Lessen the risk of having
accidents
 Specific measures that prevent laboratory
accidents
COMMON HAZARDS ASSOCIATED WITH CLINICAL o How we are…
LABORATORY WORK o What we do and use…
 Infection o How we do and use…
 Burns
 Cuts PROPER DRESS AND PPE
 Effects of toxic chemicals  The laboratory environment is hazardous by
 Explosions nature and the actual risk is largely determined by
 Electric Shock you and those working with you.
 Fire  It is the responsibility of the personnel to know
and follow the rules and being able to recognize
WHAT CAN POSE AS HAZARDS TO US IN THE
potential safety hazards.
CLINICAL LABORATORY?
 What you wear in the lab can help prevent serious
 Blood
even fatal injuries.
o number 1 hazard due to frequent
exposure in the laboratory
Proper Dress
o Best culture medium for microorganisms
 Wear pants and closed tight shoes
o Rich with nutrients (for microorganisms)
and blood cells  Remove jewelries before entering the laboratory
 Biological Agent  Tie your long hair
o Bacterial = presence of microorganisms  Bring only the things you need in the lab
in the blood; can cause infection to the  Remove all personal items (backpacks, purses,
personnel (ex. open wound) or jackets)
 Chemicals
o Drabkin’s reagent Personal Protective Equipment (PPE)
 Highly toxic to humans  For general laboratory work, wear lab coats,
 Used for manual hemoglobin safety goggles, and gloves are required.
determination  Always button your coat
o Primary laboratories are required to have  Keep the cuffs tack in to your gloves
drabkin’s reagent especially if the facility

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PROPERTY OF CHERRYANNE DOMINGO

 Wear a chemical apron when working with splash o Both should be tested weekly to ensure
hazards or reactive solutions they’re working properly and the water is
 Safety goggles can protect your eye from flying clean
debris. However, these glasses won’t protect you  If a chemical or flame exposure has occurred, yell
from splash hazards. Instead, wear chemical for help and immediately move to the nearest
resistant goggles safety shower.
 Always wear gloves. Make sure to use the o Remove the saturated clothing and
appropriate type. thoroughly drench the affected skin
o The gloves should be resistant to the under the shower.
chemicals you’ll be handling o If your clothes or skin are exposed to
o Change your gloves and wash your flame, then drench your entire body and
hands frequently especially if they come have someone call for emergency.
in contact with chemicals  The eyewash station is used for rinsing your eyes
o Puncture-resistant gloves are used in I they’re exposed to hazardous chemicals.
handling hot or cold material such as o Hold your eyes open and thoroughly
when using the autoclave, or handling rinse for at least 10 minutes.
dry ice or sharps.  There are four types of fires:
 Some chemicals produced dangerous vapors. A o Class A
respirator can protect you; however, by law, you  Ordinary combustibles (wood,
must first complete the proper training. cloth and paper)
o Ask the laboratory manager or instructor  Extinguished by water or general
for respirator training purpose extinguishers
 Always remove PPE and wash your hands o Class B
before leaving the laboratory and entering public  Organic solvents and flammable
areas. liquids
 Be aware chemicals or biological contamination o Class C
by touching items such as light switches,  Electric equipment
doorknobs, or even phones while your gloves are  Class B and C fires must be
still on. smothered with chemical foam
 Wearing PPE will minimize the risk of harm. But, extinguishers. Putting water on
clothing and PPE aren’t enough to keep you these fires will only make matters
safe. worse
 In the Philippines, laboratory workers aren’t just  Water will actually cause the fire
in their scrub suits and lab gowns are cleaned at to spread and you can even
home. electrocute yourself
 In other countries, laboratory gowns are provided o Class D
by the employers. After shift, they leave their lab  Combustible metals which aren’t
gowns for laundry. very common in the lab
o Dry Chemical Extinguishers
SAFETY EQUIPMENT  most labs contain dry chemical
 Accidents, by definition, are unforeseen. fire extinguishers which are
installed close to the exit.
 Knowledge of lab safety will help minimize the
 Effective against Class A, B, and
likelihood of accidents.
C fires
 When working in a lab for the first time, look
o If a fire occurs, and it’s too large for you
around and identify the location of the safety
to extinguish, evacuate all personnel
equipment.
immediately and call for emergency.
 Every lab must contain a safety shower and
o Don’t attempt to use a fire extinguisher
eyewash station.
unless you have been trained to do so by

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PROPERTY OF CHERRYANNE DOMINGO

certified trainers such as the local fire  Never work alone

department. o Always have at least one other person in
o A fire blanket can be used to extinguish the lab so that you can help each other in
small fires on work benches and floors. case of an emergency
 It can also be used to help a  Practice good housekeeping
person whose clothing is on fire. o A cluttered lab is a dangerous lab
 Never wrap a person while  Dispose of any trash or debris on the floor which
they’re standing. This can force could cause someone to fall
flames upward toward their head  Never place any chemical bottles on the floor, not
and neck area even temporarily
 Instead, help the person to the  Clean spills
floor, wrap the fire blanket o Check the safety data sheet for the
around them and help them roll appropriate response
until the fire is out. o For routine spill, clean it immediately and
 Each lab should have a first aid kit that contains place a “wet floor” sign
bandages and antiseptic for minor injuries.  Keep your workbench clean and organized
 Evacuation routes should be posted near the o Have only the materials your need. Store
exits. away all unneeded items
o It’s important that you know multiple  Don’t place materials near the edge of the
evacuation routes in case one is blocked. workbench where they can be easily knocked off
 Chemical fume hood  Properly dispose of broken glass
o It’s ventilated, enclosed work area that o Never try to pick up broken glass with
protects you from toxic vapors. your bare hands
o Turn on the exhaust fan o A cut or puncture caused by broken glass
o Make sure the hood is venting properly may introduce a hazardous chemical
o The opening is covered by a window directly into your bloodstream
called a sash, which can be raised and o Dispose the glass in a designated broken
lowered. glass container
o For most applications, the sash should  After you’ve finished an experiment, wash and
be opened to either 8 or 16 inches dry glassware.
o Never store chemicals under the hood  Return reagents to the storage area, and clean
and always clean and remove materials the workbench surface with ethanol or isopropyl
when you’re finished working. alcohol
 Test your safety equipment regularly to make  If a safety violation occurs, or you notice any
sure each item is ready in case there’s an unsafe condition in the lab, report it immediately
emergency. to your supervisor

BEHAVIOR CHEMICAL HAZARD

 Respect the lab and respect your colleagues  Many of the chemicals we use in the lab are
 Your behavior goes a long way to insuring that potentially dangerous, especially under high
the lab is a safe environment for everyone. heat, pressure, or when they’re mixed with other
 Follow the written Standard Operating chemicals.
Procedures step-by-step  Two main tools to identify chemical hazards:
 Never eat, drink, chew gum, or apply makeup 1. Safety Data Sheets
o To avoid contamination on your skin or 2. Chemical Labels
risk ingesting poisonous chemicals  Safety Data Sheet
o It can also contaminate your experiment o Every chemical in the lab is required to
and ruin the results have a technical document called a

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PROPERTY OF CHERRYANNE DOMINGO

safety data sheet or SDS, usually  Find out about the likely routes of
provided by the manufacturer. exposure, symptoms, as well as
o Formally known as material safety data short and long-term effects
sheets or MSDS o Section 12: Ecological
o One-stop-shop to find out about a o Section 13: Disposal
chemical’s properties, hazards, and o Section 14: Transport
safety precautions o Section 15: Regulatory Considerations
o Know the location of the safety data o Section 16: Any other pertinent
sheets in your lab and review them information
before working with a chemical for the  This is where you’ll find the date
first time when the SDS was prepared
 Globally Harmonized System of Classification along with the last known
and Labelling of Chemicals (GHS) revision
o new system provides an international  Section 12-16 are not mandatory
standard format for safety data sheets  Safety Data Sheets provide a lot of information
o All SDS must now be organized into 16 about how to use chemicals safely in the lab
sections  Reading the Label
 16 Sections o Another way to learn about chemical
o Section 1: Identification o The new GHS format requires each
 Chemical’s name, description, chemical in the lab to be labeled with:
and the manufacturer’s contact  Product name
information  Signal word (ex. “Danger” or
o Section 2: Hazard Identification “Caution”)
 Lists signal words, warnings, and  Physical health and environment
safety symbols hazard statements
o Section 3: Composition  Precautionary statements
 List of ingredients  Pictograms
o Section 4: First-aid Measures  First aid instructions
 What’s the required treatment for  Supplier’s contact information
a person who’s been exposed?  Pictograms
o Section 5: Fire-fighting Measures o Consists of a symbol on white
o Section 6: Accidental Release background, framed in a red border
Measures o Each pictogram represents a specific
 Instructions for containment and hazard
cleanup of spills or leaks
o Section 7: Handling and Storage
Requirements
o Section 8: Exposure controls and
personal protection Health
 OSHA’s exposure limits and
recommendations for PPE
o Section 9: Physical and Chemical
Properties
o Such as appearance, odor, pH, flash
point, solubility, and evaporation rate Flammability
o Section 10: Stability and Reactivity
 How to avoid hazardous
reactions
o Section 11: Toxicological Information

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PROPERTY OF CHERRYANNE DOMINGO

 Oxidizer
o Chemical that initiates combustion
through the release of oxygen
Toxicity
APPLICABLE SAFETY PRACTICES REQUIRED BY
THE OSHA STANDARD

1. Hand Washing
2. Eating, drinking, smoking, and applying
Compressed cosmetics or lip balm must be prohibited in the
gasses laboratory work area
3. Hands, pens, and other fomites must be kept
away from the mouth and mucous membranes
4. Food and drink, including oral medications and
tolerance testing beverages, must not be kept in
the same refrigerator as laboratory specimens or
Skin and eyes reagents or where potentially infectious materials
protection
are stored and tested
5. Mouth pipetting must be prohibited
6. Needles and other sharp objects contaminated
with blood and other potentially infectious
materials should not be manipulated in any way.
Unstable 7. Contaminated sharps must be placed in a
explosives puncture resistant container that is appropriate
labeled with the universal biohazard symbol or a
red container that adheres to the standard. It
must be leakproof.
8. Procedures such as removing caps when
checking for clots, filling hemocytometer
Oxidizers chambers, making slides, discarding specimens,
making dilutions, and pouring specimens or fluids
must be performed so that splashing, spraying, or
production of droplets of the specimen being
manipulated or prevented.
o Any income laboratory is still a business.
Environmental Most of the time laboratory managers
Hazards will do anything to receive higher profit
(ex. recycling sharps collector)
o Before applying for a job, ask your
employer the benefits of your work in
case of accidents
o Laboratory managers should have a
Acute toxicity record of everything that happens inside
the laboratory and provide necessary
PPEs
9. Personal protective clothing and equipment must
be provided to the laboratory staff.
10. Phlebotomy trays should be appropriately labeled
to indicate potentially infectious materials

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PROPERTY OF CHERRYANNE DOMINGO

11. If a pneumatic tube system is used to transport

specimens, the specimens should be transported
in the appropriate tube, and placed into a special
self-sealing leakproof bag appropriately labelled
with the biohazard symbol
12. When equipment used to process specimens
becomes visibly contaminated or requires
maintenance or service, it must be
decontaminated, whether service is performed.
13. Always practice the universal precaution that all
specimens must be treated as potentially
infectious.

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PROPERTY OF CHERRYANNE DOMINGO

CHAPTER 2: METHODS OF BLOOD COLLECTION 5. Clean and air-dry the site

 70% isopropanol – recommended for
CAPILLARY PUNCTURE cleaning puncture sites
 Air-drying the site:
ORDER OF DRAW o Provides maximum antiseptic
 Slides for PBS (Peripheral Blood Smear) action
 Blood Gas Specimens (CBGs – Capillary Blood o Prevents alcohol contamination
Gas) 6. Prepare equipment
 EDTA (Ethylenediamine tetra-acetic acid)
Specimens FINGERSTICK HEELSTICK
 Other Additive Specimens  Select a  Select the
 Serum Specimens fingerstick lancet appropriate heel
according to the puncture device.
*Overfilling tubes can result to microclot formation age of the patient  Select blood
and amount of collection
CAPILLARY PUNCTURE PROCEDURE blood to be devices
1. Sanitize hands and put on gloves collected. according to the
 Gloves – protect phlebotomist from  Place items w/in according tests
bloodborne pathogen exposure easy reach.
2. Position patient
 Patient’s arm must be supported on a 7. Puncture the site and discard lancet device
firm surface with the hand extended and
the palm up FINGERSTICK HEELSTICK
3. Select the puncture or incision site
 Fingerstick: Central, fleshy portion and Grasp patient’s finger with Grasp foot gently, but
slightly side or center of the middle or ring your nondominant thumb firmly with nondominant
finger and perpendicular to the grooves and index finger hand
 Heelstick: Medial or lateral planter Place the lancet flat Place the lancet flat
surface of the heel against: against the:

o Slightly side o Skin on the

center of the medial or lateral
fleshy pad of the plantar surface
3rd or 4th finger of the heel
o Perpendicular to
the fingerprint
whorls

8. Wipe away the first drop of blood

4. Warm the site (optional)  Wiping away the first drop of blood using
 Warming increases blood flow up to a gauze pad;
sevenfold o Prevents contamination of the
specimen with excess tissue
 Wrap the site for 3 to 5 minutes with
fluid
washcloth, towel, or diaper that has been
o Removes site of alcohol residue
moistened.

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PROPERTY OF CHERRYANNE DOMINGO

9. Fill and mix tubes/containers in order of draw  70% isopropyl alcohol

a) Capillet (fingerstick & heelstick)  Sterile gauze or cotton balls
 Touch one end of the capillet to the blood  Adhesive bandage/ medical tape
drop.  Biohazard sharps container
 Lower the opposite end of the tube  Disposable gloves
slightly as it fills. (Do not remove it from  Safety glasses and mask
the drop)  Transfer device
 When the tube is full, plug the opposite or
dry end with clay or other suitable VENIPUNCTURE PROCEDURE
sealant. 1. Identify the patient
b) Microcollection tube (fingerstick &  Inpatient – ask the name of the patient,
heelstick) verify the bracelet name and hospital
 Microcollection tube must be positioned number/requisition information.
below the drop.  Outpatient - ask the name of the patient,
 Scoop only the blood drop and allow it to verify the bracelet name and hospital
run inside wall of the tube. number/requisition information, and/or
 Gentle tap is occasionally needed to ask for a valid identification card.
settle blood to the bottom. 2. If fasting sample is required, ask the patient when
 Seal tubes and perform proper inversion. he or she last ate.
3. Explain the procedure to the patient.
4. Prevent the plunger from sticking by pulling it
halfway out and pushing it all the way in one time.
5. Select the proper tubes to transfer the blood to
after collection. Place in the proper order for
filling.

Capillet Microcollection tube

10. Place gauze and apply pressure
 Apply pressure with a clean gauze and
elevate foot until bleeding stops
11. Label specimen and observe handling
precautions
12. Check the site
13. Dispose used and contaminated materials
14. Transport specimen to the laboratory

VENIPUNCTURE

Sample: Venous blood collected to be aliquoted into

evacuated tubes or special collection containers

MATERIALS
 Syringe (varies in size)
 Disposable needle for syringe (21 or 22 gauge)
 Evacuated tubes or special collection containers
 Tourniquet

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PROPERTY OF CHERRYANNE DOMINGO

6. Apply the tourniquet 3-4 inches above the hand slowly pull the plunger back until the desired
puncture site. Do not put the tourniquet on too amount of blood has been obtained. Avoid
tightly or leave it on the patient longer than 1 excess probing.
minute.  5-30 degree angle from the arm surface

Note:
 The larger median cubital and cephalic veins
are the usual choice for venipuncture
 The basilic vein on the dorsum of the arm or
dorsal hand veins are also acceptable.
 Foot veins are a last resort because of the higher
probability of complications.

14. Release tourniquet

15. Lightly place gauze square or cotton ball above
the venipuncture site.
16. Gently remove the needle from the arm.
17. Activate the safety shield over the needle (if
applicable).
18. Apply pressure to the site for 3 to 5 minutes. The
patient may assist if able. This is to avoid
formation of a hematoma.
19. Aliquot blood into appropriate tubes
 By puncturing the evacuated tubes
 By manual transfer of the blood into the
tubes
20. Remove the needle from the syringe, and discard
7. Ask the patient to close his or her hand. The the needle into the sharps container.
patient must not be allowed to pump the hand. 21. Dispose the syringe and transfer device into
8. Place the patient’s arm in downward position if sharps container. Do not disconnect the syringe
possible. from the transfer device or needle before
9. Select the vein, noting the location and direction disposal.
of the vein. 22. Recheck the identification bracelet with the labels
10. Clean the venipuncture with 70% isopropyl or requisitions.
alcohol swab. Cleanse the area in a circular 23. Label all tubes
motion, beginning at the site and working
outward.
11. Put on gloves while the alcohol is drying. Do not
touch the venipuncture site.
 If you find it necessary to reevaluate the
site by palpation, the area needs to be re-
cleansed before the venipuncture is
24. Check the puncture site. Apply adhesive
performed.
bandage.
12. Draw the patient’s skin taut with your thumb. The
 If the patient is infant, the use of bandage
thumb should be 1 to 2 inches below the puncture
should be eliminated
site.
25. Remove gloves and wash hands.
13. With the bevel up, align the needle with the vein
26. Thank the patient and transport the sample(s) to
and perform venipuncture. While securely
the laboratory.
grasping the syringe with one hand, use the other

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PROPERTY OF CHERRYANNE DOMINGO

ARTERIAL PUNCTURE  A negative test result indicates inability of the

 more painful and hazardous than venipuncture ulnar artery to adequately supply blood to the
 not normally used for routine blood tests hand and therefore the absence of collateral
 Primary application: for arterial blood gas (ABG) circulation.
analysis  The radial artery should not be used. Another
 Phlebotomist must have extensive training site should be considered.
which should include the ff:
o Theory ARTERIAL PUNCTURE SITE SELECTION
o Demonstration of technique Selection Criteria
o Observation of actual procedure  Presence of collateral circulation – site is supplied
o Performance of arterial puncture with with blood from more than one artery.
supervision  Artery Accessibility
 Size of the artery
MATERIALS  Type of tissue surrounding the puncture site
 Phlebotomy kit  Absence of inflammation, irritation, edema,
 Local Anesthetic (optional) hematoma, lesion or wound in close proximity
 Special glass or plastic syringe
 Luer-tip or bubble removal cap (to maintain SITES FOR ARTERIAL PUNCTURE
anaerobic condition within the specimen) 1. Radial artery
Advantage:
MODIFIED ALLEN TEST  with good collateral circulation
 To determine if the patient has COLLATERAL  easy to palpate
CIRCULATION 2. Brachial artery
 Should be done before collecting blood  felt above the bend of the arm,
specimen from the radial artery approximately in line with the ring finger
Advantage:
MODIFIED ALLEN TEST PROCEDURE  large and easy to palpate
1. Have the patient make a tight fist.  large volume of blood can be obtained
2. Using the middle and index fingers of both hands, 3. Femoral Artery
apply pressure to the patient’s wrist, compressing  largest artery for arterial puncture
both the radial and the ulnar arteries at the same  located superficially in the groin, lateral to
time. the pubic bone
3. While maintaining pressure, have the patient  used only in emergency situation
open the hand slowly. It should appear blanched
or drained of color. VIDEOS
4. Lower the patient’s hand and release pressure on
the ulnar artery only. CAPILLARY PUNCTURES
5. Assess Result. Capillary Puncture (also known as dermal puncture) is
performed for:
(+) Result: The hand flushes pink or returns to normal  POCT (point of care testing)
color within 15 seconds. o Bleeding times
 A positive test result indicates return of the blood o Glucometers
to the hand via the ulnar artery and the presence  Capillary blood gases in neonates
of collateral circulation.  Children
 Sign to proceed with arterial puncture  Difficult draws
 Minimum volume for testing needed (e.g.
(-) Result: The hand does not flush pink or return to
hematocrit)
normal color within 15 seconds.

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PROPERTY OF CHERRYANNE DOMINGO

Capillary specimen o Do NOT choose thumb (has a

 Mixture of venous and arterial blood, along with pulse), index finger (sensitive), or
interstitial and intracellular fluids pink (insufficient tissue depth)
 Portion of arterial blood is greater than that of o CHOOSE 3rd or 4th finger
venous blood o Use palmar surface near the fleshy
o Arterial portion can be increased by center of the distant segment
warming site o Angle puncture inward towards each
other – will help blood to flow and
Equipment help to get enough sample
 Lancets
o not more than 2.0 mm depth for heel
o not more than 3.0 mm depth for adult
finger
 Microsample containers
 Heelwarmers, hotpacks, warm towels
o Increases blood flow as much as
sevenfold

Order of Draw
1. Purple
2. Green 5. Warm the puncture site
3. Red 6. Clean the puncture site with alcohol
7. Open new, sterile lancet
Capillary Puncture Outline 8. Warn patient
1. Identify the patient: by name and date of birth 9. Puncture skin – ensure that the puncture reaches
2. Wash hands, put on gloves the capillary bed
3. Assemble supplies 10. Wipe away first drop of blood with dry gauze pad
4. Choose puncture site – first drop is contaminated with alcohol and
 Areas to avoid: interstitial fluid
o Callused 11. Collect the specimen for POCT or in
o Scarred microcollection containers (Order: lavender /
o Bruised purple, green, red)
o Edematous 12. Mix specimens if appropriate
o Cold/cyanotic 13. Apply pressure and bandage
o Infected 14. Label specimen
o Previous Punctures
 Heel:
Note:
o Avoid calcaneus (heel bone) and
 Do not touch collection tube to puncture site
arch
 Excessive milking may cause hemolysis
o Avoid calloused area
 If insufficient specimen has been obtained and
o Use heel for infants less than 1 year
blood has stopped flowing, puncture must be
old
repeated at a different site using a new lancet
o Use side of the heel
 Finger:
*Link: https://www.youtube.com/watch?v=WJkkgDVX5jk
o Do NOT choose finger of an infant
less than 1 year old
o Do NOT choose finger on the side of
mastectomy

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PROPERTY OF CHERRYANNE DOMINGO

PHLEBOTOMY: DERMAL CAPILLARY PUNCTURES Procedure:

Materials: 1. Ask patient for consent and explain procedure
 Lancet 2. Tie tourniquet above puncture site
 Capillary tubes 3. Palpate using two index fingers of non-dominant
 Alcohol swabs hand
 2x2 gauze 4. Ask patient to make a fist if vein is difficult to find
 Tape 5. Remove tourniquet
 Gloves 6. Assemble needles and check expiration date
7. Screw syringe to needle
Procedure: 8. Put on gloves
1. Put on gloves after obtaining consent from patient 9. Cleanse puncture site using the circle concentric
2. Get one capillary tube method (inside out)
3. Open alcohol pad 10. Re-tie tourniquet
4. Puncture one of the 2 middle nondominant 11. Pull back safety cap and release
fingers 12. Pull back plunger a few times to loosen it
5. Point the finger down 13. Stick needle into vein
6. Cleanse the fingertip with alcohol swab and wait 14. Slowly pull back the plunger until enough blood
for it to dry sample is collected (pulling back plunger to fast
 Do not want to stick on central or medial can result to hemolysis)
finger since there’s a high chance of 15. Release tourniquet
breaking or puncturing bones 16. Grab gauze and fold it over twice
17. Pull needle out and apply pressure using gauze
 Choose lateral on either side.
18. Immediately cover needle with safety cap
7. Remove cap of pre-loaded lancet
19. Bandage patient
8. Squeeze tip of finger and push lancet into it
20. Unscrew safety cap needle and dispose properly
9. Wipe first drop of blood with a gauze
21. Attach transfer device to syringe
10. Place capillary tube below the finger with the red
22. Turn syringe upside down
line facing down
23. Press in evacuated tube on transfer device
11. Avoid touching the capillary tube to the patient’s
24. Disengage tube
skin and dab on bubble of blood until tube is full
25. Discard transfer tube and syringe
12. Cover wound with piece of gauze
26. Label the evacuated tube properly and place in
13. Deliver tube to laboratory for processing
hazard bag before sending to the lab
*Link: https://www.youtube.com/watch?v=ibU5PYOF2qg
*Link: https://www.youtube.com/watch?v=7NSEFVbzTAU

SYRINGE METHOD
EVACUATED METHOD
Syringe method
Reason for blood collection
 Performed for patients’ whose veins cannot
 Routine – to monitor treatment and guide
handle the pressure of evacuated tubes
diagnosis
Equipment:  Pre-operative
 Evacuated tube  Toxicology – monitoring medication levels
 Barrel syringe  Acutely unwell patient (eg. for septic screen
workup)
 Hypodermic needle with safety cap
Equipment:
 Transfer device
 Gloves
 Gauze
 Apron (if available)
 Alcohol swab
 Gauze
 Tape
 Tape
 Tourniquet

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PROPERTY OF CHERRYANNE DOMINGO

 Chlorhexidine wipe Contraindications - the procedure would not be

 Vacutainer performed if there is:
 Needle  Presence of thrombus or infection in central line
 Blood bottles (evacuated tube) o Excess amount of fluid
 Tourniquet o Discharge at the insertion site
 Sharps box  Fracture of disruption of central line

Procedure: Equipment:
1. Prepare equipment before going to patient  3 mL syringe – for collecting blood to perform
2. Check patient details blood tests
3. Explain procedure and gain consent  10 mL syringe – for flushing catheter and
4. Tip: ask patient were people have taken blood checking for a blood return
from them before to save time  Saline flush
5. Straighten the arm and ensure that the patient is  Blood specimen containers
comfortable  Antiseptic wipes
6. Place pad below arm  2x2 gauze
7. Apply tourniquet  Clean gloves
8. Palpate for vein  Hand sanitizer
9. Clean skin with chlorhexidine wipe
10. Prepare needle Procedure:
11. Hold skin taut with nondominant hand 1. Explain the procedure to the patient and clean
12. Pierce skin at 20 degree angle, bevel face up hands using antiseptic sanitizer (use soap and
13. Insert evacuated tubes to vacutainer one by one water if hands are soiled)
based on the proper order of draw 2. Put on clean gloves
14. Remove tourniquet 3. Open 2x2 gauze sponge and place antiseptic
15. Remove needle wipe on top
16. Place gauze above wound and ask patient to 4. Wrap gauze and antiseptic on appropriate tube
apply pressure ending and scrub for 30 seconds (to reduce
17. Label the blood samples chance of central line infection)
5. Let tube ending dry for 30 seconds to prevent
*Link: https://www.youtube.com/watch?v=-XxiRSf6n8Q stickiness from forming around the site
6. Attach a normal saline syringe to the tube ending
CENTRAL VENOUS ACCESS DEVICE
7. Flush slightly to ensure tubing patency
Indications - assessing a central venous catheter should
8. Pull back to check blood return
be performed to:
 Showing a blood return and flushing to
 To draw blood from a patient
note resistance will tell you if your line is
 To administer patent
o Medications 9. Prepare to draw blood
o Fluid
 If you cannot get blood return, refer to
o Blood products
appropriate personnel
 To provide 10. Choose a syringe size based on the amount of
o Long-term infusion therapy when blood to be drawn
periphery access is unavailable 11. Detach syringe from tubing
o Vesicant or hyperosmolar infusions 12. Cleanse the line using a 2x2 gauze and scrub for
o Complex infusion therapies 10 seconds
 To check the patency of a central venous catheter 13. Check for patency again and flush line to prevent
that is not in use clotting using a sterile saline syringe

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PROPERTY OF CHERRYANNE DOMINGO

Complications: o metabolites
 Infection o total hemoglobin
 Air embolism o CO-oximetry
 Dislodgement of a thrombus o neonatal bilirubin
 Dislodgement of the central line catheter
Equipment:
 Syringe – should include dry, electrolyte balanced
Note: You may/may not observe any of these
lithium heparin with concentration of 23 I.U./mL of
complications. Monitor your patient closely, and have
blood as preferred anticoagulant
the necessary equipment available to treat
 Safety needle
complications, should any arise
 Filter cap
 Alcohol wipe
Assessment and monitoring
 Gauze
 Monitor ease or difficulty with obtaining a blood
 Bandage
return
 Assess ease or difficulty when flushing the
Procedure (Blood collection)
catheter with normal saline
1. Confirm patient identity
 If placement is in or near right atrium, monitor for 2. Check for adequate circulation by performing
any arrhythmias Modified Allen test)
3. Locate radial artery
Documentation
4. Clean site
 Indication for the procedure  Don’t use cleaning wipes containing
 Date and time of the procedure quaternary ammonium substances such
 Type, size, and position of central venous as benzalkonium since it may affect
catheter electrolyte parameters such as sodium
 Appearance of insertion site 5. Let dry
 Ease or difficulty with obtaining a blood return 6. Preset plunger to desired sample volume
when flushing the catheter 7. Using index and mid finger, identify path of artery
 Medications administered through central line 8. Hold the syringe like pencil, with the bevel up
 Adverse outcomes 9. Flex wrist slightly (if necessary) by resting the
lower arm on the edge of the bed or by placing a
*Link: https://www.youtube.com/watch?v=_EMWuFeEphU small gauze or towel under the wrist
10. Insert in 45 degree angle, needle follows artery
ARTERIAL PUNCTURE path
Follow local hospital procedures to prepare the patient 11. Stop insertion when you see blood flash
and collection site for obtaining the blood sample 12. Hold syringe in place until desired volume is
collected
Collection sites 13. Withdraw needle and apply pressure to puncture
1. Radial artery site with gauze until bleeding stops
2. Brachial artery 14. Close safety needle and dispose based on the
3. Femoral artery hospital guidelines
15. Place filter cap on the syringe lower tip and hold
Blood gas analysis syringe vertically
 test vitals in critically ill patients 16. Tap syringe gently to force air bubble to the top
 gives information on: 17. Expel any air bubbles into filter cap
o gases 18. Mix sample in figure 8 motion for minimum of 20
o pH seconds – helps minimize clot formation and
o electrolytes dissolve lithium heparin

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PROPERTY OF CHERRYANNE DOMINGO

19. Label syringe with patient ID

Procedure (Sample analysis)

1. Transport sample to a Siemens Healthineers
blood gas system
2. Follow a two-step mixing process
a. First, by rotating wrist back and forth
(figure 8 motion)
b. Then, rolling syringe between hand 10
times
3. Remove cap from syringe and expel first few
drops of blood to gauze pad
4. Present sample to analyzer and start analysis

Note: Sample containing blood clots should not be

used since it can affect accuracy of results and have a
negative impact on the operation of blood gas analyzer

5. The results will be ready within 60 seconds

6. Remove syringe and reattach the filter cap
7. *If test should be repeated, debubble and remix
the sample prior to analysis
8. Dispose syringe according hospital guidelines

According to CLSI guidelines, blood gas testing should

be:
 Completed within 10 minutes
 Completed not longer than 30 minutes after
collection of samples

Proper handling, mixing and time to analysis helps to

ensure accurate patient results

Link: https://www.youtube.com/watch?v=l0nNt6GOEMM

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PROPERTY OF CHERRYANNE DOMINGO

THE HEMOCYTOMETER When we talk of our counting chambers, this is actually

pre-measured already. Meron siya mga etched lines sa
What is a Hemocytometer? loob that are pre-measured and take note that these
 This is a device used for manually counting cellular etches are very accurate.
elements in the body fluids. It includes the blood,
CSF, seminal fluid, synovial fluid, and whatsoever, Kung mapapansin niyo dito the ruled area is 4x4 mm and
wherein you perform manual cellular counting. this ruled area is divided into 16 secondary squares.
 This is composed of the counting chamber, thoma
pipette, and thick cover slip.

Three Counting Chambers Available in the Market

 Fuchs-Rosenthal Counting Chamber
 Neubauer Counting Chamber
 Speirs-Levy Counting Chamber

FUCHS-ROSENTHAL COUNTING CHAMBER Secondary Square

 Number of Chambers: Two
 Ruled area: 4x4 mm If you are going to use our mathematical knowledge, each
square has 1 mm in size. So yung length at width nila ay
 16 Secondary squares: 1x1 mm
1 mm. In Fuchs-Rosenthal, the depth is 0.2 mm. So ano
 Depth: 0.2 mm
ibig sabihin ng depth na yun? Kung mapapansin niyo
merong “H-shaped” trough.

(SIR BENJ)
You have two chambers wherein the ruled area is actually
H-shaped depression
4x4 mm. Ano ibig sabihin nun? If you are going to look at
the counting chamber in the microscope, ganito itsura
May H-shaped depression dito and we also have here
niya. You should see this chamber:
what we call “elevation”.

Elevation

Itong elevation na ‘to, it is elevated to appoint that when

you put the cover slip there, it will raise, or it will give you
an elevation of exactly 0.2 mm. Ganun siya ka-exact o
accurate. Itong elevation na ‘to kailangan mas mataas
siya as compared to this na nandito sa ruled areas or sa
chambers natin ng 0.2 mm.

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The ruled area kung mapapansin niyo this is actually 5x2

mm and is divided into 10 secondary squares.

5 mm

1x1
mm
2 mm
Ruled Area

SPEIRS-LEVY COUNTING CHAMBER

 Number of Chambers: Four That 10 secondary squares are measured in 1x1 mm.
 Ruled area: 5x2 mm Tulad ng Fuchs-Rosenthal, this also has a depth of 0.2
 10 Secondary squares: 1x1 mm mm.
 16 Tertiary squares: 0.25x0.25 mm
 Depth: 0.2 mm IMPROVED NEUBAUER COUNTING CHAMBER
 Number of Chambers: two
(SIR BENJ)  Ruled area: 3x3 mm
For the Speirs-Levy, there are 4 counting chambers.  Depth: 0.1 mm
 9 Secondary squares: 1x1 mm
 16 WBC Tertiary squares: 0.25x0.25 mm
 5 RBC Tertiary squares: 0.20x0.20 mm (divided
further to 16 smaller squares)

When it comes to these 4 chambers, if you are going to

look at it under the microscope, ganito magiging
kalabasan niyan:

(SIR BENJ)
Ito na kasi yung common na ginagamit natin. Yung
Speirs-Levy at Fuchs-Rosenthal hindi sila masyado
ginagamit. Sa Speirs-Levy, sinasabi this is only used for
low cellular counts. Ginagamit lang siya kapag
masyadong mababa yung cells nung sample fluid natin.
Pero if sinilip mo yung one chamber, ganito lang ang So parang ginagamit siya for CSF, synovial fluid, and it is
magiging kalabasan niyan. Pagsinilip mo pa siya further not that accurate kapag madami na masyado yung cells
o tinaasan mo pa yung magnification. na nakikita sa sample.

Unlike for Improved Neubauer Counting Chamber,

wherein it can be used for low or high. Bakit ka pag bibili
ng counting chamber na accurate lang for low cells di ba.
If meron kang Improved Neubauer Chamber that is
accurate for all. Pwede mo siyang magamit for normal,

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PROPERTY OF CHERRYANNE DOMINGO

low, at high counts. Bakit ka pa gagastos for something

that you can only get one. If you can have it all if you are
going to use the Improved Neubauer. So you can actually
use this one whether you have a low, high, or normal
count, you can still have accurate result.

Ganito itsura ng Improved Neubauer halos kapareho lang

ng Speirs-Levy natin only that if you are going to look at
Neubauer, you have two ruled area.

Sabi ko nga you have two counting chambers:

When it comes to the ruled area, that is 3x3 mm.

It also has an H-shaped trough wherein the vertical

arm, yung elevation natin will raise the cover slip to give Secondary
it 0.1 mm depth. Square

Difference (Depth):
Spears-Levy & Fuchs-Rosenthal – 0.2 mm
Improved Neubauer – 0.1 mm
The depth, sabi ko nga kanina is 0.1 mm yung taas ng
While yung elevation, it will support your cover slip kagaya ating cover slip. Take note that our ruled area/counting
lang din nung kanina. Ganito itsura niya if pagtitignan niyo chamber is divided into 9 secondary squares. Each
siya na naka-sideview. secondary square is actually measured 1x1 mm.

There are only instances wherein for example low ang

count ng cells. Kapag mababa ang count ng cells natin sa
isang sample, it will be best for you to use all the 9
secondary squares. However, in practice, you don’t use
all of these squares. What we only use are four and also
Makikita niyo na yung H-shaped will elevate you cover the central large square.
slip na 0.1 mm high. Pag sinilip mo naman siya sa
microscope ganito magiging itsura niyan: The colored blue squares are areas of the grid where
WBC are counted. Whereas, the central fifth large
square, yung naka red area that is used usualy use of
counting RBCs.

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PROPERTY OF CHERRYANNE DOMINGO

Yung “W squares” natin ay further subdivided into 16

1 2 tertiary squares. The measurement of one tertiary
square is 0.25 mm x 0.25 mm (1 mm / 4 mm = 0.25 mm)

5 0.25 mm

3 4
Itong secondary squares na ito (1,2,3,4), because these The central large square has the same dimension with
square are primary used for WBCs counting, they were W square. However, it is divided into 25 tertiary squares.
named as “W squares”. It pertains to these four corners
of the hemocytometer or your Improved Neubauer
Chamber.

Dito naman sa central large square, sabi natin itong lima

na ito diyan tayo nagbabasa ng RBCs. Kaya siya
pinangalan na “R squares”. Diyan sa lima na yan, diyan W Square Central Large Square
tayo nagbabasa ng RBCs that is in practice. 16 tertiary squares 25 tertiary squares

The measurement of one tertiary square in the central

large square is 0.2 x 0.2 mm (1 mm / 5 mm = 0.2 mm).
Take note this is actually important kasi gagamitin niyo
1 2 yan for the computation of the cells.

5
The volume of the square is = L x W x H.
4 3

Dito sa Improved Neubauer Counting Chamber, even

though may dalawa kang chambers or ruled areas, isa
lang ang ginagamit.
However, you should know by heart how to use your
Neubauer Counting Counter Chamber kasi may
instances na hindi lang lima na ‘to ang ginamit. Kumbaga
parang itong buong central square ginamit para sa RBCs
count, that is actually possible. Sabi ko nga kanina, if the
sample has a low cellular count, it is also possible that you
will used all of the secondary squares of the chamber. Kahit bilangin mo yung dalawang ruled areas, maganda
That will be more accurate especially if the cells are too siya actually pero what you are going to do is you have to
low (ex. leukopenia, or wherein yung cells natin ay below get the average of the two ruled areas. So kapag
1x109 per liter). Ang hirap makahanap ng WBCs kapag nagbilang ka sa taas at nagbilang ka din sa baba, you
ganun so it will be best na babasahin mo yung buong have to get the sum of all of the cells that you have
chamber. Pag masyado naman madami, okay lang na counted and divide it into two to get the average.
yung babasahin mo is yung 4 na blue squares lang when
it comes to WBCs. TAKE NOTE: Isang ruled area lang ang ginagamit natin
for counting. If you wish to count both ruled areas, get the
sum of the two ruled areas and divide it into two average).

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PROPERTY OF CHERRYANNE DOMINGO

Ang isang “R square” is subdivided into 16 smaller o **Sahli’s pipette – used for hemoglobin
tertiary squares. In reality, hindi na siya masyado level determination
ginagamit. The measurement of each tertiary square is
0.05 x 0.05 mm (0.2 / 4 = 0.05 mm)
 It’s divided by 4 since there are 4 tertiary squares
per row and per column of a secondary square

0.2
RBC pipette (above left) and WBC pipette (below right)
0.2

In total, the central large square has:

 25 secondary squares
 400 tertiary squares (25x16)

NEUBAUER COUNTING CHAMBER

 Number of Chambers: 2
 Ruled area: 3x3 mm
 Depth: 0.1 mm
 9 secondary squares: 1x1 mm Difference between RBC pipette and WBC pipette

 16 WBC tertiary squares: 0.25 mm x 0.25 mm

PARTS OF A PIPETTE
 16 RBC tertiary squares: 0.25 mm x 0.25 mm
 Stem- the entire length of the pipette
(divided into 16 smaller squares)
 Bulb – a bulge
 Bead – located inside the bulb, helps in mixture
of sample and diluting fluid
 Bore – hole located on the tip of pipette

(SIR BENJ)
 Di na masyadong available sa market.
 Improved version is preferred, has the same
thought
 Difference: Central large square is divided into
16, not 25 like in the Improved Neubauer
Counting Chamber

THOMA PIPETTES
Thoma Pipettes
 Used for dilution of samples
Types:
o RBC pipette – for RBC counting
o WBC pipette – for WBC counting

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(SIR BENJ) Additional information on mixing:

 Difference nung stem length makikita sa dulo  May bore (butas) sa magkabilang dulo
(bandang taas) pero pareho lang sila nung haba
 Para maiwasan yung displacement ng
sa may bandang tip.
mixture, kailangang harangan sila sa
 The RBC pipette has a smaller bore so that it magkabilang dulo
can trap WBCs and not allow the passage of
 Sample and diluting fluid should NOT be
WBCs since it’s significantly larger compared to
mixed by moving wrist left to right since:
RBCs
o hindi mahahalo nang maayos yung
 The volume of pipette is relative to size of the dalawa
bulb. Both pipettes can already contain 1 o bead could be misplaced and get
microliter, yung bulb nung RBC mas malaki kasi stuck inside the bore
it can contain 100 µL while yung bulb ng WBC ay
10 µL
 Thoma pipettes are used for dilution 5. Mix for 5 minutes to ensure that sample is well
 Thoma pipettes are TC (to contain) pipettes since mixed
it can transfer/deliver exact amount of samples
HOW TO DISPENSE SAMPLE TO
HOW TO COLLECT THE SAMPLE? HEMOCYTOMETER?
1. Aspirate blood up to 0.5 mark (depending on the 1. Before dispensing to hemocytometer,
sample) dispense/remove 3-4 drops of the mixture into a
tissue or a sink
 Kapag masyadong maraming cells sa
blood, mas konti yung sample at mas  Reason: The initial 3-4 drops contain
maraming diluting fluid sample located in the stem. The target for
sample for the test is the mixture present
 Konti yung cells, mas maraming sample
in the bulb.
at mas konti diluting fluid para mas
mababa dilution factor para di
mahirapang magbilang

↑cells ↓sample ↑diluting fluid ↑diluting factor

↓cells ↑sample ↓diluting fluid ↓diluting factor

2. Aspirate diluting fluid up to 11 µL mark (for WBC Dispensing demonstration

pipette) or 101 µL mark (for RBC pipette)
3. There should be no air in between blood and 2. Nakapatong na coverslip sa vertical line ng H-
diluting fluid since it will contribute to the volume shape
and lead to inaccurate results 3. Sa dulo ilalagay yung hemocytometer at yung
4. Mix sample with diluting fluid by: other bore
4. Sa other side ng bore nakapatong yung fingertip
 moving your wrist from forward and
natin para control yung flow ng sample from
backward or;
Thoma pipette to hemocytometer, it should be
 creating a figure 8 motion (more
well controlled
recommended)
 It is difficult for untrained individuals,
especially now that most procedures are
automated
5. The mixture should cover the whole chamber.

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PROPERTY OF CHERRYANNE DOMINGO

6. It should not overflow. Masasabi mong nag- - Don’t count cells touching bottom and
overflow kapag pumasok o nag-fill na yung right line
mixture sa trough ng Thoma pipette - Cells touching the top and left line are still
 In case it overflows, re-dispense included
 Wash and wipe the hemocytometer first
before re-dispensing
 Make sure that it is dry before washing
 DON’T use tissue to absorb excess
blood: the distribution of cells in counting
chamber should be evenly distributed.
Tendency is that the flow of cells will go
towards the tissue and there would be
uneven distribution of cells.

“Nagdidispense ako sa dalawa para masigurado na

evenly distributed yung cells, and to verify kung tama ba
yung bilang ko dun sa isa.”
 Tertiary squares: assist in manner of counting of
cells
7. After dispensing, let cells settle first by placing
 Cells that are touching the right line and the
hemocytometer on moistened chamber: petri
bottom line of first square will be counted on the
dish na nilagyan ng basang filter/tissue paper.
next square to the right, and the square below
Then, it is covered after placing hemocytometer.
 Lessens probability of inaccuracy
 Reason: Cells are still moving and they
should be allowed to settle. Water bath or
moistened chamber is used to prevent
sample from drying.
 Kapag masyadong matagal magbasa
ang medtech, the mixture might dry-up:
para nang nagbibilog sa chamber
 Signs of drying would lead to inaccurate
results and procedure should be
repeated
 The fluid would compress in the middle
once it dries up

INVERTED L RULE POISSON’S LAW OF DISTRIBUTION

 Do not count the cells touching the bottom and  a statistical tool that shows distribution of events
right line  also followed along with inverted L rule
 Done to avoid double counting of cells  can be used to prove that cells are evenly
distributed in the chamber
(SIR BENJ)  Application: the highest and lowest cell count in
 Performed counting based on protocol rule (e.g. each square should not be more than 5% or 10%
if protocol is to use 4 W squares, count WBC of the average
using 4 W squares)  Example:
 Cells should be settled since there’s a tendency o meron kang W square 1-4 with counts: 42,
that the cells are still moving and can transfer to 48, 45, 50, respectively.
other squares: it would result to inaccuracy o Lowest square (42) and highest square (50)
 Inverted L Rule should not exceed with 10 in difference.

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PROPERTY OF CHERRYANNE DOMINGO

o Proposed value of 10 is a theory, in reality it Solution:

should be not more than 5% of the average.
o The value/optimal difference will increase if Number of cells counted: 4
there are more cells and it will decrease if Volume of W square: 0.1
there are fewer cells.
o Exceeding the difference would mean that VCF = 1 = 2.5
(4) x (0.1)
there is error in dispensing and/or handling,
and inaccuracy in procedure
o DILUTION FACTOR
COMPUTATION
General Rule/Formula for Hemocytometer

 Total volume is dependent on the type of Thoma

(SIR BENJ) pipette used:
General rule: The concentration result would be written o RBC pipette = 101 microliters
in cells per cubic millimeter (cells/mm3) o WBC pipette = 11 microliters
 Reason: because we used cubic millimeters in  Why should 1 be subtracted?
Neubauer chamber o 1 should be subtracted from total volume
o Width. 3 mm since the volume in the stem of the
o Height: 3 mm Thoma pipette is not used.
o Depth: 0.1 mm o That is why we dispense 3 to 4 drops
o w x h x d = volume before we actually dispense in the
o 3 mm x 3 mm x 0.1 mm = 0.9 mm3 hemocytometer because it is not needed
 It is crucial to know the volume of each squares  Volume of sample used is usually 0.5 microliters
Secondary square: but it can be increased up to 1 if only a few cells
o Width. 1 mm are used
o Height: 1mm o You may lower down the volume of the
o Depth: 0.1 mm sample if you are expecting that the cell
o Volume: 0.1 mm3 count is too high or add more volume of
 All secondary squares have 0.1 mm3 volume the sample if you are expecting that the
 One R square would have the volume of 0.004 cell count is too low
mm3 o Volume of the sample is dependent on
the volume used (0.5 or 1)
Formula:  If you aspirated 0.5 blood
Volume = width x height x depth  If you aspirated at the 1 mark of
your Thoma pipette
VOLUME CORRECTION FACTOR (VCF) Example:
Nag-aspirate ka ng sample up to the 0.5 mark at gumamit
ka ng RBC pipette.

Solution:
Total Volume: 101
Example: Volume of sample used: 0.5
If you counted using 4 W squares, the number of square
101 - 1
used would be equal to 4. The volume of square used is DF = = 200
0.5
0.1 cubic millimeter.

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PROPERTY OF CHERRYANNE DOMINGO

Then, substitute values for Volume Correction Factor

Recall: Ano ang volume ng secondary square? 0.1
(VCF) and Dilution Factor (DF) to the main equation
mm3

Take Note: Kapag nakalagay sa problem 1:20 yung

dilution factor, yung denominator ang gagamitin natin
as the df.
Example:
4 W Squares = 50 cells
VCF = 2.5
DF = 200

Solution:
Cells/mm3 = (50)(2.5)(200) = 25,000

SHORTCUT FORMULA

Problem:
A medical technologist aspirated a sample up to 0.5 mark
of an RBC Thoma pipette and diluted the sample with
NSS. The medical technologist counted a total of 86
cells in 4 secondary squares. What is the total cell count
per mm3?

Given:
Number of cells counted = 86
Number of squares used = 4
Volume of square used = 0.1

DF = Total volume – 1 = 101 - 1 = 200

Volume of sample used 0.5

Solution:

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PROPERTY OF CHERRYANNE DOMINGO

RED BLOOD CELL COUNT RBCs under the microscope

*You could see in the picture the zone of pallor occupies
RED BLOOD CELLS 1/3 of their center making it biconcave in shape
 They are anucleate, biconcave cells with reddish
protein called hemoglobin MATERIALS NEEDED FOR RBC COUNTING
 Main Function: to transport oxygen and carbon 1. Hemocytometer
dioxide  An equipment used in counting cellular
 Under the microscope, they appear as salmon elements of body fluids. For example,
pink and they measure about 7–8 µ m in diameter RBCs, WBCs, and platelets.
 It has a zone of pallor that occupies 1/3 of their  Neubauer Counting Chamber is made up
center reflecting their biconcavity of a heavy colorless glass
 Before 1900, physicians or medical laboratory
professionals counted red blood cells in
measured volumes to detect anemia or
polycythemia
o Anemia: loss of oxygen carrying capacity
and is often reflected in reduced RBC
count or reduced RBC hemoglobin
concentration
o Polycythemia: increase in RBC count
reflecting an increase circulating RBC
mass and this condition leads to
hyperviscosity
 Historically, microscopies counted RBCs by
Improved Neubauer Counting Chamber
carefully pipetting a tiny aliquot of whole blood
and mixing it with 0.85% normal saline solution.
2. EDTA Tube
o Why? because technically your normal
 Ethylenediaminetetraacetic acid
saline matches the osmolality of blood,
consequently, the suspended RBCs  This type of acid stop clotting process
retained their intrinsic morphology and, at the same time, it retains the
neither swelling nor shrinking original shape and size of cells
o Take note that your manual RBC counts o Because the EDTA
are rarely performed nowadays in the anticoagulant can bind with the
laboratory because of inaccuracy of the calcium present in blood. Thus,
count and questionable necessity preventing the start of
o The used of more accurate manual RBC coagulation cascade in which it
procedures such as microhematocrit is the process of clot formation
and hemoglobin concentration is occurs.
desirable when automation is not  In this procedure, we will use a blood
available sample from an EDTA Tube

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PROPERTY OF CHERRYANNE DOMINGO

3. Thoma pipette o An isotonic solution is the one that has

 In this case, we use the RBC thoma the same osmolarity or solute
pipette concentration as of your RBC
 Difference between RBC pipette and o When the plasma surrounding blood cells
WBC pipette is an isotonic solution compared to the
o Dilution solution inside the blood cells, the cells
 RBC pipette: 1:200 function normally
 WBC pipette: 1:20 o The isotonic solution allows the cells
o Bulb Volume move water and nutrients in and out of
 RBC pipette: 100 units the cells. This is necessary for blood cells
 WBC pipette: 10 units to perform their function of delivering
o Bore Size oxygen and other nutrients to other parts
 RBC pipette: smaller of the body
o If we used a hypertonic environment or
 WBC pipette: larger
hypertonic solution, basically the cells
o Color of the Beads
will become plasmolyzed. Therefore, it
 RBC pipette: red
will not contain enough water to perform
 WBC pipette: white
its cellular function
 If we are already doing the RBC and
o If we used a hypotonic environment for
WBC counting, we should discard first
the RBCs, the RBCs will definitely lyse
the 5 to 6 drops for RBC counting and
spilling their contents into the
discard 2 to 3 drops for WBC counting
bloodstream or into the environment.
before putting it in the hemocytometer
This can cause dangerous side effects as
well many loss of blood cells
 Must have a high specific gravity
o RBCs has a specific gravity of above 1
o The specific gravity of your erythrocytes
is about 1.092 to 1.101
 Must be easy to prepare
 Must be cheap
4. Diluting Fluid  Must be a good preservative
 Diluting fluid destroys unnecessary cells  Must have a buffer action
and also preserves and fix the RBCs o What is a buffer action?
 What is the procedure?  This is the ability of the buffer
o Aspirate first the blood up to the solution to resist the changes in
0.5 mark of your RBC pipette pH value on the addition of small
o Wipe of excess blood at the tip of amount of an acid or a base
the pipette with a tissue paper  Must not initiate the growth of molds
o Aspirate the diluting fluid up to
101 mark for RBC counting RBC DILUTING FLUIDS
o Mix the tube using figure of 8
DACIE’S FLUID / FORMOL CITRATE
CHARACTERISTICS OF A GOOD RBC DILUTING  Dacie’s fluid is known to be the best RBC diluting
FLUID fluid because it can be kept in a long period of
 Must be an isotonic solution time.
o iso- means ‘same’  At the same time, if you use Dacie’s fluid the cell’s
o tonic- means ‘tenacity’ shape of the RBCs will not be altered
 Formalin acts as the preservative

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PROPERTY OF CHERRYANNE DOMINGO

 Composition:  Multiple myeloma

 40% solution of formaldehyde  Waldenstrom
(10.0 ml)  Macroglobulin anemia
 3% W/V trisodium citrate (990.0  Inflammatory
ml)  Other connective tissue
disorders
HAYEM’S  Another advantage of Gower’s fluid is that it
 Hayem’s diluting fluid is NOT recommended precipitates protein in cases of
because it allows the growth of yeast and as you hemoglobinemia and hyperglobulinemia
can see in the picture below it produces clumping  Composition:
of cells particularly in blood of patients suffering o Glacial acetic acid (16.65 gm)
from liver cirrhosis. o Sodium sulfate, cp crystalline (6.25 gm)
o Distilled water (100.00 ml)

Rouleaux Formation

 One advantage of Hayem’s fluid is that it can

stand long period time and it has no corrosive
effect
 Composition:
o Mercuric chloride, cp (0.50 gm)
o Sodium sulfate, cp crystalline (5.00 gm)
 or anhydrous (2.65 gm)
o Sodium chloride, cp (1.00 gm)
o Distilled water (200.00 ml)

GOWER’S
 The main advantage of using Gower’s diluting
TOISSON’S
fluid is that it prevents rouleaux formation.
 Toisson’s diluting fluid has a high specific
 Rouleaux Formation are stacks or aggregation
gravity and it stains WBC.
of RBCs that form because of the unique discoid
 Therefore, beginners in RBC counting use
shape of the RBCs
Toisson’s diluting fluid since WBCs are easily
 The flat surface of the discoid RBCs give them a
identified because the nucleus will stain blue
large surface area to make contact with and stick
 Take not that it should be filtered to prevent the
to each other
growth of fungi since Toisson’s fluid supports the
o also known as the ‘stack of coins’
growth of fungi
o Take not that you can see rouleaux
 Composition:
formation in some conditions such as
o Sodium chloride (1.00 gm)
 Infections

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PROPERTY OF CHERRYANNE DOMINGO

o Sodium sulfate (8.00 gm) o Be sure the pipette is held securely using
o Glycerine (30.00 gm) the thumb and middle finger.
o Methyl violet (0.025 gm) o If the diluting fluid goes beyond the 101
o Distilled water (200.00 ml) mark or if there is presence of bubbles
inside the bulb, discard the mixture and
start from the beginning making use of a
dry clean pipette.

BETHELL’S
 Composition:
o Sodium sulfate, crystalline (5.00 gm)
o Sodium chloride (1.00 gm)
o Glycerine (20.00 gm)
o Sodium merthiolate, 1:1000 solution
(2.00 ml)
o Distilled water (200.00 ml)

Normal Saline Solution (NSS) or Physiologic Saline

Solution
 This is used in cases of emergency or in cases of FILLING THE COUNTING CHAMBER
much rouleaux formation and autoagglutinated 5. Place the thick cover slip on top of the Improved
cells Neubauer counting chamber.
o serves as a preservative o Both the coverslip and counting chamber
o stable should be free from dirt, thumb mark,
tissue strands and the like
3.8% Sodium Citrate 6. Reshake the pipette. Discard the first 5-6 drops
of the diluted blood from the pipette and carefully
PROCEDURE OF RED BLOOD CELL COUNTING and exactly fill or charge the counting chamber by
capillary action.
PREPARE THE DILUTED BLOOD o This can be achieved by placing the tip of
1. Aspirate blood up the 0.5 mark of your RBC the pipette on the space between the
pipette cover slip and essential platform of
2. Wipe off the excess blood at the tip of the pipette counting chamber
with a clean piece of tissue paper o The angle of the pipette when charging is
o The tissue paper should not touch the 30 – 35 degrees
opening of the bore o Take not that you should be careful in
3. Immerse the pipette into the diluting fluid filling out the counting chamber. You may
contained in a clean transparent container. By experience:
syringe aspiration, draw the diluting fluid up to the  Overcharging – can be be
101 mark with a constant rotation of the pipette to readily observed by the
ensure proper mixing of blood and the diluting presence of the fluid on the
flood. This makes 1:200 dilution. moats of the counting chamber
4. Gradually bring the pipette to a horizontal position o The moats are the
and immediately mix the contents of the pipette. depressions on either
side or in between the

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PROPERTY OF CHERRYANNE DOMINGO

area on which the  The five squares have an area of 1/5

squares are mark, thus mm2
giving an H-shape  A standard pattern of counting should be
 Undercharging – it is observed adopted. Count the RBCs on the first row of the
when the failure to cover the smaller squares from left to right, drop down to
entire ruled area of the counting the second row and count from right to left, then
chamber to the third row counting from left to right, and
 Air bubbles – it indicates the lastly the fourth row counting from right to left.
presence of moisture or dirt. If
such thing occurs, clean the
cover slip and counting chamber
and repeat the process

7. Stand for 5-10 minutes to allow the RBCs to

settle.
8. Locate the ruled area for RBC counting by
focusing the microscope using the LPO initially.
Once located, shift to HPO for actual counting.

Pattern on how to read RBCs

 Take Note:
o RBCs that touch any of the lines on the
top and left borders, even if they are
outside the tertiary squares, are included
Primary Square of an Improved Neubauer Chamber in the count
o RBCs that touch any of the lines on the
Note: right and bottom borders are not
Inside the primary squares, there are nine secondary included even they are inside the square
squares. We will count the RBCs in one of the  Record the count on each of the 5 tertiary
secondary squares. squares making sure that the cell difference
between two squares is 20 or less.
o For example,
MAKING THE COUNT
R1 = 40 cells, R2 = 48 cells, R3 = 80 cells
 In the central secondary square, count the RBCs
o Therefore, the difference between R3
seen on the 5 tertiary squares designated as R
and R1 is greater than 20. If this
squares (R1, R2, R3, R4, and R5)

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PROPERTY OF CHERRYANNE DOMINGO

happens, you should repeat the it employs the use of pre-measured

procedure. volume of diluting fluid in a plastic
o You should repeat the filling of the reservoir
chamber by your diluting fluid. o A specific amount of blood is then
o Take note that all counts must be done in collected using the capillary pipette and
duplicate for us to verify the results then mixed the diluting fluid.

All counts must be done in duplicate

COMPUTATION
 General Formula for the computation of the
number of blood cells per cubic millimeter of
blood are as follows:

 Area = 1/5, Depth = 1/10, Dilution = 1/200

 To simplify it the formula is: cells counted x RED CELL COUNT: LOADING HEMOCYTOMETER
10,000 (Link: https://www.youtube.com/watch?v=Umoyb1e9LJ4)
 Take note that not all the time we have the same
1. Mix the reservoir ten times by inversion then
depth, dilution, and area. But in this case, we use
remove the capillary
1/5, 1/10, and 1/200
2. Squeeze the reservoir then mix it again
 After we computed, we can interpret the results
3. Squeeze a couple drops out of the end of the
using the reference changes
capillary and holding it with two hands, a dry tip,
go right up to the cover slip, right in the groove
REFERENCE RANGES
4. Give it a squeeze until it fills, and come away
Male 4.5-6.0 M / cumm
Female 4.0-5.5 M / cumm 5. Fill the other side with the other reservoir
Late pregnancy 3.0-5.0 M / cumm 6. Mix at least ten times by inversion
At birth 7.0 M / cumm 7. Take the capillary out
M= million, cumm = cubic millimeter 8. Squeeze the reservoir
9. Release to draw it down and mix again
 Take note, we have two units of measurement 10. Release a couple drop out of the tip
o Conventional Unit 11. The dry tip right up to the cover slip, right in the
o S.I. Unit groove
 If we want to convert it, we can multiply it using 12. Give it a squeeze, fills, and come away
this factor 0.000001  Put in a moisture chamber for just 5
o Male: 4.5-6.0 x 1012 /L minutes
o Female: 4.0-5.5 x 1012 /L
RED CELL COUNT MICROSCOPE CHECK
OTHER METHODS (Link: https://www.youtube.com/watch?v=lz9RXhF1ys4)
 Unopette System
o It is a convenient method of preparing 1. Place your hemocytometer
diluting blood sample for cell counting for

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PROPERTY OF CHERRYANNE DOMINGO

2. Take it carefully out of your moisture chamber  Cells do no conduct current

and place it on the stage of your microscope but rather they change electrical
(Stage Down – Condender Up) resistance which is then counted
3. The microscope stage should be all the way as voltage pulses
down, and the condenser up  The number of pulses
4. Then adjust your stage so that the spot of light is generated is proportional to the
right about there on the hemocytometer (where number of cells present
the rulings will be showing up)  Whereas, the amplitude of the
5. Then raised the stage pulse generated is proportional
6. You should have your 10x objective in place and to the size of the cell
raise it until it is close to your objective lens  Therefore, we can already know
o Don’t bring it up too far or you may break which are the RBCs
your cover slip 2. Optical Scatter
7. Lower the condenser and then close the iris  Light Scattering Optical Method
diaphragm  It uses a flow cytometer with
8. Lean over the microscope laser to measure light scattering
9. Looking from the microscope, you’re going to properties of cells.
lower the stage away from your objective with the  It can be a forward angle light
coarse focus knob until the rulings come into scatter which measures the
focus cells size or a side angle light
10. Place the first square that you’re going to count in scatter which provides
the center information on cell granularity
11. For your manual red cell count, put the 40x into and lobularity
place and then use the fine focus until the cells
 We can already determine which
come into focus
are RBCs
12. Then, as you count the cells, you’ll keep track of
 TM
Sysmex E 5000
them on a hand tally
 Sysmex NE 8000
13. You’ll probably need to fine focus as you count.
 Sysmex Total Hematology System (HS)
They’ll show up as dark or bright little spots in the
 Coulter Counter Model S
hemocytometer
14. As you find each cell, click it on the hand tally  Coulter Counter Model S Plus
15. After completing the count on each side of the  CounterTM STKS and CoulterTM MAXM
hemocytometer, make sure note your results on  Coulter Counter Models S Plus II, III, IV, V, VI,
a lab report form and STKR
 Technicon HTM Systems
AUTOMATED METHODS  Cell Dyn 3000/3000 SL and many more
 In the laboratory, nowadays, since there are a lot
of number of complete blood count samples to be
analyze we now use automated methods.
 Despite the number of the hematology analyzers
available from different manufacturers and their
varying levels of sophistication and complexity.
 Most of the analyzers rely on only two basic
principles of operation:
1. Electrical Impedance or Resistance
 The cells pass through an
aperture with electric current
flowing through simultaneously

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PROPERTY OF CHERRYANNE DOMINGO

KNOWLEDGE CHECK
 What is the total RBC count (in S.I. unit) of a
female patient if 400 RBCs were counted in 5 of
25 tertiary squares, and specimen dilution factor
is 200? Answer: 4.0 x 1012 / L
 Does the patient may possibly have anemia?
 A 1:200 dilution of a male patient’s sample was
made and 371 red cells (R1=65, R2=75, R3=79,
R4=87, and R5=83) were counted in an area of
0.2 mm2 What is the RBC count in Conventional
Unit? Answer: It cannot be determined (you
should repeat the procedure of filling out the
counting chambers)

**Take Note: the number of cells in R4 is 87 and the

number of cells in R1 is 65. Therefore, the difference of
the two squares are more than 20***

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PROPERTY OF CHERRYANNE DOMINGO

HEMOGLOBIN DETERMINATION  RBC count is not performed anymore in practice

because it is tedious. It is subjective to a lot of
HEMOGLOBIN errors.
 The primary function of hemoglobin within the  RBC count is phased out since it can be
RBC is to carry oxygen to and carbon dioxide determined relative to patient hemoglobin and
from the tissues. hematocrit.
 Hemoglobin is measure oxyhemoglobin or  A lot of laboratories are already automated. Only
measured indirectly by converting it to hematocrit and hemoglobin is determined
compounds like cyanmethemoglobin, acid
hematin, alkali hematin, and CYANMETHEMOGLOBIN METHOD
carboxyhemoglobin  Also known as Hemoglobin Cyanide (HiCN
 Other methods determine Hgb levels by detecting method)
oxygen and iron and as well as by determining  Reference method approved by CLSI for
blood’s specific gravity hemoglobin assay.
.  Advantages:
o The pigment cyanmethemoglobin is
stable even in diluted solution
o Certified cyanmethemoglobin
standard is commercially available
o Measured all forms of hemoglobin
except sulfhemoglobin
o The spectral curve of
cyanmethemoglobin allows the use of
different types of spectrophotometers

(SIR BENJ)
 It is the most sensitive and specific for
hemoglobin
 Sulfhemoglobin
- hemoglobins with sulfide groups instead of
oxygen
Tertiary Structure of Hemoglobin structure - can’t carry oxygen
- patients with sulfhemoglobin turn purplish
(SIR BENJ) (cyanotic), skin color described as mauve
 Hemoglobin structure lavender
o Primary – pertains to amino acid
sequences PRINCIPLE
o Secondary – helices and nonhelices  Blood is diluted in an alkaline Drabkin solution
components of potassium ferricyanide, potassium cyanide,
o Tertiary – 2D structure sodium bicarbonate, and a surfactant (nonionic
o Quarternary – 3D structure detergent).
 RBC requires hemoglobin to carry out its
functions and to form RBC (SIR BENJ)
 Oxyhemoglobin – a hemoglobin that carries  Potassium ferricyanide and potassium
oxygen cyanide - main components
 Carbaminohemoglobin  Drabkin’s solution is highly toxic to humans
- Also known as deoxyhemoglobin since it contains cyanide
- a hemoglobin that carries carbon dioxide

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PROPERTY OF CHERRYANNE DOMINGO

 There is no reagent at the same level of Drabkin’s  Structure of hemoglobin

solution (no alternative) o has 4 globin chains, each attached to
o Lysing agent (detergent): enhances pyral rings of heme
lysis of erythrocytes to release o Heme component is made of
hemoglobin Protoporphyrin IX which has attached
o The hemoglobin (Fe2+) is oxidized to iron in ferrous state
methemoglobin (Fe3+) by the potassium o Each heme can carry 1 oxygen
ferricyanide, K3Fe(CN)6. molecule; 1 hemoglobin can carry 4
o Methemoglobin – a hemoglobin with a molecules
ferric state of iron (3+)  Cyanmethemoglobin measurable at 540 nm
o The potassium cyanide (KCN) then
converts the methemoglobin to The greater absorbance value, the greater
cyanmethemoglobin (HiCN). concentration of hemoglobin.
o Sodium bicarbonate: pH 7.0 – 7.4
 Substituting dihydrogen
SPECTOPHOTOMETRY PROCEDURE
potassium phosphate (KH2PO4)
for sodium bicarbonate
(NaHCO3) shortens the time
needed for complete conversion
of Hb to HiCN from 10 minutes to
3 minutes.
 maintains the buffer of the
solution

General Chemical Formula

 Tube 1: Reagent blank
o Blanking is performed to rule out the
 The absorbance of the cyanmethemoglobin is
color of the reagent
measured in a spectrophotometer at 540 nm
o Drakbin’s Reagent was measure to rule
 The absorbance directly proportional to the
out its color
hemoglobin concentration.
o If not performed, even color of reagent
 The absorbance is compared with that of a
will be measured
standard HiCN solution.
 Tube 2: Standard
o Solution with known concentration
(SIR BENJ)
o Usually comes from manufacturer
 Rule of 3:
o Usually has 15 g/dL concentration
o Hematocrit / 3 = Hemoglobin
 Tube 3: Unknown (Patient’s Whole Blood)
o Hemoglobin / 3 = RBC
o Amount of blood is dependent on
o Only applicable for conditions of normal
procedure provided by manufacturer
people
o 5 mL of Drabkin’s + sample
o Primary laboratories use rule of 3 instead
 Tube 4: Normal Control
since the procedure is tedious and
 Tube 5: Abnormal Control
Drabkin’s solution is toxic
o Both normal and abnormal controls are
o Can be used to assess if the
used to check the reliability of the test
cyanmetheglobin method was performed
o Treated similar to unknown sample, but
right
already has known values

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PROPERTY OF CHERRYANNE DOMINGO

o For example, if normal control ranges  Patient 3 Concentration

from 8 g/dL to 12 g/dL, the range of 15 × 0.28
sample should meet that range = 10.5
0.40
o Test should be repeated if it does not  Patient 4 Concentration
enter range 15 × 0.52
 Errors are usually due to pipetting techniques = 19.5
0.40
 Patient 5 Concentration
DETERMINING THE HEMOGLOBIN 15 × 0.32
CONCENTRATION = 12
0.40

STANDARD CURVE
 A standard curve should be set up with each new
lot of reagents. It also should be checked when
Original formula alterations are made to the spectrophotometer
 Using a semi-logarithmic paper, a standard curve
is created by plotting the absorbance of standard
Derived formula solutions on the y-axis, and the concentration of
the standard solutions on the x-axis.
 Beer’s Law – absorbance of unknown over  Criteria for a good standard curve:
absorbance of standard should be equal to o Line is straight
concentration of unknown over concentration of o Line connects all points
standard o Line goes through the origin, or intersect,
of the two axes
Activity: (SIR BENJ)
 A Hemoglobin standard containing 15g/dL is  This test should be performed every time you
used. Five (5) specimens are submitted in the open a standard with a different lot number
laboratory for Hgb test. The unknown specimens,  Lot number: codes for reagents that were
together with the standard, are measured created at the same time or in the same batch
spectrophotometrically at 540 nm. The following  A standard curve should be a straight line
are the absorbance results:  We perform serial dilutions of the standard and
o Absorbance standard: 0.40 measure the absorbance of the diluted standards
o Patient 1 Absorbance: 0.46  Beer’s Law is more commonly used, however
o Patient 2 Absorbance: 0.36 standard curve should be used for machines
o Patient 3 Absorbance: 0.28  If it is plotted, the standard curve should be a
o Patient 4 Absorbance: 0.52 straight line or as straight as possible
o Patient 5 Absorbance: 0.32  X (horizontal) = concentration
 Y (vertical) = absorbance
Identify the hemoglobin concentration of the Five (5)
specimens. A standard containing 15 g/dL is used, the following dilutions
are made
Solution: Tub Tub Tub Tub
Tube 5
e1 e2 e3 e4
Cyanmethemoglobi 1 2 3 4
 Patient 1 Concentration n Standard mL mL mL mL
5 mL
15 × 0.46 Cyanmethemoglobi
= 17.25 4 4 4 4
n reagent 4 mL
0.40 mL mL mL mL
(Drabkins)
 Patient 2 Concentration Mix tubes and stand for 10 minutes
15 × 0.36 Undilute
= 13.5 Dilution 1:5 2:5 3:5 4:5
d
0.40

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PROPERTY OF CHERRYANNE DOMINGO

3 6 9 12 Y-intercept 0
Concentration 315g/dL
g/dL g/dL g/dL g/dL Slope 0.26666667
Read absorbance at 540 nm. Record results.
Absorbance 0.08 0.16 0.24 0.32 0.40
 Using the standard curve, we can get the
Using a graphing paper, plot the absorbance reading at the y-
axis and the concentration at the x-axis concentration of the hemoglobin
Determiine the hemoglobin concentration of the control  Derived formula for determining hemoglobin
specimens and the patient specimens from the Standard concentration:
Curve
𝑦−𝑏
𝑥=
 Serial dilution in hematology is different from 𝑚
clinical chemistry
 Serial dilution in hematology – different tubes with Activity:
different dilutions Determine the hemoglobin concentration using the
standard curve
 Using this Hemoglobin standard curve and the
measured absorbance at 540nm, identify the Hgb
concentration of these unknown samples:
o Patient 1 (A= 0. 45)
o Patient 2 (A=0.30)
o Patient 3 (A = 0.42)
o Patient 4 (A= 0.25)
o Patient 5 (A= 0.55)

Solution:
 Patient 1 Concentration
0.45 − 0
𝑥=
0.02667
𝒙 = 𝟏𝟔. 𝟖𝟕
 Patient 2 Concentration
0.30 − 0
𝑥=
0.02667
𝒙 = 𝟏𝟏. 𝟐𝟓
 Patient 3 Concentration
0.42 − 0
 If the curve is bended, then the performance of 𝑥=
0.02667
the procedure was erroneous or there is
𝒙 = 𝟏𝟓. 𝟕𝟓
something wrong with the standard
 Patient 4 Concentration
 You have to determine the slope
0.25 − 0
 Since it’s a straight line, the slope with almost be 𝑥=
equitable to 0 (zero) *y-intercept ata dapat to kaso 0.02667
slope sinabi? 𝒙 = 𝟗. 𝟑𝟕
 Patient 5 Concentration
 Can be used to solve for slope: 0.55 − 0
𝑥=
0.02667
𝒙 = 𝟐𝟎. 𝟔𝟐

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PROPERTY OF CHERRYANNE DOMINGO

Recap/Summary: o Reagent-specimen solutions should not

 Standard curve is prepared using your be discarded into sinks
standard o A licensed waste disposal service should
be contracted to discard the reagent
 Get different dilution of standard and get its
o A special container should be used for
concentration
disposal
 Standard has a known concentration
 Another method that has been used in some
 Pano nakuha 5 g/dL? Kasi 15 x1 divided by 5
automated instruments involves the use of
is 3. Dun sa tube 2, 15 (original
sodium lauryl sulfate (SLS) to convert
concentration) x2 divided by 5 is equal to 6
hemoglobin to SLS-methemoglobin. This method
 We can get y-intercept through excel
does not generate toxic wastes.
 Y-intercept is almost equal to zero so it can
 Carboxyhemoglobin takes 1 hour to convert to
be omitted
cyanmethemoglobin and cause erroneous results
 Substituting this formula y = 0.0267 + 3E-16,
in specimens from heavy smokers
we can now get the hemoglobin
o Carboxyhemoglobin is a type
concentration of the patient
hemoglobin where carbon monoxide
 Slope is usually constant as long as you use
binds to hemoglobin.
the same lot number of standard
o Carbon monoxide is from smoking,
 Transpose x to other side of equation and exhaust of automobiles
transfer y to get formula
 Turbidity will cause a falsely high/ elevated result
𝑦−𝑏 o High WBC count (greater than 20 x
𝑥=
𝑚 109/L) - in cases of leukemia
 Concentration (x) is equal to absorbance (y) o High platelet count (greater than 700 x
minus 0 (b is almost equal to zero) divided by 109/L)
the slope (m)  Remedy: reagent-specimen
solution be centrifuged and the
REFERENCE VALUES supernatant measured
 Only RBC is lysed by surfactant.
However, WBCs can also absorb
light from spectrophotometer if
it’s too many
o Lipemia – contains a lot of fat,
namumuti-muti
 Most of the time, SI units are used  Remedy: Sample blank - adding
 Multiply conventional unit by 10 to get 0.01 mL of the patient’s plasma
concentration to SI to 5 mL of cyanmethemoglobin
 Men have higher hemoglobin levels than women regent and using this solution as
due to the following reasons: a blank
o Menstruation of women  Also use sample as your blank to
o Men have greater muscle mass; require rule out lipemia
more oxygen o Hemoglobin S (Hb S) and Hemoglobin
C (Hb C) – variants of hemoglobin, there
SOURCES OF ERRORS AND COMMENTS were substitutions in the amino acid
 Cyanmethemoglobin reagent is sensitive to sequence
light. It should be stored in brown bottle or in a  Remedy: make a 1:2 dilution
dark place. with distilled water (1 part
 Because Drabkin’s reagent contains cyanide, it specimen plus 1 part water) and
is highly toxic and must be used cautiously. multiply the results by 2.

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PROPERTY OF CHERRYANNE DOMINGO

o Abnormal globulins, such as those found 7. Take the reading of the lower meniscus and
in patients with plasma cell myeloma or report the hemoglobin level in g/dL (CU) or g/L
Waldenstrom macroglobulienia – (SI) – kung saan yung inabot na volume sa
abnormal protein Sahli’s tube, yun yung hemoglobin level
 Remedy: add 0.1 g of potassium
carbonate to the  It has similar procedure and procedure with alkali
cyanmethemoglobin reagent hematin method. Acid hematin = Acid, Alkali
hematin = Alkaline
OTHER METHODS OF HEMOGLOBIN
DETERMINATION ALKALI HEMATIN METHOD
 Not commonly used since these have a lot of Principle: the use of an alkaline solution (NaOH) for
possible errors, especially human/performance hemoglobin determination; produces a true and relatively
errors stable solution of hematin

ACID HEMATIN METHOD Procedure:

 Principle: involves the conversion of hemoglobin 1. Place 5mL of NaOH on a reaction tube
to acid hematin by using 0.1N HCl. The yellowish- 2. Deliver 0.05 mL of blood to the tube
brown solution is compared to the color standard 3. Heat in a boiling water bath for 4-5 minutes
in the comparator block - Allows RBC to further lyse
 Types of hemoglobinometer: Sahli-Hellige, 4. Cool and read against an appropriate standard
Sahli-Adam’s, Sahli-Haden
Disadvantage: The bloods of newborn and young infants
contain alkali resistant fetal hemoglobin (Hg F). This may
be a significant source of error in hemoglobin
determination
 Hg F resistant to lysis due to sodium hydroxide
Sahli’s
tube TALLQUIST OR SCALE
Sahli’s
pipette  This method is based on the principle of color
comparison
 Uses simple chart consisting of different degrees
Comparator of redness to serve as a comparator.
c block  Blood is collected and a drop is placed on a piece
of thick absorbent paper
Procedure:  The color of the slightly dried blood on the paper
1. Deliver 0.1N HCl up to the 2 mark of the Sahli’s is then compared to the color chart.
or square tube  Hemoglobin is reported in terms of percentage
2. Aspirate 0.02 mL of blood using Sahli’s pipet (%).
(similar to Thoma pipet but without bulb)  This has percentage error of 30 – 50% (error
3. Expel the volume of blood to the Sahli’s tube should be at least 5% for it to be statistically
4. Mix and stand for at least 10 minutes significant, lower than that is better)
5. Add distilled water to the mixture drop by drop
ensuring mixing after each addition with the
stirring rod
6. Continue adding water until the color of the
solution matches that of the standard on the
comparator block (amber colored, used to
compare color of solution)

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PROPERTY OF CHERRYANNE DOMINGO

CARBOXYHEMOGLOBIN
 not done as a routine examination. It is only
performed when carbon monoxide poisoning is
suspected
 Carboxyhemoglobin – carbon monoxide binds
to hemoglobin; carbon monoxide has 240 times
greater affinity to hemoglobin compared to
oxygen to hemoglobin
 Despite how many oxygen molecules are
present, carbon monoxide will bind to hemoglobin
 Carbon monoxide
Hemoglobin scale - silent killer gas; odorless and colorless
- carbon monoxide poisoning effect;
 Not performed because: grogginess, dizziness, sleepiness
- From exhaust of vehicles
o Drying of blood in paper is hazardous
- Smokers have urge to smoke when stressed
o Comparison of color is very subjective
since they get carbon monoxide which affects
OXYHEMOGLOBIN neurons and limits its functions; allows them
to focus on certain topic/things
 Sodium Carbonate
- 10%-20% carboxyhemoglobin can be
- a photometric determination of hemoglobin
tolerated by the body; excess will lead to side
by measuring oxyhemoglobin. Simple and
effects
quick procedure but there is no possibility of
preparing a stable oxyhemoglobin standard
GASOMETRIC METHOD
 Photoelectric oxyhemoglobin
Van Slyke Oxygen Capacity
- pulse oxygen saturation as well as pulse rate
 This method measures the amount of oxygen
is measured through the finger by attaching
using a Van Slyke manometric apparatus
the photoelectric oxyhemoglobin monitor
 The level of hemoglobin is determined by
- yung nilalagay sa daliri pag nagpapacheck-
up computation
- pulse oxygen saturation is measured in the 1 gram of Hemoglobin = 1.34 mL O2
finger through photo electric detector and
infrared light
CHEMICAL METHOD
Kennedy’s method; Wong’s method
 This method measures the amount of iron
present.
 This method is based on the fact that most of the
iron is found in the red blood cell and combined
with the hemoglobin molecule.

1 gram of Hemoglobin = 3.47 mg Fe2+

SPECIFIC GRAVITY METHOD

Pulse oximeter (photoelectric oxyhemoglobin) Copper sulfate method
 This method employs the use of copper sulfate
with a known specific gravity (1.052 – 1.054).
o The blood specific gravity is 1.053 (due
to hemoglobin component of RBC)

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PROPERTY OF CHERRYANNE DOMINGO

 30 mL container; perform up to 25 tests; solution  Hemoglobin electrophoresis using cellulose

changed daily acetate at an alkaline pH (8.2 to 8.6) is a
 A drop of blood is added to the copper sulfate screening test for the detection of variant or
solution. abnormal hemoglobins
o Distance between the drop of blood and  Further confirmation of these variants can be
solution: 1 cm achieved using citrate agar electrophoresis
 If the drop falls, the level of hemoglobin is said to  Used to determine specific variants of
be acceptable hemoglobin based on migration in
o Acceptable drop of blood will sink in the electrophoresis gel
solution within 15 seconds (Hb > 12.5  Electrophoresis
g/dL) o Performed based on migration of
o Paglumulutang-lutang yung blood sa different proteins due to their charges;
gitna, specific gravity is within 1.052- proteins are amphoteric (possess
1.054; accept donor different charges based on environment;
o If it sinks, specific gravity is beyond; charge is different when in alkaline vs
accept donor acid environment)
o If it floats, it’s less than 1.052 (Hb, 12.5 o Placed in agarose gel to make migration
g/dL); reject donor easier
 This method is used in screening potential blood o Add dye to observe protein movement
donors o Expose samples to electric charge in
 Very qualitative order for them to move
 Used when performing mass blood donation
 Color of copper sulfate: Blue
 Blue changes to green when too much blood is
added; indicates that you should change the
solution

Electrophoresis at pH 8.6

 At pH 8.6 (basic state), hemoglobins are

negatively charged. If exposed to electric charge,
they migrate to anode.
 Only common hemoglobins change to negative
charge
 Hemoglobin H and Hemoglobin Bart’s
- quantitative defects in hemoglobin
o Hemoglobin H – 3 gene deletion of
alpha chain
o Hemoglobin Bart’s - 4 gene deletion
of alpha chain; no alpha chain
Copper sulfate solution used for Specific Gravity Method produced
 Usually, patients don’t last
HEMOGLOBIN ELECTROPHORESIS for 1 or 2 days
 To detect abnormal hemoglobins  Mnemonic:
(Hemoglobinopathies) o Accelerated, Fast, Slow, Origin

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PROPERTY OF CHERRYANNE DOMINGO

o *A Fat Santa Claus

 Security Guard on Duty – S, D, G
in slow
 CEO – hemoglobins in origin

Electrophoresis at pH 6.2

 At pH 6.2, the migration varies. Some becomes

positively charged while others become
negatively charged
 (+) charged = migrate to cathode (anionic)
 (-) charged = migrate to anode (cationic)

 Examples on how to determine hemoglobin

patterns:
o Pattern A – has hemoglobin A2, S, A
o Pattern B - has a lot of hemoglobin S

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PROPERTY OF CHERRYANNE DOMINGO

HEMATOCRIT DETERMINATION & RULE OF THREE o Better formation of the packed RBCs for
hematocrit measurement
HEMATOCRIT
 It is the volume of packed red blood cells that PROCEDURE
occupies a given volume of whole blood after 1. Fill two plain capillary tubes approximately
centrifugation of a blood sample. three-quarters (3/4) full with blood
 It is also known as Packed Cell Volume (PCV) anticoagulated with EDTA or heparin.
or Erythrocyte Volume Fraction (EVF)  In Henry’s Book, 2/3 filled, 0.05 mL
 It is reported either as a percentage (%), cell blood, filled portion 5 cm
volume percent (CV%), volume percent (Vol%) or  Nevertheless, they are both
in liters per liter (L/L) correct (Rodaks and Henry)
o This percentage is your conventional  Length: 7-7.5 cm
unit for hematocrit measurement  Bore size: 1.2 mm
o For the SI unit, it can be converted to  The capillary tubes will be filled through
liters per liter (L/L) capillary action
 Hematocrit Determination  Two capillary tubes – because of a
o It is one of the simplest and valuable test duplicate microhematocrit determination
in hematological investigation and to assure precision and
o It is useful in detecting cases of anemia, reproducibility of the results (part of the
polycythemia quality control); it can also be used for
 Anemia: low hematocrit value; balancing the centrifuge
low packed cells volume relative  If the blood is already anticoagulated with
to the plasma EDTA or heparin, we must use a non-
 Polycythemia: increase in the heparinized capillary tube
packed cell volume/hematocrit  Excess anticoagulant – can
value cause a false result in the
hematocrit determination
 A non-heparinized (blue ring) capillary
tube is used if blood is collected on
anticoagulated tube
 If blood is from a skin puncture, use
heparinized (red ring) capillary tube
 It has an anticoagulant heparin

 In the normal blood, after centrifugation, what we

can see is a packed RBCs here measured as the
hematocrit, buffy coat consisting of the WBCs
and platelets, and the plasma.

ADAM’S MICROHEMATOCRIT METHOD

 It is routinely used in the laboratory
 Advantage of Microhematocrit Method vs.
Macrohematocrit Method
o Less time consumed doing the procedure
o Requires less blood sample

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PROPERTY OF CHERRYANNE DOMINGO

2. Seal the end of the tube with the colored ring o Kaya sometimes you will see
using nonabsorbent sealing clay rpm (revolutions/rotations per
 Seal by placing the dry end into the tray minute), instead of ‘g’
with sealing compound at a 90-degree o Relative Centrifugal force is a
angle more accurate measurement
 Rotate the tube slightly and remove it because it accounts for the
from the tray radius of the rotor
 The plug should be at least 4 mm long  Kasi pwede magiba-iba
 Other references mentioned that it yung radius ng ating
should be 4 – 6 mm long or at least 4 microhematocrit centrifuge
mm long depende sa liit o laki nito
 Mas magandang
measurement ang relative
centrifugal force (g) than
rotations per minute (rpm)

3. Balance the tubes in a microhematocrit 5. Determine the hematocrit by using a

centrifuge with the clay ends facing outside microhematocrit reading device
away from the center, touching the rubber  This is what we will see after
gasket centrifugation:
 The sealing clay end should be the one
facing outside away from the center
o Why? because the centrifugal
force is acting outwardly away
from the center of rotation
o Kapag ang nakalagay is yung
end na walang sealing clay
(facing outward), mag-leleak
yung blood. Pagkakuha niyo
ubos na yung sample niyo.
4. Tighten the head cover on the centrifuge and
close the top. Centrifuge the tubes at 10,000 g to
15,000 g for 4 - 5 minutes
 ‘g’ means relative centrifugal force
 Relative Centrifugal Force is actually
based on revolutions per minute

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PROPERTY OF CHERRYANNE DOMINGO

 Additional Information c) Place it at the black line at the top of the

o If we will look in the layer of the instrument in the little plastic
buffy coat, mauuna ang platelets d) Take the bottom wheel and move it until the red
followed by the WBCs line reads 100
o For the packed RBCs, the top e) Holding the bottom plate in place, you are going
layer is composed of to move the top plate until the spiral line meets
reticulocytes / nucleated RBCs the top of the column of plasma
bago yung mga mature RBCs f) Then moving both plates together, move the
6. The values of duplicate hematocrits should agree spiral line until it reaches the intersection of the
within 1% (0.1 L/L) red blood cell column and the plasma
 Take Note: We should not include the g) The number that you see at the bottom of the
buffy coat when taking the reading wheel is the hematocrit (ex. 43%)
because it will result false increase h) Remove the capillary. Push it forward and just
hematocrit determination grasped it at the end.
i) Remember to dispose all the sharps including the
slides, pasteur pipettes, capillary tubes, and
needles in the sharps container.

VIDEO
HEMATOCRIT RECTANGLE READER
Link: https://www.youtube.com/watch?v=fngyZJv8vA4
Example of a microhematocrit reading device
*This is an example of a different hematocrit reader
VIDEO a) Put the zero where the clay and the red cells
MICROHEMATOCRIT DEMO meet. We’re going to put the 100 at the top of the
Link: https://www.youtube.com/watch?v=tU-FzfcUO2w plasma
**Hindi masyado marinig yung audio*** b) Look at it from directly on top
c) Tilt it a little bit for production problems
(Sir Clarenz) d) Take the tray and move it along until the bottom
a) Mix the anticoagulated tube of the meniscus of the plasma is on this 100% line
b) Fill 3/4 or 2/3 of the capillary tube without moving the zero from the zero line
c) Wipe the excess blood from the outside of the e) Take the centerpiece and move it till the line is at
tube before sealing it the top of the red cells then we go right over here
d) The seal should be 4-6 mm long. Seal it properly. and read it off of the scale
e) Place it on the centrifuge. The sealed end should
be the one facing outwards. (Sir Clarenz)
f) Centrifuge the tubes at 10,000 g for 4 - 5 minutes  The usage of this type of hematocrit reader is for
us to position the centrifuge tube so that the base
VIDEO of the red cell intersects with the zero line.
MEASURING MICROHEMATOCRIT  We need to move the slide until the top of the
Link: https://www.youtube.com/watch?v=IAOLhHFVvXU plasma intersects with the 100% line

a) We are going to use the microhematocrit reading READACRIT CENTRIFUGE

device to measure the hematocrit which you can
 uses precalibrated capillary tubes and has
estimate simply by looking at the column of blood
built-in hematocrit scales, which eliminates the
and estimating what proportion the packed red
need for separate reading devices.
blood cells is at the total blood volume.
 The use of SUREPREP Capillary Tubes
b) Take your micro capillary tube and with the
eliminates the use of sealants.
bottom of the tube or the bottom column of blood.

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PROPERTY OF CHERRYANNE DOMINGO

 They have a factory-inserted plug that seals ang pagkakaorient natin nung slide.
automatically when the blood touches the plug Dapat yung sealing clay nakaface
outward. Kapa pabaliktad naman yun,
then the blood will also leak resulting to a
false decrease hematocrit because a
higher number of RBCs will be loss
compared to the plasma. Because the
packed RBCs in the lower part of the
tube, will leak during centrifugation.
b) An increased concentration of anticoagulant
(short draw in an evacuated tube) decreases the
hematocrit reading
 Remember for hemoglobin
determination, even though we have an
excess anticoagulant, the hemoglobin
will not be affected by the increase
concentration of anticoagulant
 However, in hematocrit, it can cause a
REFERENCE VALUES
false low result
 Possible cases of increase concentration
of anticoagulant
o We have a short draw in an
evacuated tube. For example, if
 Estimation of hemoglobin and red blood count is
the evacuated tube requires 5 ml
possible on the basis of the hematocrit value
of blood but we only collected
under normal circumstances.
around 3ml. This can result to an
 There is a variation for male and female for RBCs
increase in the anticoagulant
and hemoglobin. Likewise, ganun din po sa
value. That increase
hematocrit determination
anticoagulant (ex. EDTA) will
 We can use our hematocrit to estimate cause shrinkage of the cells
hemoglobin and red blood cell counting under which will result to a decrease of
normal circumstances hematocrit. As the red cell
 1% Hematocrit – 0.34 g/dL Hemoglobin or forms a packed RBC, it will have
107,000 RBC/cumm a decrease result.
 Again, just like your RBCs and hemoglobin o If you have an anticoagulated
determination, a low hematocrit value means tube at nag-collect pa tayo sa
there is anemia. An increase in hematocrit value capillary tube na meron din
means there is polycythemia. anticoagulant.
 Take note that there are also other conditions that c) A decreased or increased result may occur if
we may consider and the values maybe a case of the specimen was not mixed properly.
an error like a false increase or a false decrease  We should first mix the anticoagulated
value. tube because if kumuha tayo ng blood
tas nakuha natin yung portion na puro
SOURCES OF ERROR AND COMMENTS red cells then it will show an increase
a) Improper sealing of the capillary tube causes hematocrit. Kapag mas maraming
a decreased hematocrit reading as a result of plasma naman yung na-collect natin,
leakage of blood during centrifugation. then that would result to false decrease
 Ganun din pag linagay natin siya sa ating
microhematocrit centrifuge. Hindi tama

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PROPERTY OF CHERRYANNE DOMINGO

result so kailangan na-mix natin siya cells meron po naiwan na

properly plasma. Hence, we call it
d) Insufficient centrifugation or a delay in trapped plasma
reading results after centrifugation causes  Trapping of the plasma will
hematocrit readings to increase. cause a false increase in the
 We should also take into account the microhematocrit measurement
time and the speed of the centrifugation, particularly a 1%-3% increase in
as well as the time when the results are the hematocrit measurement
read are also important. o Trapping of the plasma causes the
 Insufficient centrifugation will result to microhematocrit to be 1% to 3% (0.01 to
an increase in the hematocrit reading 0.03 L/L) higher than the value obtained
kasi hindi pa kumpleto yung pagform ng using automated instruments that
packed RBCs so marami pang plasma calculate the hematocrit and are
doon sa loob niya unaffected by the trapped plasma.
 Hindi mo agad binasa yung results,  Sa automation kasi it is
hindi na rin tama yung formation ng measured by calculation. Not
packed RBCs which will result to falsely directly minemeasure yung
increase hematocrit readings packed RBCs. Hence, if you will
e) The buffy coat of the specimen should not be use automation, it will be
included in the hematocrit reading because this unaffected by the trapped
falsely elevates the result plasma unlike sa manual or
 If you include the buffy coat consisting of semi-automated methods,
the WBCs and platelets that will result to wherein we are measuring the
a falsely elevated result as well. hematocrit directly,
f) A decrease or increase in the readings may be makakaapekto sa result kapag
seen if the microhematocrit reader is not used merong presence ng trapped
properly. plasma. Again, resulting to a
g) Many disorders, such as sickle cell anemia, false increase result around 1% -
macrocytic anemias, hypochromic anemias, 3%.
spherocytosis, and thalassemia, may cause o Automated Methods (Coulter Counter,
plasma to be trapped in the RBC layer even if Autoanalyzer, & other automated
the procedure is performed properly. machines)
o If you have these disorders magkakaroon  Hematocrit is computed
ng variations in the cell sizes. Yung  The machine computes the it by:
biconcave and the shape of the RBC will Hematocrit = RBC x MCV
now vary  The RBC is computed using an
 Erythrocytosis – variation in the automation through:
sizes  Electrical Impedance:
 Poikilocytosis - variation in the basic principle of
shape of RBC coulter counter;
o Trapped Plasma: amount of plasma that wherein, blood cells
still remains in the RBC portion after the are non-conductors of
microhematocrit has been spun. electricity. As they
o Increased in macrocytic anemias, passed, they create an
spherocytosis, thalassemia,hypochromic impedance or
anemia, and sickle cell anemia resistance of current
 Hindi okay yung pagkaform niya which are measured by
ng RBCs that in between the red pulses

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PROPERTY OF CHERRYANNE DOMINGO

 Optical Light  ESR: refers to the speed of

Scattering: wherein settling of RBCs in
blood cells create a anticoagulated blood
forward (measures the o 10 – 0 (White) is used for Hematocrit
cell size) and side  The anticoagulant of choice is double oxalate
(measures the cell
granularity) angle light
scatter
 MCV is under the RBC indexes
 It can be computed
manually
 MCV = hematocrit x 10
RBC
 In automation, MCV is
determined through the
use of RBC histogram
h) A temporarily low hematocrit reading may result
immediately after a blood loss because plasma
is replaced faster than are the RBCs.
 Greater plasma than the packed RBCs
i) The fluid loss associated with dehydration
causes a decrease in plasma volume and falsely
increases the hematocrit reading.
 Dehydration can be caused of diarrhea, PROCEDURE
vomiting, or burn. Again, for the measurement of hematocrit, we will use the
 Falsely increase hematocrit kasi mas WHITE calibration (10 to 0)
1. Fill the Wintrobe tube with blood using a Pasteur
mababa yung plasma volume and it will
pipet
show a relative increase in the hematocrit
j) Proper specimen collection is an important 2. Centrifuge the tube at 3,000 rpm for 30 minutes
consideration. The introduction of interstitial o Adam’s: 10,000 – 15,000 g for 4-5 min
fluid from a skin puncture or the improper o Wintrobe: 3,000 rpm for 30 minutes
flushing of an intravenous catheter causes 3. Read the volume of packed red blood cells
decreased hematocrit readings. 4. Compute for the hematocrit level using the
formula:
 Kung hindi proper yung pag-perform ng
for example, skin puncture, wherein there
Hematocrit = height of packed red blood cells X 100
is an introduction of interstitial fluids
height of whole blood used
that will cause a decreased in hematocrit
readings.
Take note: lower meniscus dapat
What are the layers of macrohematocrit?
MACROHEMATOCRIT METHODS
 First Layer – Fatty Layer
WINTROBE METHOD o Not visible unless the patient is lipemic
 Second Layer – Plasma
 This method employs the use of Wintrobe tube
o Length: 11.5 cm  Third Layer – Buffy Coat
o Bore 3 mm o Can be used to estimate the WBCs
because 1 mm of buffy coat is
 The tube has two calibrations:
o 0 – 10 (Red) is used for Erythrocyte approximately 10, 000 WBCs per cubic
Sedimentation Rate (ESR) millimeter
 Fourth Layer – Packed RBCs

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PROPERTY OF CHERRYANNE DOMINGO

2. Dilute the blood with the diluting fluid to the bulb

about half full
3. Seal the tube and centrifuge at 2,500 rpm for 15-
30 minutes
4. Read the volume of packed red cells

SANFORD-MAGATH
 Anticoagulant of choice is 1.3% sodium oxalate;
tube is calibrated at 1 mm per division. The tube
is about 5 inches long and a funnel-like mouth

PROCEDURE
1. Place 1 mL of anticoagulant into the tube
2. Add 5 mL of venous blood and mix
3. Centrifuge at high speed for 15 minutes
The methods differ based on: 4. Read the volume of packed red cells
 Anticoagulant
 Type of tube BRAY’S
 Some parts of the procedure  Anticoagulant of choice is heparin, tube is flat-
bottomed and calibrated on both sides similar to
HADEN’S MODIFICATION the Wintrobe tube. Calibration is from 10-50
 Anticoagulant of choice is 1.1 % sodium oxalate mm, each division is 1mm, and the capacity is
in distilled water. The method uses a calibrated 5mL
tube
PROCEDURE
PROCEDURE 1. Fill the tube with heparinized blood
1. Place 1mL of 1.1% sodium oxalate into the tube 2. Let it stand at a vertical position for 1 hour
2. Add 5mL of blood. Mix well. 3. Read the volume of red blood cells from the
3. Centrifuge for 20 minutes at 3,000 rpm lower right side calibration
4. Read volume of Packed Red Blood cells
*no centrifugation*
Hematocrit (%) = volume of packed red cells x 20
RULE OF THREE
If less than 5mL of blood is used, use the following
formula:

HCT (%) = volume of packed red blood cells X 100

volume of whole blood used  This rule only applies to specimens that have
normocytic normochromic RBCs
VAN ALLEN METHOD o Mag-co-conform yung result natin sa
 Anticoagulant of choice is 1.6% sodium oxalate ‘Rule of Three’ kapag ang red cells ay
in distilled water. The method uses a tube with nasa normal state (normal size,
bulb and calibration of 1 to 10cm (10-100mm) shape, and hemoglobin)
o Any variations in that the ‘Rule of Three’
PROCEDURE will not conform.
1. Fill the tube with blood up to the 10th mark of the  Example: poikilocytosis,
tube macrocytic red cells, sickle cells,
etc.

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PROPERTY OF CHERRYANNE DOMINGO

 This is a quick visual check of the results of the o We should do some further investigations
RBCs, hemoglobin, and hematocrit. Wherein, such as reviewing the blood film,
in some way, we can estimate RBCs, microscopically identify the shapes and
hemoglobin, and hematocrit from one another. size of RBCs, and investigate possible
 Through the use of “Rule of Three”, we can sources of error.
estimate the hemoglobin by multiplying the RBC o Upon examination of the blood film,
by 3 and we can estimate the hematocrit by nakita sa patient na ‘to that the cells are
multiplying the hemoglobin by 3 described to be microcytic
 Hematocrit ranges +/-3 nung ating (hemoglobin hypochromic red cells.
x 3) na answer  Small RBCs less than 7 microns
 Kapag nagkaroon ng discrepancy sa ‘Rule of in size
Three’, it may indicates the presence of  Central pallor is more than 1/3 of
abnormal RBCs kapag may mga variation in the cell diameter
sizes or shapes sa RBCS or it can also indicate  Characteristically seen in iron
that there may be causes of error in the RBC, deficiency anemia
hemoglobin, or hematocrit. So pwede magkaroon o What is the possible reason for the
ng false increase or false decrease result. false increase of hematocrit?
 Possibly because of the
microcytic hypochromic red cells
 Presence of microcytic red
cells can result to formation of
trapped plasma
 Trapped plasma can cause
false increase of hematocrit
measurement
o For example, paano kapag yung RBC
naman is around 3.5 M/cumm?
*4 M/cumm is same as 4 M/microliter  The hemoglobin should be 10.5
g/dL; hematocrit should be
CASE 1 between 28.5 – 34 .5
 Rule of Three  Pasok yung 32% sa range
o Hemoglobin = 4 x 3 = 12 g/dL  Tama na yung RBC at HCT, pero
o Hematocrit = 12 x 3 = 36% ang naging problema na ngayon
 36 – 3 = 33 is yung hemoglobin. Nagkaroon
 36 + 3 = 39 ng decrease in hemoglobin
 Range: 33% – 39%  False decrease hemoglobin
o It conforms to the Rule of Three. Pasok measurement
yung 36% sa range.
CASE 3
CASE 2  Rule of Three
 Rule of Three o Hemoglobin = 4 x 3 = 12 g/dL
o Hemoglobin = 3 x 3 = 9 g/dL o Hematocrit = 12 x 3 = 36%
o Hematocrit = 9 x 3 = 27%  36 – 3 = 33
 27 – 3 = 24  36 + 3 = 39
 27 + 3 = 30  Range: 33% – 39%
 Range: 24% – 30% o Tama ang RBC at hematocrit, ang
o It does not conform to the Rule of nakaiba ay hemoglobin. Mataas ang
Three. The hematocrit is above the 30%. naging result ng hemoglobin (15 g/dL)

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PROPERTY OF CHERRYANNE DOMINGO

o It does not conform to the Rule of

Three.
o Upon investigation, nakita natin yung
specimen here is lipemic. Again, kapag
lipemic yung plasma, it will result to
falsely elevated hemoglobin.
 Other example na pwede
magkaroon ng false increase
hemoglobin:
 Kapag may bilirubin
content
 high WBCs content
more than 20
 high platelet content
more than 700
 presence of other
hemoglobinopathies
(ex. hemoglobin S and
hemoglobin C)

We have many cases pa na pwede mag-violate sa ‘Rule

of Three’. It can be because of abnormal condition, or
falsely increase or decrease results. Kaya kailangan we
need to do further investigation to identify if ano po
yung cause kung bakit nag-fail magconform sa ‘Rule of
Three’.

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PROPERTY OF CHERRYANNE DOMINGO

REFERENCES

Unit 1.1 Laboratory Safety:

University of Santo Tomas powerpoint presentation: Unit 1.1. Laboratory Safety
Notes from the discussion by Mr. Benjie M. Clemente, RMT, MLS (ASCPi)cm, MPH

Methods of Blood Collection

University of Santo Tomas powerpoint presentation: Methods of Blood Collection
Notes from https://www.youtube.com/watch?v=WJkkgDVX5jk
Notes from https://www.youtube.com/watch?v=ibU5PYOF2qg
Notes from https://www.youtube.com/watch?v=7NSEFVbzTAU
Notes from https://www.youtube.com/watch?v=-XxiRSf6n8Q
Notes from https://www.youtube.com/watch?v=_EMWuFeEphU
Notes from https://www.youtube.com/watch?v=l0nNt

The Hemocytometer
Presentation and Discussion by Mr. Benjie M. Clemente, RMT, MLS (ASCPi)cm, MPH

RBC Count
Presentation and Discussion by Mr. Earl Adriane A. Cano, RMT, AMRSPH, MSMT(c)

Hemoglobin Determination
Presentation by Mr. Clarenz Sarit M. Concepcion, RMT, MPHI
Notes from the discussion of Mr. Benjie M. Clemente, RMT, MLS (ASCPi)cm, MPH

Hematocrit Determination & Rule of three

Presentation and Discussion by Mr. Clarenz Sarit M. Conception, RMT, MPHI

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PROPERTY OF CHERRYANNE DOMINGO
PROPERTY OF CHERRYANNE DOMINGO

RED BLOOD CELL INDICES  Example:

 It includes: 16 𝑥 10
o Mean Cell Volume (MCV) 𝑀𝐶𝐻 =
5
o Mean Cell Hemoglobin (MCH)
 calculates hemoglobin content of RBCs o Hemoglobin = 16 g/dL
o Mean Cell Hemoglobin Concentration o RBC count = 5 x 1012/L
(MCHC) o MCH = 32 pg
 calculates hemoglobin concentration of  Interpretation:
RBCs o Reference Interval: 26 – 32 pg
 serves as a quality control check  Normochromic – normal hemoglobin
 used for initial classification of anemias  In peripheral blood smear, the central
pallor is expected to be 1/3 of the cell
MEAN CELL VOLUME diameter.
 Average volume of the RBC o Less than 26 pg
 Expressed in femtoliters (fL) or 10-15 L  Hypochromic – low hemoglobin
concentration
Formula for Mean Cell Volume:  In peripheral blood smear, the zone of
𝐻𝐶𝑇 𝑥 10 pallor is more than 1/3 of the cell
𝑀𝐶𝑉 =
𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 diameter
o Greater than 32 pg
Hematocrit (%)  Hyperchromic
RBC count (x 1012/L)

 Example: MEAN CELL HEMOGLOBIN CONCENTRATION

45 𝑥 10  Average concentration of hemoglobin in each
𝑀𝐶𝑉 = individual RBC
5
 The units used are grams per deciliter or as a
percentage (%)
o Hematocrit = 45 %
 Serve as better measurement in classification in
o RBC count = 5 x 1012/L
anemias
o MCV = 90 fL
 Interpretation: Formula for Mean Cell Hemoglobin Conc.:
o Reference Interval: 80 – 100 fL 𝐻𝐺𝐵 𝑥 100
10
 Normocytic 𝑀𝐶𝐻𝐶 =
𝐻𝐶𝑇
o Less than 80 fL
 Microcytic – smaller cell size / volume Hemoglobin (g/dL)
o Greater than 100
80 fL
fL Hematocrit (%)
 Macrocytic – large cell volume
 Example:
MEAN CELL HEMOGLOBIN 16 𝑥 100
𝑀𝐶𝐻𝐶 =
 Average weight of hemoglobin in a RBC 48
 Expressed in picograms (pg) or 10-12 g
 Not considered in classification of anemias o Hemoglobin = 16 g/dL
o Hematocrit = 48 %
Formula for Mean Cell Hemoglobin: o MCHC = 33.33 g/dL
𝐻𝐺𝐵 𝑥 10  Interpretation:
𝑀𝐶𝐻 = o Reference Interval: 32-36 g/dL
𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡
 Normochromic
Hemoglobin (g/dL)  The central pallor is 1/3 of the cell
RBC count (x 1012/L) diameter.

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PROPERTY OF CHERRYANNE DOMINGO

o Less than 32 g/dL o presence of

 Hypochromic – low amount of hemoglobinopathies like
hemoglobin; it will be observed in the Hgb S and Hgb C
peripheral blood smear as having a o presence of abnormal
lager central pallor more than 1/3 of the globulins such as seen in
cell diameter multiple myeloma and
 Occur in conditions ssuch as: Waldenstrom’s
 Iron deficiency anemia macroglobulinemia
 Anemia of chronic inflammation wherein there is increase
 Hemoglobinopathy and of IgG and IgM
Quantitative hemoglobin defects respectively.
such as Thalassemia  Another cause of falsely increase
 Qualitative and quantitative MCHC could be because of the
hemoglobin defects such as Hb E presence of cold agglutinin or in cases
disease & trait of cold agglutinin diseases
 Sideroblastic anemia  wherein there is an autoimmune
o Greater than 36 g/dL that made the red blood cells
 Hyperchromic –increased hemoglobin aggregate.
concentration  can be found in cases of
 The term hyperchromic misnomerA.
hypochromic isisaamisnomer. infection due to Mycoplasma
A celldoes
cell does not
not really
really contain
contain aa pneumoniae, a primary atypical
hemoglobin value that is more than 36 pneumonia wherein there is a
g/dL. production of anti-I which
 This is only applicable if the red cell resulted to production of cold
present in the Peripheral Blood Smear agglutin or a case of cold
are spherocytes or the cells become agglutinin disease.
spherocytic  this would make the red cells
 which decreases the surface aggregate and when the cells
area to volume ratio of the red aggregate, there is an increase
cell in MCHC computation.
 the red cell appears full; the  Likewise, there is also an
result becomes hyperchromic increase in MCV, if there is a
 not because of the increase in case of cold agglutinin
hemoglobin concentration  while the RBC count, if
 If the MCHC ranges from 36 to 38 g/dL, automation is performed, will
check for presence of spherocytes. show a decreased count.
 If the MCHC is greater than 38 g/dL,  one of the automation principle
investigate for an error in the is based on the red cell size, the
hemoglobin values. aggregated red cells will not be
 Baka nagkaroon tayo ng false counted resulting to a
increase in the hemoglobin decreased RBC, increased
value kaya tumaas drastically MCV, and increased MCHC.
yung MCHC value.
 Possible causes of error of
increase hemoglobin value: A
case of turbidity due to:
o lipemic sample
o a high WBC count of more
than 20
o a high platelet count of
more than 700

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PROPERTY OF CHERRYANNE DOMINGO

RED BLOOD CELL INDICES, RED BLOOD CELL CLASSIFICATION OF ANEMIA BASED ON RED
MORPHOLOGY AND DISEASE STATES BLOOD CELL MEAN VOLUME (MCV)

RED BLOOD
MCV MCHC
CELL FOUND IN
(fL) (g/dL)
MORPHOLOGY
 Iron deficiency
anemia
 Anemia of
inflammation
Microcytic;
< 80 <32  Thalassemia
Hypochromic
 Hb E disease
and trait
 Sideroblastic
anemia
 Hemolytic
Anemia
 Myelophthisic
Normocytic; anemia
80-100 32-36  Algorithm for morphologic classification of
Normochromic  Bone marrow
failure anemia based on their mean cell volume
 Chronic renal  Correlate RBC indices to the different types of
disease anemia
 Megaloblastic  Microcytic: MCV < 80 fL (Hypochromic)
anemia o Sideroblastic anemia
 Chronic liver o Iron deficiency anemia
Macrocytic; disease
>100 32-36 o Anemia of chronic inflammation
Normochromic  Bone marrow
o Thalassemia
failure
 Myelodysplast o Hb E disease and trait
ic syndrome  Macrocytic: MCV > 100 fL (Normochromic)
o Nonmegaloblastic
 Red blood cell indices particularly the MCV and MCHC  Aplastic anemia
value can be correlated to the red blood cell  Chronic liver disease
morphology if its microcytic, normocytic, macrocytic, or  Alcoholism
if its hypochromic or normochromic. It can be o Megaloblastic
correlated with various types of anemia.  Vitamin B12 deficiency
 Folate deficiency
Note  Result in defect in DNA
 Megaloblastic anemia due to vitamin B12 maturation and red blood cell
deficiency or folate deficiency development
 Normal MCV and normal MCHC is seen in  Myelodysplasia
healthy individuals but it does not necessarily  Erythroleukemia
indicate that the patient is healthy. There are also  Some drugs
types of anemia that exhibits normocytic /  Normocytic: MCV 80 – 100 fL (Normochromic)
normochromic red cell morphology. o Reticulocyte ↑
o Bone marrow failure or aplastic anemia  Hemolytic anemia
can be a case of macrocytic;  Intrinsic
normochromic and normocytic; o Membrane defects
normochromic o Hemoglobinopathies
o Chronic renal disease because of low o Enzyme deficiencies
erythropoietin production  Extrinsic
o Immune causes
o Nonimmune RBC injury:
 Microangiopathic
 Macroangiopathic

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PROPERTY OF CHERRYANNE DOMINGO

 Infectious agents
 Other injury: drugs,
chemicals, venoms,
extensive burns
o Reticulocyte N or ↓
 Aplastic anemia
 Anemia of renal disease
 Myelophthisic anemia
 Infection (parvovirus B19)
 Anemia of chronic inflammation

PERIPHERAL BLOOD FILM FROM PATIENTS

Peripheral Blood Film from a Patient with Aplastic

Anemia

 Normocytic; normochromic red cell

 Low RBC count
 Low presence of WBCs or platelets
 Pancytopenia – low RBC, low WBC, low platelets
o Common case for a peripheral blood film of a
patient with aplastic anemia

Peripheral Blood Film from a Patient with Hypochromic,

Microcytic Anemia

 Smaller red blood cell sizes / microcytic red cell

 Compared with nuclear diameter of lymphocyte;
RBC shows smaller diameter
 There is hypochromia because of increased central
pallor Peripheral Blood Film from a Patient with Megaloblastic
 Presence of target cells Anemia
 There is a variation in cell sizes or anisocytosis
o Show an elevated red blood cell distribution  Oval macrocytes
width case of microcytosis and hypochromia  High MCV - Macrocytic; normochromic
 May indicate iron deficiency anemia  Large RBCs
 Hypersegmented neutrophil having a
segmentation of more than 5
 Pancytopenia – low RBC, low WBC, low platelets
o Common condition found in a peripheral blood
film from a patient with Megaloblastic anemia

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PROPERTY OF CHERRYANNE DOMINGO

o MCH
5 𝑥 10
𝑀𝐶𝐻 =
2.8

 Hemoglobin = 50 g/L = 5 g/dL

 RBC count = 2.8 x 1012 / L
 MCH = 17.86 pg
Peripheral Blood Film from a Patient with Hereditary
Spherocytosis o MCHC
5 𝑥 100
 Presence of spherocytes 𝑀𝐶𝐻𝐶 =
20
 Specific gene mutation
 Different types of gene mutation but it will result to  Hemoglobin = 50 g/L = 5 g/dL
defect in the vertical membrane protein interaction  Hematocrit = 20 %
particularly in the ankyrin and spectrin  MCHC = 25 g/dL
o This would result to an RBC losing the
unsupported lipid membrane over time  A stained blood film of this patient would most likely
 It would result to formation of reveal a red cell picture that is?
spherocytes
 Spherocytes will have a decrease surface area to o Peripheral Blood Film from a Patient with
volume ratio; cells appear to be full with hemoglobin Hypochromic, Microcytic Anemia
 This will be measured as having an increased  MCV: less than 80 fL – Microcytic
MCHC or hyperchromia  MCH: less than 26 pg – Hypochromic
 The spherocytes does not contain more than 36 g/dL  MCHC: less than 32 g/dL –
o There is decrease surface area to volume ratio Hypochromic
and the cells became spherocytic hence it
appears to have an increased hemoglobin
concentration
 MCHC value between 36 to 38 g/dL should be check
for presence of spherocytes.

SAMPLE COMPUTATION
 What is the MCV, MCH, and MCHC if the patient’s
hematocrit is 20 %, the RBC is 2.8 x 1012 / L and the
hemoglobin is 50 g/L?

o MCV
20 𝑥 10
𝑀𝐶𝑉 =
2.8

 Hematocrit = 20 %
 RBC count = 2.8 x 1012/L
 MCV = 71.43 fL

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PROPERTY OF CHERRYANNE DOMINGO

References

RBC Indices
University of Santo Tomas powerpoint presentation: RBC indices
Presentation and discussion by Mr. Clarenz, Sarit M. Concepcion, RMT, MPHI

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PROPERTY OF CHERRYANNE DOMINGO

BLOOD FILM PREPARATION complete blood count or hemogram which is

required for the enumeration and checking of
INTRODUCTION your standard elements.
▪ This can be used for your WBC
• In clinical laboratory setting, we mostly utilize
differential count
our blood smear or blood film.
• analyzing, identifying and
• So, blood film examination will be subjected for
classifying its morphology
microscopic examination of your blood which
• give an estimate of your cell
is spread on a glass slide
count especially in platelet
• Some methods will utilize cover slip and this will
estimates.
be useful in giving information regarding the o Examples of assays that can be conducted
all formed elements of the blood or the cellular using PBS:
elements which are found in your blood.
o WBC differential count
• It is important to discuss this procedure because o Morphology
it will give a considerable amount of o Platelet estimates
information about the condition of your • Microscopic examination of the blood smear on a
patient. glass slide aids in providing information regarding
o Therefore, it has to be:
formed elements of the blood.
▪ properly prepared
• The reliability of the information obtained depends
▪ properly stained
on the well-made and well stained films that are
▪ properly evaluated
systematically examined.
• In this procedure, many laboratories use blood
o It has to be prepared immediately as
films which are made by your wedge technique possible.
or wedge smear technique using your sample
o As a medical laboratory scientist, medical
which is EDTA anticoagulated blood.
technologist, blood films should be prepared
o This sample which is prepared using
systematically, correctly, and immediately as
your wedge technique is stained
possible.
using your Wright or Wright-Giemsa
▪ Wag patatagalin sa lab because that
staining technique.
can subject to erroneous outcomes
o After preparing your smear and
or results.
staining it, this will be subjected for
• Hence, smear preparation should be mastered by
evaluation. Thus, the evaluation will
every medical technologist.
follow a systematic approach:
▪ first using 10x magnification
PERIPHERAL BLOOD FILM PREPARATION
▪ followed by 40x high-dry
• Materials needed:
magnification or 50x
1. Glass slides & coverslip (for other method)
depending on the protocol of
o In this video and for this discussion, we
the laboratory
have utilized wedge prep technique
▪ and finally using the oil
▪ most common methodology of
immersion objective on the
preparing your smear
microscope.
2. Pasteur pipette
• Evaluation of the peripheral blood film is an integral 3. Anticoagulated blood
part of CBC or hemogram 4. Tube holder
o Required for the enumeration and checking 5. Pencil for labeling
of cellular elements 6. PPEs
o This preparation of PBS is very important 7. Cannister with yellow bag
especially when analyzing cellular elements, 8. Sharps container
which is an integral part of your CBC or 9. Oil cloth

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PROPERTY OF CHERRYANNE DOMINGO

VIDEO
MATERIALS NEEDED FOR PERIPHERAL BLOOD
SMEAR PREPARATION
• Glass slides
o Preferably with frosted edge for easier
spread of blood throughout the slide
• Pencil for labeling
• Pipette PROCEDURE
• Blood 1. Place a small drop of blood (2-3 mm in diameter)
• Tube holder about 1 cm from the end of a clean, dust-free slide.
• Cannister with yellow bag
• Sharps container
• Other necessary materials (alcohol, tissue, gloves)
• For the setup, it is recommended to use oil cloth so - 1cm -
if there are ever spills for blood it will not easily spill
in the working area.
2. With the thumb and forefinger of the right hand, hold
the end of the spreader against the surface of the
METHOD 1: TWO-SLIDE OR WEDGE METHOD
smear slide at 30-45 degree angle.
• Push-type wedge preparation
• The simplest and the most common method of
smear preparation
• It uses 2 slides: 30 – 45o
o one for the smear o
o one that serves as a spreader
• High-quality, beveled-edge microscope slides are 3. Allow the blood to spread evenly on the vertex,
recommended. between the two slides.
• The one that has frosted edge are required or 4. Quickly and smoothly push forward to the end of the
recommended to be used in this method. film slide, creating a wedge film
o A pulled-type – the spreader slide is pushed o Do this smoothly and at a constant angle;
into the drop of blood and pulled along the hold the spreader firmly onto the slide
length of the slide to make the film.
o Some books would use the term pulled-type.
This is another – probably, a subtype of
wedge method.
5. Air-dry the slide.

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PROPERTY OF CHERRYANNE DOMINGO

5. When the slide is held up to the light, the feathery

edge/thin portion of the film has rainbow
appearance.
6. The whole drop of the blood is picked up and spread.

Note
• Maintaining a consistent angle between the
slides and an even, gentle pressure is essential.
- For both procedure number 2 and 3.

VIDEO
HEMATOLOGY – MAKING A PERIPHERAL BLOOD
SMEAR BY ROBERT BOUNDS
Link: https://youtu.be/cI9GObT73lY
• In this video I’m going to show you how to make a • When tilting the slide, your PBS, you can observe a
peripheral blood smear and for this you’re going to feathery edge with a rainbow appearance at the
need some slides. edge if properly smeared.
• You are also going to need some anticoagulated
blood preferably EDTA.
• I’m using a cat piercer to make the blood drop.
• First thing you need to do is install your cat piercer
and then go ahead and get two slides out.
• First slide you’re going to add a small drop of blood
to.
• The second slide you’re going to place it on top and
slowly bring it back until it makes contact with the
drop and then rapidly push it forward.
• And hopefully after doing this, the smear will be about
two-thirds way down the slide and you’ll get a nice • Tongue-shaped, well-prepared smear with a
feathered edge at the end. transition from thick to thin.
• Also, you can make a slide thicker or thinner • For the evaluation, the portion wherein it is observed
depending on the angle of the second slide. is at edge which is the zone of morphology.
• Now, if you want to make it thicker you need to hold • The super edge or far edge part of the smear,
it at a higher angle which makes the smear thicker. although blood can be observed at that side, hindi na
• As you can see after I add the drop, I’m going to take sila overlapping hiwa-hiwalay sila, but what you can
the slide and hold it in a much larger angles and this observe is distorted morphology – and we don’t like
makes it much shorter. it, doon lang tayo sa zone of morphology.
• This would be useful for somebody with a low
hemoglobin and that’s how you do it. UNACCEPTABLE PERIPHERAL BLOOD FILMS AND
ITS CAUSES
FEATURES OF PROPERLY MADE PERIPHERAL - Knowing the reasons or causes of your unacceptable
BLOOD FILM PBS, will help you later on in creating or preparing
1. The film must cover 2/3 to 3/4 the length of the slide. your film.
2. The film is finger shaped.
o Slightly rounded at the feathery edge, not
bullet shaped.
3. The lateral edges of the film are visible.
4. The film is smooth without irregularities, holes or
streaks.

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PROPERTY OF CHERRYANNE DOMINGO

• Many ridges or waves, in this case the yung nasa

dulong slide.
• The spreader can be moved in an uneven motion
due to hesitation or probably there is too much
pressure exerted while you are spreading your
smear onto the slide.

REMINDERS ONCE YOU HAVE CREATED THE

1. Caused by: chipped/rough edge on spreader slide SMEAR
- Toothed rough edge • The film should have a smooth, even appearance
2. Caused by: hesitation in forward motion of and be free from ridges, waves, or holes.
spreader slide • A good film includes a thick portion and a thin
- Possibly due to shaking portion and a gradual transition from one to the
3. Caused by: spreader slide pushed too quickly other.
4. Caused by: drop of blood was too small • Moving the pusher slide forward too slowly
5. Caused by: drop of blood was not allowed to spread accentuates poor leukocyte distribution
across the width of the slide o by pushing larger cells, such as monocytes
6. Caused by: dirt or grease on the slide; may also be and granulocytes, to the very end and sides
due to elevated lipids in the blood specimen of the film.
- Presence of bubbles o Observe and practice spreading in correct
- Sometimes pathologic causes or patient pressure
samples causes could be attributed to the • The faster the film is air dried, the better is the
elevated lipids in the blood sample spreading of individual cells on the slide.
7. Caused by: uneven pressure on the spreader slide o Slow drying (like in humid environment) will
8. Caused by: time delay; drop of blood began to dry result to the formation of artifactual cells
- Some parts or side of you smear were not evenly • The slide may be labeled with a lead pencil on the
distributed frosted end or directly on the thicker end of the blood
film.
o We do not use a pen or a marker because
those materials can be later on washed out
using your reagents.

HOW SHOULD YOU TROUBLESHOOT IF THE

WEDGE-PREP SMEAR ISN’T GOOD?
• The thickness of the film can be adjusted by:
o changing the angle of the spreader slide
• What can be observed is many holes on your smear.
o changing the speed of spreading
o This can indicate a dirty slide.
o using a smaller or larger drop of blood.
• Some would have thin smear because of:
• At a given speed,
o small drop of blood
o increasing the angle of the spreader slide
o the angle of spreader
will increase the thickness of the film.
o slide is too small
• At a given angle,
o very low patient hematocrit – can also be
o increasing the speed with which the
attributed to too thin smears
spreader slide is pushed will increase the
• Too thick smears can be attributed to:
thickness of the film.
o a large drop of blood
o angle of your spreader which can be too big
o if patient has high hematocrit levels
• The smear ends in a straight line instead of feathery
edge.
o This can be caused by unreliable spreader or
the spreader was not near at the end of
sample slide.

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PROPERTY OF CHERRYANNE DOMINGO

Note
• Factors to be considered when troubleshooting
your PBS:
o Amount of blood placed on your smear
o Angle of spreader and slide
o Speed of smearing or spreading
• Other factor to be considered is the quality of
slide
o Check whether slide is chipped on the
edges – should not be used for PBS
o Ensure that glass slide to be used is
tested and cleansed before proceeding.
• A skillful medical technologist performing this method
Not unless you are using a high-yield type
should be able to manipulate fragile coverslips
of slides, usually used by professional
without breakage.
hematologists and medical laboratory
o Advantage:
scientists which are assigned in
▪ It produces an excellent leukocyte
hematology section in preparing your
distribution, which lends itself to
PBS, para naiiwasan na natin yung
more accurate determination of
pagcheck pa ng slide natin. But I think as
differential counts.
a medical technologist, before you
▪ Minimal amount of bone marrow is
proceed to PBS preparation, you have the
needed
responsibility to check first your slides and
▪ Many smear can be made from a
your spreader.
small bone marrow sample
• Patient condition
▪ Only small amount of storage
o High hematocrit level (in cases of
space required.
polycythemia vera)
o Disadvantage:
o Low hematocrit level (in cases of anemia)
▪ Labeling, transport, staining and
o High lipid content in blood can cause
storage of these small breakable
erroneous results in preparing smear
smear present many problems.
• Mahirap maglabel dahil
METHOD 2: TWO-COVER SLIP OR EHRLICH’S TWO- masyadong maliit.
COVERGLASS ▪ Requires clean coverslip
• It is an older technique that is now used only rarely ▪ Coverslips are fragile
for PBS, but it is sometimes still used for making
bone marrow aspirate smears. PROCEDURE
• Why is it used in bone marrow smear preparation? 1. Get two cover glasses. Hold one coverglass on its
o Because later we will discuss that you only adjacent corners with the thumb and index finger.
have or you are just required to use small 2. Place one small drop of blood on top of the held
amount of your sample tapos mas madali din coverglass.
yung ating pagfile or pagstore ng ating mga 3. Position the other coverglass on top of the coverglass
smears unlike yung ating mga malalaking with blood so that the two form a 16-sided figure as
slides that could take some space in the illustrated below. (Refer to letter B of the image)
laboratory. o As this is done, the blood begins to spread.
• No. 1 or 1 ½ cover glasses 22 mm square are 4. Before the blood completely spreads, separate the
recommended. coverglasses by doing a rapid, even, horizontal,
• If done correctly, both coverslips will have quality and lateral pull.
smear that will appear similar to thumb print. o Avoid squeezing the coverglasses together.
• Making smears on coverslips requires manual 5. Air-dry the coverglass.
dexterity.
o Mas maliit, mas manipis din ang ating slides
– ang ating glasses.

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PROPERTY OF CHERRYANNE DOMINGO

METHOD 3: SPINNER METHOD • In the laboratory with the advent of the automation,
• Blood films that combine the advantages of easy we make use of machines. This would prevent us
handling of the wedge slide and uniform distribution from the common mistakes that were mentioned a
of cells of the cover glass preparation may be made while ago.
with special types of centrifuges known as spinners. • Eto rin mas madali, within few seconds you will be
• The spinner slide produces a uniform blood film, in able create a series of slides. Kasi totoo lang class,
which all cells are separated (a monolayer) and mahirap talaga mag produce or gumawa ng smear
randomly distributed. especially if it is not practiced by a medical
• Smear prepared using this method have white blood technologist consistently, mahirap talaga siya. And it
cells that can be easily identified at any sport in the takes time for you to create these smears on your
film. own.
o Remember: On a wedge smear, • However, although with expertise of our medical
▪ disproportions of monocytes occur laboratory scientist – medical technologist in the
at the tip of the feather edge setting, we make use of automated machines in
▪ the neutrophils are seen just in from preparing your smears.
the feather edge • The Sysmex SP-1000i is an automated slide making
▪ and of both neutrophils and and staining system.
monocytes may occur at the lateral o This is used in making automated smear
edges of the film. preparation and staining.
• Dito kay spinner, it would create a uniform blood film. • Dependent in hematocrit values
• Remember that it will have a uniform or monolayer of o it adjusts the size of the drop of blood used
cells which are distributed accurately all throughout and the angle and speed of the spreader slide
your slide or smear. in making a wedge preparation.
• One of its advantages would be the preparation is • Film is produced for every 30 seconds
uniform without risking of breaking the cells like • With printed label:
other automated smear preparation system or with o patient name
the other methods, for example, the wedge method. o number
o date
• The slide is dried, loaded into a cassette, and moved
to the staining position, where stain and then
buffer and rinse are added at designated times.
• When staining is done, the slide is transferred to a
dry position, then to a collection area where it can
be used for microscopic evaluation.

DxH SLIDEMAKER STAINER II CELLULAR

ANALYSIS SYSTEM
• Prepares slides automatically based on orders
received from the LIS
AUTOMATED SMEAR PREPARATION • Create and stain smears from capped whole blood
tubes, open-top tubes or pediatric whole blood tubes
• Create up to four slides from a single 90 µl sample
aspiration and up to 12 slides from a single sample
presentation
• Create multiple stain smear slides, according to
any parameter results, such as a low WBC or Blast
suspect flag

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PROPERTY OF CHERRYANNE DOMINGO

▪ In some methodologies, in some

books, nagmemention ng ibang
anticoagulant.
▪ Heparin – causes blue background
▪ Oxalate and Fluoride – distort
cellular morphology
o Staining Jar or Dip Method
▪ Manual Technique
▪ Slides are manually smeared by
dipping into the reagents found in
coplin jar
▪ Some laboratories only flood the
smears, but in this procedure found
in your hematology laboratory
manual, we make use of a coplin jar
to dip smears onto these reagents.

• In my own experience as a medical laboratory

scientist in the Philippines, depende kung ano yung
laboratory mo but most of the – probably the best
training for a medical technologist would be to be
subjected for manual procedure of preparing your
smear and then later on, once you have perfected
the preparation of your smear, probably we can
transition to the automated systems. So once these
systems are down, you will be able to perform the
test without hesitation but still with accuracy and
precision pa rin.

STAINING OF PERIPHERAL BLOOD FILM STAINING JAR OR DIP METHOD: WRIGHT STAINING
• Purpose of staining: identify the cells and recognize PROCEDURE
morphology easily through microscope. 1. In horizontal position, immerse the slide in the jar
• Ethylenediaminetetrataceetic acid (EDTA) is the containing methanol (solution 1) for 30 seconds
anticoagulant of choice for hematology cell counts • Methanol will serve as the fixative.
and cell morphology. o The cells are fixed to the glass slide by the
• Romanowsky stain – Pure Wright stain or a Wright- methanol in the stain
Giemsa stain 2. Dip in eosin stain (solution 2) for 6 seconds
• This is a methyl-alcoholic solution of your eosin and a o Free eosin is acidic and stains basic
complex mixture of your triazine including your components, such as hemoglobin or
methylene blue, azure blue, and other derivatives. eosinophilic granules, red.
• This staining technique is certified by the Biological 3. Dip in methylene blue stain (solution 3) for 4
Stain Commission and is commercially available as a seconds
solution, or pwede rin naka powder and then you will o Free methylene blue is basic and stains
just prepare it once you need it. acidic cellular components, such as RNA,
o Most commonly used for staining peripheral blue.
blood and bone marrow smears 4. Dip in buffer solution/aged distilled water
o Are polychrome stains – contain eosin and (solution 4) for 45 seconds
methylene blue o 0.05 M sodium phosphate (pH 6.4) or aged
o Wright Staining method uses EDTA distilled water (distilled water placed in a
anticoagulated blood glass bottle for at least 24 hours; pH 6.4 to
6.8)

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PROPERTY OF CHERRYANNE DOMINGO

5. Clean the back of the slide with a clean gauze o And then let it dry. Make sure to dry your
6. Allow the slide to dry in a tilted position smears in a tilted position.
▪ Make sure na hindi tatama yung
merong smear doon sa side ng ating
Note
basket, in this case. Make sure that
• Procedures may vary based on different
you know where your blood smear is
laboratory protocols. It is necessary to check
placed so that this will not be affected
the Lab’s SOP before performing the PBS
after, hindi siya masisira.
staining

AN OPTIMALLY STAINED PERIPHERAL BLOOD

FILM SHOULD HAVE THE FOLLOWING
CHARACTERISTICS
1. The red blood cells (RBCs) should be pink to salmon
in color.
2. Nuclei should appear dark blue to purple.
3. Cytoplasmic granules of neutrophils are lavender to
lilac.
4. Cytoplasmic granules of basophils are dark blue to
black.
5. Cytoplasmic granules of eosinophils are red to
orange.
6. The area between the cells should be colorless,
clean, and free of precipitated stain.

VIDEO
STAINING JAR OR DIP METHOD OF STAINING
YOUR PERIPHERAL BLOOD SMEAR
• Setup
o Solution 1 – fixative
o Solution 2 – acid stain
o Solution 3 – basic stain
o Solution 4 – buffer
• Procedure
o Solution 1 will be the first reagent where we
will place our peripheral blood smear.
o This will be followed by your solution 2 which
is your eosin.
o Your solution 3 which is your methylene blue.
o And next is your buffer.
o So first we will fix our slide using your fixative,
methanol. Make sure to take note of the time
which is 30 seconds.
o Hold the smear firmly and horizontally as you
placed it inside the coplin jar.
o Get ready to dip it to your solution 2, your
eosin, the acidic dye for 6 seconds.
▪ When you are checking the quality of your smear, you
o This will be followed on your solution 3. So
can actually discern it using just macroscopically
dip your smear on solution 3, which is
observing the slide, but of course you cannot see the
methylene blue, the basic dye for 4 seconds.
cellular products or the formed elements in the blood,
o This will be followed by dipping your smear in
not unless you check it under microscope.
your buffer solution. This can also be an aged
▪ Sometimes ang ginagamit nalang natin if you want to
distilled water for 45 seconds.
check it quickly, you make use of your HPO instead.

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PROPERTY OF CHERRYANNE DOMINGO

And then once you are fine with the picture of your maoobserve natin. Therefore in this
blood smear, that’s the time you can evaluate using case, it is helpful to observe the
your OIO. neutrophil or eosinophil granules.
• Causes
THINGS TO REMEMBER o Stain or buffer too alkaline (most common)
• Each step in PBS preparation is crucial and o Inadequate rinsing
necessary to ensure accuracy in interpreting cellular o Prolonged staining
morphology. ▪ Or the medical technologist can
• The best staining results are obtained from freshly probably overstain this smear using
made slides that have been prepared within 2 to 3 your basic dye
hours of blood collection. o Heparinized blood sample
• Once you are preparing for your PBS it is best ▪ Giving it a gray/blue/green
prepared using your freshly extracted blood. Or if in background on your smear
case may konting delay, make sure that you have • What should you do?
created your PBS within 2-3 hours of blood collection. o Staining for a shorter time or using less
And of course, using your blood prepared using your stain and more diluent may correct the
correct anticoagulant which is your EDTA, do not problem.
forget that! o If these steps are ineffective, the buffer may
be too alkaline, and a new one with a lower
KNOW THE PROBLEMS AND ITS CAUSES TO pH should be prepared.
TROUBLESHOOT YOUR PBS o Replace the reagents with new ones in its
• Inadequately stained red cells, nuclei, or optimal state
eosinophilic granules may be due to under staining or ▪ Remember to observe the stains not
excessive washing. just the slide. Baka dalawang linggo
• Presence of precipitates on the film due to unclean na yan naandiyaan. So probably you
slides prepare a new sets of your reagents
• Drying during the period of staining or your stains.
• Inadequate washing of the slide at the end of the
staining period
• Failure to hold the slide horizontally during initial
washing
• Inadequate filtration of the stain
• Permitting dust to settle on the slide or smear

SCENARIO 1 SCENARIO 2
• Problems • Problems
o Red blood cells appear gray/blue/green o Red blood cells too pale or bright red in
▪ Can cause discoloration of red color/orange
blood cells. Sabi natin kanina, pink o Nuclear chromatin is pale blue
to salmon pink in color would be the o White blood cells barely visible
best color of our red blood cells o Eosinophils have sparkling brilliant red
under the microscope. granules
o Nuclear chromatin deep blue to black • Causes
o White blood cells are too dark o Stain or buffer too acidic (most common)
o Neutrophil granules are deeply overstained, o Underbuffering (time too short)
large and prominent o Over-rinsing
o Eosinophil granules are blue/gray, not o So remember here ha, there should be an:
orange ▪ insufficient staining
▪ Yung basophil natin since it is a basic ▪ prolonged washing time
dye-loving, you cannot observe it ▪ mounting the coverslip before the air-
directly. Talagang maitim yung kulay dry
or dark blue in color yung ▪ the acidity of the stain is too high

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PROPERTY OF CHERRYANNE DOMINGO

▪ the buffer may have excessive • To show the morphology of the blood, and for you
acidophilia kaya siya nag kulay red. to check the appearance of your blood that you have
• What should you do? prepared from your peripheral blood smear, you can
o Prevent the exposure of stains or buffer to use your microscope or if in case your laboratory
acid fumes as this may result to over acidic have this setup, better, para makita natin if the zone
reagents of morphology would be visible and also if the cells
o Low pH of the buffer/methyl alcohol may lead are correctly stained.
to the development of formic acid as a result • You may add the thinnest part of your smear. This
of oxidation on standing. is how your cells look like, hiwa-hiwalay sila and also
o Replace the reagents with new ones in its you cannot observe the normal morphology of your
optimal state red blood cells. Mukha silang hypochromic and then
macrocytic cells.
• But if you will move towards the part of your feathery
edge, you can see the zone of morphology wherein
your red blood cells are not packed together, not
overlapping, you can observe the normal central
pallor of your cells, and then you can also observe
and check and identify the different WBCs on this
OTHER STAINS view.
• Other Romanowsky-type stains: • When you are preparing for differential counting,
o Giemsa, Leishman’s, Jenner’s, May- the knowledge of finding the zone of morphology in,
Grünwald, MacNeal’s particularly, nasa feathery edge siya ng PBS that is
▪ Note that Giemsa stain is excellent very essential in conducting differential count.
for malarial parasite (thick and thin • In the differential count discussion, this will be further
smear prep) and other parasite discussed especially how would you find WBCs –
staining procedures. paano ba yan, meron ba siyang manner of utilizing
the field that we can see here under the microscope?
VIDEO Dapat ba under the microscope or dapat ba naka
CHECKING YOUR PREPARED PERIPHERAL BLOOD LCD? We will discuss this further in the next
FILM discussions.
• That is the video demonstrating the prepared smear
after staining.
• It is important to be knowledgeable of other methods
of staining so yung kaninang diniscuss natin we made
use of your staining jar or dip method. This would
require the medical technologist to immerse the
blood smear into the jar or coplin jar containing
your reagent.
• Other method we have your staining dish method, ito
naman would involve placing the smear in rack
position in a dish and then ilalagay mo yung stain.
• We have prepared here a video showing to you using And then in a minute or definite time, the smear will
our microscopes in hematology laboratory, wherein be stained already.
you can check if your smears are good or not good. • And then of course automated method, which were
• Ano ba yung side saan tayo magiidentify, presented to you a while ago, so pwede tayong
mageevaluate ng cells? gumamit ng hema stainer automatic slide stainers,
• Take note, this is just an introduction to the mga Sysmex SP-1000i, yung Beckman Coulter, that
subsequent discussion which will be your differential was presented to you in our video.
cell counting. This differential cell counts will discuss • So those are the preparations methodology in
further the utilization of our prepared smear but in this providing PBS for hematology section.
case titignan lang yung slide na ginawa natin.

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PROPERTY OF CHERRYANNE DOMINGO

References

BLOOD FILM PREPARATION:

University of Santo Tomas powerpoint presentation: BLOOD FILM PREPARATION
Prepared by: Diana Leah M. Mendoza, RMT, MPH, MLS(ASCPi)cm

McPherson, R. and Pincus, M., 2017. Henry's Clinical Diagnosis And Management By Laboratory Methods. 23rd ed.
Elsevier

Rodak, B. and Carr, J., 2017. Clinical Hematology Atlas. 5th ed. London: Elsevier Health Sciences.

Keohane, E., Smith, L. and Walenga, J., 2019. Hematology. 6th ed. St. Louis, Missouri: Elsevier

Sadang, MG. and Llanera, F., 2015. Laboratory Manual in Hematology. Quezon City, Philippines: C&E Publishing, Inc.

Videos from:
• https://youtu.be/cI9GObT73lY Robert Bounds
• Ann Kathleen Gonzales, MLS(ASCPi)cm
• Clarenz Sarit Concepcion, RMT, MPH, MLS(ASCPi)cm
• Diana Leah Mendoza, RMT, MPH, MLS(ASCPi)cm
• Ron Christian Sison, RMT, MPH, MLS(ASCPi)cm

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PROPERTY OF CHERRYANNE DOMINGO

WHITE BLOOD CELL COUNT CHARACTERISTICS OF A GOOD WBC DILUTING

FLUID
According to Ma’am Magcaling:  Must be a hypotonic solution
 Also called as Leukocytes o To crenate the RBC; to hemolyze RBC
 Help protect the body against infection o 1% HCl
 Low WBC Count = viral infection  Must be easily prepared
o In laboratories, this is prepared by the
 High WBC count = bacterial infection
laboratory technicians.
 WBC is part of the Complete Blood Count (CBC)
 Must be readily available
 Must be a good preservative
 The WBC Count is the number of WBCs in 1mm 3 of  Must be cheap
blood. o In laboratories it will cost you 1,000 php or
 The blood is accurately diluted with a fluid producing less for 500 mL which is already prepared.
complete hemolysis of red blood cells.
o Diluted with a hypotonic solution in order to PIPET METHOD: PREPARATION OF DILUTED
hemolyze RBCs BLOOD
o To see WBC, it must be diluted with HCl  Aspirate blood up the 0.5 mark of WBC pipet
(diluting fluid) o From the EDTA tube
 Wipe off excess blood; making sure it’s not touching
MATERIALS the tip of pipet
 Anticoagulated blood  Place the diluting fluid in a container. Perform dilution
o Most commonly used: EDTA by syringe aspiration making a 1:20 dilution
 WBC pipet o During face-to-face classes, students are
 1% HCl required to bring small vials because HCL is
o Diluting fluid from a stock solution and in order to prevent
 Aspirator contamination, a small amount is transferred
o Rubber tubing connected to the WBC that is enough for the dilution from the stock
pipette with syringe solution into the vials.
 Syringe used is 1 mL or tuberculin o The WBC pipette, containing the aspirated
syringe blood is then transferred to the vial (in a
o What was used before was a sucking tube, vertical position) to aspirate HCl up to the
also a rubber tubing connected to the WBC 11 mark.
pipette without the use of a syringe but is  When aspirating, pull the plunger of
pipetted by mouth. the syringe slowly to prevent bubbles
 Not practice anymore due to the and air. If this happens, repeat
danger of ingesting the blood and procedure.
reagents. o After aspiration of HCl, remove the rubber
 Microscope tubing.
o For viewing and counting  Gradually bring pipet to horizontal position &
 Improved Neubauer CC immediately mixing contents.
 Thick coverslip o Mix in a circular motion.
 Tissue paper
o Important for the wiping of excess blood in
the WBC pipette before being put in the
diluting fluid.

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PROPERTY OF CHERRYANNE DOMINGO

Additional info from lab manual:  Place thick coverslip on top of CC.
 Preparation of Diluted Blood  Reshake pipet. Discard the first 2-3 drops of diluted
1. Aspirate blood up to the 0.5 mark of the blood. Angle of pipet while charging is 30°-35°.
WBC pipette. o Between the capillary portion of 0.5 -1, this
2. Wipe off the excess blood at the tip of the is mostly HCl which should first be discarded.
pipette with a clean piece of tissue paper.  Do not overcharge the CC.
Make sure that the tissue paper does not  Let the CC stand for 5-10 minutes to allow the WBCs
touch the opening of the bore. to settle.
3. Place the diluting fluid in a clean o Pag viniew kagad, medyo malikot pa ang
transparent container, then immerse the WBC at hindi mabibilang.
pipette. Use syringe aspiration to draw the  Locate the ruled area for WBC counting by focusing
diluting fluid up to the 11 mark with constant the microscope using LPO
rotation of the pipette. This will ensure
proper mixing of the blood and the diluting Additional info from lab manual:
fluid. This makes a 1:20 or 1/20 dilution.  Filling the Counting Chamber (Charging)
4. Gradually bring the pipette to a horizontal 1. Place the thick coverslip on top of the
position and immediately mix the contents. improved Neubauer counting chamber.
Make sure that the pipette is held securely Both the coverslip and counting chamber
with the thumb and the middle finger. must be free from dirt, thumbmarks, tissue
 NOTE: If the diluting fluid goes beyond the 11 strands, and the like.
mark or if bubbles are present inside the bulb, 2. Reshake the pipette. Discard the first 2-3
discard the mixture and start from the drops of the diluted blood from the pipette
beginning. Use a clean dry pipette. and carefully and exactly fill or charge the
counting chamber by capillary action. This
can be achieved by placing the tip of the
pipette on the space between the coverslip
and the central platform of the counting
chamber. The angle of the pipette while
charging is 30°-35°.
 NOTE: Overcharging can be readily observed
by the presence of fluid on the moats of the
counting chamber. Undercharging, on the other
hand, can be observed if the entire ruled area of
the counting chamber is not adequately covered.
Air bubbles in the counting chamber indicates
the presence of moisture or dirt. If this occurs,
clean the coverslip and the counting chamber
and repeat the process.
3. Let the chamber stand for 5-10 minutes to
allow the WBCs to settle.
4. Locate the ruled area for WBC counting by
focusing the microscope using the LPO.

PIPET METHOD: MAKING THE COUNT

 Count the WBCs seen on the 4 secondary squares
designated as W squares with an area of 4 mm2
PIPET METHOD: FILLING/LOADING THE COUNTING  A standard pattern of counting should be adopted.
CHAMBER  Record the count on each of the 4 secondary squares
 Let it stand for 2 minutes before charging to counting making sure that the cell difference between the
chamber. squares is 12 or less.
 In the counting chamber they call it charging, filling,  Do actual count & compute.
or loading.

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PROPERTY OF CHERRYANNE DOMINGO

COMPUTATION

Formula:
𝑡𝑜𝑡𝑎𝑙 𝑛𝑜 𝑜𝑓 𝑊𝐵𝐶𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
𝑁𝑜. 𝑜𝑓 𝑊𝐵𝐶𝑠⁄𝑚𝑚3 =
𝑎𝑟𝑒𝑎 𝑥 𝑑𝑒𝑝𝑡ℎ 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛

𝑡𝑜𝑡𝑎𝑙 𝑛𝑜 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

=
4 𝑚𝑚2 𝑥 1⁄10 𝑚𝑚 𝑥 1⁄20

= 𝑊𝐵𝐶𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 50
According to Ma’am Magcaling:
 Improved Neubauer Counting Chamber
Normal Values:
1. A = W1 square
𝑪𝒐𝒏𝒗𝒆𝒏𝒕𝒊𝒐𝒏𝒂𝒍 𝑼𝒏𝒊𝒕:
2. B = W2 square
3. C = W3 square 5,000 − 10,000 𝑊𝐵𝐶𝑠/𝑚𝑚3
4. D = W4 square
 Count niyo lang lahat ng WBC na nakikita niyo 𝑺𝑰 𝑼𝒏𝒊𝒕:
diyaan. 5.0 − 10.0 𝑥 109 𝑊𝐵𝐶𝑠/𝐿
 Ang difference niya would be 12 or less lang.
Hindi siya pwede mag exceed. So hanggang 12 𝐶𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 = 0.001
or less.
 Iaadd niyo lang lahat, sum up niyo lang si A, B,
PRACTICE 1
C, at D.
 W1: 48
 W2: 51
 W3: 53
Additional info from lab manual:
 W4: 55
 Filling the Counting Chamber (Charging) 48 + 51 + 53 + 55
1. Count the WBCs seen on the four 𝑁𝑜. 𝑜𝑓 𝑊𝐵𝐶𝑠⁄𝑚𝑚3 =
4 𝑚𝑚2 𝑥 1⁄10 𝑚𝑚 𝑥 1⁄20
secondary squares designated as W
squares with an area of 4mm2.
𝑜𝑟
2. A standard pattern of counting should be
adopted. Count the WBCs on the first row
= (48 + 51 + 53 + 55) 𝑥 50
of the tertiary squares from left to right, drop
down to the second row and count from = 𝟏𝟎, 𝟑𝟓𝟎 𝑾𝑩𝑪𝒔/𝒎𝒎𝟑
right to left, then to the third row counting
from left to right, and on to the fourth row  Conversion to SI:
counting from right to left.
 NOTE: WBCs that touch any of the lines on the 0.001 𝑥 10, 350
top and left borders, even if they are outside the = 𝟏𝟎. 𝟑𝟓 𝒙 𝟏𝟎𝟗 𝑾𝑩𝑪𝒔/𝑳
secondary square, are included in the count
while those that touch any of the lines on the  Interpretation: Above normal value
right and bottom borders are not included even
if they are inside the square.
3. Record the count on each of the four
secondary squares making sure that the
cell difference between the squares is 12 or
less.
 NOTE: All counts must be done in duplicate.

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PRACTICE 2  Technicians are the one who usually dilutes these

 W1: 45 fluids.
 W2: 40  Commercially available
 W3: 43
 W4: 48 Additional info from lab manual:
 Tuerk’s
45 + 40 + 43 + 48 1. Glacial acetic acid: 2.00 mL
𝑁𝑜. 𝑜𝑓 𝑊𝐵𝐶𝑠⁄𝑚𝑚3 = 2. Methyl violet: 1.00 mL
4 𝑚𝑚2 𝑥 1⁄10 𝑚𝑚 𝑥 1⁄20
3. Distilled water: 100.00 mL
𝑜𝑟

= (45 + 40 + 43 + 48) 𝑥 50 According to Sir Benjie:

 Remember the discussion regarding the
= 𝟖, 𝟖𝟎𝟎 𝑾𝑩𝑪𝒔/𝒎𝒎𝟑 hemocytometer.
 Any cell count that uses hemocytometer,
 Conversion to SI: universal siya so kahit anong gamitin ninyong or
0.001 𝑥 8,800 kahit anong bilangin niyong cells sa
= 𝟖. 𝟖 𝒙 𝟏𝟎𝟗 𝑾𝑩𝑪𝒔/𝑳 hemocytometer you can follow yung shortcut
method na tinuro ko sa inyo before.
 Interpretation: Within normal value

According to Ma’am Magcaling:

 Nowadays kasi we do not do this anymore
kunyari sa lab kasi, work in the laboratory hindi
na namin siya ginagawa ngayon kasi we use the  If you know that formula kahit anong cells ang
automated analyzers na. bilangin niyo, whatever squares ang gamitin
 So pag automated yung ginamit niyo nag niyo, tas kahit ilang square ang gamitin,
gegenerate na siya ng WBC count tapos in SI macocompute ninyo siya.
unit na.  Yung tinuro ni ma’am Magcaling, yung times 50,
 We usually do WBC counting nalang when we that is the standard. So, if ever kunwari sinabi na
do manual method of body fluid cell count. We WBC counting na standard, that would be fine.
usually do this in clinical microscopy nalang.  However, there are instances kasi kunwari,
 Pero in hematology, may automated analayzers nagbilang ka lang sa dalawang square or
naman na. pero kailangan pa rin matutunan nagbilang ka sa dalawang chambers, tama?
Tapos tig-apat na squares, ganun, also hindi
yung manual method kasi minsan nagbbreak
down ang mga machines so kunwari may STAT applicable yung times 50.
analysis kayo nagmamadali ang doctor at  So ayun, basta anuhin niyo nalang sa RBC
kailangan ng WBC count, kailangan niyo counting, ganun din naman yung ano natin.
matutunan yung manual count. Habang sira  They are almost the same actually, nagkakaiba
yung machine naggagawa niyo yung manual lang doon sa use of the RBC pipette and WBC
count niyo so may results kayong mabibigay sa pipette kasi of course, technically, sa RBC
mga doctors or clinicians. pipette mas malaki yung dilution factor natin
doon kasi mas madami ang RBC. Sa WBC
count, ang ginagamit natin is WBC pipette kasi
WBC DILUTING FLUIDS
konti lang ang WBC.
 1-3% Acetic Acid with Gentian Violet
o Glacial Acetic Acid: 2.0 g
o Gentian Violet: 1.0 ml
o Distilled water: 100.0 ml
 1% Hydrochloric Acid
o Hydrochloric Acid: 1.0 ml
o Distilled water: 100.0 ml

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References

White Blood Cell Count:

University of Santo Tomas powerpoint presentation: White Blood Cell Count
Notes from the discussion by Ms. Anna Lissa V. Magcaling, RMT
Mr. Benjie M. Clemente, RMT, MLS (ASCPi)cm, MPH

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PROPERTY OF CHERRYANNE DOMINGO

CORRECTED WBC COUNT Continuation:

 We expose our whole blood into a hypotonic
solution, the WBC diluting fluid, which is 1% HCl
or 2-3% acetic acid – these are acids that will lyse
the RBC.
 However, nucleated RBCs hemoglobin are mostly
Hgb F that resists lysing from acids. Even when
exposed to an acidic solution these nucleated
RBCs will not be lysed.
 When viewed under the microscope in a
hemocytometer, these cells look similar to WBCs
(Demonstrated by the comparison between a
lymphocyte and a nucleated RBC in the picture,
Synchronous Discussion: wherein both cells look similar to one another)
 Nucleated red blood cells (nRBCs) are the o When unstained, it would be more difficult
precursors to normal mature RBCs (erythrocytes) to differentiate the two from each other and
and are very similar to WBCs, and thus nucleated could lead to counting of nucleated RBCs
RBCs are often counted as WBCs and result in a as WBC
false increase in WBCs. o Even automated analyzers count these
cells as WBCs
 Most of the automated analyzers have the
Discussion by Sir Benjie: principle of electrical impedance and flow
 Nucleated RBC cytometry
o Smooth purplish cytoplasm with o Electrical impedance – uses electricity
pyknotic nucleus  Basic principle: resistance of the
o Orthochromic normoblast/Metarubricyte cell with regards to electricity
stage  Ex. You have here your tube, kapag
o Predecessor of reticulocytes dumaan dito yung blood cell, ang
 In normal adults, the earliest stage gagawin is magcoconduct or
that you can see in a peripheral maglalagay ng electricity kung saan
smear or PBS are reticulocytes with nagfflow yung cells natin.
a normal value of 0.5-1.5% of the  In the absence of cells, there is
total WBC count. continuous flow or conduct of
o There are instances that nucleated RBC, electricity, but if it passes through a
specifically the orthochromic RBC, go out of cell it results to a resistance in the
the peripheral blood smear especially in area where the cell is located –
severe anemia resistance of the area is measured,
o There are also instances where nucleated size, component, depending on
RBCs go out of the peripheral blood smear parang may naka set na electrical
especially if the patient is a newborn. This resistance that would identify the
is a normal case for newborns. cells.
 Problem: when it comes to counting of WBCs,  Amount of electricity resisted by the
analyzers or automated machines and even our wells will be the factor of its
eyes can count this nucleated RBCs as WBCs. identification.
 Why? Because this nucleated RBC cells resist
lysing.

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FORMULA
Continuation:
o Flow cytometry 𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡
 Kapag ang cell dumaan sa tube, 𝑢𝑛𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 𝑥 100
magkakaroon ng forward light =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑁𝑅𝐵𝐶𝑥 𝑝𝑒𝑟 100 𝑊𝐵𝐶𝑠 + 100
scatter at right angle scatter
 Yung scatter na yan would
determine cell granularity at cell CASE 1
size A 19-year-old man came to the Emergency Room with
 Bottomline is once nag aspirate ng severe joint pain, fatigue, cough, and fever.
blood, bago mapunta siya sa
dalawang tubes na ito, Review the following laboratory results:
magkakaroon ng separate tubing.  WBC count: 21.0 x 109/L
 WBC count – ieexposed pa
 RBC count: 3.23 x 10 12/L
rin sa usual na diluent
 Hemoglobin: 9.6 g/dL
which is the acid
 Platelet count: 252 x 109/L
o For analyzer 2-3%
 Differential count
acetic acid is
o 17 band neutrophils
commonly used
o 75 segmented neutrophils
 RBC count
o 5 lymphocytes
 Still counts nucleated RBCs as
o 2 monocytes
lymphocytes
o 1 eosinophil
 False increase in WBC count
o 26 nRBCs
o Question: Paano mo malalaman na meron
kang nucleated RBC?
What is the corrected WBC count in SI unit?
o Answer: Malalaman mo nalang yan kapag
 SOLUTION
nag perform ka ng manual differential count
21.0 𝑥10^9/𝐿𝑥 100
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 =
26 + 100
WHY ARE WE COMPUTING FOR CORRECTED WBC
COUNT?
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 = 𝟏𝟔. 𝟕𝒙𝟏𝟎𝟗 /𝑳
 Automated hematology analysers are unable to
differentiate nucleated red blood
CASE 2
 Nucleated red blood cells are not lysed by the diluting
If the total leukocyte count is 10.0 x 10^3/uL and 25
fluid
nRBCs are seen per 100 leukocytes on the differential.
Total WBC = WBC + nucleated RBC
What is the corrected leukocyte count?
↓
 SOLUTION
False increase in WBC count
↓
Perform the WBC differential count
10.0 𝑥 103 µ𝐿 𝑥 100
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 =
↓ 25 + 100
Look and count the nucleated RBCs per 100 WBCs
↓ 𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 = 𝟖𝒙𝟏𝟎𝟑 /µ𝑳 𝒐𝒓 𝟖𝟎𝟎𝟎/µ𝑳
>5 nRBCs
↓ CASE 3
Calculate for the corrected WBC count The peripheral blood shown is from a 10-month-old baby
boy with the following results on an automated impedance
counter.
 WBC count: 35.0 x 10^9/L
 RBC count: 2.5 x 10 ^12/L
 Hemoglobin: 45 g/L

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 Hematocrit: 0.16 L/L

 Platelet count: 250 x 10^9/L
 Reticulocyte count: 8.0%
 110 nucleated RBC/100 WBCs and many target cells
seen in peripheral blood smear
 Other laboratory results:
 Serum iron elevated
 Total iron-binding capacity (TIBC) decreased
 Serum ferritin elevated

 SOLUTION

35.0 𝑥 109 ⁄𝐿 𝑥 100

𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 =
110 + 100

Discussion by Sir Benjie:

 When it comes to your corrected WBC count, this
can only be done manually. Wala pa tayong
machines na available that would give us a
corrected WBC count kasi even machines nalilito
kung ano ba ang nucleated RBC at WBC. Usually
nagpplug lang sila, nagssasabi lang sila na
nucleated RBC are present. Usually pag nagplug
ng ganun yung machines natin you have to
perform now corrected WBC counting and diyan
tayo mas advatangeous regarding to other
countries kasi most of them hindi marrunong mag
manual.
 Kaya gusting gusto nila mg Pinoy sa ibang bansa
dahil nga super trained tayo mag ano ng manual
procedures. Sino ba naming hindi eh wala naman
tayong machines.

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References

Unit 1.1 Laboratory Safety:

University of Santo Tomas powerpoint presentation: Unit 1.1. Laboratory Safety
Notes from the discussion by Ma’am Gemee C. Estrada, RMT, MLS (ASCPi), MD
Mr. Benjie M. Clemente, RMT, MLS (ASCPi)cm, MPH

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LEUKOCYTE DIFFERENTIAL COUNTS AND BEFORE DOING THE DIFFERENTIAL COUNT…

ABSOLUTE COUNTS  The technologist should first check if the smear/blood
 Differential count usually go hand in hand with your film is:
WBC counting o Well made (with a feathery edge, covers 2/3
o so kapag nagperform ka ng WBC count of the slide, etc.)
automatic na nagbibigay ka ng differential o Stained properly
count. o Cells are evenly distributed
 Leukocyte differential count – identify / differentiate  When it comes to manual differential counting,
which are your cells. nagrerely tayo sa blood smear / blood film.
o Different leukocytes will perform different
functions MAKING A GOOD BLOOD SMEAR
o Differential count would give us an idea  Preferably the specimen of choice is a capillary
nung state ng patient natin blood specimen
o For example: increased WBC o Collected properly using the needle prick
 Next thing that you have to do is to method then the smear made on the spot
check for the differentials; dun using clean glass slides
makikita kung ano ba yung problem  EDTA collected specimens can also be used for
 For example: smear preparations
 mataas ang neutrophils – o The smears should be made as soon as the
phagocytosis; bacterial blood is collected
infection ang state the o To prevent:
patient  artifact formation
 mataas ang monocytes –  platelet satellitosis
phagocytosis but more on  adherence of platelets in the
chronic state of infection periphery of neutrophils
 mataas ang eosinophils –  usually the recommended
allergic reaction or parasitic anticoagulant is citrate para
infection (helminthic hindi kumapit sa periphery
infection) ng neutrophils

WHAT IS DIFFERENTIAL COUNT?

 This is an examination where the leukocytes are
identified and counted. The result of the count is  vacuolation of red blood cells
expressed in percentages (%).  Once the smear has been made it has to be air dried
 When it comes to performing differential count, you quickly (especially from EDTA anticoagulated
have to count 100 WBCs. specimens).
o Ilan ang neutrophils? eosinophils?  Follow the proper staining procedure.
basophils? monocytes? lymphocytes?  Do not stain blood films that are not well dried.

Notes from Lab Manual:

 Differential white cell count is the determination of
the percentage of each type of white blood cells
(WBCs) in the peripheral blood. It consists of the
enumeration of the relative proportion of the
various types of WBCs as seen in stained blood
smears.

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 Also evaluate of the platelets are evenly distributed

in the smear and in the tail-end/feathery edge.
Platelets tend to occupy the edges of a smear.

 The recommended area to evaluate in the smear

(yellow box)
o Recommended area for counting
 Sa gitna ng thick and thin periphery
 50% overlapping and 50% non-
overlapping ng red blood cells

RECOMMENDED WAYS TO SCAN A SLIDE

 Check for 100 WBCs
 You can scan the slide by using these kung san ka
masaya; ang hindi okay is kapag yung field naulit mo
 Kaya natin to ginagawa so you will not repeat the
same field
o Kapag na-count mo ulit yung WBC na
andyan that will give you a big problem with
regards to your differential count

 Way of scanning your slide; the method is called

crenellation technique
o Crenellation - eto yung sa battlefield ng
EVALUATION OF THE BLOOD FILMS castle; sa tower ng castles may pa ganyan;
 Once the smears are done for staining the slide is diyan nilalagay yung canons; yung mga tao
evaluated under the microscope first at 5x (scanner) nasa likod ng area na yun para protected sila
to check if the distribution is even.
 The leukocytes are already identifiable at 10x (Low
Power Objective) but better visualized at 40x (High
Power Objective) and 100x (Oil Immersion
Objective).
o We perform WBC differential counting in
100x (Oil Immersion Objective).

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DIFFERENTIAL COUNT LEUKOCYTES

 Identify every leukocyte that you will see and make a
tabulation using a counter. NEUTROPHILS

 Size: Average of 12 um.

o Generally:
 smaller than monocytes and
o Deep counter eosinophils
 parang may piano dito na keys na  larger than lymphocytes and
pinipindot natin basophils.
 then may picture dito ng WBCs  Nuclei: has a multilobed (3-5 lobes)
depending on the arrangement,  Cytoplasm: has lilac-colored granules
that’s usually the differential count
 tapos nandito yung tally natin Notes from Sir Benjie:
 now eto kapag nagreach ng  The first thing that you have to look at is the
100, tutunog siya *ting* that cytoplasm pwede ring nucleus muna.
would signify na umabot ka  Ang usual kong unang nakikita is the neutrophils
na ng 100 WBCs kung ilang lobes meron sila then afterwards yung
cytoplasm.
 Remember: Neutrophils - presence of 3 - 5 lobes
o Sa 6th edition ng Rodak’s, 2 - 5 lobes
 tapos kung ilan yung mga counts mo o Pero kapag 2 lobes lang ang neutrophils,
dito sa mga cells na to, yun na yung it is known as Pelger - Huet anomaly
percentage nila, syempre 100  Characteristic: pince nez
naman yung kinacount nating cells  Kamukha ni baymax

Note:
Formula for percentage (%):
# 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 100
%=
𝑇𝑜𝑡𝑎𝑙 𝑐𝑒𝑙𝑙𝑠
Example: o Stick with 3 – 5 lobes, 2 lobes would mean
Neutrophils counted = 50 Pelger-Huet anomaly
Total cells = 100  Cytoplasm – lilac-colored granules
Neutrophils = 50 %

 100, 200, 500 or 1000 leukocytes are counted but

only 100 is usually counted for practical purposes.
 The count for each leukocytes is expressed in
percentages.

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 Variant: Band Neutrophil

o Younger forms of a neutrophil  Number of lobes:
o Frequently the nuclei assumes a U-shape o Usually bilobed
o Reported separately in the differential count o Sometimes has three lobes
o Very rare with more than three lobes
 Normal Values: 3-5%
Notes from Sir Benjie:
 Increased in drug allergies and parasitic infections.
 Most of the laboratory protocols in the Philippines
will identify band as a separate neutrophil.
 There are studies that would say band neutrophils Notes from Sir Benjie:
can be counted as neutrophils  Cytoplasm - very characteristic yung red
o There are no differences between the granules
two when it comes to function. o kitang-kita pagka-reddish or orangey
 Dependent on the protocol of the laboratory o because of highly basic granules
whether to count it as separate neutrophil or  major component of granules:
isasama sa count ng neutrophil mismo. Major basic protein – carries
 function of eosinophils which is
anti-helminthic
 Normal Value: 60-70%  kapag nilagay mo sa stain it will
 Usually increased in bacterial infections. uptake acidic dye kaya siya
 Number of lobes: nagiging orangey
o 10-30% have two lobes  Nucleus – bilobed
o 40-50% have three lobes  Usually for allergies and parasitic infection
o 10-20% have four lobes
o No more than 5% have five lobes.
BASOPHILS
EOSINOPHILS

 Size: Average 13 um.

 Size: Generally smaller than neutrophils, eosinophils
o Generally:
and monocytes
 slightly larger than the neutrophil
 Nuclei: bilobed or trilobed
 larger than the basophil
 Cytoplasm: has large basophilic (dark blue)
 smaller than a monocyte
granules that are haphazardly arranged.
 Nuclei: bilobed
 Cytoplasm: large red granules

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MONOCYTES

 Size: 14 to 20 um.
 Normal Value: 0-1%
o The largest of the leukocytes
 Sometimes can be confused with neutrophils with o Two to three times the diameter of an RBC.
toxic granulations.
 Nuclei:
o Toxic granulations however are smaller
o single nuclei
compared to the larger deep blue stained
o partially lobulated
granules of the basophil.
o deeply indented or horseshoe shaped.
 Cytoplasm: abundant light gray cytoplasm
Notes from Sir Benjie: sometimes with vacuolation
 Hindi siya palaging present with regards to the
peripheral smear. Sobrang onti lang ng instances
na makakakita ka ng basophil (once daw kay sir)
o Neutrophils and lymphocytes lagi nakikita
o Eosinophils kokonti rin pero basophils
bibihira talaga
 Usually ang major characteristic ng basophil is the
presence of large basophilic (dark blue) granules  Normal Values: 1- 4%
o Halos di mo na makita ang nucleus ng cell
kasi it is obscured by the large basophilic Notes from Sir Benjie:
granules  Monocytes and Lymphocytes are mononuclear
o These cells have high acidic granules and their cytoplasm is smooth
o When you dip it with your stain, it will  Monocyte
uptake the basic dye which is methylene o The presence of vacuoles in their
blue kaya it is really dark blue cytoplasm
 When can you see basophils?  Vacuoles – the area wherein
o Tumataas yung basophils natin if in case nagkaroon na ng killing of the
meron kang allergic reactions. microorganism (marami nang
o It goes hand in hand with your napatay na bacteria)
eosinophils. o If wala pa siyang vacuoles, bago pa lang
o Kapag tumaas yan ng 5%, malaking yung cell, kaka-release pa lang niya sa
problema na yan. Di yan masyadong circulation
tumataas ng 5% o Or tendency is natapakan lang ng
 Kung ganun ka-taas, malapit na nucleus yung vacuole
mamatay pasyente mo  What if kung walang vacuole yung monocyte?
 Toxic granulations can be seen in cases of severe o Check for their nucleus, walang ganyang
infection. kalalim na identation sa lymphocyte.
o Neutrophils with darkly pigmented o Yung identation ng lymphocyte parang
granules naflatten lang ng onti
 masyadong maliit yung granules
na kitang kita mo pa yung nucleus
o Basophils malalaki yung granules na
nag-oobscure sa nucleus

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LYMPHOCYTES  Skip the cell; wag na isama

sa 100; identify the next cell
o 3) Lymphocyte

 Tip: Use the red blood cells as a reference to the size.

 Normal Value: 20-30%

Notes from Sir Benjie:

 Size: 6-10 um.  Sa lymphocyte, hindi ganun kalalim ang
o Generally smaller than the neutrophil, identation, nagflatten lang siya sa area na yan
eosinophil, monocyte and the basophil.  The nucleus of your lymphocyte would somehow
 Nuclei: already occupy the whole space of your cell
o round nuclei  Ganun kalaki yung nucleus nila.
o deeply stained blue with visible chromatin
o sometimes the nuclei is indented
 Cytoplasm: scant to abundant cytoplasm generally
without granules
 Variants
1 2
 Tip: Use the red blood cells as a reference to the
size.

BLAST CELLS / IMMATURE CELLS

 Size: usually larger than the normally seen

 Lymphocyte or Monocyte? leukocytes.
o 1) Monocyte  Nuclei: have a large blue nuclei
o 2) It could possibly be a Monocyte or a large  Cytoplasm: scant cytoplasm
Lymphocyte  When one sees these cells are counted as
 There are instances that even unidentified and should be referred for further
trained eyes can have difficulty in evaluation.
identification of cells
 Nucleus is somehow that of a Notes from Sir Benjie:
monocyte; however, the  Most of the time, kapag naka-encounter tayo ng
appearance / color is that of a blast cells, nirerefer sa pathologist.
monocyte (** yan talaga sinabi ni sir;
 Nilalagay sa remarks na blast cells are present,
medj magulo si sir dito :< )
consult hematologist
 Cytoplasm is somehow irregular, a
o hindi na iniidentify yung blast cells
characteristic of monocyte
 Kung isa lang that would be fine,
pero kung 3 or more, malaking
problema na yun
 Skiptocyte – unidentifiable cell

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ABSOLUTE COUNT General Formula:

 This is the relation of the percentage of a particular Percentage of the Leukocyte x WBC Count
leukocyte to the total leukocyte count (WBC count)
Absolute Neutrophil Count
Notes from Sir Benjie: = (% Neutrophils) x (WBC Count)
 In absolute count, you will provide the whole
value of your cell. Absolute Eosinophil Count
 With regards to your differential counting what = (% Eosinophils) x (WBC Count)
you have reported are the percentages of your
WBCs; tawag natin dun is relative counting. Absolute Lymphocyte Count
 For example: = (% Lymphocytes) x (WBC Count)
o Neutrophil = 50%
o Lymphocyte = 30%
o Eosinophil = 10% Example 1:
o Monocytes = 10% A certain patient has a leukocyte of 7.0 x 109/L and in the
 Relative count that is relative to your WBC count differential count which has 70% neutrophils.
 The next thing that you have to check is absolute Absolute Neutrophil Count
count ANC = (% Neutrophils) x (WBC Count)
o Relative increase ANC = (0.70) x (7.0 x 109/L)
 Increase is due to a decrease in ANC = 4.9 x 109/L
other WBCs  Pasok sa normal values
o Relative decrease
 Relative count is always equal to 100 % Notes from Sir Benjie:
 For example, based dun sa values natin from 10%  These reference values will differ based on
bumaba naging 3% ang eosinophil references, protocols of the laboratory, machines
o Then, tumaas ang monocyte dahil sa and reagents na gamit nila
relative decrease ng eosinophil at  These values came from your reference book.
naging 17% (**di minention anong book :< pero iba siya sa
o Based sa relative count, masasabi mo na manual)
ang monocyte is increased
 baka merong chronic infection ABSOLUTE RELATIVE
CELL
o Check first for the absolute count ng COUNT COUNT
patient before ka magsabi ng diagnosis 2.3 – 8.1
NEUTROPHILS 50 – 70%
mo X109/L
0 – 0.06
 How will you perform absolute counting? BANDS 0 – 5%
X109/L
o Relative count of your cell x WBC count 0 – 0.4
 Multiply then check if pasok pa ba EOSINOPHILS 1 –- 3 %
X109/L
yung count ng ating individual cell 0 – 0.2
BASOPHILS 0 – 2%
sa normal value ng kanilang X109/L
absolute count 0.01 – 1.3
MONOCYTES 2 – 11%
 By then, dun ka pa lang pwede X109/L
0.8 – 4.8
magdiagnose LYMPHOCYTES 18 – 42%
X109/L

Example 2
Leukocyte count = 13 X109/L; Neutrophil count = 60%
 Relative count: Normal
 Absolute count:
ANC = (0.60) x (13 x 109/L)
ANC = 7.8 X 109/L
 Normal

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Example 3  Absolute counts give a picture of the actual number

Leukocyte count = 5 X109/L; Neutrophil count = 80%; of a particular leukocyte.
Lymphocyte count = 15%; Monocyte = 5% Example:
 Relative count: Neutrophil is increased Who has a worse predicament? Patient A or B?
 Absolute count: o Patient A – 68% neutrophils in the differential
ANC = (0.80) x (5.0 x 109/L) count
ANC = 4.0 X 109/L o Patient B – 90% neutrophils in the differential
 Absolute count is normal count
Now we put some WBC counts.
Given these values compute the absolute
Notes from Sir Benjie:
neutrophil count. Which patient now has the
 If you based this only on relative count, you can
worse predicament? Patient A or B?
interpret that ang taas ng neutrophil mo merong
o Patient A: 32.0 x 109/L
bacterial infection ang patient.
o Patient B: 7.0 x 109/L
o You can give a wrong diagnosis sa patient
o Pinagastos mo na patient mo
(** di sinagutan to ni sir :<, sinagutan na lang namin hehe)
o Nagcontribute ka pa sa possibility of
antibiotic resistance
Patient A
 You always have to check for the absolute
 Relative count: Normal
counting of your cells before you provide
 Absolute count:
diagnosis
ANC = (0.68) x (32.0 x 109/L)
o Sa absolute count dapat magbased
ANC = 21.76 X 109/L
 When it comes to laboratories rin kasi, ang
 Absolute count is not normal
nirereport lang nila is relative count, nirereport
Patient B
lang ang percentages
o May mga doctors na di pre-med ang  Relative count: Neutrophil is increased
Medtech tapos hindi nila alam yung  Absolute count:
absolute counting nagkakaroon ng ANC = (0.90) x (7.0 x 109/L)
relative increase sa differential count ANC = 6.3 × 109/L
tapos dinadiagnose na nila as bacterial  Absolute count is normal
infection
 Pero normal naman neutrophil
mo
o So bakit ka nagkaroon ng ganitong case
kung saan tumaas neutrophil natin?
 Medyo bumaba yung
lymphocytes and monocytes to
the point na tumaas ang relative
count ng ating neutrophil
 We always have to make your
differential count 100%
 So kahit ano pa yan dapat 100%
ang relative count
 Tumaas itong neutrophil dahil
bumaba yung relative count ng
lymphocyte and monocyte

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Notes from Lab Manual:

STAINING JAR OR “DIP” METHOD  Station 4 – Deionized water (1000
 Procedure of Staining mL)
o Dip in solution 1  Station 5 – Phosphate buffer (500
 Methanol, the fixative mL)
 30 seconds  Station 6 – Warm air
o Dip in solution 2 o Hema-Tek 1000 Slide Stainer
 Eosin, the acidic dye  The bottles of the Stain-Pak
 6 seconds (stain, buffer and rinse solutions)
o Dip in solution 3 are opened by making a small
 Methylene blue, the basic dye hole and a cannula is inserted
 4 seconds into each solution. A pump tube
o Dip in buffer solution / aged distilled water set is installed to transport each
 45 seconds solution.
 Procedure of Differential Counting  Tubing 1 – stain solution
o Prepare a stained blood smear.  Tubing 2 – buffer solution
o Place one drop of cedar wood oil on the  Tubing 3 – rinse solution
feathery edge of the stained blood o Hema-Tek 2000 Slide Stainer
smear.  This stainer employs the same
o Examine the smear using the LPO of the principle as Hema-Tek 1000. The
microscope. Focus on the area where the innovation is improved staining
red blood cells are not too overlapping system through the use of new
or too scanty. pumps and volume controls. The
o Shift to OIO. Using the strip differential operator can electronically adjust
methods, count 100 white blood cells the stain, buffer, and rinse
while differentiating them. solutions.

TYPES OF STAINS USED IN DIFFERENTIAL

WHITE BLOOD CELL COUNTING
 Romanowsky stains
o Contains methylene blue or its oxidative
product, such as azure B
o Contains eosin B or eosin Y
o The dyes produce multiple colors when
used on cells and cellular components
 This is the reason why the stains
OTHER METHODS OF STAINING are considered polychromatic.
 Staining dish methods o Wright’s stain
o Involves placing the blood smear on a  Most satisfactory in general
rack positioned on a dish routine hematologic studies
 Automated method  Composition:
o Hemasteiner automatic slide stainer  Oxidized methylene blue
 Freshly prepared staining  Eosin azures
solutions are used daily for every o Giemsa stain
4-8 hours during operation.  Excellent stain for the
 Station 1 – Methanol (500 mL) demonstration of inclusion bodies
 Station 2 – Wright’s or Wright’s - and intracellular parasites as well
Giemsa stain (500 mL) as for staining WBCs
 Station 3 – Stain - Buffer mixture  Composition:
 Wright’s stain (80 mL)  Eosin Y with azure blue
 Phosphate buffer (500  Methylene blue in
mL) methanol with glycerin

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o Leishman, Jenner, and May-Grunwald

 Similar to Wright’s stain except
for the method used to oxidize
methylene blue
 Panoptic stains
o Panoptic stains consist of Romanowsky
stain and another dye
 Wright’s – Giemsa
 Jenner – Giemsa
 May – Grunwald – Giemsa

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PROPERTY OF CHERRYANNE DOMINGO

Notes from Lab Manual:

WHITE BLOOD CELLS

NV OF NV OF NV OF NV OF
RELATIVE RELATIVE ABSOLUTE ABSOLUTE
WBC DESCRIPTION
COUNT – COUNT – COUNT – COUNT –
CU (%) SI (L/L) CU (/cumm) SI (109/L)

Nucleus: broken into

segments
Neutrophilic
Cytoplasm: contains 50 – 70 0.50 - 0.70 2,300 - 8,100 2.3 - 8.1
segmenter
small, pinkish
granules

Nucleus: compact
and usually round
Lymphocyte 18 – 42 0.18 - 0.42 800 - 4,800 0.8 - 4.8
Cytoplasm: light blue
and scanty

Younger form of
Neutrophilic
neutrophil with C, S,
band 0–5 0 - 0.5 0 – 600 0 - 0.6
U, or horseshoe-
(stab or staff)
shaped nucleus

Nucleus: spongy and

sprawling with brain-
like convolutions
Monocyte 2 – 11 0.02 - 0.11 450 - 1,300 0.45 - 1.3
Cytoplasm: gray
Vacuoles –
sometimes present

Nucleus: usually
bilobed
Eosinophil Cytoplasm: contains 1–3 0.01 - 0.03 0 – 400 0 - 0.4
large, coarse, reddish
or orange granules
Nucleus: usually
indistinct and
obscured by the
Basophil granules 0–2 0 – 0.02 0 – 100 0 - 0.1
Cytoplasm: contains
large purplish-black or
dark blue granules

*NV = normal values

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References

LEUKOCYTE DIFFERENTIAL COUNTS AND ABSOLUTE COUNTS:

University of Santo Tomas powerpoint presentation: Leukocyte Differential Counts And Absolute Counts
Prepared by: Miguel Francisco C. Cajipe, MD, RMT, DPSP-CP

Sadang, MG. and Llanera, F., 2015. Laboratory Manual in Hematology. Quezon City, Philippines: C&E Publishing, Inc.

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PROPERTY OF CHERRYANNE DOMINGO

LEUKOCYTE ALKALINE PHOSPHATASE SCORING CHRONIC MYELOGENOUS LEUKEMIA (CML)

TEST
According to ma’am Ponciano:
Importance:
 A neoplasm that consist, or the peripheral
 It is used to differentiate chronic myelogenous
smear contains also mature neutrophils
leukemia from leukemoid reaction.
 Question: How would you differentiate the
neutrophil present in chronic myelogenous
LAPS TEST
leukemia?
 PRINCIPLE:
 Answer: Neutrophils in CML doesn’t
o Alkaline leukocyte phosphatase catalyzes
differentiate fully so they lack the secretory
the hydrolysis of phosphate esters in alkaline
granules like your alkaline phosphatase so you
solution. 1-Naphthol released from 1-
cannot observe the activity leukocyte alkaline
naphthyl-phosphate is coupled with
phosphatase.
diazonium salt forming an insoluble pigment
at the site of activity.
 CHARACTERISTICS:
LAP SCORING TEST 1. Increased WBC count (greater than
 Used in patients with increased WBC count 50,000/cumm)
 Reactive process (leukemoid reaction) 2. Translocation of chromosome 9 and 22
 Chronic myelogenous leukemia (Philadelphia chromosome)
3. Increased number of mature neutrophils in
LEUKEMOID REACTION PBS
 But incomplete in maturation.
According to ma’am Ponciano: 4. Decrease amount of leukocyte alkaline
 Has almost the same characteristics in the phosphatase
peripheral blood smear with chronic  So, the activity would now have a
myelogenous leukemia. decreased amount of leukocyte
alkaline phosphatase in its
 CHARACTERISTICS: cytoplasm or the activity would be
1. Increased WBC count (Less than lesser than that of your leukemoid
50,000/cumm) reaction.
2. Presence of toxic granulation on the
cytoplasm of the cell LAPS PROCEDURE
 Remember that neutrophils have fine MATERIALS NEEDED
granules on its cytoplasm and it is  Heparinized capillary blood
neutral in color o Capillary blood is preferred.
3. Absence of basophilia o If you have anticoagulated blood, it should
4. Increase number of mature cell (neutrophils) have heparin as its anticoagulant.
5. Increase LAP scoring  Alkaline phosphatase test kit
 Increased because it contains the o Fast blue RR salt (fast violet B salt)
secretory granule leukocyte alkaline  Primary stain
phosphatase which is lacking in the o Mayers hematoxylin solution
neutrophils of chronic myelogenous  Counterstain
leukemia. o Citrate concentrate*
o Acetone*
 Mixture of citrate concentrate and
acetone will serve as fixative.
o Coplin jars

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o Glass slides and microscope 1. Scan the bone smear or peripheral blood smear using
 Coplin jars, glass slides and OIO at the thin area where RBC are not overlapping
microscope doesn’t come with the kit with each other.
but you will be needing this 2. Count 100 granulocytes (band and segmenters)
laboratory apparatus. consecutively
o Exclude lymphocytes, monocytes, and your
PREPARATION OF SMEARS basophils.
1. Prepare bone marrow and blood smears (preferably o Grade according to the intensity of the color
without anticoagulant or with heparin) that is developed.
2. Blood smears should be stained within 8 hours after 3. Reference interval: Normal = 10-100 (depends upon
collection the reagent kit used)
o Because the activity of leukocyte alkaline
phosphatase will be affected. Leukemoid = increased
3. Dry smears for 1 hour before fixation. CML = decreased
o Should be completely fixed – completely dry
blood smears or bone marrow smears.
4. Immerse smears for 30 seconds in fixative (citrate a-
acetone).
o For fixation use citrate-acetone mixture.
5. Rinse in deionized water.

STAINING AND COUNTERSTAINING PROCEDURE

1. Prepare stains immediately before use and discard
after use.
o Freshly prepared.
o Discard after use. No need to stack the stains
that are to be used in the procedure.
2. Immersed slides on the prepared stain and incubate
at 23-26 degrees C for 30 mins away from direct light.
o Direct light would activate your leukocyte
alkaline phosphatase activity.
3. After incubation, wash slides gently with deionized
water for 2 minutes without drying.
4. Place slides in Mayer’s hematoxylin solution
(counterstain) for 10 minutes
The top two neutrophils in this image are scored as
5. Rinse with deionized water for 3 minutes
1 and 2+ respectively while the bottom neutrophil is
6. Examine the slide under the microscope
scored as 0 (no cytoplasmic staining). Note the
Note: nucleated RBC next to the top two neutrophils does
 If positive with LAPS score not stain.
 If RR salt was used – red violet color is
observed (nucleus)
 If fast violet B salt - blue nucleus is observed
According to ma’am Ponciano:
 Example of this in peripheral blood. Cells reacted
with leukocyte alkaline phosphatase, your
CALCULATION AND OBTAINING RESULTS staining procedure.
 No stain = negative. Did not take up the stain.
According to ma’am Ponciano:
 Plus 1 is only 1/4 of the cell is stained.
 You have to grade the intensity of the color that
 Plus 2 if 1/2 of the cell. If you join the stained
was developed on the nucleus of the cell so
area, it occupies half of the cell then it is graded
what you will do is like a leukocyte differential
as plus 2.
count.

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POSSIBLE SOURCES OF ERROR

1. Certain drugs such as cortisol, and oral
contraceptives
2. Stress related
3. Osteoblasts and endothelial cells stain strongly
o Osteoblasts if you are having or examining
your bone marrow smear.
o Because it is stained strongly, you could
interpret that as one of the leukocytes or
neutrophils that are stained with the activity
leukocyte alkaline phosphatase.
4. Not following strictly the procedure
The top neutrophil in this image is a 3+ while the o Shortcut of the procedure.
second neutrophil would be scored as 4+ i.e. intense o Incubate for less or longer than 30 minutes.
cytoplasmic staining with the nucleus obscured. 5. Expertise of the microscopist
o Must be very familiar on how to grade cells in
LAPS scoring test.
According to ma’am Ponciano:
 Now, plus 4, this is very subjective. Remember ADDITIONAL CLINICAL APPLICATION
when grading the activity of leukocyte alkaline 1. Increase in postsurgical operation until after 1 week
phosphatase, you grade it as plus 4 if the 2. Differentiation of choriocarcinoma and hydatidiform
nucleus of the cell is fully stained. mole
 So eto buong buo na yung cell that is stained
with your staining procedure or your H-mole = high/elevated values
Hematoxylin and fast RR blue. Choriocarcinoma = normal values
 If the stained area of the cell is 3/4, that is graded
as plus 3.
ACTIVITY WORK
1. Bone marrow smears was submitted in the laboratory
Computation: 100 granulocytes (band / segmenters) for LAPs scoring. Request form was filled up properly
was counted with all the data necessary to prepare a request form.
The Medical technologist took notice that the
Formula: submitted specimen was collected 24 hours after it
was collected. Will the Med tech proceed with the
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑟𝑎𝑡𝑖𝑛𝑔,
examination. Support your answer with 2-3 sentences
𝑔𝑒𝑡 𝑡ℎ𝑒 𝑠𝑢𝑚 𝑜𝑓 𝑡ℎ𝑒 𝑟𝑎𝑡𝑖𝑛𝑔
2. Compute for LAPS value with the given data. Make
an interpretation of test result.
o 60 cells= 0
o 35 cells= 1+
o 5 cells =2++

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References

Unit 1.1 Laboratory Safety:

University of Santo Tomas powerpoint presentation: Unit 1.1. Laboratory Safety
Notes from the discussion by ASST. PROF. FELICITAS E..PONCIANO, MSMT

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PROPERTY OF CHERRYANNE DOMINGO
PROPERTY OF CHERRYANNE DOMINGO

MALARIAL SMEAR PREPARATION AND Notes from Parasitology core group Malaria ppt:
EXAMINATION
• GENERALITIES
o Phylum apicomplexa
Notes from Ma’am Tesalona’s ppt: o Alternating sexual and asexual stages
• Kingdom: Protista o Final Host: Mosquito (Female anopheles)
• Phylum: Apicomplexa § Sexual stage
• Class: Sporozea o Intermediate Host: Man
• Subclass: Coccidia § Asexual stage
• Suborder: Haemosporina o Habitat: Liver, RBCs
• Genus: Plasmodium § Intracellular
o MOT: Bite of mosquito
According to Yambao (Para Lab - Pingol & Unas): o IS to FH: Gametocytes
PHYLUM APICOMPLEXA § Cell specializing in the transition
• GENERALITIES between the human and the mosquito
o Intracellular protozoans host
o Possess apical complex § Gametocytes arise from erythrocytic
o May require intermediate host to complete the life asexual stages
cycle o IS to IH: Sporozoites
o Undergoes both sexual and asexual reproduction § Product of a complex developmental
• CLASSIFICATION process in the mosquito vector
o Present in blood
Notes from Ma’am Tesalona’s ppt:
§ Plasmodium
§ Babesia • Arthropod transmitted (female anopheles
o Intestinal coccidians mosquito)
§ Partially acid fast intestinal coccidians • Mainly transmitted thru skin inoculation
o Tissue coccidians (extraintestinal) • No definite organ for locomotion
§ Toxoplasma • Intraerythrocytic parasites
§ Sarcocystis • Life cycle consist of two phases (sexual and
asexual phase)
PLASMODIUM • Requires two hosts to complete its cycle
• Etiologic agent of malaria
• GENERALITIES • FORMS
o Intracellular parasites o Early Trophozoite/Ring Form
o Vector borne parasite: Female Anopheles § Ring-shaped with a red chromatin dot
o Major vector in PH: Anopheles minimus § Scant amount of blue cytoplasm
flavirostris o Trophozoite Form
o MOT: § Large chromatin mass with prominent
§ Bite of mosquito (major) ameboid cytoplasm, which is spread through
§ Blood transfusions the eythrocyte
§ Congenital o Schizont
o Undergoes sexual (sporogony) and asexual § Chromatin has divided into two or more
(schizogony) reproduction masses of chromatin with small amounts of
o Infective stage to man: Sporozoite cytoplasm, called merozoites
• Plasmodium spp. o Gametocyte
o Plasmodium falciparum § Fills the entire red blood cell
o Plasmodium vivax § Characterized by a large chromatin mass
o Plasmodium ovale and a blue cytoplasm with pigment
o Plasmodium malariae § Round to banana-shaped
o Plasmodium knowlesi (zoonotic)

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STAGES FOUND IN BLOOD APPEARANCE OF RBC APPEARANCE OF PARASITE
Plasmodium falciparum
Normal. Delicate cytoplasm.
Ring Multiple infection of RBC more 1-2 small chromatin dots.
common than in other species. Occasional applique (accolle) forms.
Seldom seen in peripheral blood.
Trophozoite Compact cytoplasm.
Normal. Dark pigment.
Rarely, Maurer’s Clefts (under certain Seldom seen in peripheral blood.
staining conditions). Mature = 8-24 merozoites.
Schizont
Dark pigment.
Clumped in one mass.
Crescent/sausage shape.
Chromatin in a single mass
Gametocyte Distorted by parasite. (macrogametocyte) or diffuse
(microgametocyte).
Dark pigment mass.
Plasmodium vivax
Normal to1-1/4 X.
Large cytoplasm with occasional
Round.
Ring pseudopods.
Occasionally fine Schuffner’s Dots.
Large chromatin dot.
Multiple infection of RBC common.
Large ameboid cytoplasm.
Trophozoite Large chromatin.
Fine, yellowish-brown pigment.
Large.
May almost fill RBC.
Schizont Enlarged 1-1/2-2 X. Mature = 12-24 merozoites.
May be distorted.
Yellowish-brown, coalesced pigment.
Fine Schuffner’s Dots.
Round to oval.
Compact.
May almost fill RBC.
Gametocyte Chromatin compact.
Eccentric (macrogametocyte) or
diffuse (microgametocyte).
Scattered brown pigment.
Plasmodium malariae
Sturdy cytoplasm.
Ring Normal to 3/4 X.
Large chromatin.
Compact cytoplasm.
Large chromatin.
Trophozoite
Occasional band forms.
Coarse, dark-brown pigment.
Mature = 6-12 merozoites with large
nuclei, clustered around mass of
Schizont Normal to 3/4 X.
coarse, dark brown pigment.
Rarely, Ziemann’s Stippling (under
Occasional rosettes.
certain staining conditions.
Round to oval.
Compact.
May almost fill RBC.
Gametocyte
Chromatin compact, eccentric
(macrogametocyte) or more diffuse
(microgametocyte).

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Scattered brown pigment.

Plasmodium ovale
Normal to 1-1/4 X.
Round to oval.
Sturdy cytoplasm.
Ring Occasionally Schuffner’s dots.
Large chromatin.
Occasionally fimbriated
Multiple infection of RBC common.
Compact with large chromatin.
Trophozoite
Dark-brown pigment.
Mature = 6-14 merozoites with large
Schizont nuclei, clustered around mass of dark-
Normal to 1-1/4 X. brown pigment.
Round to oval. Round to oval.
Some fimbriated. Compact.
Schuffner’s dots. May almost fill RBC.
Gametocyte Chromatin compact, eccentric
(macrogametocyte) or more diffuse
(microgametocyte).
Scattered brown pigment.

According to Ma’am Tesalona’s ppt & Parasitology core group Malaria ppt:
P. falciparum P. vivax P. ovale P. malariae P. knowlesi
Affinity towards Affinity towards
AFFINITY TO Infects mature or
Infects red cells young or young or Infects red cells
RED BLOOD older red blood
of all ages immature red immature red of all ages
CELLS cells
cells cells
RBC enlarged
EFFECT ON No alteration in *Oval, fringed, Smaller than
RBC enlarged
RBC SIZE size fimbriated red normal
cell
CYTOPLASMIC Schuffner’s
Maurer’s dots James’ dots Ziemann’s dots
INCLUSIONS granules
Malignant
tertian malaria /
FEBRILE /
Estivo-autumnal Benign tertian Ovale tertian Quartan malaria
ERYTHROCYTIC
malaria / (48 hours) (48 hours) (72 hours)
CYCLE
Black water fever
(36-48 hours)
STAGES SEEN Ring,
IN THE gametocyte,
All All All
PERIPHERAL other stages are
BLOOD rare

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STAGES OF MALARIAL PARASITES

P. falciparum P. vivax P. ovale P. malariae
SCHIZONT/CRYPTOZOITE
(Stage that initially develops in hepatic cells)
TROPHOZOITE / RING FORM Applique / accole Single compact Assumes a band
Small hyaline ring
(Vegetative state containing 1 Small ring forms ring shape.
that appears
nucleus and develops within with double nuclear (like trophozoite of Single large
ameboid
RBC) dots P. malariae) compact ring.
SCHIZONT
(Trophozoite in which the nucleus has divided)

6-12
MEROZOITE 8 arranged around
6-32 12-24 average of 8
(Cell resulting from the final a central block of
average of 20-24 average of 16 arranged in rosette
division of schizont/cryptozoite) pigment
or daisy formation

HYPNOZOITE
(Dormant stage that persist in liver or hepatic cells, but only in the case of Plasmodium vivax and Plasmodium ovale)

GAMETOCYTE Crescent/sausage/ Round to oval. Round to oval.

Round to oval.
(Sexually differentiated but banana shaped Same size as Smaller than RBC.
Fills enlarge RBC.
immature cells) larger than RBC RBC.

PLASMODIUM SPP.

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P. falciparum

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P. vivax

P. malariae

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P. ovale

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Notes from Ma’am Tesalona’s ppt: • Occurence of Relapse and Recrudescence

• Phases of Life Cycle • Pathogenesis
o Asexual or Schizogony - develops in an o P. falciparum
intermediate host § Presence of “sticky knobs” on infected RBCs
o Sexual or Sporogony - develops in a definitive (cytoadhesion).
host § PfEMP-1 (most adhesive protein).
o Complications
• Host requirement
o Man - intermediate host § Cerebral Malaria
§ Blackwater Malaria
o Female Anopheles mosquito - definitive host
§ Nephrotic Syndrome
• Mainly transmitted thru skin inoculation
• Epidemiology
According to Yambao (Para Lab - Pingol & Unas): o As of 2017, there are 219 million cases worldwide
PLASMODIUM KNOWLESI (WHO Malaria Report).
• Zoonotic o Most cases are still coming from:
• Species affecting long tailed macaques § African Region (92%).
• Endemic areas: o Widest distribution:
o Malaysia § P. falciparum
o Philippines • Africa
o Other South East Asian countries • SEA
• Similar to P. malariae • Western Pacific Region
§ P. vivax
MALARIA • Americas
o Philippine Setting:
Notes from Ma’am Tesalona’s ppt: § P. falciparum: most prevalent
• DISEASE
o Chronic malaria leads to anemia, associated Notes from Ma’am Tesalona’s ppt:
with impaired physical and mental growth and • GEOGRAPHICAL DISTRIBUTION
development in children. o Plasmodium falciparum & Plasmodium
o No exact features malariae
o Prodromal symptoms: weakness and § Asia and Africa
exhaustion, desire to stretch and yawn, o Plasmodium vivax
aching bones, limbs and back, loss of § Latin America, India and Pakistan
appetite, nausea and vomiting and a sense of o Plasmodium ovale
chilling § Exclusively found in Africa
o Plasmodium knowlesi
§ Philippines and most Southeast Asia
• Remains the leading parasitic disease that cause
mortality worldwide
• Classical Paroxysms: characteristic periodicity LABORATORY DIAGNOSIS
o Chills (15-60 mins) • Sample: Capillary Blood
o Fever (2-6 hours) • Gold Standard: Microscopy
o Sweating (2-4 hours) o Preparation of Thick and Thin Smears.
• Other signs and symptoms o Stain Used: Giemsa (pH 7.2).
o Anemia • Other Methods:
o Splenomegaly o Quantitative Buffy Coat
o Headache o Rapid Diagnostic Tests
o Body pain o Serology (IFAT)
o Nausea o Molecular Methods
o Vomiting o Culture
o Pallor

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BASIC LABORATORY TECHNIQUES IN MALARIA

Notes from Ma’am Tesalona’s ppt:
MICROSCOPY
• LABORATORY DIAGNOSIS
o Examination of thick and thin blood film (gold THICK SMEAR THIN SMEAR
standard) Layer of red blood cells Single layer of red blood
§ Thick smear 10- 20 times thicker. cells.
• rapid diagnosis Parasites are within red
cell ghosts (staining Used as label to identify
• must be dehemoglobinized prior to
unfixed red cells causes patient.
staining hemolysis).
§ Thin smear Used to identify parasite
Used to detect parasites
• for specie identification species, after they have
and estimate parasite
• must be fixed prior to staining been seen in the thick
density (malarial count).
§ Stains: Giemsa, Wright’s and Delafield’s film.
Hematoxylin Gives sensitivity to Gives specificity to
diagnosis. diagnosis.
o QBC or Quantitative buffy coat method
Put label on the thickest
o Para Sight F test Never fix thick smear.
part of the thin smear.
§ Dipstick test for simple and rapid
*Dehemoglobinized: rupture RBCs to view the malarial
examination of Plasmodium falciparum
parasites.
infection
o Serologic tests (IHA, IFAT and ELISA)
PREPARATION OF BLOOD SMEARS FOR MALARIA
§ These tests are used in epidemiological
o Step 1
studies and cannot differentiate between
§ Clean the 3rd or 4th finger from the thumb
current and previous infections
with cotton soaked in 70% alcohol, using firm
o PCR
strokes to remove dirt and grease from the
TREATMENT finger.
• Chloroquine: mainstay drug for malaria. § Airdry the finger.
• Artemisinin based Combination Therapies (ACTs) § Using a sterile lancet, puncture the ball of the
such as Arthemeter-Lumefantrine (Coartem). patient’s finger.
• Other Drugs § Apply gentle pressure to the finger to
o Quinine express the first drop of blood and wipe it
o Primaquine away with dry cotton.
o Prophylactic Drugs o Step 2
§ Mefloquine § Apply gentle pressure to the finger then
§ Doxycycline collect 3 small drops of blood on one side of
§ Atovaquone/Proguanil a clean slide and 1 small drop next to the 3
• Resistance drops, leaving some space between the thick
o Duffy negative and thin smears to be made.
o Hemoglobinopathies, Sickle cell § Handle clean slides only by the edges.
o G6PD deficiency § Wipe the remaining blood away from the
patient’s finger with dry, clean cotton.
PREVENTION AND CONTROL o Steps 3-4
• Early diagnosis and treatment. § Make the thin smear first.
• Personal protection measures (use of insecticide § Place slide on a flat, firm surface.
treated nets, insect repellants). § Bring down a second slide (spreader) on the
• Use of Chemoprophylaxis. 1 small drop at an angle of 45°, allow the
blood to run along its edge.
• Larvicides (use of B. thuringiensis)
§ Firmly push the angled side away from the
• Larviparous fish.
blood towards the other end of the slide.
• Health Education.
o Step 5
• Vaccine: no effective vaccine yet.
§ After spreading the thin smear, make the
thick smear.

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§ With the corner of the second slide, quickly § Fix thin smear with methanol.
join the drops of blood, and spread them in a § Air dry smears.
circular motion until it is about the size of a 5 § Flood or immerse thick and thin smears in
centavo coin. freshly prepared 10% Giemsa Stain.
§ The thickness should be such that it is just § Wash smears.
possible to see newsprint through it. § Air dry.
o Step 6 • Rapid Method of Staining (used for between 1-5 slides
§ Using a lead pencil, label the smear with the o Air dry smear before fixing the thin smear with
name or number of the patient and date on methanol. Do not fix or expose the thick smear to
the thick portion of the thin film. methanol vapor.
§ Airdry slide on a flat, level position. o Flood thick and thin smears with 10% Giemsa
o Step 7 solution for 10-15 minutes.
§ Fix the thin smear with methanol by using a o Gently wash smear and air-dry.
dropper or by dipping it in the solution for a • Regular Method of Staining (used for between 10-20
few seconds. slides)
§ Air dry the smear. o Air dry smear before fixing the thin smear with
§ Do not fix the thick smear. methanol. Do not fix or expose the thick smear to
o Step 8 methanol vapor.
§ Stain thick and thin smears with Giemsa or o Immerse thick and thin smears with 3% Giemsa
Wright’s stain for 10-15 minutes then wash solution for 30-45 minutes.
under running tap water. o Gently wash smear and air-dry.
§ 10% Giemsa Stain preparation: 1 part stain
+ 9 parts distilled, rain, or tap water. ADDITIONAL VIDEOS FROM OTHER
o Step 9 SECTIONS
§ Air-dry smears in a vertical position then MALARIA THICK SMEAR PREPARATION
examine under oil immersion
• https://www.youtube.com/watch?reload=
• Common Faults in Blood Smear Preparation 9&v=WPP7AjmStBg&feature=youtu.be&f
o Too big thin smear, thick smear badly positioned. bclid=IwAR2JOh8mhf0eH8jeTSJX8hbPYj
o Edge of spreader slide chipped. R3egzx94w5M4H65vdQd8MhElQYso1iM
o Blood smears spread on a greasy slide. zI
o Too much blood. MALARIA THIN SMEAR PREPARATION
o Too little blood.
• https://www.youtube.com/watch?reload=
• Other Common Faults 9&v=acoALifVvb8&feature=youtu.be&fbcl
o Insects eat dry blood and damage the film. id=IwAR2qxytciNOjZNZPWekZ5LsuJS2f
o Smears made on badly scratched slides. wXb8_PsT6ojZJbwJAPOjjHXmSrDKupg
o Thick unevenly dried.
o Autofixation.
o Wet smears sticking to one another.
• Staining
o Components of Buffer Solution
§ 1 g Na2HPO4 (anhydrous disodium
hydrogen phosphate).
§ 0.7 g KH2PO4 (anhydrous potassium
dihydrogen phosphate).
§ 1000 mL (1L) distilled or deionized water.
§ Adjust pH to 7.2.
o Components of Giemsa Stock Solution
§ Giemsa Powder: 3.8 g.
§ Methanol: 250 mL.
§ Glycerol: 250 mL.
o Procedure

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Notes from Hema Lab Manual:

TEST FOR MALARIAL PARASITES

• Diagnosis is made from peripheral blood
• Best obtained in the morning and afternoon and carried out for three days
• Microorganisms which parasitize RBCs may be missed unless the laboratory is notified that malaria is
suspected.
• The disease is transmitted through a mosquito bite (female Anopheles mosquito).
• The parasite lives in the bloodstream
• The parasite goes through a stage of development known as schizogony, which means sexless.
• It infects the human host by entering the RBCs, causing hemolysis.
• This results in toxicity as manifested by chills and fever.
• Treatment consists of the administration of anti-protozoan drugs.
• If the patient is not having an attack, the malarial parasite cannot be detected.
• Hence, it is necessary that the patient should be experiencing chills and fever at the time blood is drawn.
• A thick blood smear preparation is the method of choice for diagnosis.
• In case of heavy infection, the parasites may be identified even on an ordinary thin smear.

PROCEDURE FOR THIN SMEAR PREPARATION

1. Make a skin puncture
2. Prepare two blood smears as in differential count
3. Stain smears with Wright’s stain. Allow to dry.
4. Examine under OIO

PROCEDURE FOR THICK SMEAR PREPARATION

1. Make a finger puncture
2. Place a large drop on the glass slide. Using the corner of another slide, spread the blood so that it covers an area
about the size of a 25-centavo coin.
3. Allow the smear to dry.
4. Stain the smear with Giemsa stain for 30 minutes.
5. Dip the stained smear in buffer solution to rinse. Air-dry.
6. Examine under OIO.

MANNER OF REPORTING
• If malarial parasites are seen, report as positive.
• If no malarial parasites are seen, report as negative.

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Notes from Parasitology Lab Manual:

PREPARATION OF BLOOD SMEARS FOR DIAGNOSIS OF MALARIA

• Microscopy remains the gold standard in the diagnosis of malaria
o Microscopy - specific, cost-effective and practical for routine laboratories in the country.
o Important aspect is a properly prepared and stained blood smear.
o Thick and thin smear are prepared for malaria diagnosis
o Giemsa stain adjusted to a pH of 7.2 is ideal for staining.
• Take Note: The best time to collect and prepare blood smears is during the height of fever

PROCEDURE FOR PREPARATION OF THIN AND THICK SMEARS

1. Identify the patient properly.
2. Record the patient’s information on a registry or logbook. The contents of the logbook will depend on the protocol
of the clinical laboratory.
3. Wear protective gloves. Make sure all the materials for blood collection and smearing should be in easy reach.
4. Hold the patient’s less dominant hand with the palm facing upwards and select the middle finger or ring finger.
For pediatric patients, the big toe can also be selected. In all times, never select the thumb and index finger for
peripheral blood collection.
5. Clean the selected finger with an alcohol swab or cotton moistened with 70% alcohol. Clean the finger thoroughly
to remove dirt and oil.
6. Dry the finger with a clean cotton cloth and stroke in a firm manner the selected finger to promote blood circulation.
7. Puncture the finger or toe using a sterile lancet with a quick stabbing action.
8. Express the first drop of blood by applying pressure to the finger or toes. Wipe the first drop of the blood with a
dry cotton.
9. Work quickly and handle the slides only by the edges and collect the blood sample as follows.
10. Gently apply pressure to the finger and collect a single drop of blood on the middle portion of the slide. This is
intended for the thin smear.
11. Gently apply more pressure to express more blood and collect 2-3 larger drops of blood about 1 cm away from
the drop of blood for the thick smear.
12. Wipe the remaining blood off the finger with a dry cotton.
13. For the thin smear, place the slide with blood on a flat, firm surface. Using a second slide as a spreader, touch the
small drop with the edge of the spreader to allow the blood to run right along the edge.
14. Push the spreader along the slide in a firm manner making sure the angle is maintained at 45 degrees. Make
sure the spreader edge is in even contact during smearing.
15. For the thick smear, handle the slide on its edges and make the blood smear by using the corner of the spreader
to join the drops of blood. Spread them to make an even thick smear. Spread the blood in circular form for 3-6
movements. The diameter of thick smear should be approximately 1 cm.
16. Dry the thick and thin smears. When drying make sure the smear is level and protected from dust, insects such as
flies, sunlight and extreme heat.
17. Label the slides using pencil. Label by writing the patient’s name or code and date on the frosted end or on the
thicker portion of the thin smear.

STAINING OF THICK AND THIN SMEARS USING GIEMSA (RAPID METHOD)

• Staining is done in order to visualize malarial parasites using the Giemsa stain, a Romanowksy stain composed
of methylene blue and eosin.
o Note: This method is preferred for staining 1-5 slides at a time and used for rapid diagnosis of malaria.

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Notes from Parasitology Lab Manual (continuation):

• Procedure:
o Note: Before staining, make sure the blood smears are dry. For thick smears, these should be
dehemoglobinized and not fixed with methanol prior to staining. For thin smears, no
dehemoglobinization is done prior to staining.

1. Dehemoglobinize the thick blood smear by dipping it in tap water. Dehemoglobinization is done to remove
hemoglobin that can result in poor staining and can obscure the malarial parasites. For the thin smear, apply 2-
3 drops of methanol to fix it. Let it dry afterwards.
2. Place the slide on the staining rack and gently flood the slide with the prepared Giemsa working solution. Make
sure it covers the whole slide.
3. Stain the blood smears for 5-8 minutes. This could be extended up to 1 hour. Experience with the Giemsa stain
brand being used will help in determining the estimated time.
o Note: The use of Wright-Giemsa stain is acceptable but not Wright stain alone. Preferably, the smear is stained
with a 3% Giemsa working solution (pH 7.2) for 30-45 minutes. Other stains include Leishman’s stain
and Jarwant Singh Battacharya (JSB) stain. JSB is the standard method used by laboratories under the
National Malaria Eradication Programme in India. JSB I uses Methylene Blue JSB II uses Eosin stains.
(CDC.gov)
4. Gently remove the excess stain by applying drops of water on the slide. Avoid pouring the stain directly off
the sides of the slide. This could cause the metallic green surface scum of the stain to stick to the smear making
microscopy difficult.
5. Dry the slides on a drying rack.

• Additional Note:
o According to CDC, detection of malarial parasite is best done on a thick smear because blood is more
concentrated and RBCs are lysed during the dehemoglobinazation process.
o Thick smear is more sensitive in detecting the malarial parasites because the blood is concentrated,
allowing a greater volume of blood to be examined.
§ However, thick smears are more difficult to read, and thin smears may be preferred by laboratories that
have limited experience.
§ Generally, thicker smear is useful in quantitating the parasite and thereby establishing the extent of
parasitemia.
o Thin smear on the other hand facilitates species identification because apart from the idea that one can
describe the morphology of the Plasmodium sp., The appearance of both the uninfected and infected red
cells may be noted and thus better help in malarial diagnosis.
o However, microscopic examination must be done carefully and efficiently because some structures maybe
mistaken like a malarial parasite. These include platelets, artifacts, fungal spores and some stain debris.
§ Among these, the most common is the blood platelets superimposed on red cells which can be readily
identified because of the absence of pigments and any true ring form.
§ Clumps of bacteria or platelets may also be confused with schizont stage of the parasite (Henry,
2009).
o Persons suspected of having malaria but whose blood smears do not demonstrate the presence of parasites
should have blood smears repeated approximately every 12-24 hours for 3 consecutive days. If smears
remain negative, then the diagnosis of malaria is unlikely. (CDC. gov)

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Notes from Parasitology Lab Manual (continuation):

• MANNER OF REPORTING
o Quantitation of Malarial Parasite
§ Estimated Count (Plus System):
• Less precise as variation in the thickness of the film results in false variation in parasite count.

+ = 1-10 parasites/100 thick field

++ = 11-100 parasites/100 thick field
+++ = 1-10 parasites/thick field
++++ = more than 10 parasites/thick field

§ Actual Count:
Formula for Parasite count / uL:
# 𝑜𝑓 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
= × 8,000
200 𝑊𝐵𝐶
= # of parasites counted × 40

• GUIDE IN BLOOD COLLECTION AND SMEAR PREPARATION

o Blood collection for thick or thin blood film
1. Select the finger to puncture (usually the third or fourth finger)
2. Puncture the side of the ball of the finger. Do not make the puncture too close to the nail bed.
3. If the blood does not well up from the puncture, gently squeeze the finger.
4. Always grasp the slide by its edges.
5. To control the size of the blood drop on the slide, touch the finger to the slide from below.
o Blood collection for thick and thin blood film on the same slide.
1. Touch the blood drop with a clean slide.
2. Using the corner of another slide, spread the blood drop into the shape of a circle or square of 1 cm2.
3. Gently squeeze the patient’s finger again, and touch the edge of a clean slide to the newly formed blood
drop.
4. Take this slide and hold the edge that has the blood drop at an 45° angle against the surface of the first
slide. Wait until the blood completely spreads along the edge of the second slide.
5. While holding the second slide at the same angle, rapidly and smoothly push the slide forward.
6. To control the size of the blood drop on the slide, touch the finger to the slide from below.

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RETICULOCYTE COUNT Notes from Sir Benjie’s Discussion:

• Basically when we discuss hematopoiesis or
RETICULOCYTE
erythropoiesis particularly, nadaanan natin dun
yung reticulocyte.

RETICULOCYTES
• Reticulocytes are the earliest precursor of red
cell which can be seen in the bone marrow.
• Within this reticulocytes, makikita natin that we
have this so called reticulum.
o These reticulum are basically the remnants
of the RNA.
• From the metarubricyte, the nucleus will extrude.
Lalabas yung nucleus sa red blood cell, and of
course, there are residuals of RNA left in the
RBC.
• Those cells na marami pang residuals ng RNA
are basically known as your reticulocytes.
• These reticulocytes are stained by supravital
• The penultimate erythroid cells in the maturation dyes.
sequence. o Supravital – staining of live cells prior to
• Young erythrocyte which is formed when the nucleus fixation
of the late normoblasts are lost through extrusion o Normally, we fix first the cells to preserve
• Young RBC which contains a small network of the morphology, but for supravital staining,
basophilic materials (residual RNA) called we stain live cells.
”reticulum” o Supravital dyes will be taken up by the cell
• Residual RNA inside the cell is visible when stained and most likely, these dyes will form
with a supravital dye. precipitate sa reticulum.
• Supravital dyes: o These blue threads are the reticulum.
o New methylene blue o The ones pointed by the arrow are
o Brilliant cresyl blue reticulocytes. These are cells na
maraming reticulum, na maraming residual
RETICULOCYTE COUNT RNAs based on our supravital staining.
• Used as an index of bone marrow activity and o Yung mga cells na walang reticulum, these
RBC production are already matured red blood cells.
• Also used to monitor therapeutic measures for o The targets are those that manifest
anemia reticulum in their cytoplasm.
• Specimens: o Supravital stains used for the
o EDTA-anticoagulated whole blood determination of reticulocytes:
o Peripheral blood ▪ New methylene blue
▪ collected by means of pricking ➢ differs from methylene blue
because NMB has additional
components
▪ Brilliant cresyl blue

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Notes from Sir Benjie’s Discussion METHODS

(continuation): • Microscopic Methods (performed manually by
counting reticulocytes under the microscope)
RETICULOCYTE COUNT o Wet Method
• The reason why we are performing reticulocyte ▪ Rapid Method of Schilling
count is to check for RBC production ▪ Osgood-Wilhelm
• Sino ba yung nagpoproduce ng RBCs? ▪ Sabin
o Bone marrow o Miller Disc Method
o Dry Method
• Once we are testing for reticulocyte count, we
▪ New methylene blue
are actually measuring the index of your bone
▪ Cook
marrow activity.
▪ Meyer
• When we discuss hematopoiesis or particularly
▪ Tureen
erythropoiesis, erythropoiesis follow the negative
▪ Seiverd
feedback mechanism,
• Automated Method: Sysmex R-1000 (uses a
• Negative feedback mechanism
machine)
o Someone is decreased and that decrease
o Sysmex R-1000 is an automated reticulocyte
will trigger the release of the other
analyzer which uses the principle of flow
• Hypoxia – decrease in oxygen detected by
cytometry to determine the relative
kidney triggers RBC production
reticulocyte count and absolute reticulocyte
• Our blood flows into the blood vessel. if there is
count.
decrease in oxygen, the release of EPO or
erythropoietin will be triggered.
WET METHOD
• Erythropoietin is a hormone that will trigger or
• PROCEDURE:
stimulate RBC production. EPO will be
1. Place a drop of freshly filtered stain on a
transported to the bone marrow, then received by
clean dry glass slide.
CFU-Meg-E. It will differentiate to CFU-E and will
2. Add an equal volume of blood. Mix by
differentiate to pronormoblast, etc.
stirring.
• With tissue hypoxia, there should be increase
3. Cover the mixture with clean coverslip
in bone marrow activity because
lined with petroleum jelly or Vaseline.
erythropoiesis is triggered. If there is hypoxia,
4. Proceed with counting.
reticulocyte count is increased.
• Monitor therapeutic measures for anemia MILLER DISC METHOD
• Anemia – disorder or disease wherein oxygen • In the miller disc method, an optical disc is inserted
delivery is impaired into the eyepiece or ocular of the microscope to
• When patient is suffering from anemia, retics obtain the reticulocyte count.
count is requested to check if the bone marrow • The disc is composed of two squares.
is responding to hypoxia, if the retics count is
• The area of square B is 1/9 the area of square A.
decreased or within normal range, bone marrow
Alternatively, square B may be positioned at the
does not respond to the anemia, that is
center of square A.
uncompensated anemia.
• RBCs – counted in the smaller square.
• Specimens:
• Reticulocytes – counted in the larger square.
o EDTA-anticoagulated whole blood
• The selection of the counting area is the same as
▪ EDTA preserves the morphology of
the differential WBC count wherein the RBCs are
the red blood cell well.
positioned side by side with each other. Not too far
▪ When it comes to hematology, it is
from each other and not too overlapping.
always used as an anticoagulant
• A minimum of 112 cells should be counted in the
except for ESR and coagulation
small square because this is equivalent to 1,008 red
studies (citrate is used for both)
blood cells in the large square, and satisfies the
o Peripheral blood
requirement of the College of American Pathologist.

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PROPERTY OF CHERRYANNE DOMINGO

• According to the requirement of the College of • PROCEDURE:

American Pathologist, a manual reticulocyte count 1. Mix equal volumes of blood and freshly
should be based on at least 1,000 red blood cells. filtered stain in a tube. Allow this mixture to
stand at room temperature for 15-30 minutes.
2. Remix the preparation and prepare smears.
Allow the smears to dry.
3. Examine under OIO.
4. Count the reticulocytes seen in 10
successive fields of vision.
5. Compute

Formula for Dry Method:

# 𝑜𝑓 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠
𝐑𝐞𝐭𝐢𝐜𝐮𝐥𝐨𝐜𝐲𝐭𝐞𝐬 %: = 𝑥 100
1000
Description of the image: Miller Ocular Disc Counting Grid
Numerator: # of Reticulocytes = sum of the
as Viewed through a Microscope. reticulocytes counted in the 10 successive fields
of vision
Formula for Miller Disc Method:
Denominator: 1000 = assumption that there are
𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝐴
about 100 RBCs per field of vision, so 100 RBCS
ሺ𝑙𝑎𝑟𝑔𝑒 𝑠𝑞𝑢𝑎𝑟𝑒ሻ 𝑥 100 times 10 fields of vision will give you 1000
𝐑𝐞𝐭𝐢𝐜𝐮𝐥𝐨𝐜𝐲𝐭𝐞𝐬 %: =
𝑅𝐵𝐶𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝐵
ሺ𝑠𝑚𝑎𝑙𝑙 𝑠𝑞𝑢𝑎𝑟𝑒ሻ 𝑥 9 Factor: 100 = reticulocyte count is expressed in
percentage
• Example:
16 𝑥 100 # 𝑜𝑓 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠
𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 % = 𝐑𝐞𝐭𝐢𝐜𝐮𝐥𝐨𝐜𝐲𝐭𝐞𝐬 %: =
115 𝑥 9 10
• Example #1:

F1 = 0 F6 = 2
F2 = 1 F7 = 2
F3 = 3 F8 = 2
F4 = 0 F9 = 0
F5 = 2 F10 = 3
o Large square = 16 reticulocytes 15
o Small square = 115 RBCs 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 % = 𝑥 100
1000
o % Retics = 1.5%
▪ round off to the nearest tenths o % Retics = 1.5% or 15 x10-3
▪ 1 decimal place lang usually ginagamit;
other ref. - more than 1 decimal place; • Example #2:
it does not matter significantly (Sir Benj)
F1 = 5 F6 = 3
DRY METHOD F2 = 3 F7 = 4
• Most commonly employed manual technique in the F3 = 4 F8 = 5
clinical laboratories here in the Philippines. F4 = 6 F9 = 6
F5 = 2 F10 = 2
40
𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 % = 𝑥 100
1000

o % Retics = 4% or 40 x10-3

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PROPERTY OF CHERRYANNE DOMINGO

Notes from Sir Benjie’s Discussion: Notes from Sir Benjie’s Discussion
(continuation):
METHODS
• MILLER DISC METHOD
MICROSCOPIC METHODS o A specialized eyepiece na napapalitan
• WET METHOD depending on the magnification na gusto
o Slide: NMB + add drop of blood mo
o Then, cover with coverslip then examine o There are other specialized eyepieces
under the microscope such as yung merong graduations or
o Problem: cells are not fixed in the slide; pointers.
lumalangoy lang yung red cells sa field. o Under the microscope, may makikita
o Mahirap basahin. kayong dalawang square sa loob.
o Directly nilalagay sa slide (unlike sa dry
method na miminix muna sa test tube)
• DRY METHOD
o Most commonly used
o In a test tube, add equal amount of NMB
and whole blood, mix, then, allow it to
stand for about 15-30 minutes at room
temp. to allow the retics to uptake the dye o When using miller disc, diyan ka na
o Place it on a slide (para kang gumagawa mismo sa square or field of vision
ng blood film). Then, examine under the magbibilang.
microscope. o Hindi ka na magbibilang ng 10 fields.
• Procedure for reading the dry and wet method are o Pumunta ka lang sa area kung saan 50 %
the same wherein retics are counted in 10 are overlapping and 50% are
successive fields of vision. nonoverlapping. Then, dun ka magbilang.
• Retics are examined under OIO and counted in o SQUARE B – MATURED RBCS
areas wherein 50 % of your RBCs are o SQUARE A – RETICULOCYTES
overlapping and 50% are nonoverlapping. ▪ Including reticulocytes in Square B
o Thick area – 100% of RBCs are o COMPUTATION:
overlapping ▪ The whole formula is multiplied by
o Thin area – 100% of RBCs are 100
nonoverlapping (hiwahiwalay) ▪ Square B is 1x1 of the whole square
o At 1 field – there are about 100 RBCs. (1/9 the area of square A). There
• With this, if there is 50% overlapping and 50% are 9 SQUARE B (small squares)
nonoverlapping RBCs, it is estimated that there sa buong SQUARE A. kaya siya
are 100 RBCs in that field. minumultiply by 9.
• Count retics in 100 RBCs and count in 10 ▪ Reticulocytes are counted based on
successive fields the number of RBCs because we
• COMPUTATION: are trying to get the percentage.
o Principle of computation: How many ▪ In a swamp of your RBCs, gaano
retics are seen in 1000 RBCs? karami doon ang reticulocytes?
o In the formula, 1000 pertains to 1000 That’s the purpose of having the
RBCs retics count in percentage, you get
o Computing for percentage of reticulocyte the relative frequency.
therefore is multiplied to 100.
𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝐴
ሺ𝑙𝑎𝑟𝑔𝑒 𝑠𝑞𝑢𝑎𝑟𝑒ሻ 𝑥 100
# 𝑜𝑓 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 𝐑𝐞𝐭𝐢𝐜𝐬 %: =
𝐑𝐞𝐭𝐢𝐜𝐮𝐥𝐨𝐜𝐲𝐭𝐞𝐬 %: = 𝑥 100 𝑅𝐵𝐶𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝐵
1000 ሺ𝑠𝑚𝑎𝑙𝑙 𝑠𝑞𝑢𝑎𝑟𝑒ሻ 𝑥 9

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PROPERTY OF CHERRYANNE DOMINGO

REFERENCE RANGE of 2% to 3% to compensate for the mild

• CONVENTIONAL anemia.
o Adults: 0.5-1.5% o In patients with a hematocrit of less than
o Babies: 2.0-6.0% 25%, the count should increase to 3% to 5%
• SI to compensate for the moderate anemia.
o Adults: 5-15 x 10-3/uL • Example:
o Babies: 20-60 x 10-3/uL o 64 reticulocytes counted
• Conversion factor: Conventional unit x 10 = SI unit o Patient’s HCT = 29%
• Neonates have higher reticulocyte count because % 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑥 𝑃𝑎𝑡𝑖𝑒𝑛𝑡 ′ 𝑠 𝐻𝐶𝑇 ሺ%ሻ
=
they have more hematopoietic tissues in their bone 45
marrow.
o Corrected Reticulocyte Count = 4.1%
Notes from Sir Benjie’s Discussion:
• Adults usually have a lower reticulocyte count. Notes from Sir Benjie’s Discussion:
• 100% of bone marrow of babies are active in • Main purpose: to compensate with the
hematopoiesis and still contributing to patient’s HCT
erythropoiesis. Kaya mas marami yung retics nila • Corrections are performed if the patient’s HCT
• 50% na lang nacocontribute sa bone marrow ng falls below the normal reference range 35%
adults kaya bumababa yung ref range for retics (other ref.: 38%)
count. • Correction is needed because the counting of
• This would also vary depending on the age and reticulocytes is based on relative frequency.
sex of the patient. • Computation is based on # of red cells. Gaano
o Females have a physiologic increase of karaming reticulocyte sa pool of matured red
reticulocyte count because of ovulation blood cells? Ilan reticulocytes sa 1000 na RBCs?
(monthly menstrual cycle – shed blood) Ginawa natin was estimation, we counted
• As a physician, if your patient is female, ask if reticulocytes sa 50% overlapping red blood cells
kakatapos lang magmens or gaano katagal from and 50% nonoverlapping red blood cells kasi at
the last drop of blood or menstruation, and try to that area, there are 100 estimated red blood cells.
correlate the physiologic functions of the patient • Problem: If the patient has low HCT (less than
to avoid misdiagnosis. 35%), maglalayo sa isa’t-isa yung RBCs. The
• SI = Absolute Reticulocyte Count red blood cells are far from each other, so sa
isang field, hindi 100 yung RBCs na andun.
• We are correcting reticulocyte count based on the
CORRECTED RETICULOCYTE COUNT HCT of the patient.
• In patients with low hematocrit levels, the percentage • Patient’s HCT divide it by 45 (normal HCT) then
of reticulocytes may be falsely elevated because the multiply it by % RETICULOCYTE
Reticulocytes
ABSOLUTE COUNT
whole blood contains fewer RBCs, and so
• It refers to the actual number of reticulocytes in 1 liter
reticulocyte count in percent needs to be corrected.
or 1 microliter of blood.
• It can also be reported as the number of cells per
Formula for Corrected Reticulocyte Count:
microliter, if the RBC count is expressed in number
% 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑥 𝑃𝑎𝑡𝑖𝑒𝑛𝑡 ′ 𝑠 𝐻𝐶𝑇 ሺ%ሻ of cells per microliter instead of the SI unit.
=
45

Denominator: 45 = the average normal Formula for Absolute Reticulocyte Count:

hematocrit of individuals % 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑥 𝑅𝐵𝐶 𝐶𝑜𝑢𝑛𝑡 ሺ𝑆𝐼ሻ
=
100
• REFERENCE RANGE:
o The corrected reticulocyte count depends on • REFERENCE RANGE:
the degree of anemia. o 20 x 109/L to 115 x 109/L
o Patients with a hematocrit of 35% should
Notes from Sir Benjie’s Discussion:
have an elevated corrected reticulocyte count
• The computation is divided by 100 kasi yung
reticulocyte count natin is in percentage.

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PROPERTY OF CHERRYANNE DOMINGO

RETICULOCYTE PRODUCTION INDEX (RPI) • Example:

• Reticulocytes that are released from the bone o Corrected Retic Count = 4.1%
marrow prematurely are called “Shift” o Patient’s HCT = 29%
reticulocytes. These are released earlier than usual 𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝐶𝑜𝑢𝑛𝑡
to compensate for anemia. 𝐑𝐏𝐈: =
𝑀𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑇𝑖𝑚𝑒
• When erythropoiesis is evaluated, a correction
should be made for the presence of shift o RPI = 2.05
reticulocytes if “polychromasia” is reported in the
RBC morphology. Notes from Sir Benjie’s Discussion:
• Most non shift are normal reticulocytes become • RPI: Gaano karaming reticulocyte yung
mature erythrocytes within 1 day after entering the prinoproduce ng bone marrow natin in a day?
blood circulation. • SHIFT RETICULOCYTES
• However, the shifted reticulocytes stay longer as o also known as stress cells
reticulocytes in the peripheral blood circulation, and o somewhat larger red blood cells
so, they contribute to the reticulocyte count for more o appear polychromatic or polychromasia /
than 1 day. darker in peripheral blood stain with
• For this reason, reticulocyte count is falsely Wright stain
increased when polychromasia is present o Stain used: Wright stain
• Automated technique: measured by Immature • COMPUTATION:
Reticulocyte Fraction o Patients with higher HCT has a shorter
• Manual technique: adjustment by calculating RPI maturation time
o Kapag yung value ng reference interval is
Formula for RPI: between 2 to 3, parang hindi siya ganun
𝐻𝐶𝑇ሺ%ሻ ka-ganda, hindi rin ganun ka-pangit.
# 𝑜𝑓 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 𝑥
𝐑𝐏𝐈: = 45 o Ma maganda if the value is greater than 3.
𝑀𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑇𝑖𝑚𝑒 • Main purpose: To check if the bone marrow
response is adequate for anemia.
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝐶𝑜𝑢𝑛𝑡
𝐑𝐏𝐈: =
𝑀𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑇𝑖𝑚𝑒
SOURCES OF ERROR AND COMMENTS
• REFERENCE INTERVAL:
• If a patient is anemic or polycythemic
o RPI greater than 3 : adequate Bone Marrow
o adjust accordingly blood and dye proportion
response
o instead of 1:1 ratio
o RPI less than 2: inadequate Bone Marrow
• Since the reticulocytes have lower specific gravity
response
than the mature erythrocytes, be sure to mix the
• In calculating the RPI, the patient’s hematocrit is
blood and stain preparation in the vial or tube 15 to
used to determine the appropriate correction factor
30 minutes incubation time before making a blood
or maturation time in days (substituted in the
smear.
formula).
• Moisture, poor drying
o forms refractile bodies which may be seen as
MATURATION reticulum / may be confused with reticulocytes
PATIENT HCT
TIME (DAYS)
• RBC inclusions that stains supravitally such as Heinz
40-45% 1 bodies, Pappenheimer and Howell Jolly are
35-39% 1.5 confused with retics
25-34% 2 • If Miller Disc is used:
15-24% 2.5
o heed the “edge rule”
<15% 3
o similar to the manual blood cell count
procedure to avoid bias

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PROPERTY OF CHERRYANNE DOMINGO

Notes from Sir Benjie’s Discussion: VIDEO

• If the patient is anemic and polycythemic, the HOW TO COUNT RETICULOCYTES
Link: https://www.youtube.com/watch?v=q3oNCz_U1sY
patient needs to adjust blood and dye proportion
• RETICULOCYTE COUNTS
o Normally, 1 part of blood: 1 part of dye.
o In the cases of polycythemic patients • INSTRUMENTS:
wherein the patient has pancytosis, adjust o Blood sample
natin. o Retic stain
▪ Damihan natin yung dye o Tube
▪ 1 part of blood: 2 parts of dye o Capillaries
o In the cases anemic patient, adjust natin. o Slides
▪ Damihan natin yung blood o 37 C water bath
▪ 2 parts of blood: 1 part of dye o Microscope
o Parafilm
• RBC inclusions that stains supravitally such as
o Counter
Heinz bodies, Pappenheimer and Howell Jolly
o Immersion oil
are confused with retics
o Heinz Bodies • METHOD:
▪ Inclusions formed because of the o Put one drop of retic stain
precipitation ng toxic agents or o Mix the blood before using it.
substances o Put two drops of blood sample.
▪ Primarily because nafoform ang o Mix the tube very well.
heinz bodies bec. of G6PD o Cover with parafilm.
deiiciency o Label the tube with the patient’s initials
o Howell-Jolly bodies o Place it in the incubator for 15 minutes.
▪ Remnants of DNA o Make your smear.
▪ Damihan natin yung dye o Let the smear dry before examination.
o Pappenheimer bodies o Magnify first at LPO.
▪ Accumulation of iron sa ating o Put immersion oil.
mitochondria ng RBCs o Magnify at 100x and count.
o Move to go to the other field.
• Stained using Supravital Staining: Heinz bodies,
o You have to count reticulocytes in each field.
Howell-Jolly bodies, and Reticulocytes
o Clean the place at the end of class.
• If Miller Disc is used: heed the “edge rule”
o included yung RBCs na nasa edge
o as long as 50% of the cell pumasok sa Note from ppt:
loob counted siya sa loob ng box • Based on our Laboratory Manual, incubation is
done at room temperature for 15 to 30 minutes.

VIDEO
RETICULOCYTE COUNT
Link: https://www.youtube.com/watch?v=_0LvErkZE84
• STAIN: New methylene blue and Brilliant cresyl blue
• MATERIALS:
o Glass Slide
o Test Tube 12 x75
o 0.5% New methylene blue
o Hematocrit tube
o Light microscope

NOTE from HEMA trans:

• Hello :> Naka-thai kasi yung buong vid, yung
materials lang yung naka-english. Panoorin
niyo na lang yung vid if u want hehe about
siya sa materials and procedure.

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PROPERTY OF CHERRYANNE DOMINGO

Notes from Hematology Lab Manual:

• Normal Values:
RETICULOCYTE COUNT o Adults: 0.5-1.5%
• Reticulocytes are young RBCs which are formed o Babies: 2.0-6.0%
when the nucleus of the late normoblasts are lost
through extrusion. The cytoplasmic ribonucleic TECHNIQUES FOR DRY METHOD
(RNA) is retained. • New methylene blue
• The term “reticulocyte” is derived from the fact • Cook
that the cell contains a small network of • Meyer
basophilic materials called reticulum which is • Tureen
demonstrable only by the use of supravital • Seiverd’s
stain. The more mature the reticulocyte is, the
greater is the ribosomal content of the cell. OTHER METHODS OF RETICULOCYTE COUNT
• Reticulocyte count is used as an index of bone Microscopic Methods
marrow activity and RBC production. It is also • WET METHOD
used to monitor therapeutic measures for o Rapid method of Schilling, Osgood-Wilhem,
anemia. and Sabin
• The specimen of choice is EDTA- o Procedure:
anticoagulated whole blood because the final 1. Place a drop of freshly filtered stain on
result is not affected even after 24 hours, a clean dry glass slide.
irrespective of storage temperature. Peripheral 2. Add an equal volume of blood. Mix by
blood can also be used. stirring.
3. Cover the mixture with clean coverslip
DRY METHOD lined with Vaseline.
• Procedure: 4. Proceed with counting.
1. Mix equal volumes of blood and freshly • MILLER DISC METHOD
filtered stain in a tube. Allow this mixture o Miller disc is an optical aid inserted into the
to stand at room temperature for 15-30 eyepiece of the microscope. This allows for
minutes. a more accurate count.
2. Remix the preparation and prepare o The disc ruling consists of a center square
smears. Allow the smears to dry. containing a secondary square ruled area
3. Examine under OIO. that is 1/9 the area of the larger square.
Note: Mature RBCs stain gray-blue
while reticulocytes are identified by
the presence of deep blue
filamentous web or granules within
the cells. Any cell that contains two or
more particles of blue-stained
materials is classified as a
reticulocyte.
4. Count the reticulocytes seen in 10 Formula for Miller Disc Method:
successive fields of vision or while 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝐴
enumerating 1,000 mature RBCs. ሺ𝑙𝑎𝑟𝑔𝑒 𝑠𝑞𝑢𝑎𝑟𝑒ሻ
𝐑𝐞𝐭𝐢𝐜𝐬 %: = 𝑥 100
5. Compute 𝑅𝐵𝐶𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝐵
ሺ𝑠𝑚𝑎𝑙𝑙 𝑠𝑞𝑢𝑎𝑟𝑒ሻ 𝑥 9
Formula for Dry Method:
# 𝑜𝑓 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 Automated Method
𝐑𝐞𝐭𝐢𝐜𝐬 %: = 𝑥 100
1000 • Sysmex R-1000 is an automated reticulocyte
# 𝑜𝑓 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 analyzer which uses flow cytometry for the
𝐑𝐞𝐭𝐢𝐜𝐬 %: =
10 determination of percent leukocyte as well as the
absolute reticulocyte count.

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PROPERTY OF CHERRYANNE DOMINGO

References

Reticulocyte Count:
University of Santo Tomas powerpoint presentation: Reticulocyte Count
Notes from the discussion by Asst. Prof. Ruby Garcia-Meim, RMT, MSMT
Mr. Benjie M. clemente, RMT, MLS (ASCPi)cm, MPH

Sadang, M. G. and Llanera, F. 2015. Laboratory Manual in Hematology. Quezon City, Philippines: C&E Publishing, Inc.

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PROPERTY OF CHERRYANNE DOMINGO

ABSOLUTE EOSINOPHIL COUNT Additional info from lab manual:

 It is a blood test that quantitavely measures the  Absolute eosinophil count is the number of
number of 1 type of white blood cell, which is the eosinophils in 1 mm 3 of blood.
Eosinophil.
 Eosinophils MATERIALS
o Become active when you have certain  Anticoagulated blood
allergic diseases, parasitic infections,  WBC pipette
and other medical conditions such as:
 Randolph’s diluting fluid
 Acute hyper eosinophilic syndrome
 Aspirator
(rare, but sometimes fatal
 Microscope
leukemia like condition)
 Improved Neubauer CC
 Early stages of Addison’s disease
 Thick coverslip
RANDOLPH METHOD  Tissue paper
PROCEDURE
 The diluting fluids used for absolute eosinophil
According to Sir Benjie: count are:
 Randolph’s fluid will lyse cells except eosinophil. o Randolph’s diluting fluid
o Phloxine
o Pilot diluting fluid
1. Aspirate blood up to 1 mark of WBC pipette.  In this procedure, we made use of the Randolph’s
2. Draw Randolph’s diluting fluid up to the 11 mark to diluting fluid which is composed of:
make a 1:10 dilution. o Acetone (serves as a fixative)
3. Mix the pipette well and let it stand for 10-15 minutes o Distilled water (to lyse the red blood cells)
to allow the eosinophil cells to take up the stain. o Eosin (to stain the eosinophil)
4. Remix the dilution. Discard 2-3 drops of the diluted  Notice the color of the diluting fluid is orange. In the
blood then charge both sides of the counting principle of absolute eosinophil count on diluting the
chamber. After charging the counting chamber, allow
blood with Randolph’s diluting fluid, the red blood
the cells to settle just like in WBC count.
cells and other leukocytes are lysed, except
5. Count the eosinophils in all 9-secondary squares
eosinophils.
with an area of 9 sq. mm.
 Eosinophils take up the eosin stain and would
appear orange or reddish-orange.

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Continuation...
 As long as you memorize, you know the
measurements ng ating neubauer counting
chamber, wala kayong talo. Kaso compared dito
sa long method, kasi ganon din di ba? The long
method would require you to memorize all of
your measurements.
 The answer would be 222.2 based on our
computation kanina. So based on our
computation kanina, if you have the standard,
 The cells are counted in all 9 secondary squares
sabi natin kanina the number of cells times the
under low power objective (LPO).
dilution factor divided by the number of squares
 Unlike in WBC or RBC, absolute eosinophil is easy
times the volume of squares.
to count. You will only be counting the cells that stain
orange or reddish-orange because these are the
eosinophils.
 Note also that some debris would stain orange.
These are not included in the counting. You would
know if it is a debris or dirt with the shape or form.  The number of cells we have is 20, times the
o WBCs – round with nucleus inside the cell dilution factor which is 10 divided by the number
o Eosinophils – round, reddish-orange of squares na ginamit natin.
structure with nucleus inside the cell  Ilan squares ginamit natin? 9. Times the volume
of the squares, that is 0.1. So the answer would
be 222.2222.

 Now, ang pinaka shortcut dito would be to

multiply the number of cells by 11.11. You would
arrive at 222.2222 pero that is if you are using
the standard.
 Count all eosinophils found in all 9 secondary
 Again, that is if we are using the standard. As
squares of the improved neubauer counting
long as you know this, if binago natin yung mga
chamber.
factors doon.
 All cells touching the lines on the top and left borders
 For example, we aspirated up to the 1 mark.
are included, while the lines touching the right and
What if 0.5 lang ang inaspirate na blood kaso
bottom borders are not included.
onti nalang yung sample na natitira. Di ba? At
According to Sir Benjie: least with this formula, you know paano mo siya
 Question: Ilan ang volume ng squares? imamanipulate.
 Answer: This one (primary square) is 3mm by 3  Siyempre babaguhin mo siya sa dilution factor.
mm, that means one secondary square is 1 mm Kasi ang dilution factor mo, if you aspirated up to
by 1 mm and the depth nitong neubauer counting the 0.5 mark lang. Tama? That would be 0.5
chamber is 0.1 mm. So what is the volume of this divided by 10. Okay? So ang magiging dilution
(secondary square)? factor mo would be? Tama ba? 0.5 divided by
o Volume is 0.1 10. Ay hindi, baliktad. Total volume minus 1
 Question: Anong pinagkaiba ng dalawang divided by 10.
formula?  Ay hindi, baliktad. Total volume minus 1 divided
 Answer: Actually, wala. Okay? Mas by 10. Your dilution factor would be 20.
komportable lang ako doon sa shortcut ko.  naguluhan ka rin? HAHA ganito see next page

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PROPERTY OF CHERRYANNE DOMINGO

Continuation... Additional info from lab manual:

 Diba ang formula for DF is Total Volume minus  Other Methods of Absolute Eosinophil Count
1 divided by volume of sample used. So bale if 1. Pilot eosinophil count
0.5 mark lang yung sample, (11-1) / 0.5 = 20 2. Direct eosinophil count by Friedman
mwa yun ibig sabihin ni sir :>  Diluting Fluids for Absolute Eosinophil Count
 9 squares ang ginamit natin kanina times the 1. Phloxine
volume of the squares, 0.1 Composition:
 So 9 times 0.1 that is 0.9 (9 x 0.1 = 0.9) o Propylene glycol – hemolyze
 So the number of cells, 20, multiplied by the RBC
dilution factor which is 10, that is 200, divided by o 1% phloxine – stains eosinophil
0.9 only
 Question: Is 20 the dilution factor? o 10% sodium carbonate – lysis
 Answer: Yes, 20 yung dilution factor. Yung 20 of other WBC (accentuator)
na dilution factor is if 0.5 ang ginamit natin na 2. Pilot
blood instead na 1. Composition:
 Sabi natin we have to aspirate up to the 1 mark. o Propylene glycol
Eh what if onti lang yung sample natin? o 1% water solution of phloxine
 So ang naaspirate mo lang is up to the 0.5 mark o 10% water solution of sodium
tapos dilute mo na siya up to the 11 mark. carbonate
 The dilution factor would now be 20. o Heparin – prevents clumping
3. Randolph’s
 Kasi diba ang standard natin na dilution factor is
Composition (1):
10. And also what if tinamad ka, nagbilang ka
o Acetone – fixative 2.0 mL
lang sa tatlong squares?
o Distilled water – lyses RBC
 At least you have to adjust din your computation.
96.0 mL
The number of squares na ginamit mo would be
o Eosin – stains eosinophil 2.0
3 times 0.1.
mL
Composition (2):
COMPUTATION o Phloxine
o Propylene glycol
Formula: o Sodium carbonate
𝐴𝑏𝑠𝑜𝑙𝑢𝑡𝑒 𝑒𝑜𝑠𝑖𝑛𝑜𝑝ℎ𝑖𝑙 𝑐𝑜𝑢𝑛𝑡 (/𝑐𝑢 𝑚𝑚) o Heparin or sodium citrate

𝑡𝑜𝑡𝑎𝑙 𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

=
𝑎𝑟𝑒𝑎 𝑥 𝑑𝑒𝑝𝑡ℎ 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛
𝑡𝑜𝑡𝑎𝑙 𝑛𝑜. 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑
=
9 𝑥 1/10 𝑥 1/10
= 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 100/9
= 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 11.11
𝑪𝒐𝒏𝒗𝒆𝒓𝒔𝒊𝒐𝒏 𝑭𝒂𝒄𝒕𝒐𝒓 (𝑪𝑭): 0.001
𝑵𝑽: 150 − 300 𝑐𝑒𝑙𝑙𝑠/𝑚𝑚3 (conventional)
0.150 − 0.300 𝑥 109 /𝐿 (SI)

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PROPERTY OF CHERRYANNE DOMINGO

References

Absolute Eosinophil Count:

University of Santo Tomas powerpoint presentation: Absolute Eosinophil Count
Notes from the discussion by Asst. Prof. Vivian G. Villegas
Mr. Benjie M. Clemente, RMT, MLS (ASCPi)cm, MPH

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PROPERTY OF CHERRYANNE DOMINGO

ERYTHROCYTE SEDIMENTATION RATE Continuation…

• Rapid, nonspecific test for inflammatory diseases. • That means when it comes to your ESR all you
o Ordered alongside with other laboratory have to do is to stand your red bloods or stand
procedures to prove and disprove the your blood – whole blood.
presence of inflammatory diseases and • Stand mo lang yung whole blood niyo for about
monitor the course of inflammatory an hour and then measure niyo distance ng
conditions such as rheumatoid arthritis, plasma, siyempre yung pinaka-top ng plasma
infections and some malignancies hanggang sa pinakadulo dulo ng red blood cell.
o Basically, this is like an initial test or a Para ka lang gumawa ng hematocrit without
screening test centrifugation.
• The distance in millimeters that the RBCs settle in 1
hour.
• When an anticoagulated blood is allowed to stand at UNDERLYING MECHANISM IN ESR
room temperature for a period of 1 hour, the RBCs • RBCs have a net negative surface charge and tend to
settle toward the bottom of the tube. repel one another.
• However, if we have an episode of inflammation of
the body, the body would usually respond by
According to Sir Benjie:
producing number of proteins such as fibrinogen.
• Not that really requested kasi aside from sayang
• Now this fibrinogen which is positively charged will
sa pera, it is not specific at all.
counteract or neutralize the negatively charged
• If a patient is suffering from inflammation or any
surface of the RBC
inflammatory disease, tendency is that the ESR
• Since its is now neutralized, it will now start forming
of the patient will be increased.
stacks of RBCs like stacks of coin so it will now form
• Aside from that, ESR has a large range of
which is known to us as rouleaux formation.
normal reference range kaya a slight increase in
• Rouleaux formation can be observed in the peripheral
your ESR is really not that significant.
blood smear.
• For example ang range natin dito would be 0 to
• Rouleaux formation will facilitate the RBCs to settle
30, kapag ang ESR mo is 35, it is not that
faster
significant.
• More fibrinogen you have, the more rouleaux will form
• Tapos kapag naging significant man siya, pwede
which will result to a higher ESR
mo masabi doon that the patient is suffering from
inflammatory disease, however, di mo masasabi
STAGES IN ESR:
what type of inflammatory disease or what type
1. In the initial 10 minutes, there is little sedimentation
of inflammation it is.
as rouleaux form
• Actually if the patient is suffering from a simple
• Rouleaux – described as stacking of coins
na lagnat, if the patient is feverish, it is expected
or stacking of your red blood cells, wherein
already that their ESR is increased.
nagpapatong-patong yung red blood cells
• Those are the possible limitations with regards
natin
to ESR, however, still here in the Philippines,
2. For about 40 minutes, settling occurs at a constant
somewhat talagang marami pa rin nagrerequest
rate of your red blood cells
na physicians with regards to ESR.
3. Sedimentation slows in the final 10 minutes as cells
• Ano ba ibig sabihin nito? From the term pack at the bottom of the tube
erythrocyte sedimentation rate, this is actually
the rate of your red blood cell to settle at the
bottom. So gaano kabilis yung red blood cell
natin to settle – or parang bumaba.
•

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FACTORS AFFECTING THE ESR RATE

• Blood proteins and lipids
• Red cell factors
• WBCs
• Drugs
• Clinical conditions
• Pre-analytic factors such as Specimen handling and
technique
According to Sir Benjie:
• Question: What is the charge of your cell
membrane ng ating red blood cell? One of the
components of your RBC is made up of
glycophorin A that is very much rich with sialic
acid and because of that sialic acid, it gives the
RBC membrane what charge?
• Answer: Negative. Take note that your RBCs
are highly negatively charged. That is one
reason why hindi sila nagststack – why they do
not interact with each other. Hindi sila nagdidikit-
dikit sa ating peripheral blood because the RBC
membrane is highly rich with sialic acid that
contributes to the negativity ng ating RBCs.

According to Sir Benjie:

• If you can remember yung tinatawag natin na
zeta potential ng RBC that pertains to the net
Additional info from lab manual: negativity of your RBC membrane. The higher
• Erythrocyte sedimentation rate (ESR) refers to the zeta potential of your red blood cell, the
the speed of the settling of red blood cells in better.
anticoagulated blood. It is the measure of the • Kasi if you can recall the law of magnetism, like
distance and speed of fall of RBC’s in the plasma poles repel. Since your red blood cells are highly
in a tube of a standard bore and length after negatively charged, yung RBC membrane nila
standing perpendicularly. The most important are highly negatively charged dapat nagrerepel
factor influencing ESR is the action of plasma sila sa isa’t isa, hindi sila magpapatong-patong.
proteins. Hindi sila magdidikit-dikit.
• Stages of ESR: • That is one of the greatest factor that affects your
1. Initial period of aggregation of rouleaux red blood cell, their zeta potential. Kapag
formation – 10 minutes bumaba ang zeta potential ng ating red blood
2. Period of fast settling – 40 minutes cells, tendency is mabilis silang magpapatong-
3. Final period of packing 10 - minutes patong, mas mataas ang ESR.
• In cases of blood proteins and lipids, tendency is
MATERIALS that yung lipids natin or yung proteins natin are
• Unclotted blood somewhat thick. Proteins are thick that it can
• Wintrobe tube mask the zeta potential of your red blood cells or
• Pasteur pipette/syringe with cannula it can mask the the RBC membrane, lowering
the zeta potential. That means baba ang zeta
• Timer
potential, tataas ang ESR.
• Westergren tube
• Red cell factor – shape or size of RBC. If they
• Westergren rack
are macrocytic, ESR will be decreased. If sickle,
• Rubber aspirator
mas mataas ang ESR kasi mas madali silang
• Test tube rack
magpapatong-patong.

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PROPERTY OF CHERRYANNE DOMINGO

MOST COMMONLY USED METHODS OF ESR Continuation...

• Modified Westergren TABLE 6.1. DIFFERENCES BETWEEN
• Wintrobe WINTROBE AND WESTERGREN METHODS
• Disposable kits Westergren Wintrobe
• Automated Length of time 30 cm 11.5 cm
Diameter of 2.5 mm 3 mm
bore
Additional info from lab manual:
Bottom of tube Open Flat and
WINTROBE-LANDSBURG METHOD closed
• In majority of cases, the Wintrobe-Landsberg Calibration 0 – 200 mm 0 – 100 mm
method is quite an accurate method. Its Anticoagulant 3.8% Hellen and
advantages outweigh its few drawbacks. This of choice trisodium Paul’s double
method uses the Wintrobe tube, which is citrate oxalate
calibrated on two sides (0-10 and 10-0) Advantage/s Most sensitive Smaller
method for amount of
• Procedure:
serial study of blood is
1. Fill the Wintrobe tube with blood with a chronic needed. Other
Pasteur pipette or a cannula attached to diseases tests can be
a syringe. performed
2. Place the tube in a vertical position on a after ESR
rack. determination
(hematocrit
3. After letting the tube stand for one hour,
and LE cell
record the ESR in millimeters. preparation)
Disadvantage/s Large amount Less sensitive
of blood is due to a
necessary shorter column

OTHER METHODS OF ESR DETERMINATION

WESTERGREN METHOD
I. MACRO METHODS
• The Westergren method is considered the most
A. Graphic or Cutler – anticoagulant of choice is
sensitive for ESR determination. It can be used
3% sodium citrate, uses Cutler tube which has
for the serial study of chronic diseases like
a 5 mL capacity; graduation of 0-50 mm
tuberculosis, carcinoma, and others.
Procedure:
• Procedure:
1. Add 0.8 mL of blood to 0.5 mL of 3%
1. Fill the Westergren tube with blood using
sodium citrate.
a rubber aspirator.
2. Close the tube with paraffin-coated
2. Let the tube stand vertically on a
cork. Mix.
Westergren rack.
3. Allow the tube to stand for 1 hour,
3. Record ESR in millimeters after an hour.
observing every 5 minutes.
4. Compare results with the normal values
in the sedimentation chart.

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PROPERTY OF CHERRYANNE DOMINGO

Continuation... Continuation...
A. Linzenmeier method – anticoagulant of choice Procedure:
is 3% sodium citrate; uses Linzenmeier tube 1. Draw 5% sodium citrate by turning the
which is 65 mm in length, 5 mm in diameter, and top-screw to the left until the upper
has a capacity of 1 mL (with a mark at 18 mm). meniscus of the solution reaches the
Procedure: lower mark A.
1. Add 0.8 mL of blood to 0.2 mL of 3% 2. Draw up the blood until the height of the
sodium citrate. liquid reaches the upper meniscus of
2. Mix and pour the mixture into the tube graduation B.
up to the 1 mL mark. 3. Clean the tip of the tube with ether, then
3. Allow the tube to stand in an upright continue to draw up the mixture into the
position until the RBC’s settle at the 18 bulb by turning the top-screw to the left
mm mark. until the lower meniscus of the blood
4. Note the time for this event to take columns ends just a few millimeters
place. Record in minutes. below the lower opening of the bulb. Do
not draw blood into the bulb completely.
4. Shake the tube carefully.
5. Force the blood up and down twice very
slowly by turning the screw to the left,
then to the right.
6. Set the tube vertically on a rack and
B. Bray’s method – anticoagulant of choice is 1.3% read the ESR at the end of one hour.
sodium citrate; uses Bray’s tube which is flat-
bottomed and calibrated on both sides like the
Wintrobe tube. Bray’s tube can also be used for
hematocrit and LE cell preparation.
Procedure:
1. Place 1.3% sodium oxalate up to the 5
mm mark on the left side calibration.
2. Add venous blood up to the 50 mm B. Smith Micro – used for infants and children
mark. when venipuncture may not be practical
3. Mix by inverting the tube. Avoid bubble Procedure:
formation. 1. Fill the special pipette with 5% sodium
4. Set the tube vertically and read the rate citrate and expel 0.04 mL.
of settling every five minutes for 30 2. Draw capillary blood with the same
minutes and the total fall after one hour. pipette. Three successive batches of
5. Plot the graph and record the maximum 0.1 mL are collected and expelled into
sedimentation rate (MSR). It is usually the tube containing the citrate solution.
sufficient to make readings at 10, 20, Thorough shaking is necessary to
30, and 60 minutes. ensure adequate mixing and prevent
II. MICRO METHODS coagulation.
A. Micro Landau (a modification of Linzenmeier- 3. The blood is transferred to the special
Raunert) – anticoagulant of choice is 5% sodium sedimentation tube using a capillary
citrate; uses a Micro-Landau tube which is pipette and the test is completed in the
calibrated 0-50 mm and has two graduation usual manner.
marks, one at 12.5 mm and another at 62.5 mm, C. Crista or Hellige-Vollmer
with a small bulb similar to RBC and WBC
pipettes.

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PROPERTY OF CHERRYANNE DOMINGO

Continuation... According to Sir Benjie:

III. AUTOMATED METHODS • This is a kind of tube wherein meron kang butas
A. Automated ESR System by Vega Biomedical sa magkabilang end. For this, para lang din
• The automated ESR system is a fully siyang pipette.
automated instrument for ESR
determination. One mL of blood is
collected on an evacuated tube
containing liquid sodium citrate. The
tube is then placed in the Ves-Matic
analyzer where it is automatically
mixed, allowed to settle, and read.
Results are comparable to the
Westergren method. The
determination takes only 22 minutes
to finish.
1. Mini-Ves – four samples at one time • Pipette is labeled from 0 to 100.
2. Ves-Matic – 20 samples at one time; • Aspirate blood up to the 0 mark and afterwards
prints results place a stopper sa area na ito (see image
3. Ves-Matic 60 – 60 samples at one below).
time; prints results; and identifies
sample by a barcoder reader.

• Usually used stopper is covered with parafilm

MODIFIED WESTERGREN ERYTHROCYTE
and then afterwards put in rubber by letting it
SEDIMENTATION RATE
stand in the rubber to prevent spilling of blood.
• Most commonly used method and recommended by
• Westergren is difficult to use due to improper
the International Council for Standardization in
sealing and the tendency of blood spilling.
hematology (ICSH) and the CLSI
• It has a taller column height so that is about 200 mm
column height which will allow for the detection of WINTROBE ERYTHROCYTE SEDIMENTATION RATE
highly elevated ESR. • Procedure:
• Procedure: 1. Mix collected blood in EDTA tube. Fill the
1. Collect blood in EDTA tube. Dilute at four (4) wintrobe tube.
parts blood to one (1) part 3.8% sodium 2. Fill the Wintrobe tube using a Pasteur pipette
citrate or 0.85% NaCl (ex: 2 mL blood that to the 0 mark.
would require 0.5 mL diluent); alternatively, 3. Allow to stand for 1 hr at room temperature
we can also collect blood in special into a Wintrobe rack.
sedimentation test tubes. 4. Record the number of millimeters the RBCs
• Done in order to maintain the net negativity or the zeta have fallen in a span of one hour. Read the
potential of the RBC. However, there are already tube from the bottom of the plasma meniscus
designed tubes of 3.8% sodium citrate which is the to the top of the sedimented red cells.
black top. So no need to collect EDTA tube.
2. Place the diluted specimen in a 200-mm
column → internal diameter - 2.55 mm.
• Why is it 2.55 mm / more? To prevent capillary action
3. Stand for 1 hour at RT (18° to 25° C).
• Make sure that the table where it is placed is not
moving. Little movement could give you erroneous
results.
4. Record the number of millimeters the RBCs
have fallen in 1 hour. Reported as x/mm/hr.
• x = Number

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PROPERTY OF CHERRYANNE DOMINGO

According to Sir Benjie: • The higher the temperature, the higher the ESR. Mas
• Wintrobe is a graduated test tube. And mainit, mas lesser and viscosity ng plasma. The
mapapansin niyo with regards to your colder, parang kumakapal yung viscosity ng plasma,
graduations, meron kang graduations sa left and hence bumabagal yung pag sediment ng ating
sa right. erythrocyte.
• Yung sa left, it starts from 0 to 10. Yung sa right • Tilting the pipette → increased ESR
graduated from 10 to 0, pababa. • Even a slight tilt of the pipette causes the ESR to
• Now, para saan ba ang Wintrobe? Itong increase. Tube should be perfectly vertical.
Wintrobe yung left side, is used for ESR. Yung • According to literature, a single decrease – parang a
right side is used for macro hematocrit method. slight change, kasi dapat ang pipette is at 90°,
literatures would say plus minus 1° sa angle would
already cause 15% ng change sa ESR. Ganun ka
significant ang angle, kailangan 90° ang angle.
• Blood specimens must be analyzed within 4 hours of
collection if kept at RT (18°- 25° C)
• Bubbles in the column of blood invalidate the test
results.
• The blood must be filled properly to the zero mark.
• A clotted specimen → falsely decrease ESR
• Clot trap fibrinogen hence, no rouleaux formation
occur
• Vibrations on the laboratory bench. → falsely
increase ESR
o Note: Accdg sa asynch, falsely decrease daw
pero sa Rodak’s falsely increase.
• Hematologic disorders → decrease the ESR
Take note: • Severe anemia → falsely elevated
• Left = ESR = 0 – 10 • Change in the RBC to plasma ratio which will favor
o Sa taas nagsisimula ang bilang. the rouleaux formation
o Ang tinitignan dito ay kung gaano
kababa yung binaba ng red blood cell.
• Right = Hct = 10 – 0
o Sa baba nagsisimula ang bilang.
o Ang tinitignan dito ay yung height ng
red blood cell

SOURCES OF ERROR
• Increased concentration of anticoagulant → falsely
low ESR
According to Sir Benjie:
• This is due to the sphering of the RBC which inhibits
• You already have here a rubber stopper na
rouleaux formation
ipapasok mo nalang yung pipette. Ipapasok mo
• The 3.8% sodium citrate would give us or would
yung pipette sa tube and then yung blood natin
maintain the net negative charge ng ating red blood
because of air pressure, magkakaroon ng
cell. Increasing the concentration of the anticoagulant
displacement tataas yung blood natin paakyat.
would give alteration to the negativity of the RBC.
• Use of Sodium oxalate, potassium oxalate, heparin → • Disposable commercial kits are available for ESR
falsely elevated ESR testing. Several kits include safety caps for the
• Significant change in the temperature of the room columns that allow the blood to fill precisely to the
zero mark. This safety cap makes the column a

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PROPERTY OF CHERRYANNE DOMINGO

closed system and eliminates the error involved in According to Sir Benjie:
manually setting the blood to the zero mark. • Question: Is there such a thing as decreased
• So meron siyang parang stopper dito, covered na ESR? How would you measure decreased ESR
siya di siya open tulad ng Wintrobe at Modified if the lowest limit for your reference value is 0?
Westergren Walang negative na ESR. If you have
spherocytosis usually the ESR would be
AUTOMATED ERYTHROCYTE SEDIMENTATION decreased kasi malalaking bilog and red blood
RATE cells natin kapag nagpataong-patong sila,
• Ves-Matic mataas kaya maraming plasma pa rin ang
o Designed to measure 20 blood samples mereretain doon sa loob.
simultaneously
o The results are said to be comparable to the
Westergren method CLINICAL CONDITIONS
o The results are available in approximately 20 ELEVATED ESR
minutes. • Anemia
• ESR STAT-Plus • Macrocytosis
o Centrifugation-based method o Large cells will settle faster
o It provides result in 5 minutes. • Malignancy
o smaller required sample volume and shorter • Multiple Myeloma
testing time • Pregnancy
o suitable for a pediatric patient population • Rheumatoid Arthritis
• Sedimat • Rheumatic fever
o It uses the principle of infrared measurement. • Acute & Chronic Infections
o It is capable of testing one to eight samples
randomly or simultaneously and provides DECREASED ESR
results in 15 minutes • Acanthocytosis
• Anisocytosis (marked)
REFERENCE VALUES
• Hemoglobin C
Male (Westergren) • Microcytosis
0 − 50 𝑦 ∶ 0 − 15 𝑚𝑚/ℎ𝑟 o Small cells will settle more slowly
> 50 ∶ 0 − 20 𝑚𝑚/ℎ𝑟 • Polycythemia
Female (Westergren) o Increase in viscosity
0 − 50 𝑦 ∶ 0 − 20 𝑚𝑚/ℎ𝑟 • Sickle cells
• Spherocytosis
> 50 ∶ 0 − 30 𝑚𝑚/ℎ𝑟
• Thalassemia

USES VIDEO
1. Aid in detecting inflammatory responses MODIFIED WESTERGREN METHOD
2. Monitor disease course or activity • For today, we will do the erythrocyte sedimentation
3. Screen for occult inflammatory or neoplastic rate using Modified Westergren method.
conditions • Materials:
o Collected EDTA blood
o Properly barcoded and then labeled
o Dispette
o Filling reservoir
• First off, let’s mix this EDTA blood properly. Mix by
gentle inversion.
• And then fill this up until the mark line, here. There is
an indicator up to where we will fill it up with blood.
• Gently remove the cap and then fill it up with the
properly mixed blood up until the mark with the line.

ENCISO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 7

PROPERTY OF CHERRYANNE DOMINGO

• Fill it up slowly. You can also use a pipette in filling it According to Sir Benjie:
up and we can just transfer the blood directly into the • Basically ganun lang gumawa ng ESR
tube. pagdating ninyo sa laboratory. That is if
• Let’s cap this one first... and gently put back the cap. disposable yung ginagamit niyong Westergren.
Make sure that it is tightly sealed. • So, parang isasalpak niyo lang, because of air
• Invert it gently. Make sure it is properly mix. displacement or air pressure, aakyat na yung
• Gently invert it for 8 times. Make sure it is properly blood hanggang sa pinakataas, and don’t worry
mixed. kasi may stopper naman doon sa taas.
• And then we put the dispette holding it in one hand, • Let it stand for 1 hour and afterward read nalang
holding it firmly. So we just puncture the upper most yung movement ng red blood cell up to the
part. bottom.
• Slowly puncture the cap membrane. Then stop, • For this case yung ginawa ni ma’am Bangawil(?)
gently push it downwards. makikita niyo na yung displacement is around
• The blood will just go up, up until the 0 mark. naandito (see image) kaya niya ni-read na 11.
• So there you go, up until the 0 mark. Careful not to • Reporting natin diyan would be 11 mm/hr.
have bubble formation.
• Then after that we just gently put it in the rack.
• Let it stand for 60 minutes or 1 hour. Leave it
undisturbed.
• After 1 hour, we will check for the ESR result.
• I have here one that is prepared already, so just for
us to check after an hour.
• We will read the erythrocyte sedimentation rate, read
it out from the interphase of the plasma, the upper
part of the interphase of the plasma and the red cell.
• For this one, our ESR is 11 mm/hr.

ENCISO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 8

PROPERTY OF CHERRYANNE DOMINGO

References

Unit Erythrocyte Sedimentation Rate:

University of Santo Tomas powerpoint presentation: Erythrocyte Sedimentation Rate
Notes from the discussion by Mr. Benjie M. Clemente, RMT, MLS (ASCPi)cm, MPH

ENCISO, ESCASA, ESTOLAS, ESTRADA, EUSEBIO, FELICIANO, MORALES, MORENO |3L-MT 9