OLFU Safety in Hematology Laboratory

2021 – 2022
LAB 1
RMT 2023
CLINICAL HEMATOLOGY 1
1st Semester
Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT
Date: September 15, 2021 TRANS 1 HEMA311
LAB

Outline 1. Fire Hazards

At the end of the session, the student must be able to learn:  Enforcement of a non-smoking policy
I. Laboratory Hazards  Placement of fire extinguishers every 75 feet, checked
A. Occupational Hazard monthly and maintained annually.
1. Fire Hazards  Placement of fire detection system and manual fire alarm near
2. Chemical Hazards exit doors which is less than 200 ft away and should be tested
i. MSDS Diamond every three months.
3. Electrical Hazards  Written fire prevention and response procedures and fire drills
4. Sharp Hazard
B. Physical Hazard **related to chemical and electrical hazards
1. Ergonomic Hazard **emergency fire drills are important to avoid risks
2. Vibration Hazard **REQUIRED inside facility:
3. Noise Hazard smoke detector system
C. Other Hazards automatic fire sprinkler
1. Biological Hazard floor plan
2. Radiation Hazard -includes designated fire exits
II. Biosafety Level
A. Safety Precautions
1. OSHA Standards Table 1.0: Types of Fires and Fire Extinguishers
i. Handwashing Procedure
III. Laboratory Waste Management
A. Segregation
1. Biodegradable
2. Non-Biodegradable
3. Hazardous Waste
i. Special waste
ii. Biological Waste
iii. Chemical Waste
iv. Radioactive Waste
B. Waste Disposal Standard
1. Blood Containing Waste
i. Standard Waste Protocol (PHIL)
C. Disposal
1. Flushing Down the Drain to the Sewer System Table 2.0: IN CASE of Fire and Proper Use of Fire Extinguisher
2. Incineration
3. Landfill Burial PROPER USE FIRE
IN CASE OF FIRE
4. Recycling EXTINGUISHER
IV. Occupational Safety and Health Act Rescue Pull the pin
A. Government Regulatory Agency Aim the nozzle at the base of
1. Department of Labor: 29 Code of Federal Activating fire alarm
the fire
Regulations Parts 1900-1910 Containing the fire
i. Hazard Communication Standard Squeeze the lever slowly
*closing window and doors
ii. Hazardous Waste Operations
Extinguishing the fire Sweep from side to side
iii. Occupational Exposure to bloodborne
pathogens Standards
2. Department of the Interior, Environmental Protection
Agency: 40 Code of Federal Regulations Parts 200-399 2. Chemical Hazards
i. Clean Air Act and Clean Water Act  Labelling of all chemicals properly.
ii. Toxic Substances Control Act  Follow handling, storage requirements.
iii. Comprehensive Environmental Response,  Use adequate ventilation.
Compensation and Liability Act (CERCLA)  Spill response procedures should be included in the safety
3. Voluntary Agencies/Accrediting Agencies procedures.
i. The Joint Commission  MSDS should be available and reviewed by laboratory
ii. College of American Pathologist personnel.
iii. Centers for Disease Control and Prevention
iv. Clinical and Laboratory Standards Institutes **also refers to reagents, and acids used inside the laboratory.
**storage of chemicals should be observed to avoid this
hazards risk.
always adequate ventilation

I. LABORATORY HAZARDS MSDS Diamond (Material Safety Data Sheet)

**also known as NFPA Rating Explanation Guide which contains the
common identifier along with a rating number (from 0-4) inside a colored
A. Occupational Hazard field to indicate hazard rating.
**Occupational Hazards are hazards and risks of illnesses or
accidents in the workplace.

Acuña, J., Largueza, J.M -- TRANSCRIBER

[HEMA311]1.01 Safety in Hematology Laboratory Prof. Antonio C. Pascua, Jr., RMT, MSMT

C. Other Hazard

1. Biological Hazards

 Refers to the bacteria, fungi, parasites, and virus.

 Dealing with all microorganisms.
**Bacillus subtilis – most common type of contaminant

2. Radiation Hazards
 Most common in radiology laboratory
** x-rays, MRI’s
IORATORY HAZARDS
BLUE -- HEALTH HAZARD RED – FLAMMABILITY HAZARD II. BIOSAFETY LEVEL
4 – Deadly Flash Points
3 – Extreme Danger 4 – Below 73°F
2 – Hazardous 3 – Below 100°F
1 – Slightly Hazardous 2 – Below 200°F TYPES OF EXAMPLES
0 – Normal Material 1 – Above 200°F LEVEL RISK PRECAUTIONS
AGENTS OF AGENTS
0 – Will not burn

WHITE – SPECIFIC HAZARD YELLOW – INSTABILITY HAZARD Standard

Oxidizer – OX 4 – May detonate microbiological
practice.
Acid – ACID 3 – Shock and heat may detonate Those not
Bacillus subtilis **ASEPTIC
Alkali – ALK 2 – Violent chemical change known to
Mycobacterium TECHNIQUE
Corrosive – COR 1 – Unstable if heated I Minimal
cause
gordonae (tap No special
Use no water – W 0 -- Stable disease in
water) equipment.
healthy
Radiation Hazard -- ☢ soil microbes  PPE’s
adults
 Good immune
3. Electrical Hazards system
 Use of adapters, gang plugs and extension cords are
prohibited Biolological
 Stepping on cords, rolling heavy equipment over cords should Safety Cabinet
be prohibited (BSC) I or II.
E. coli,
 Before repair or adjustment of electrical equipment, unplug Common PPE’s
Salmonella,
first the equipment making sure that the hand is dry and no II Moderate human Autoclave
HIV, HBV,
jewelry should be present pathogens. Limited access
influenza
Most microlabs
**most common hazard inside the laboratory fall in this
**DIRECT – can cause shock or even category.
INDIRECT – usually results to fire and explosion Those that
Bacillus
machines or equipment should be near/close to the power may cause
anthracis,
source. Francisella,
serious or
 using of electrical extensions lethal
Brucella, Same as above
Mycobacterium plus negative air
disease via
tuberculosis, flow, sealed
4. Sharp Hazards III High inhalation.
Rickettsia windows.
 sharp objects such as syringe with needle, knives for **droplets
rickettsii, proper
histopathology analysis, and lancets. **airborne
Coxiella ventilation
 contaminated needle is dangerous (accidental pricks) Effective
burnetelli, mold
treatment
stages of
 Needle Puncture available.
systemic fungi
 Containers should be puncture proof Requires use of
 Improper disposal is the major cause of needle prick incident class III BSC;
 Replaced once the container is ¾ full full-body air
** Fully- filled container is prohibited. Those that supplied positive
** 0.01%-0.1% chance of getting infection from needle stick pose high pressure suit;
injury risk of life- independent unit
threatening with specialized
B. Physical Hazard Ebola virus,
disease. ventilation &
Lassa virus,
May be waste
 An agent, factor, or circumstance that can cause harm without others that
IV Extreme transmitted management to
contact. cause
by prevent release
It includes: hemorrhagic
aerosols. into environment
 Ergonomic Hazard fevers
NO Biosafety
- Can affect posture VACCINE Cabinet
 Vibration Hazard or  Waste
- From the analyzers THERAPY. Management
 Noise Hazard Control
- From the analyzers or machines  proper
ventilation
** CoViD-19 lies between Level III and Level IV

Acuña, J., Largueza, J.M -- TRANSCRIBER

[HEMA311]1.01 Safety in Hematology Laboratory Prof. Antonio C. Pascua, Jr., RMT, MSMT

Chemical Waste
Radioactive Waste
A. Safety Precautions
** AGAR disposal
- Disinfect
 Exposure to blood and body fluids is the most common risk - Autoclave before disposal
associated in hematology laboratory. ** autoclave is an essential tool inside the
 Bloodborne pathogens are pathogenic microorganisms laboratory.
present in blood causing infection or diseases.
**WHO, DOH B. Waste Disposal Standard
1. OSHA Standards (Occupational Safety and Health 1. Blood Containing Waste
Administration)
.  Objects contaminated with blood should be autoclaved before
 OSHA provides standards to maintain safe work environment. disposal.
 The following practices are enforced inside the laboratory:  Blood should be treated before disposal; treatment involves
1. Handwashing the use of aldehyde, chlorine compounds, phenolic
2. Food, drink and medications not allowed compounds or thru autoclaving before pouring down the sink
3. Applying cosmetics are prohibited with running water.
4. Fomites or any surfaces must be kept away from
mouth and all mucous membranes. Standard Waste Protocol (Philippines)
**FOMITES- surfaces that are likely to cause an
infection. COLOR TYPE OF WASTES
5. Contaminated sharps must be disposed properly
6. Personal Protective Equipment must be worn at all RED Sharps, needles
times following the proper donning YELLOW Infectious wastes
7. Equipment should be check and maintained YELLOW WITH BLACK BAND Chemical Wastes
**OSHA Philippines works hand-in-hand with DOLE ORANGE Radioactive Wastes
Non-infectious wet wastes,
Handwashing Procedure GREEN
biodegradable wastes
Non-infectious dry wastes, non-
 Wash your hands BEFORE: entering workplace, handling BLACK
biodegradable wastes
equipment, before filling up napkin dispensers, eating WASTE CONTAINER/BAGS ARE COLOR CODED
 Wash your hands AFTER: going to toilet, meal, smoking, SOURCE: DEPARTMENT OF HEALTH
cleaning, handling wastes, removing gloves, touching parts of
the body, every patient interaction, handling chemicals C. Disposal
 HOW TO WASH YOUR HANDS?  Flushing down the drain to the Sewer System
 Turn on tap, wet hands with warm water then apply liquid ** specific sewer system
soap, lather and rub at least for 20 seconds.  Incineration
 Clean each nail, between each finger, front and back of the ** Level III or Level IV Biosafety level
hands up to the wrist then rinse off soap using water pointing  Landfill burial
downwards.  Recycling
 Dry hands using disposable paper towel.
IV. Occupational Safety and Health Act
 Turn off the water tap using another disposable paper towel.

**Handwashing must be done for 1 minute or 10 seconds  Republic Act 11058 ”An Act Strengthening
each step. Compliance with Occupational Safety and Health
**clothes should not touch any area of the sink Standards and Providing Penalties for Violations
**PROPER DONNING Thereof”
- Handwashing ** Aims to make sure that all workers in any
 Shoe Cover field are protected in all hazards.
 Gown
 Mask or Respirators GOAL: Provide all employees (clinical laboratory personnel
 Goggles or Face Shield included) with a safe work environment.
 Gloves A. Government Regulatory Agency
**PROPER DOFFING 1. Department of Labor: 29 Code of Federal
Gloves Regulations Parts 1900-1910
 Goggles  Hazard Communication Standard
 Gown  Hazardous Waste Operations
 Mask  Occupational Exposure to bloodborne
- Handwashing pathogens Standards
**Another DOFFING technique 2. Department of the Interior, Environmental
Gown and Gloves (for disposable gowns) Protection Agency: 40 Code of Federal Regulations
- Handwashing Parts 200-399
 Goggles  Clean Air Act and Clean Water Act
 Mask  Toxic Substances Control Act
- Handwashing  Comprehensive Environmental
Response, Compensation and Liability
III. LABORATORY WASTE MANAGEMENT Act (CERCLA)
3. Voluntary Agencies/Accrediting Agencies
 The Joint Commission
A. Segregation
 College of American Pathologist
 Separation of waste
 Centers for Disease Control and
1. Biodegradable
Prevention (CDC)
2. Non-biodegradable
 Clinical and Laboratory Standards
3. Hazardous Waste
Institute.
Special waste
Biological Waste

Acuña, J., Largueza, J.M -- TRANSCRIBER

[HEMA311]1.01 Safety in Hematology Laboratory Prof. Antonio C. Pascua, Jr., RMT, MSMT

 Safety in the Laboratory is BASIC but

needs COMMON SENSE 

Acuña, J., Largueza, J.M -- TRANSCRIBER

 OLFU Blood Collection Technique
2021 – 2022
LAB 2
RMT 2023
CLINICAL HEMATOLOGY 1
1st Semester
Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT
Date: September 21, 2021 TRANS 2 HEMA311
LAB

Outline Can be obtained either through a catheter placed in an artery or by using

At the end of the session, the student must be able to learn: a needle and syringe to puncture an artery
I. Blood Collection Technique
A. Arterial Puncture - Performed in emergency cases: accident, sudden heart
1. Puncture Sites attack
i. Radial Artery
ii. Brachial Artery - A catheter or using needle or syringe → must be PRE
iii. Femoral Artery HEPARINIZED
2. Complications
i. Arteriospasm 1. Puncture Site
ii. Hematoma • Radial artery
iii. Nerve Damade o Thumb No need for tourniquet
iv. Fainting o 90 degree
B. Skin Puncture o Dangerous and critical
1. Sites of Puncture • Brachial artery - At arm. Harder to locate
i. Finger • Femoral artery
ii. Earlobe •
iii. Lateral Portion of the Plantar Surface of the
2. Complications
Heel or Toe • Arteriospasm - help them relax
2. Sites to avoid • Involuntary contraction of the artery
3. Indications of Skin Puncture • Hematoma
i.Newborn/Infant • Excessive bleeding
ii. Geriatic • Nerve damage - prevented by choosing an appropriate
iii. Test that requires small amount of blood sampling site and avoiding redirection of the needle - select
4. Advantages of Skin Puncture the appropriate veins for venipuncture
5. Order of Draw • Fainting - prevented by ensuring that the patient is supine
6. Complications with feet elevated before beginning the blood draw. “Supine
C. Venipuncture position”
1. Things to Remember
2. Method of Collection Figure 1.1
i. Single Collection
ii. Multiple Colletion
iii. Winged Collection
3. Sites of Puncture
4. Sites to avoid
5. Complications
6. Venipuncture in Special Situations
7. Additives in Collection Tubes
i. Clot Activators
ii. Anticoagulants
iii. Antiglycolytic Agent
iv. Separator Gel

I. BLOOD COLLECTION TECHNIQUE

**Blood is one of the most commonly submitted and processed

specimens in the laboratory. Examination of blood in the different
sections of the laboratory provides a picture of the condition of the
patient. It is important that blood samples are collected and
processed properly so that accurate, precise, and reliable results B. Skin Puncture
are obtained. A mixture of capillary, venous, and arterial blood with interstitial (tissue)
fluid and intracellular fluid.
**Regulations of the Occupational Safety and Health
Administration (OSHA) that took effect on March 6, 1992, outlined
in detail what must be done to protect health care workers from 1. Sites of Puncture
exposure to bloodborne pathogens, such as the pathogens that • Finger - Non dominant, 3rd or 4th finger - 3-4 mm deep
cause hepatitis C, hepatitis B, hepatitis D, syphilis, malaria, and • Earlobe - Abnormality in WBC (presence of callus)
human immunodeficiency virus (HIV) infection • <1yo Lateral portion of the plantar surface of the heel or toe-
ideal for newborn
**Standard precautions must be followed at all times, with special 2. Sites to Avoid
attention to the use of gloves and hand washing. • inflamed and pallor areas
• cold and cyanotic areas
A. Arterial Puncture • congested and edematous areas
Done to collect arterial blood which is used for blood gas analysis • scarred and heavily calloused areas

Collected from an artery, primarily to determine arterial blood gases

Performed by: DOCTORS OR RESPIRATORY THERAPIST

Acuña, J., Largueza, J.M -- TRANSCRIBER

[HEMA311] 1.02 Blood Collection Technique Prof. Antonio C. Pascua, Jr., RMT, MSMT

Figure 1.2 Decrease the possibility of hemolysis and to prevent blood

from adhering to the sides of the tube.
• “Winged” Collection - Butterfly set

Figure 1.3

Image source: WHO Guidelines on Drawing Blood

3. Indications of Skin Puncture

• Newborn/infant - Less amount of blood
• Geriatric - To avoid collapsing
• Test requiring small amount of blood
4. Advantages of Skin Puncture Image source: WHO Guidelines on Drawing Blood
It is easy to obtain (it can be difficult to obtain blood from the veins,
especially in infants). There are several collection sites on the body, and Figure 1.4
these sites can be rotated. Testing can be done at home and with little
training.
5. Order of Draw
• Hematology- *platelets are not aggregated
• Clinical chemistry blood banking
6. Complications
•collapse of veins if the tibial artery is lacerated from
puncturing the medial aspect of the heel
•osteomyelitis of the heel bone (calcaneus)
•nerve damage if the fingers of neonates are punctured
•haematoma and loss of access to the venous branch used
•scarring
•localized or generalized necrosis

•skin breakdown from repeated use of adhesive strips

Image source: WHO Guidelines on Drawing Blood
C. Venipuncture
• manner of inserting a needle attached to a syringe to a palpable vein
to collect blood for laboratory testing 3. Sites of Puncture
• Specimen collected: venous blood
collecting venous blood **Needles for drawing blood range from 19 to 23 gauge.
• Most widely used blood sample in all laboratory tests **The most common needle size for adult venipuncture is 21
gauge with a length of 1 inch
1. Things to Remember • Median - Most stable - connects the basilic and cephalic
• Proper identification of patient
veins in the antecubital fossa
• Tourniquet application- only 1 min, 3-4 inches away used to provide a
barrier against venous blood flow to help locate a vein. • Cephalic - Lateral area “away” - Blood spurts. - Quiet
**A tourniquet can be a disposable elastic strap, a heavier unstable - the cephalic vein, located on the outside (lateral)
Velcro strap, or a blood pressure cuff. Latexfree tourniquets are aspect of the antecubital fossa on the thumb side of the
available for individuals with a latex allergy. hand;
• Disinfection
• Basilic - Easily rolls - More chances of hematoma - w/ nerve
• Angle of needle insertion- 30-40 degree
endings - the basilic vein, located on the inside (medial)
• Bevel UP
aspect of the antecubital fossa
• Needle length • Occluded veins - Multiple puncture part - Impared circulation
• Position of the patient • Fistula - Abnormal connection - Result from injury/surgery. -
• Label Connection of organs/vessel - Dialysis patient
• Disposal • IV fluid site - If no choice at all. - Stop for 5-10 mins - First 5
2. Method of Collection ml, disregard
• Single Collection - Syringe collection - consists of a barrel, Older children (18months to 3 yo)
graduated in milliliters, and a plunger. • Femoral vein
• Multiple Collection - ETS. reduces the risk exposure of • Long saphenous vein
blood. • Popliteal vein
**OSHA recommends the use of plastic tubes whenever • Ankle vein *Antecubital fossa
possible. Most glass tubes are coated with silicone to help

2 Acuña, J., Largueza, J.M -- TRANSCRIBER

[HEMA311] 1.02 Blood Collection Technique Prof. Antonio C. Pascua, Jr., RMT, MSMT

Figure 1.5 ** The cephalic and basilic veins should only be used if the
median cubital or median veins are not prominent after
checking both arms. The basilic vein is the last choice due to
the increased risk of injury to the median nerve and/or
accidental puncture of the brachial artery, both located in
close proximity to the basilic vein.

4. Sites to Avoid
• Sites with hematoma
• Occluded veins
• Edematous area
• Sites with burns, scar, tattoo
• Sites with Fistula
• IV fluid sites

5. Complications
• Ecchymosis (Bruise)
3 yo to adult life -most common complication. Caused by leakage of a small
• Wrist vein amount of blood in the tissue around the puncture site
• Dorsal vein of hand -can prevent applying direct pressure to the venipuncture site with
• Dorsal vein of ankle *Antecubital fossa a gauze pad
-Bending the patient’s arm at the elbow to hold the gauze pad in
Figure 1.6
place is not effective in stopping the bleeding and may lead to bruising.
• Hematoma
-a results when leakage of a large amount of blood around the
puncture site causes the area to rapidly swell.
-remove the needle immediately and apply pressure at least 2
minutes.
-may result in bruising of the patient’s skin around the puncture
site.
-can also cause pain and possible nerve compression and
permanent damage to the patient’s arm.
-most commonly occur when the needle goes through the vein or
when the bevel of the needle is only partially in the vein and when the
phlebotomist fails to remove the tourniquet before removing the needle
Figure 1.7 or does not apply enough pressure to the site after venipuncture.
-can also form after inadvertent puncture of an artery.
• Pain
• Syncope and fainting
• Iatrogenic anemia
• Infections
• Edema
• Allergies
-use hypoallergenic tape or apply pressure manually until the
bleeding has stopped completely
• Petechiae
Antecubital fossa -small red spots indicating that small amounts of blood have
• Two patterns of veins escaped into the skin.
1. H pattern - Median capital vein - Cephalic vein - Basilic -indicate a possible hemostasis abnormality and should alert the
vein phlebotomist to be aware of possible prolonged bleeding.
2. M pattern - Median - Accessory cephalic - Basilic vein
• Hemoconcentration
Figure 1.8 -increased concentration of cells, larger molecules, and analytes
in the blood as a result of a shift in water balance
-caused by leaving the tourniquet on the patient’s arm for too
long. The tourniquet should not remain on the arm for longer than
1 minute. If it is left on for a longer time because of difficulty in
finding a vein, it should be removed for 2 minutes and reapplied
before the venipuncture is performed
• Hemolysis
- rupture of red blood cells with the consequent escape of
hemoglobin—a process termed hemolysis—can cause the
plasma or serum to appear pink or red

3 Acuña, J., Largueza, J.M -- TRANSCRIBER

[HEMA311] 1.02 Blood Collection Technique Prof. Antonio C. Pascua, Jr., RMT, MSMT

Table 1.1 the glucose level is delayed. The most commonly used
antiglycolytic agent is sodium fluoride.
- Tubes containing sodium fluoride alone yield
Adverse event Cause Management serum.
Hematoma Poor collection Apply pressure - Tubes containing sodium fluoride and an
techniques and firm badge anticoagulant (such as EDTA or oxalate) yield
plasma
Fainting due Anxiety Recline chair • Separator Gel. Is an inert material that undergoes a
to: a Lowered blood Discontinue temporary change in viscosity during the centrifugation
hypothalamic volume and collection Loosen process; this enables it to serve as a separation barrier
response other associated clothes Give fluid
between the liquid (serum or plasma) and cells. Because this
resulting in causes
gel may interfere with some testing, serum or plasma from
bradycardia,
these tubes cannot be used with certain instruments or for
vomiting,
sweating blood bank procedures.
Syncope Physical stress Fluid
Inadequate fluid administration
intake

6. Venipuncure in Special Situations

• Edema Swelling caused by an abnormal accumulation of
fluid in the intercellular spaces of the tissues is termed
edema. The most common cause is infiltration of the tissues
by the solution running through an incorrectly positioned
intravenous catheter. Edematous sites should be avoided for
venipuncture because the veins are hard to find and the
specimens may become contaminated with tissue fluid.
• Obesity In obese patients, veins may be neither readily
visible nor easy to palpate. Sometimes the use of a blood
pressure cuff can aid in locating a vein. The cuff should not
be inflated any higher than 40 mm Hg and should not be left
on the arm for longer than 1 minute.9 The phlebotomist
should not probe blindly in the patient’s arm because nerve
damage may result.
• Burned, Damaged, Scarred, and Occluded Veins Burned,
damaged, scarred, and occluded veins should be avoided
because they do not allow the blood to flow freely and may
make it difficult to obtain an acceptable specimen.
• Mastectomy Patients The CLSI requires physician
consultation before blood is drawn from the same side as a
prior mastectomy (removal of the breast), even in the case of
bilateral mastectomies.9 The pressure on the arm that is on
the same side as the mastectomy from a tourniquet or blood
pressure cuff can lead to pain or lymphostasis from
accumulating lymph fluid. The other arm on the side without
a mastectomy should be used.

7. Additives in Collection Tubes

• Clot activators. Blood specimens for serum testing must
first be allowed to clot for 30 to 60 minutes prior to
centrifugation and removal of the serum.10 A clot activator
accelerates the clotting process and decreases the
specimen preparation time. Examples of clot activators
include glass or silica particles (activates factor XII in the
coagulation pathway) and thrombin
• Anticoagulants. Prevents blood from clotting.
- Ethylenediaminetetraacetic acid (EDTA),
citrate, and oxalate remove calcium needed for
clotting by forming insoluble calcium salts.
- Heparin prevents clotting by binding to
antithrombin in the plasma and inhibiting thrombin
and activated coagulation factor X.
** Tubes with anticoagulant must be gently inverted immediately

• Antiglycolytic Agent. Inhibits the metabolism of glucose by

blood cells. Such inhibition may be necessary if testing for

4 Acuña, J., Largueza, J.M -- TRANSCRIBER

 OLFU Red Blood Cell Count
2021 – 2022
LAB 3
RMT 2023
CLINICAL HEMATOLOGY 1
1st Semester
Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT
Date: September 29, 2021 TRANS 3 HEMA311
LAB

Outline ** The most commonly used counting chamber is the Levy chamber with
At the end of the session, the student must be able to learn: improved Neubauer ruling.
I. RED BLOOD CELL COUNT **Thoma pipette is also essential in manual cell counting procedures to
II. MATERIALS NEEDED (MANUAL) accurately dilute blood specimens with fluids needed for specific cell
A. Hemacytometer counting procedures. A red cell pipet and a white cell pipet are the thoma
B. Thoma Pipette pipets used in cell counting. A red cell pipet can contain 101units of
C. Suction Device
volume whereas white cell pipet can contain 11units.
D. Thick Coverslip
E. Cell Counter
F. RBC Diluting Fluid
III. PROCEDURE
A. Additional Procedure
IV. POST LABORATORY
V. SOURCES OF ERRORS AND COMMENTS
VI. NORMAL VALUES

I. RED BLOOD CELL COUNT

• The number of WBCs in 1 liter or 1 microliter of blood

• Manual RBC counts are rarely performed because of the
inaccuracy of the count and questionable necessity. Use of
other, more accurate manual RBC procedures, such as the
micro hematocrit and hemoglobin concentration, is desirable
when automation is not available. Primary square: 2
• Highest in the morning and lowest in the evening. Each primary square has 9 SECONDARY SQUARE
• Increased: myeloproliferative disorder such as polycythemia The four corners of the secondary square is for WBC
• Decreased: anemic cases, bleeding WBC secondary square has 16 TERTIARY SQUARE.
***counting of red blood cells will give us the reflection of how our For RBC in the center has 25 TERTIARY SQUARE
body is capable of delivering oxygen. Each 25 TERTIARY SQUARE has 16 QUATERNARY SQUARE.
• ***ELEVATION OF RBC COUNT = excessive production of
**each secondary square has 1mm x 1mm ➔ Total of 9mm2
bone marrow Manual cell counts are performed using a
hemocytometer for counting chamber, and manual dilutions
made with calibrated, automated pipettes and diluents 2. RBC DILUTING FLUID
(commercially available or laboratory prepared). The principle • NSS
for the performance of cell counts is essentially the same for • 3.8% Na citrate
• Dacies
white blood cells, red blood cells, and platelets; only the
• Hayem’s
dilution, diluting fluid, and area counted vary. • Toisson’s
• Will not confirm any forms/ types of diseases. Only a • Bethell’s
SCREENING TEST • Gower’s
• Identifying how capable the body is to transport oxygen to ***ISOTONIC solution (RBC)
different parts of our body. ***HYPOTONIC (WBC)

III. PROCEDURE
II. MATERIALS NEEDED (MANUAL) 1. Draw blood up to 0.5 mark using the RBC Pipette.
**COMPLICATED AND SUBJECTIVE Note: if the blood was drawn too far above the 0.5 mark, the
• Hemacytometer procedure should be repeated using a new pipette, excess
• Thoma Pipette blood causes an inaccurate result.
• Suction device 2. Wipe the outside walls of the pipette with clean gauze.
• Thick coverslip Note: do not allow capillary attraction to draw fluid from the
• Cell Counter tip onto the gauze. Gauze tends to absorb the liquid portion
• Diluting fluids of the blood, causing an inaccurate result.
3. Dip the pipette into diluting fluid, and then draw the diluting
1. HEMACYTOMETER
More convenient, accurate, organize fluid into the pipette, slowly, until the mixture reaches the
101 mark. Gently rotate the pipette to ensure a proper
• “Heart of manual cell count”
• Different Types
amount of mixing. The dilution is 1:200
4. Remove the tubing from the pipette, and then mix it on a
- Improved Neubauer
horizontal axis for 5 minutes.
- Neubauer 5. Discard the first 3-4 drops of the diluted sample.
- Fuchs –Rosenthal 6. Charge both sides of the hemocytometer counting chamber
- Speirs–Levy -Tuerk’s with a drop of the diluted sample and allow to stand for few
- Bass –Jones minutes.
7. While keeping the hemacytometer in a horizontal position,
place it on the microscope stage.
Acuña, J., Largueza, J.M -- TRANSCRIBER
 [HEMA311] 1.03 Red Blood Cell Count Prof. Antonio C. Pascua, Jr., RMT, MSMT

8. Using HPO, count the red cells in the 5R’s squares of the
central secondary square.
V. SOURCES OF ERRORS AND COMMENTS
9. Calculate the number of RBC per luter of each of the
hemocytometer. Average the results and report this number. • Dust and fingerprints may cause difficulty in distinguishing
*** additional procedure: the cells
STEP 1: ***directly aspirate the blood from EDTA • Diluting fluid should be free of contaminants
tube • If the count is low, a greater area may be counted
STEP 3: The mixture will confine at the bulb • Chamber must be charged properly to ensure accurate
**** no bubbles count
**** no spaces • Allow cells to settle for 10mins before counting
STEP 4: *** 5 minutes mixing • Use of other, more accurate manual RBC procedures, such
STEP 5: discard the first 3-5 drops as the microhematocrit and hemoglobin concentration, is
STEP 6: *** put the slide to petri dish with moist desirable when automation is not available
tissue paper or gauze to keep the
moisture ( 5 – 10 minutes incubating)
STEP 7: ***microscope VI. NORMAL VALUES
*** PARFOCAL FOCUSING •Female: 3.6 – 5.6 x 1012/L
*** Scanner = primary square •Male: 4.2 – 6.0 x 1012/L
LPO = secondary square •At Birth: 5.0 – 6.5 x 1012/L
HPO = tertiary square
STEP 8: *** cells touching LEFT and TOP triple
lines are included in count
Cells touching RIGHT and

BOTTOM triple lines are NOT included in count

****aspirate blood directly from EDTA tube

Rules
• count all cells inside the square
• count all the cells in the middle of L line “ L RULE”
***the difference between each primary square must only be 5-10%

IV. POST LABORATORY

*** CELLS COUNTED = get the AVERAGE RBC count of

2 counting area (top and bottom)
DILLUTION FACTOR = solute ÷ total volume
DEPTH = constant value 0.1 mm
Area mm2 = RBC area you count
** RBC tertiary square 1st, 5th, 13th, 21st, 25th
( i.e every tertiary square= 1 (tertiary area) ÷ 25
(squares)) = 0.04
0.04 x 5 (number of RBC area you count) = 0.02 (area
factor
Unit for Final RBC Count (x1012/L)
***rule of 3 is only applicable for normocyctic and normochromic

2 Acuña, J., Largueza, J.M -- TRANSCRIBER

 OLFU Hemoglobin Determination
CLINICAL HEMATOLOGY 1 LAB 4 2021 – 2022
RMT 2023 Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT
1st Semester

Date: October 28 , 2021 TRANS 4 HEMA311

LAB

Outline Sahli graduated tube: with markings with an increment of 2 every line
At the end of the session, the student must be able to learn: that is equivalent to 0.2 (for blood and HCl)
I. Hemoglobin
II. Methods
i. Acid Hematin Procedure
ii. Cyanmethemoglobin
iii. Other Methods

I. Hemoglobin
Hemoglobin is the most significant component of RBC.
 Globin
 Protoporphyrin IX
 Iron
 Higher in the morning (due to physical activities)
 Lower in the evening (resting time)
 Carries oxygen from the lungs to the tissues and takes carbon ***Equivalent Hgb value concentration: check the line in graduated
dioxide tube
 Screen for anemia and may detect RBC breakdown/ Brown color = red cells have been lysed.
hemolytic anemia
 Higher in high altitude 2. Cyanmethemoglobin
 Increase in strenuous muscular activity
Normal Values:
 At birth: 15-20 g/dL Principle:
 Women: 12-16 g/dL
 Men: 13-18 g/dL
High Low
Sickle cell anemia Anemia
 Absorbance of cyanmethemoglobin at 540 nm (set
Thalassemia Blood loss wavelength) is directly proportional to the Hb concentration
Iron, folate and Vitamin  Uses a standard curve (spectrophotometer)
Transfusion reaction
B6, B12 deficiencies ***indirect colorimetric
Hemolysis ***manually or automated is still acceptable
Dehydration
Polycythemia vera Materials and Instruments
High altitude  Anticoagulated blood (venous blood)
 Test tubes
 Pipette
 Spectrophotometer
II. Methods  Drabkin’s Reagent
 Parafilm

1. Acid Hematin

Principle: Colorimetric Procedure

Comparator block: Subjective

***Compare the “color” of solution to standard

Materials and Instruments

***Sahli pipette: Straight micropipette with a single mark = 0.02 mL

blood

Acuña, J., Largueza, J.M -- TRANSCRIBER

[HEMA311] 2.01 Hemoglobin Determination Prof. Antonio C. Pascua, Jr., RMT, MSMT

Reagent: Drabkin’s Reagent

3. Other Methods
 Potassium ferricyanate
Hgb === methemoglobin === cyanmethemoglobin Gasometric (Van Slyke oxygen capacity)
***Non-ionic detergent is for RBC lysing
*** Whatever absorbance you get is not yet the final  1 gm hb = 1.34 mL O2
hemoglobin concentration; needs to be compared with
standard curve Specific Gravity method ( Copper sulfate)
Ab’s = curve
All forms of derivatives can be measured by  sp.gr = 1.053
cyanmethemoglobin except SULFHEMOGLOBIN  approximation of hemoglobin concentration
15 seconds until the RBC blood drop settles at the bottom
***Turbidity increases; absorbance of light also increases indicates normal hemoglobin
***the heavier the blood, hemoglobin is normal

Sources of Error Chemical (Kennedy’s, Wong’s)

 1gm Hb = 3.47 mg Iron

References:
 Rodak’s Hematology 6th Edition
 Turgeon Clinical Hematology Theory and Procedures 5th
Edition
 Laboratory Sidenotes of Prof. Antonio C. Pascua Jr.,
RMT, MSMT - OLFU Valenzuela CMLS

Acuña, J., Largueza, J.M -- TRANSCRIBER

 OLFU Hematocrit
2021 – 2022
LAB 5
RMT 2023
CLINICAL HEMATOLOGY 1
1st Semester
Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT
Date: November 2, 2021 TRANS 5 HEMA311
LAB

Outline
At the end of the session, the student must be able to learn:
I. Hematocrit
A. Principle
II. Method for Hematocrit Determination
A. Macrohematocrit
• Disadvantages
• Materials
• Procedures
B. Microhematocrit
• Centrifugation
• Materials
• Procedures
III. Sources of Error
IV. Trapped Plasma
V. Normal Values

I. Hematocrit

Ratio of the volume of packed red blood cells to the volume of

i. Disadvantages
whole blood.
Packed cell volume means RBC’s only The problem with this is, it is time consuming. It must be centrifuged for
Reported as percentage of L/L 30 minutes. Also large amount of blood must be used. As you centrifuge
the blood, there will be trapped plasma in the container so this amount of
Example 0.45 – 45% plasma could still remain on the RBC portion of the blood after you
0.47 – 47% centrifuged it.
0.40 – 40% If there is a left over plasma in between, Hct will be falsely elevated. So
SI UNIT for Hct is L/L this is a common problem in the macro method.
 Also an important parameter to differentiate anemia.
 The least choice parameter is RBC count. a. Wintrobe and Landsberg: double oxalate (most common
 Priority would always be HCT and HGB is the use of Wintrobe tube)
 Amount of space occupied by RBC’s in a given volume of b. Van Allen: sodium oxalate
blood. c. Haden : sodium oxalate
A. Principle d. Sandford-Magath: sodium oxalate
e. Bray: heparin
 Whole blood is centrifuged top determine packing of Red Blood
Cells. The length of the Wintrobe tube is 115 mm and the diameter of the
 When blood is centrifuged, blood components are separated bore is 3mm.
depending on their densities.
 Two main divisions of blood are the solid and liquid part. The
solid part I composed of the formed elements, RBC, WBC, and ii. Materials
Plt. The liquid portion is composed of Plasma. iii.
 At the top is plasma with water and other chemical constituents iv. W
Wintrobe tube with rubber caps
of the blood. The solid portion of blood is heavier/denser thus
Long stem pipette
settles at the bottom of the tube. Among the 3 formed elements,
Centrifuge
the densest is the RBC since it has Hgb which contains oxygen.
Anticoagulated blood (at least 3 mL of whole blood
 Buffy coat is composed of all other elements of the blood. At the Cotton
top of the buffy coat is the platelets, then the monocytes,
lymphocytes, the granulocytes which is heavier among the first
two because of the granules and at the bottom of the buffy coat iii. Procedures
are the reticulocytes or if there are any immature blood cells. v.
 On hematocrit, we ONLY measure the RED BLOOD CELLS. We vi. W
1. Mix the blood
DO NOT INCLUDE the buffy coat, as it name implies PACKED 2. With the use of a long stem pipette, full the
RED CELLS.
Wintrobe tube to the10 mark
 Hct is only a computated value for automated analyzer. In
manual determination it depends on the RBC Count and Mean Note: No air bubbles should be present on the surface
Cell Volume, and can be measure through macro and micro of the blood, it may be removed by touching it with
method. tissue paper or with a pipette. If the bubbles are present
in the middle of the tube, aspirate the blood and
II. Method for Hematocrit Determination proceed to step 2 again.
3. Centrifuge the blood for 30mins.
1. Macrohematocrit Determination 4. Read the height of the packed red blood cells on
the scale at the right side of the tube, which is
• You will be using larger amount of blood. Venipuncture then graduated from 0-10 cm from the bottom to top,
transfer to EDTA Tube. read upward.
• Whole Blood is transferred to a Wintrobe tube and centrifuged If the packed RBC is in the 3.5 mark, multiplyby 10 then
for 30 minutes at 2000-3000 rpm.
your Hct will be 35% or 0.35.
• Wintrobe tube- a special container for the measurement of
There are times that there a presence of slant RBC so
ESR (0-10 down) and Hct (0-10 up). This is on a single tube
thus, it has 2 markings. You just have to fill the tube with blood make sure that you only include those parallel to the
up to the 10th mark. line.

1
Acuña J., Largueza J. M. - TRANSCRIBER
 [HEMA311] 2.02 Red Blood Cell Count Prof. Antonio C. Pascua, Jr., RMT, MSMT

2. Microhematocrit Determination

 Less blood, skin puncture only. III. Sources of Error

 Specimen is EDTA Blood (directly obtained)
 Capillary blood Increased Decreased
 Sample is transferred to a capillary tube and
centrifuged to a microhematocrit centrifuge.  Insufficient  Improper sealing of
Note: If you are going to use blood from EDTA tube, use a capillary tube centrifugation time- the capillary tube-
with a blue mark (non-heparinized/no anticoagulant) RBC will not be packed there could be leakage.
If you are going to use blood from skin puncture, use a capillary tube  Inclusions of buffy  Increased
with red band (heparinized). coat concentration of
 Disorders such as anticoagulant- EDTA
sickle cell anemia, can cause crenation or
marcocytic anemia, destruction of RBC.
i. Centrifugation hypochromic anemia  Prolonged
 Dehydration- portion of centrifugation- RBC
 Better method for Hct.
plasma is water, if the may be destroyed
 Heavier centrifugal force means more packed RBC.
patient is dehydrated  Acute blood loss
 Heavier centrifugal force will avoid unwanted trapped
plasma. then it will yield lesser
 Ten thousand to Fifteen thousand gravitational force, plasma.
centrifuged for only 5-10 mins.
 75 mm length with 1 mm bore diameter Additional note: During blood collection process, prolonged application of
 A lipemic patient will have lipid components on the tourniquet may cause falsely increased Hct due to Hemoconcentration.
top, but in most cases, only the plasma is present. For any reason that you may had difficulty in blood collection, interstitial
 A specialized centrifuge for microhematocrit could be fluid may interfere into the specimen and may cause falsely decreased
used, it is called READACRIT – a centrifuge with Hct.
markings/lines.
In cases of anemia, there is nothing we could do about it happens
Microhematocrit Reading naturally.
 Horizontal line- align the end portion of packed cell.
Do not include the clay or wax. Align at 0. IV.Trapped Plasma
 Diagonal line- Align the last portion of the plasma Amount of plasma that still remains in RBC portion after the
here. microhematocrit has been spun. This could increase the Hct by 1-3%.
 Adjustable diagonal line- adjust this line to the
packed cell, exluding the buffy coat. V. Normal Values
Note: Make sure that before you put the capillary tube in the
centrifuge, seal one end with wax, but clay will do. At birth: 24-60%
Female: 36-48%
Example: Male 40-55%
The reading is 32, then you will report it as 32%. The accepted value
for the reading could be less than or more than one. So 31% and
33% is accepted.

ii. Materials
*** CELLS COUNTED = get the AVERAGE RBC
Heparinized capillary tube count of2 counting area (top and bottom)
Microhematocrit reader DILLUTION FACTOR = solute ÷ total
Microhematocrit centrifuge volumeDEPTH = constant value 0.1 mm
Lancet Area mm2 = RBC area you count
Cotton ** RBC tertiary square 1st, 5th, 13th, 21st, 25th
Alcohol ( i.e every tertiary square= 1 (tertiary area)
÷ 25(squares)) = 0.04
0.04 x 5 (number of RBC area you count) = 0.02
iii. Procedures (areafactor
Unit for Final RBC Count (x1012/L)
1. After necessary preparation, perform a capillary puncture and ***rule of 3 is only applicable for normocyctic and normochromi
produce a rounded drop of blood.
2. In a horizontal position, put one end of capillary tube on the drop V. SOURCES OF ERRORS AND COMMENTS
of blood and fill the tube for about ⅔ full. Note: The tube will be
filled with a capillary action. If using tubes with colored ring at • Dust and fingerprints may cause difficulty in
one end, fill from the opposite end. distinguishingthe cells
3. Seal one end of the capillary tube with clay by placing the dry • Diluting fluid should be free of contaminants
end of the tube into the clay in a vertical position. • If the count is low, a greater area may be counted
4. After sealing with clay, seal it again with wax. • Chamber must be charged properly to ensure
5. Assemble the tube in the microhematocrit centrifuge in such a accuratecount
way that the unsealed end is near the center of the centrifuge. • Allow cells to settle for 10mins before counting
6. Spin at 10,000 to 15,000 rpm for 5 mins • Use of other, more accurate manual RBC Procedures,
7. Using the microhematocrit reading device, determine the Hct. suchas the microhematocrit and hemoglobin
Note: Exclude the buffy coat in determining the Hct. concentration, is desirable when automation is not
8. The reading on the window of the Hematocrit reader available
corresponds to the Hct value.
DO NOT FORGET TO WIPE OFF THE FIRST DROP OF BLOOD, AS
TISSUE JUICES MAY INTERFERE.

2
Acuña J., Largueza J. M. - TRANSCRIBER
 OLFU
Erythrocyte Sedimentation Rate LEC
CLINICAL HEMATOLOGY 1 2021 – 2022
1st Semester

RMT 2023 Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT TRANS 6 HEMA311
Date: November 10, 2021 LEC

Outline B. Stage of ESR

At the end of the session, the student must be able to learn:
I. Erythrocyte Sedimentation Rate
a. Principle 1. Initial rouleaux formation:10
b. Stage of ESR mins
c. Methods of ESR
d. Procedure (Westergren) *RBC form a stock of coin. The more protein in plasma,
e. Procedure (Wintrobe) the more promotes Rouleaux Formation.
f. Sources of Error
g. Automated ESR 2. Rapid Settling of RBC’s: 40 mins
II. Osmotic Fragility Test 3. Final Sedimentation of RBC’s: 10 mins
a. Principle
b. Materials **total of 60 mins. ESR should be read on 60 mins time,
c. Procedure (Sanford Method) less than 60 could cause falsely decreased result, more
d. Reference Value than 60 could cause falsely elevated result.
e. Data and

I. ERYTHROCYTE SEDIMENTATION RATE

C. Methods of ESR
 The non-specific measurement used to detect and monitor and
inflammatory response to tissue injury.  Westergren Method- ratio is 4 volume of blood to 1 volume of
 Used for screening on any possible inflammation. Termed also as sodium citrate on black top tube(9:1 from blue top tube). Most
Sedimentation Rate-measures the rate of setting of the RBC in commonly used method for ESR. Recommended by the
diluted human plasma or whole blood. international Council for the Standardization on Hematology and
 Ordered with another test to detect inflammatory conditions. the CLSI.
 May include RA, with certain infections, malignancy.  Modified Westergren- EDTA is the anticoagulant used, it must be
 It is not recommended for asymptomatic individuals because of its dilutred to the ratio of 4 volume of blood to 1 volume of 0.9% sodium
low specificity and sensitivity. citrate.
 Distance in millimeters at which the RBS’s fall in 1 hour.  Wintrobe- oxalate-anticouagulated whole blood

A. Principles
 When anticoagulated blood is allowed to stand at room
temperature for a period of time, the RBC settle towards the
bottom of the tube.
 If the tube is left undisturbed, RBC’s will settle down. This will be
reported in mm.
 Graduation found in Wintrobe and Westregren is in mm.
 The phenomenon depends on an interrelationship of variables,
such as the plasma protein composition, the concentration of
erythrocytes and the shape of RBC.
 You can have the plasma component of the blood.
 ESR will be affected by the RBC’s. Since it has negative charge,
they’ll repel one another and this phenomenon is called zeta
potential. This is partial or totally counteracted if there is increase
in quantities of positively charged proteins on plasma, RBC will
settle down rapidly.
 Faster Sedimentation Rate means higher ESR.

Acuña J., Largueza J.M. – TRANSCRIBER

[HEMA311] 2.03 Red Cell Abnormalities Part 2 I Prof. Antonio C. Pascua, RMT, MSMT

D. Procedure (Westergren)

E. Procedure (Wintobe)
F. Sources of Error

• Increased anticoagulant

• Use of sodium/potassiumoxalate and heparin

• Significant change in the temperature of the room

• Slight tilt of the pipette

• Blood specimens must be analyzed within 4 hours of

collection if kept at room temperature

• Bubbles in the column of blood

• Clotted specimen

• Tubes must not be subjected tovibrations

G. Automated ESR

This is not yet widely acceptable since it cannot completely finish the stages of
sedimentation. The only advantage is smaller blood sample is needed and uses
shorter time.
 Ves-Matic System (Diesse, Inc., Hialeah, FL)- optoelectronic sensor
which measures the change in opacity of a column of blood as
sedimentation of blood progresses.
 Sedimat 15 (Polymedco, Cortlandt Manor, NY)- uses the principle of
infrared measurement
 ESRSTATPLUS System (Hema Technologies, Lebanon, NJ)- based
on centrifugation

• Westergren
• M <50yo = 0-15 mm
• M >50yo = 0-20 mm
• F <50yo = 0-20 mm
• F >50yo = 0-30 mm
• Children = 0-10 mm
• Wintrobe
• Male = 0-9 mm
• Female = 0-20 mm
• Children = 0-13 mm

Acuña J., Largueza J.M. – TRANSCRIBER

[HEMA311] 2.03 Erythrocyte Sedimentation Rate I Prof. Antonio C. Pascua, RMT, MSMT
 Thin or flat cells have an increased surface/volume ratio

II. OSMOTIC FRAGILITY

TEST
 A measure of the ability of the RBC to take up fluid without
lysing D. Reference Value
 Employed to help diagnose different types of anemia, in which
the physical properties of red cells are altered.

A. Principle Reference Value

 Red cells suspended in hypotonic solution of sodium chloride initial = 0.42-0.44%

take up water, swell, become spheroidal, and after reaching the
critical volume, eventually burst.
 Cells that are thicker than normal have a decreased complete = 0.32-0.34%
surface/volume ratio.

B. Materials

 Ad

C.Procedure (Sanf

 Increased OFT (decrease resistance): found

in hemolytic anemias and hereditary
spherocytosis and whenever spherocytes
are found
 Decreased OFT (increase resistance):
occurs following splenectomy and in liver
disease, sickle cella nemia IDA and
thalassemia

References:

 Rodak’s Hematology: Clinical Principles and

Applications, 5th Edition
 Clinical Hematology:Theory and Procedures, 5th Edition,
Turgeon, M.L.
 Laboratory Sidenotes of Prof. Antonio C. Pascua,
RMT,
AcuñaMSMT – OLFU VALENZUELA
J., Largueza J.M. – TRANSCRIBER
[HEMA311] 2.03 Erythrocyte Sedimentation Rate I Prof. Antonio C. Pascua, RMT, MSMT

Acuña J., Largueza J.M. – TRANSCRIBER

 OLFU WHITE BLOOD CELL COUNT
2021 – 2022
LAB 8
RMT 2023
CLINICAL HEMATOLOGY 1
1st Semester
Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT
Date: December 7 , 2021 TRANS 8 HEMA311
LAB

Outline **Thoma pipette is also essential in manual cell counting procedures to

At the end of the session, the student must be able to learn: accurately dilute blood specimens with fluids needed for specific cell
A. WHITE BLOOD CELL COUNT counting procedures. A white cell pipet is the thoma pipet used in white
B. MATERIALS NEEDED (MANUAL) cell counting. A red cell pipet can contain 101units of volume whereas
a. Hemacytometer white cell pipet can contain 11units.
b. 1-3% Acetic Acid
c. 1 % HCL
d. Turk’s diluting fluid
C. PROCEDURE
D. POST LABORATORY
E. SOURCES OF ERRORS

I. WHTE BLOOD CELL

● The number of WBC’s in 1 liter or 1microliter of blood

(regardless if granulated or agranulated) WBC count
accounts for all the white cells. In manual counting it is
hard to identify which is which.
● Utilizes to indicate infection, during infection physiologically
normal na yung wbc count is increased good sign since your
body is helping you to eliminate infections.
● In manual white blood cell counting, you have to know the
terms, leukocytosis and leukopenia, which is the elevated
number of WBC above the normal range and the reduced
number of WBC, respectively. People with leukopenia has
higher risk of infection,.

II. MATERIALS NEEDED (MANUAL)

**COMPLICATED AND SUBJECTIV
Hemacytometer
Thoma Pipet
Suction device Primary square: 2
Thick coverslip Each primary square has 9 SECONDARY SQUARE
Cell Counter The four corners of the secondary square is for WBC
Diluting fluids WBC secondary square has 16 TERTIARY SQUARE.
For RBC in the center has 25 TERTIARY SQUARE
1. HEMACYTOMETER Each 25 TERTIARY SQUARE has 16 QUATERNARY SQUARE.
More convenient, accurate, organize **each secondary square has 1mm x 1mm ➔ Total of 9mm2
•“Heart of manual cell count”
•Different Types
- Improved Neubauer 2. WBC DILUTING FLUID

- Neubauer • 1-3% ACETIC ACID

- Fuchs –Rosenthal
• 1% HCl

- Speirs–Levy -Tuerk’s
• TURK’S DILUTING FLUID IS MADE UP OF:
- Acetic acid
- Bass –Jones
- Distilled water
** The most commonly used counting chamber is the Levy chamber with
improved Neubauer ruling. - Gentian Violet Stain
***ISOTONIC solution (RBC)
***HYPOTONIC (WBC) red cells must be lysed

III. PROCEDURE

Largueza, J.M -- TRANSCRIBER

[HEMA311]3.01 White Blood Cell Count Prof. Antonio C. Pascua, Jr., RMT, MSMT

***use of Turk’s Diluting Fluid

You may use this as a supplement reference in the WHITE BLOOD CELL
COUNT Procedure

https://fankhauserblog.wordpress.com/1980/02/01/white-blood-cell-count/

V. SOURCES OF ERRORS AND COMMENTS

USE L RULE, count all WBC’s inside the 4 secondary squares
Dust and fingertips may cause difficulty in
distinguishing the cells
Diluting fluid should be free ofcontaminants
Of the count is low, a greater area may be counted
Chamber must be charged properlyto ensure accurate
count
Allow cell to settle for 10 mins before counting
***The accuracy of the manual WBC count can be assessed by
performing a WBC estimate on a Wright-stained peripheral blood film
made from the same specimen

N-RBC’s

Falsely counted as WBC

Not lysed by WBC diluting fluids

USE HPO to confirm the WBC Correct the WBC Count by dividing uncorrected WBC x100 by the
number of nRBC per 100 WBC + 100
IV. POST LABORATORY
VI. NORMAL VALUES

•4.0-11.0 x 109/L
•at birth: 10.0-30.0 x 109/L

EXAMPLE

Multiple to 0.001 to convert to the SI Unit

Largueza, J.M -- TRANSCRIBER

[HEMA311]3.01 White Blood Cell Count Prof. Antonio C. Pascua, Jr., RMT, MSMT

Leukocytosis Leukopenia

● Bacterial ● Viral infections(HIV)

infection ● Brucellosis
● Appendicitis ● Typhoid fever
● Leukemia ● Rheumatoid
● Pregnancy arthritis
● Uremia ● Cirrhosis
● Ulcers ● Typhoid and
paratyphoid
** in most cases sa infection, inc ● Anemia:
ang wbc.
- Aplastic anemia
● Exudates: - Megaloblastic
anemia
- Empyema /
pyothorax or ● Hypersplenism
purulent pleuritis ● Drugs:
- Tuberculosis - cytotoxic drugs
- Malignant ● Radiation
neoplasms ● Rarely
- Lymphoma leukemia
- Mesothelioma ● transudates:
- Pancreatitis - Congestive
heart failure
- Rheumatoid
arthritis - Cirrhosis with
ascites

ADDITIONAL NOTES

Whole blood anticoagulated with

ethylenediaminetetraacetic acid (EDTA) or blood from a
skin puncture is diluted with 1% bufferedammonium
oxalate or a weak acid solution (3% acetic acid or 1%
hydrochloric acid).

Alternately, a 1:100 dilution may be used counting the

number of cells inthe entire counting area (9 large squares,
9 mm2) on both sides of the chamber (Table 14-1). As an
example, if an average of 54 cells were counted in the
entire counting area on both sides of the chamber
- Cell count:
** 1% ammonium oxalate. Df is 1:20,area counted is
4mm2
** 1% ammonium oxalate. Df is1:100, area counted is
9mm2

- Extremely elevated total leukocyte(WBC) counts of 50.0 ×

109 /L or higher are consistent with a diagnosis of
empyema.

- In general, WBC counts less than

1.0 × 109 /L are associated withtransudates, and WBC
counts greater than 1,000 × 109 /L are associated with
exudates.

Largueza, J.M -- TRANSCRIBER

 OLFU WHITE BLOOD CELL DIFFERENTIAL
2021 – 2022
COUNT LAB 9
RMT 2023 CLINICAL HEMATOLOGY 1
1st Semester

Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT

TRANS 9 HEMA311
LAB
Date: December 14 , 2021

Outline
At the end of the session, the student must be able to learn:
A. LEUKOCYTE DIFFERENTIAL COUNT 7. After staining, rinse the smear with neutral pH distilled
B. MATERIALS water
C. PROCEDURE IN SMEAR PREPARATION 8. Wipe the back of the slide. Air dry the slides
D. STAINING PROBLEMS
E. DIFFERENTIAL COUNT 9. As drying the stained smear, make sure it is in vertical
F. ABSOLUTE COUNT position

STAINING OF SMEARS

 Before staining, make sure that the slide is dry

A. LEUKOCYTE DIFFERENTIAL COUNT  Stains:
o Pure Wright Stain
• The white blood cell differential count determines the o Wright-Giemsa stain
number of each type of white blood cell, present in the
blood.  Purpose: make the cells more visible and to allow their
morphology tobe evaluated
USES OF BLOOD SMEAR EXAMINATION
 Fixative: Methanol
 pH: 6.4 – 6.8; staining is always pH dependent
 Used for WBC Differential Count
WEDGE TECHNIQUE: easiest way
 Can also count WBC but for estimation only
Too much blood may result to thick smear
 Platelet estimation >3 mm in diameter: thick smear : falsely elevated
Estimate means inaccurate value.

EDTA: specimen of choice because it preserves the cell THICK THIN

morphology
OTHER SPECIMEN: skin puncture is also applicable but for small Pressure Decrease increase
amounts of blood only.
FRESHLY COLLECTED BLOOD: within 2 hours, smears must be Angle (30 - 45 increase decrease
prepared. degree)

PLATELET SATELLITISM: platelets adhere at the surface of Speed Increase (short and decrease
neutrophils due to the used of EDTA and the antibodies may result dark red)
in adhesion
CITRATE: specimen of choice for platelet counting. Specimen >3mm increase decrease
Only used citrate if there is presence of satellitism
If there is presence of increase in Hct : thin smear should be
prepared
B. MATERIALS THICK AREA: cells are clumping
THIN AREA: cells are deteriorated
• Cell counter THICK TO THIN AREA SMEAR TRANSITION: cells are equally
• Glass slides distributed
• Microscope As much as possible do not allow the end of the blood touch
• Cedar wood oil the sides of the slide
• Spreader slides
• Wright’s stain

C. PROCEDURE IN SMEAR PREPARATION

STEPS IN PREPARING SMEAR
1. After preparing the smear, fix it with methanol for 2-3
minutes
2. Allow to dry
3. Proceed with staining, flood the smear with Wright or
Wright-Giemsastain for atleast 3 minutes
4. After 3 minutes, add the buffer.
5. Allow to buffer to stay for around 3 minutes
6. Mixing of the buffer and stain will produce green-metallic
sheen

Acuña J, Largueza, J.M -- TRANSCRIBER

 D. STAINING PROBLEMS
TOO ACIDIC STAIN TOO ALKALINE STAIN
1. Thin blood smear 1. Thick blood smear
2. Insufficient staining time 2. Prolonged staining
3. Prolonged buffering or 3. Insufficient washing
washing 4. Alkaline
4. Old stain pH of
stain
compone
nts

MACROSCOPIC EXAMINATION

HEPARINIZED BLOOD SAMPLE: imparts bluish discoloration in

smear background
GRAINY: RBC Agglutination
: Cold agglutinin: Anti-I Antibody
Figure 1-3: Examples of the Blood Smear
MICROSCOPIC EXAMINATION

Thick smear: overlapping of cells

High Power Objective (HPO) Field: specific area
: 10 HPO (white cells count estimation)

Computation
Average count cells x 2,000
SI unit : multiply to 0.001 x10^9/L

Oil Immersion Objective: used for white cells differential counting

: Platelet estimate: 10 OIO field

Figure 4: Observing direction of the blood smear. Computation

Average count x 20,000
BLOOD SMEARS UNDER MICROSCOPE Cells/uL x0.001 x10^9/L
Minimum white cell count must be 100 but due to some
condition and hindi naabot yung 100, you can lower it down to
50
Battlement technique : zigzag like : performed it on the part where
cells are equally distributed

E. DIFFERENTIAL COUNT

 One hundred WBCs are counted and classified through

the use of push-down button counters
 Results are reported as percentage
FEATURES OF A WELL-MADE WEDGE PERIPHERAL BLOOD  Principle: a stained smear is examined to determine the
FILM percentage of each type of leukocyte present and assess
1. The film is two thirds to three fourths the length of the slide the erythrocyte and plateletmorphology.
2. The film is finger shaped, very slightly rounded at the  Manual : 100 WBC
feather edge, notbullet shaped; this provides the widest  The more white cells counted, the more cells to be
area for examination differentiated
3. The lateral edges of the film are visible  Eosinopenia and Basopenia: technically they don’t
exist because basophil and eosinophil are normal in
4. The film is smooth without irregularities, holes or streaks
low count
5. When the slide is held up to the light, the thin portion  MANNER OF REPORTING : % (percent) : relative WBC
(feather edge) ofthe film has a “rainbow” appearance.
differential count
6. The whole drop of blood is picked up and spread.
7. There is a transition from thick to thin portion (area
where cells areequally distributed

Acuña J, Largueza, J.M -- TRANSCRIBER

[HEMA311]3.02 White Blood Cell Count Differential Count Prof. Antonio C. Pascua, Jr., RMT, MSMT

Figure 5: Comparison of Normal WBC in the blood

F. ABSOLUTE COUNT

Computation
% relative count X WBC count

Unit: x10^9/L

**** Sum of absolute count must be equal to WBC

count
Scanner and LPO: coarse adjustment
OIO and HPO: fine adjustment

Addition of oil must be in between OIO and HPO

objective (no need to bring down the film/stage

EXAMPLE ABSOLUTE BLOOD COUNT

 WBC absolute count:
o WBC count = 6 x 109/L
o Multiply the relative count with the
WBC count:
o Neutrophil: 50% x 6 = 3x109/L
o Lymphocyte: 34% x 6 = 2.04x109/L
o Monocyte: 10% x 6 = 0.6x109/L
o Eosinophil: 5% x 6 = 0.3x109/L
o Basophil: 1% x 6 = 0.06x109/L
 The sum of the values should and must be
equal to the value of the WBC count

NOTE: The Trans might be different from the

discussion of Sir Anton since no handouts was
uploaded in the Canvas. Reading the book or other
reference is highly recommended. Thank You and
Happy Holidays

Largueza, J.M -- TRANSCRIBER