[TRANS] LESSON I: SAFETY IN HEMATOLOGY

LABORATORY

SAFETY PRECAUTIONS
• Exposure to blood and body fluids is the most common risk
associated in hematology laboratory.
• Bloodborne pathogens are pathogenic microorganisms
present in blood causing infection or diseases.
occupational safety health administration

OSHA STANDARDS
• OSHA provides standards to maintain safe work
environment.
• The following practices are enforced inside the
laboratory:
1. Handwashing.
2. Food, drink and medications not allowed.
3. Applying cosmetics are prohibited. IN CASES OF FIRE: RACE
4. Fomites or any surfaces must be kept away from mouth
and all mucous membranes.
5. Contaminated sharps must be disposed properly.
6. Personal Protective Equipment must be worn at all
times following the proper donning.
7. Equipment should be check and maintained.

HANDWASHING
• wash your hands before: entering workplace, handling
equipment, before filling up napkin dispensers, eating
• wash your hands after: going to toilet, meal, smoking,
cleaning, handling wastes, removing gloves, touching parts
of the body, every patient interaction, handling chemicals
• How to wash your hands?
o Turn on tap, wet hands with warm water then apply
antimicrobial soap, lather and rub at least for 20 HOW TO USE A FIRE EXTINGUISHER: PASS
seconds.
o Clean each nail, between each finger, front and back
of the hands up to the wrist then rinse off soap using
water pointing downwards.
o Dry hands using disposable paper towel.
o Turn off the water tap using another disposable paper
towel.

OCCUPATIONAL HAZARD

•
untoward circumstance that may take place in
hazard a given setting.
• examples: exposure to pathogens, calamities
risk • likelihood of something to happen.
NOTE: We can’t eradicate hazards but we can lower the risk. CHEMICAL HAZARD
• Labelling of all chemicals properly.
FIRE HAZARD • Follow handling, storage requirements.
• Enforcement of a non-smoking policy. • Use adequate ventilation.
• Placement of fire extinguishers every 75 feet, checked • Spill response procedures should be included in the safety
monthly and maintained annually. procedures.
• Placement of fire detection system and manual fire alarm • MSDS should be available and reviewed by laboratory
near exit doors which is less than 200 ft away and should personnel.
be tested every three months.
• Written fire prevention and response procedures and fire COLOR CLASSIFICATION
drills. yellow reactivity
• Location of fire extinguisher should be always visible and white specific hazard
easy access. blue health hazard
red fire hazard

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 TRANS: Hematology

GOVERNMENT REGULATORY AGENCIES

• Department of Labor: 29 Code of Federal Regulations
Parts 1900-1910
o Hazard Communication Standard
o Hazardous Waste Operations
o Occupational Exposure to bloodborne pathogens
Standards
• Department of the Interior, Environmental Protection
Agency: 40 Code of Federal Regulations Parts 200-399
o Clean Air Act and Clean Water Act
o Toxic Substances Control Act
o Comprehensive Environmental Response,
Compensation and Liability Act (CERCLA)
• Voluntary Agencies/Accrediting Agencies
o The Joint Commission
o College of American Pathologist
o Centers for Disease Control and Prevention (CDC)
o Clinical and Laboratory Standards Institute

ELECTRICAL HAZARD
• Use of adapters, gang plugs and extension cords are
prohibited.
• Stepping on cords, rolling heavy equipment over cords
should be prohibited.
• Before repair or adjustment of electrical equipment, unplug
first the equipment making sure that the hand is dry and no
jewelry should be present.
• Grounding.
• Never use frayed wirings.

NEEDLE PUNCTURE
• Containers should be puncture proof.
• Improper disposal is the major cause of needle prick
incident.
• Replaced once the container is ¾ full.

WASTE DISPOSAL STANDARD

BLOOD CONTAINING WASTE

• Objects contaminated with blood should be autoclaved
before disposal.
• Blood should be treated before disposal; treatment involves
the use of aldehyde, chlorine compounds, phenolic
compounds or thru autoclaving before pouring down the
sink with running water.

COLOR TYPE OF WASTE

yellow infectious and pathologic wastes
green non-infectious wet wastes
black non-infectious dry wastes
red sharps and pressurized containers
violet pharmaceutical waste
orange radioactive waste

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 TRANS: Hematology

[TRANS] LESSON II: GENERAL BLOOD GAUZE PADS

COLLECTION EQUIPMENT AND SUPPLIES • a loosely woven cotton fabric that is applied with pressure
to the arm immediately upon withdrawal of the needle
BLOOD DRAWING STATION following venipuncture.
• dedicated area of a medical laboratory or clinic equipped for
performing phlebotomy procedures on patients, primarily ADHESIVE BANDAGE
outpatients sent by their physicians for laboratory testing. • placed over the puncture site to stop the bleeding, when a
patient has fragile skin, such as small children or the elderly,
PHLEBOTOMY CHAIRS adhesive should not be placed directly on the skin.
• should be comfortable for the patient and have adjustable • patients should be advised to hold the arm straight, apply
armrests to achieve proper positioning of either arm. pressure for 3 to 5 minutes, and remove the bandage in 15
• have adjustable armrests that lock in place to prevent the to 20 minutes.
patient from falling should fainting occur.
NEEDLES
EQUIPMENT CARRIERS • phlebotomy needles must be sterile, disposable, and
• make blood collection equipment portable designed for single use only.
• important in a hospital setting and other instances in which • parts of a needle:
the patient cannot come to the laboratory o hub/plastic section
• examples: handheld carriers, phlebotomy carts equipment ▪ end that attaches to the blood collection
device
GLOVES o shaft
▪ long cylindrical portion
• are available in a variety of materials and in many sizes and
o lumen
styles.
▪ internal space of the needle
• types: latex (powdered or non-powdered), nitrile,
o bevel
neoprene, polyethylene, synthetic vinyl
▪ allows needle to easily slip into the skin and
• IMPORTANT NOTE: vein
o non-sterile gloves can be used for phlebotomy
• needles vary in length and diameter/gauge.
procedures.
• IMPORTANT NOTE:
o powder in gloves can contaminate samples; powder-
o Phlebotomist will select the appropriate size needle for
free (OSHA recommended).
each venipuncture, based on the physical
o patients with latex allergies: a different type of glove
characteristics of the patient’s vein and the volume of
must be used
blood required for the ordered test(s).
o changed after each patient (single use).
• needle gauge
o number indicated by a number that is related to the
TOURNIQUET
diameter of the lumen.
• a device that is applied or tied around a patient’s arm prior
o relationship is indirectly proportional (the higher the
to venipuncture to restrict blood flow; causes venous filling
number the smaller the diameter of the needle).
and stretches vein walls making the vein more prominent.
• types: latex, nitrile, vinyl (strap tourniquet)
COMMON VENIPUNCTURE NEEDLE GAUGE WITH
• IMPORTANT NOTE:
o can be used more than once but is not recommended NEEDLE TYPE AND TYPICAL USE
(disinfected with 70% alcohol or 10% bleach). GAUGE NEEDLE TYPE TYPICAL USE
o discarded if dropped or contaminated with blood or special needle collection of donor units,
other visible components. 15-17 attached to autologous blood donation,
o patients with latex allergies: use a different type of collection bag and therapeutic phlebotomy
tourniquet used primarily as a transfer
needle rather than for blood
18 hypodermic
ANTISEPTICS collection: safety issues
• are substances used to prevent sepsis (microorganisms or have diminished use
their toxic products within the bloodstream). sometimes used when
• prevent or inhibit the growth and development of large-volume tubes are
microorganisms but do not necessarily kill them. multi-sample collected or large-volume
20
• most common antiseptic: 70% isopropyl alcohol hypodermic syringes are used on
• other antiseptics: patients with normal-size
o 70% ethyl alcohol veins
o Benzalkonium chloride considered the standard
o Chlorhexidine gluconate venipuncture needle for
o Hydrogen peroxide (H2O2) multi-sample routine venipuncture on
21
o Povidone–iodine (0.1%–1% available iodine) hypodermic patients with normal veins or
o tincture of iodine for syringe blood culture
o alcohol pads, hand sanitizers collection
• IMPORTANT NOTE: used on older children and
o disinfectants multi-sample adult patients with small
22
▪ chemical substances or solutions that are hypodermic veins or for syringe draws
used to remove or kill microorganisms on on difficult veins
surfaces and instruments. used on the veins of infants
23 butterfly and children and on difficult
or hand veins of adults

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 TRANS: Hematology

• types of needles: • IMPORTANT NOTE:

o hypodermic needles o most commonly used AC in the hematology section;
▪ attached to a syringe. preserve cellular morphology and prevents platelet
▪ length: 1-1.5 in aggregation (for complete blood count)
o multi-sample needles o excess EDTA: will cause platelet satellitism
▪ used in evacuated tube systems (ETS); allow
multiple tubes of blood to be collected during CITRATES
a single venipuncture. • inhibits clotting by chelating or binding calcium.
▪ length: 1-1.5 in
• most common is: sodium citrate
o winged-infusion/butterfly needles
• sodium citrate
▪ consists of a needle attached to tubing with a
o 3.2% buffered sodium citrate (light blue top)
connector on the end.
o 3.8% buffered sodium citrate (black top tube)
▪ length: ½ in
• safety features:
LIGHT BLUE TOP BLACK TOP
o must provide immediate permanent containment and
TUBE TUBE
be activated using one hand, which must stay behind
(coagulation (original method
the needle at all times.
testing) of ESR)
o includes resheathing devices, such as shields that
cover the needle after use; blunting devices; and Concentration 3.2 % g/dL 3.8% g/dL
equipment with devices that retract the needle after Molarity 0.109 M 0.129 M
use. Blood to AC
9:1 4:1
Ratio
TUBE HOLDERS/BARREL/ADAPTER
• used in ETS (evacuated tube system); together with a multi- • IMPORTANT NOTE:
sample needled. o underfilled tubes: prolonged result
o require immediate mixing after collection to prevent
• clear, plastic, disposable cylinder with a small threaded
activation of the coagulation process and micro-clot
opening at one end (often also called a hub) where the
formation, which invalidates test results.
needle is screwed into it and a large opening at the other
end where the collection tube is placed. The large end has
flanges or extensions on the sides that aid in tube HEPARIN
placement and removal. • both an in vivo (naturally occurring) and in vitro
anticoagulant.
EVACUATED TUBES • acts as a co-factor of: anti-thrombin
• are used with both ETS and the syringe method of obtaining • inhibits clotting by preventing activation of thrombin.
blood specimens. • concentration: 0-2 mg/dL or 15-20 mL
• either glass or plastic (plastic is recommended for safety
reasons). OXALATES
• comes in varieties of sizes and colors (tube stopper color • inhibits clotting by precipitating calcium (formation of
depends on the additive present and type of test). calcium oxalates)
• size: 2 mL to 15 mL • most common is: potassium oxalate – added to tube with
• filled with “vacuum”/negative pressure – allows tube to sodium fluoride (gray stopper)
be filled automatically • concentration: 1-2 mg/mL
• IMPORTANT NOTE: • double balanced oxalate (2:3 parts)
o premature loss of vacuum can occur from: proper o potassium oxalate – shrinks cells (invented by Paul
storage, opening of the tube, dropping of the tube, Heller)
advancing the tube too far onto the needle o ammonium oxalate – swells cells (invented by
venipuncture, bevel becomes partially out of vein Wintrobe)
o premature loss of vacuum can lead to: • IMPORTANT NOTE:
o an underfilled tube may affect testing. o oxalates distorts cellular morphology, RBC’s become
o always check expiration date of evacuated tube prior to crenated.
blood collection.
ANTIGLYCOLYTIC AGENTS
BLOOD COLLECTION ADDITIVES
• substance that prevents glycolysis, the breakdown or
metabolism of glucose (blood sugar) by blood cells.
ANTICOAGULANTS • most common is sodium fluoride – maybe used in
• substances that prevent clotting of blood. combination with potassium oxalate (gray top tube).
• most act by inhibiting the action of: calcium • if glycolysis is not prevented, the glucose concentration in a
blood specimen decreases at a rate of 10 mg/dL per hour.
EDTA (ETHYLENEDIAMINETETRAACETIC ACID) • concentration: 10 mg/mL
• inhibits clotting by chelating or binding calcium (calcium • tubes with antiglycolytic agents:
salts).
• types:
o Di Potassium EDTA (K2)
o Di Sodium EDTA (Na2)
o Tri Potassium EDTA (K3)
• concentration: 1.5 mg/mL

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 TRANS: Hematology

CLOT ACTIVATORS
• is a substance that enhances coagulation in tubes used to
collect serum specimens.
• enhances platelet activation.
• examples:
o silica particles
o serum separator tubes (SST); blood clots 15-30 mins
o thrombin- 5 mins
o cellite, ellagic acid, diatomite, kaolin

THIXOTROPIC GEL
• is an inert (non-reacting) non-synthetic substance initially
contained in or near the bottom of certain blood collection
tubes.
• moves between the cells and the serum or plasma after
centrifugation.
• found in serum separator tubes (SST).

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 TRANS: Hematology

TUBE STOPPER COLOR AND USE IN THE LABORATORY

COLOR TUBE
ADDITIVE INVERSIONS TESTS IN THE LABORATORY
CODE COLOR
• serum determinations in chemistry;
silicone coated (glass) – no • examples of tests: Glucose, Cholesterol,
0
additive Triglycerides, HDL, Potassium, Amylase,
alkaline phosphatase (ALP), BUN, CK,
RED
liver enzymes
silicone coated (plastic) – clot • serology tests: RPR, Monospot, RF,
5
activator ANA
• blood clotting: 60 minutes
• serum determinations in chemistry; serum
clot activator and gel for serum
GOLD 5 testing (same as red top)
separation (SST)
• blood clotting: 30 minutes

LIGHT lithium heparin and gel for

8 • plasma determination in chemistry
GREEN plasma separation (PST)

thrombin based clot activator

5-6w/o gel: • for STAT serum determinations in
ORANGE with gel for serum separation
8x chemistry
(SST)

clot activator 8
• for trace elements; toxicology, nutritional
ROYAL BLUE
chemistry determinations
K2 EDTA 8

sodium heparin 8 • for plasma determination in chemistry

GREEN • example of tests: ammonia,
lithium heparin 8 chromosome screening

potassium oxalate and sodium

8
fluoride
• for glucose determination/glycolytic
GRAY sodium fluoride/Na2EDTA 8 testing
sodium fluoride (serum tube) 8

TAN K2 EDTA (plastic) 8 • for lead determinations

Sodium Polyanethol Sulfonate • blood culture specimen collections in

8
(SPS) – 0.025% microbiology
YELLOW
• blood bank studies, HLA phenotyping,
Acid Citrate Dextrose (ACD) 8 DNA and paternity testing
• whole blood hematology tests
liquid K3 EDTA (glass) 8 • examples: Complete blood count,
Platelet count, Reticulocyte count, ESR,
LAVENDER
Hemoglobin A1c
spray-coated EDTA K2 EDTA
8 • K2EDTA → routine blood banking tests,
(plastic)
donor screening
K2 EDTA and gel for plasma
WHITE 8 • molecular diagnostic tests, DNA testing
separation (PST)
• whole blood hematology determinations
• routine blood banking testing and donor
screening
PINK spray coated K2 EDTA (plastic) 8
o with special cross-match label for
patient information required by the
AABB
• for coagulation testing (PT and APTT)
buffered sodium citrate 3-4
• CTAD for selected platelet function
LIGHT BLUE
CTAD (Citrate, Theophyline, assays and routine coagulation
3-4 determination
Adenosine, Dipyridamole
RED/LIGHT • use as a discard tube or secondary
none (plastic) 0
GRAY specimen tube

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HEMATOLOGY 1 | HEMA31111LAB
WEEK 3: Capillary Puncture| 1st SEMESTER |Trans 3
TEACHING AND LEARNING ACTIVITIES

• The students will demonstrate the proper

procedures of collecting blood through skin or
capillary puncture. They will be assigned in
pairs and will demonstrate the collection of
blood through capillary puncture in front of
the instructor. The students will answer the
guide questions after the experimentation.

PRE-ANALYTICAL PHASE

• Depending on the laboratory tests to be

performed, small or big amounts of blood are
needed. Few drops of blood are used for tests
such as bleeding and clotting time
determinations, blood typing, blood smear
preparation, etc. which may obtained from a
skin or capillary puncture utilizing the ear lobe
or tip of finger in adults and the big toe or
heel in infants.

ANALYTICAL PHASE CAPILLARY TUBES/MICROHEMATOCRIT TUBES

❖ Materials: ❖ disposable, narrow-bore plastic or plastic-clad

✓ Sterile blood lancet glass capillary tubes that fill by capillary action
✓ 70% Alcohol and typically hold 50 to 75 uL of blood (0.05
✓ Cotton or gauze pad mL – 0.075 mL)
✓ Capillary tube/Microhematocrit tube ❖ Length: 75 mm or 7.5 inches; Internal bore:
✓ Microcollection containers (microtainer) 1.3 mm
✓ Sealant (clay) ❖ Red mark: Heparin (ammonium heparin)
❖ Blue mark: no additive
LANCETS AND INCISION DEVICES

• Sterile, disposable, sharp-pointed or bladed

instrument that either punctures or makes an
incision in the skin to obtain capillary blood
specimens.
• Selection depends on the age of the patient,
collection site, volume of specimen required,
and the puncture depth needed to collect an
adequate specimen without injuring bone.
• Laser lancets
✓ Vaporizes water in the skin to
produce a small hole in the capillary
bed without cauterizing delicate
capillaries.
✓ no risk of accidental sharps injury, and
no need for sharps disposal

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HEMATOLOGY 1 | HEMA31111LAB
WEEK 3: Capillary Puncture| 1st SEMESTER |Trans 3
MICROCOLLECTION CONTAINERS

• Also called microtubes special small plastic

tubes; used to collect the tiny amounts of
blood obtained from capillary punctures
• Often referred to as “bullets” because of their
size and shape

SEALANT

• Plastic or clay sealants that come in small

trays are used to seal one end of
microhematocrit tubes (5-6 mm or 4-6 mm)

PROCEDURE:

1. REVIEW AND ACCESSION TEST REQUEST

2. PATIENT IDENTIFICATION
3. POSITIONING OF THE PATIENT
4. SELECTION OF APPROPRIATE PUNCTURE SITE
❖ Site must be warm, pink or normal
color, and free of scars, cuts, bruises,
or rashes.
❖ It should not be cyanotic (bluish in
color), edematous (swollen), or
infected
❖ Swollen or previously punctured sites
should be avoided, because
accumulated tissue fluid can
contaminate the specimen and
negatively affect test results. Specific
locations for capillary puncture
include fingers of adults and heels of
infants.

PUNCTURE SITES

• ADULTS AND OLDER CHILDREN: (> 1yr old)

✓ Palmar surface of the distal or end
segment of the middle or ring finger

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HEMATOLOGY 1 | HEMA31111LAB
WEEK 3: Capillary Puncture| 1st SEMESTER |Trans 3
of the non-dominant hand (less
calloused)
✓ Site must be perpendicular to the
grooves in the whorls (spiral pattern)
of the fingerprint
• NFANTS/YOUNG CHILDREN: (< 1 yr old)
✓ Medial Lateral plantar surface of the
heel
✓ Puncturing of the bone can cause
osteomyelitis and osteochondritis

5. WARMING OF THE SITE

• Warming the site increases blood flow as
much as seven times (causes arterialization);
important when performing heel stick on
newborns
• IMPORTANT NOTE:
✓ Increased blood flow makes
specimens easier and faster to obtain
and reduces the tendency to
compress or squeeze the site, which
can contaminate the specimen with
tissue fluid and hemolyze red blood
cells
✓ Essential when collecting capillary pH
or blood gas specimens
(arterialization)
• To avoid burning patients, the devices provide
a uniform temperature that does not exceed
42°C
• Warming is done for 3-5 minutes using a
washcloth, towel, or diaper that has been
moistened with comfortably warm water or
using a commercial heel warming device
6. PREPARE PATIENT -> APPLY ANTISEPTIC
7. PUNCTURING THE SITE
• Finger puncture: Grasp the patient’s finger
between your non-dominant thumb and index
finger. Hold securely in case of sudden
movement. Place the lancet device flat
against the skin in the central, fleshy pad of
the finger, slightly to the side of center and
perpendicular to the fingerprint whorls

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HEMATOLOGY 1 | HEMA31111LAB
WEEK 3: Capillary Puncture| 1st SEMESTER |Trans 3
• Heel puncture: Grasp the foot gently but
firmly with your non-dominant hand. Encircle
the heel by wrapping your index finger around
the arch and your thumb around the bottom.
Wrap the other fingers around the top of the
foot. Place the lancet flat against the skin on
the medial or lateral plantar surface of the
heel
• IMPORANT NOTE:
✓ PUNCTURE DEPTH:
8. WIPE FIRST DROP OF BLOOD
• Position the site downward and apply
gentle pressure toward the site to
encourage blood flow. Wipe away the first
drop of blood with a dry gauze pad.
• IMPORTANT NOTE:
✓ First drop is typically contaminated
with excess tissue fluid, and may
contain alcohol residue that can
hemolyze the specimen and also keep
the blood from forming a well-
rounded drop.
9. FILLING AND MIXING OF TUBES/CONTAINERS
• Continue to position the site downward to
enhance blood flow and apply gentle,
intermittent pressure to tissue
surrounding a heel puncture site or
proximal to a finger puncture site
• IMPORTANT NOTE:
✓ Do not use a scooping motion against
the surface of the skin and attempt to
collect blood as it flows down the
finger. Scraping the scoop against the
skin activates platelets, causing them
to clump, and can also hemolyze the
specimen
10. PLACE GAUZE AND APPLY PRESSURE
11. LABEL AND OBSERVE SPECIAL HANDLING
INSTRUCTIONS
12. CHECK THE SITE AND APPLY BANDAGE
• The site must be examined to verify that
bleeding has stopped. If bleeding persists
beyond 5 minutes, notify the patient’s
nurse or physician. If bleeding has
stopped and the patient is an older child
or adult, apply a bandage and advise the
patient to keep it in place for at least 15
minutes.
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SUMMARY:
1. REVIEW AND ACCESION TEST REQUEST
2. PATIENT IDENTIFICATION
3. POSITION THE PATIENT
4. SELECTION OF APPROPRIATE PUNCTURE SITE
5. WARMING OF THE SITE
6. PREPARE EQUIPMENT
7. PUNCTURING THE SITE
8. WIPE FIRST DROP OF BLOOD
9. FILLING AND MIXING OF TUBES/CONTAINERS
10. PLACE GAUZE AND APPLY PRESSURE
11. LABEL AND OBSERVE SPECIAL HANDLING
INSTRUCTIONS
12. CHECK THE SITE AND APPLY BANDAGE
ADDITIONAL NOTES:
CAPILLARY PUNCTURE
• Also called as: Dermal puncture, skin
puncture
• Procedure in which the skin is punctured
with a lancet to obtain blood in the
capillaries/capillary bed in the dermal
layer of the skin for laboratory testing
• COMPOSITION OF CAPILLARY BLOOD:
✓ Arterial blood, Venous blood,
Interstitial/Tissue fluid
✓ Increased: Glucose, WBC
✓ Decreased: total proteins, calcium,
potassium, hemoglobin, hematocrit,
platelets
✓ IMPORTANT NOTE: hemolysis and
tissue fluid contamination can
increase potassium levels (potassium
is inside RBC’s)
• Blood collection preferred for: infants, very
small children, elderly, obese, or severely
burned patients
• Is employed if the test requested requires a
small amount of blood
• IMPORTANT NOTE:
✓ Capillary specimen collection is
especially useful for PEDIATRIC
PATIENTS in whom removal of larger
quantities of blood can have serious
consequences
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HEMATOLOGY 1 | HEMA31111LAB
WEEK 3: Capillary Puncture| 1st SEMESTER |Trans 3
INDICATIONS OF CAPILLARY PUNCTURE:
• For adults and older children (>1 yr old):
✓ Available veins are fragile or must be
saved for other procedures such as
chemotherapy
✓ Several unsuccessful venipunctures
have been performed and the
requested test can be collected by
capillary puncture
✓ The patient has thrombotic or clot-
forming tendencies
✓ The patient is apprehensive or has an
intense fear of needles
✓ There are no accessible veins (e.g. the
patient has IVs in both arms or the
only acceptable sites are in scarred or
burned areas)
✓ To obtain blood for POCT procedures
such as glucose monitoring
• For infants and very young children (< 1 yr
old):
✓ Infants have a small blood volume;
removing quantities of blood typical
of venipuncture or arterial puncture
can lead to anemia. According to
studies, for every 10 mL of blood
removed, as much as 4 mg of iron is
also removed
✓ Large quantities removed rapidly can
cause cardiac arrest. Life is
threatened if more than 10% of a
patient’s blood volume is removed at
once or over a short period
(iatrogenic anemia) -> doctor’s fault
✓ Obtaining blood from infants and
children by venipuncture is difficult
and may damage veins and
surrounding tissues.
✓ Puncturing deep veins can result in
hemorrhage, venous thrombosis,
infection, and gangrene.
✓ An infant or child can be injured by
the restraining method used while
performing a venipuncture.
✓ Capillary blood is the preferred
specimen for some tests, such as
newborn screening tests
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CAPILLARY PUNCTURE IS NOT APPROPRIATE
WHEN/CONTRAINDICTION:
• Patient is severely dehydrated
• Shock (increased leakage of plasma)
• Poor circulation
• Tests that requires more amount of blood:
ESR, Coagulation studies, blood cultures
• Specimens must be collected quickly to
minimize the effects of platelet clumping and
micro-clot formation and to ensure that an
adequate amount of specimen is collected
before the site stops bleeding. Hematology
specimens are collected first because they are
most affected by the clotting process. Serum
specimens are collected last because they are
supposed to clot. The CLSI order of draw for
capillary specimens is as follows:
✓ Blood gas specimen (sample is arterial
blood)
✓ Slides (unless the specimen is placed
in an EDTA tube)
✓ EDTA microcollection tubes (affected
by clotting process and platelet
aggregation)
✓ Other additives (Heparin)
✓ Serum microcollection tubes (allowed
to clot)
• IMPORTANT NOTE:
✓ Puncture/incision releases tissue
thromboplastin/tissue factor which
activates coagulation
✓ SCOOPING: activates platelet
clumping and hemolysis
✓ EXCESSIVE MILKING -> can cause
hemolysis
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HEMATOLOGY 1 | HEMA31111LAB
WEEK 4: Venipuncture| 1st SEMESTER |Trans 4
PRE-ANALYTICAL PHASE
• Larger amount of blood for chemistry and
other hematological analyses are obtained by
venipuncture from the median basilic or
median cephalic veins of the antecubital fossa
and from the dorsal surface of the hand or
foot in adults or the external jugular vein or
femoral veins in infants.
ANALYTICAL PHASE
• Sterile syringe
• Sterile needle (gauge 21)
• Tourniquet
• Cotton
• 70% Isopropyl alcohol
• Evacuated tubes
• Micropore tape
• Yellow bag
PROCEDURE
1. REVIEW AND ACCESSION TEST REQUEST
2. APPROACH, IDENTIFY, AND PREPARE PATIENT
3. POSITIONING THE PATIENT AND
TOURINIQUET APPLICATION
4. SELECTING THE VEIN
5. CLEAN AND AIR DRY THE SITE
6. PREPARE EQUIPMENT
7. ANCHORING AND NEEDLE INSERTION
8. ESTABLISH BLOOD FLOW, RELEASET
TOURNIQUET, ASK PATIENT TO OPEN FIST
9. FILLING OF TUBES
10. REMOVE NEEDLE AND PLACE GAUZE
11. .DISRARD COLLECTION UNIT, SYRINGE OR
NEEDLES
12. LABELLING OF TUBES
13. OBSERVE SPECIAL HANDLING INSTRUCTIONS
14. CHECK PATIENT’S ARM AND APPLY BANDAGE
REVIEW AND ACCESSION TEST REQUEST
• This is the first step for the laboratory in the
pre-analytical (before analysis) or pre-
examination phase of the testing process
• Test requisition:
✓ form on which test orders are
entered; become part of a patient’s
medical record and require specific
information to ensure that the right
patient is tested, the physician’s
orders are met, the correct tests are
REYNA| MT 3-YA-4
performed at the proper time under
the required conditions, and the
patient is billed properly
• Accessioning the test request
✓ accession is the process of recording
in the order received
✓ When a test request is accessioned it
is assigned a unique number used to
identify the specimen and all
associated processes and paperwork
and connect them to the patient.
APPROACH, IDENTIFY, AND PREPARE PATIENT
• PATIENT IDENTIFICATION most important step
in the venipuncture procedure
✓ Obtaining a specimen from the wrong
patient can have serious, even fatal,
consequences, especially specimens
for type and cross-match prior to
blood transfusion
✓ Misidentifying a patient or specimen
can be grounds for dismissal of the
person responsible and can even lead
to a malpractice lawsuit against that
person.
• IMPORTANT NOTE:
✓ When identifying a patient, ask his or
her full name and date of birth
✓ CLSI recommendation – ask patient
to spell his/her last name
✓ Check the ID band or bracelet if
applicable
✓ 3 way ID – patient’s verbal
statement, checking of the ID band,
visual comparison of the labeled
specimen band before leaving the
bedside
✓ Sleeping patients – wake person
gently; do not startle patient as it will
affect test results
✓ Unconscious patients - Ask a relative
or the patient’s nurse or physician to
identify the patient and record the
name of that person
✓ Infants and children – a nurse or
relative may identify the patient
• PREPARING THE PATIENT
✓ Explain procedure; Never attempt to
explain the purpose of a test to a
patient. Because a particular test can
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be ordered to rule out a number of
different problems, any attempt to
explain its purpose could mislead or
unduly alarm the patient
✓ Addressing needle phobia:
➢ Have the patient lie down
during the procedure, with
legs elevated
➢ Apply an ice pack to the site
for 10 to 15 minutes to numb
it before venipuncture.
➢ Have only the most
experienced and skilled
phlebotomist perform the
venipuncture. 4. SELECTING THE VEIN
✓ Verify diet restrictions and latex
❖ Antecubital fossa – most preferred
allergy
venipuncture site
POSITIONING THE PATIENT AND TOURINIQUET ❖ A patient will generally have the most
APPLICATION prominent veins in the dominant arm
❖ To locate a vein, palpate (examine by touch or
• Inpatients normally have blood drawn while feel) the area by pushing down on the skin
lying down in their beds with the tip of the index finger
• Outpatients at most facilities are drawn while ❖ In addition to locating veins, palpating helps
sitting up in special blood-drawing chairs determine their patency (state of being freely
• TOURNIQUET APPLICATION open), size and depth, and the direction or the
✓ Tourniquet is applied: 3-4 inches path they follow
above the puncture site no longer ❖ Do not select a vein that feels hard and cord-
than 1 minute like or lacks resilience, as it is probably
✓ If it is closer to the site, the vein may sclerosed or thrombosed. Such veins roll
collapse as blood is removed. If it is easily, are hard to penetrate, and may not
too far above the site, it may be have adequate blood flow to yield a
ineffective representative blood sample
✓ Hand veins- the tourniquet is applied ❖ CLSI recommendation - when a tourniquet is
proximal to the wrist bone used during preliminary vein selection, it
✓ When the tourniquet is in place, ask should be released and reapplied after 2
patient to clench or make a fist minutes
✓ IMPORANT NOTE:
➢ Pumping of the fist should be VENIPUNCTURE SITES
prohibited causes veins to
❖ Antecubital Fossa- Antecubital (means
move; changes in blood
front of the elbow), fossa- means a
components
shallow depression
(hemoconcentration,
✓ is the shallow depression in the
potassium and ionized
arm that is anterior to (in front of)
calcium)
and below the bend of the elbow
❖ H-Shaped Antecubital Veins
✓ is displayed by approximately 70%
of the population and includes the
median cubital vein, cephalic vein,
and basilic vein

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✓ Median cubital vein - located
near the center of the antecubital
fossa
➢ preferred vein because it is
typically large, close to the
surface
➢ closer to the surface and the
most stationary
➢ easiest and least painful to
puncture; least likely to bruise
❖ Cephalic vein
➢ Located in the lateral aspect
(outer side) of the antecubital
area; second choice
➢ Often harder to palpate than
medical cubital vein
➢ Fairly well anchored
➢ Often the only vein felt in obese
patients
❖ Basilic vein
➢ located on the medial aspect
(inner side) of the antecubital
area; last choice
➢ Not well anchored and rolls easily
➢ Increased risk of puncturing a
median cutaneous nerve branch
or the brachial artery
➢ Not recommended unless no
other vein in either arm is more
prominent
❖ M-Shaped Antecubital Veins
➢ Median vein/intermediate
antebrachial vein
➢ Median cephalic
vein/intermediate cephalic vein 5. CLEAN AND AIR DRY THE SITE
➢ Median basilic vein/ intermediate
• The Recommended antiseptic for cleaning a
basilic vein
venipuncture site is 70% isopropyl alcohol,
❖ OTHER SITES:
which is typically available in sterile,
➢ Great Saphenous vein
prepackaged pads referred to as alcohol prep
➢ Femoral vein
pads.
➢ Jugular vein
• Clean the site with a circular motion, starting
at the point where you expect to insert the
needle, and moving outward in ever-widening
concentric circles (circles with a common
center) until you have cleaned an area
approximately 2 to 3 inches in diameter
• IMPORTANT NOTE:
✓ Allow site to dry naturally, do not dry
the alcohol with unsterile gauze, do
not fan or blow the site

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✓ The evaporation and drying process holder reflux (flow of blood from the tube
helps destroy microbes, prevents back into the vein) and a possible adverse
specimen hemolysis from alcohol patient reaction from additives can occur if
contamination, and avoids a burning tube blood is in contract with the needle.
sensation when the needle is inserted

6. PREPARE EQUIPMENT

7. ANCHORING AND NEEDLE INSERTION

• To anchor antecubital veins, grasp the

patient’s arm with your free hand, using your
fingers to support the back of the arm just
below the elbow.
• Place your thumb a minimum of 1 to 2 inches
below and slightly to the side of the intended
venipuncture site and pull the skin toward the
wrist
• The bevel of the needle should be facing up;
Position the needle above the vein so it is
lined up with it and paralleling or following
its path
• ANGLE OF INSERTION: 15-30 degrees
(depending on the depth of the vein)

8. ESTABLISH BLOOD FLOW, RELEASET TOURNIQUET,

ASK PATIENT TO OPEN FIST

• To establish blood flow when using the ETS

system, the collection tube must be advanced
into the tube holder until the stopper is
completely penetrated by the needle
• If the vein has been successfully entered,
blood will begin to flow into the tube. If you
are using a syringe, a flash of blood in the
syringe hub indicates that the vein has been
successfully entered
• Blood flow into the syringe is achieved by
slowly pulling back on the plunger with your
free hand

9. FILLING OF TUBES

• Following the order of draw, place ETS tubes

in the holder and advance them onto the
needle. ETS tubes fill automatically until the
tube vacuum is exhausted or lost. A syringe is
filled manually by slowly and steadily pulling
back on the plunger until the barrel is filled to
the appropriate level.
• Keep arm in a downward position so that
blood fills ETS tubes from the bottom up and
does not contact the needle in the tube
10. REMOVE NEEDLE AND PLACE GAUZE
• After the last tube has been removed from
the holder or an adequate amount of blood
has been collected (if you are using a syringe),
fold a clean gauze square into fourths and
place it directly over the site where the
needle enters the skin. Hold the gauze lightly
in place but do not press down on it until the
needle is removed.

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• Apply pressure to the site for 3 to 5 minutes
or until the bleeding stops. Failure to apply
pressure or applying inadequate pressure can
result in leakage of blood and hematoma
formation
• Do not ask the patient to bend the arm up.
The arm should be kept extended or even
raised (Ecchymoses/sis)
ADDITIONAL NOTES:
11. DISCARD COLLECTION UNIT, SYRINGE OR
NEEDLES
• OSHA regulations prohibit cutting, bending,
breaking, or recapping blood collection
needles or removing them from tube holders
after use
12. LABELLING OF TUBES
• Tubes must be labeled in the presence of the
patient immediately after blood collection,
never before
• Information included:
✓ Patient’s first and last name
✓ Patient’s identification number
(inpatient) or date of birth
(outpatient)
✓ Date and time of collection
✓ Phlebotomist’s initials
✓ Pertinent additional information, such
as “fasting”
13. OBSERVE SPECIAL HANDLING INSTRUCTIONS
• Place specimens that must be cooled (e.g.
ammonia) in crushed ice slurry
• Put specimens that must be kept at body
temperature (e.g., cold agglutinin) in a 37°C
heat block or other suitable warming device
Wrap specimens that require protection from
light (e.g., bilirubin) in aluminum foil or other
light-blocking material or place them in a
light-blocking container
until it does. If the patient continues
to bleed beyond 5 minutes, the
appropriate personnel such as the
patient’s physician or nurse should
be notified
EVACUATED TUBE SYSTEM
• It is a closed system in which the patient’s
blood flows through a needle inserted into a
vein and then directly into a collection tube
without being exposed to the air or outside
contaminants
• Involves the use of multi-sample needles,
adapter, follows the “order of draw” for
evacuated tubes
• Most common and efficient system that is
preferred by CLSI for collecting blood samples
THE ORDER OF DRAW
• Refers to the order in which tubes are
collected during a multiple-tube draw or are
filled from a syringe. CLSI recommends the
following order of draw for both ETS
collection and in filling tubes from a syringe:
1. Blood culture tubes (yellow stopper)
2. Coagulation tube (light blue stopper)
3. Serum tube with or without activator
(red, gold, red-gray marbled, orange,
yellow-gray stopper)
4. Heparin (green or light green stopper)
5. EDTA tube (lavender or pink stopper)
6. Sodium fluoride with or without EDTA or
oxalate (gray stopper)

14. CHECK PATIENT’S ARM AND APPLY BANDAGE

• Examine the venipuncture site to determine if

bleeding has stopped. (Bleeding from the vein
can continue even though it has stopped at
the surface of the skin)
• Instruct the patient to leave the bandage on
for a minimum of 15 minutes, after which it
should be removed to avoid irritation
• IMPORTANT NOTE:
✓ If bleeding has not stopped, the
phlebotomist must apply pressure

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FAILED VENIPUNCTURE
• If you are unable to obtain a specimen on the
first try, evaluate the problem and try again
below the first site, on the opposite arm, or
on a hand or wrist vein
• If the patient’s veins are small or fragile, it
may be necessary to use a butterfly or syringe
on the second attempt (twice)
• If the second attempt is unsuccessful, ask
another phlebotomist to take over
• Unsuccessful venipuncture attempts are
frustrating to the patient and the
phlebotomist.
PROBLEM SITES DURING VENIPUNCTURE
• BURNS, SCARS, AND TATTOO
➢ Healed burn sites and other areas
with extensive scarring may have
impaired circulation and can
therefore yield erroneous test results.
➢ Newly burned areas are painful and
also susceptible to infection. Tattooed
areas can have impaired circulation,
may be more susceptible to infection,
and contain dyes that can interfere
with testing.
• DAMAGED VEINS
➢ Some patients’ veins feel hard and
cord-like and lack resiliency because
they are occluded or obstructed
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➢ These veins may be sclerosed
(hardened) or thrombosed (clotted)
from the effects of inflammation,
disease, or chemotherapy drugs.
• EDEMA
➢ Edema is swelling caused by the
abnormal accumulation of fluid in the
tissues. It sometimes results when
fluid from an IV infiltrates the
surrounding tissues
• HEMATOMA
➢ swelling or mass of blood (often
clotted) that can be caused by blood
leaking from a blood vessel during or
following venipuncture
➢ Venipuncture through an existing
hematoma is painful and can result in
collection of a specimen that is
contaminated with hemolyzed blood
from outside the vein and unsuitable
for testing
• MASTECTOMY
➢ Blood should never be drawn from an
arm on the same side as a
mastectomy (surgical breast removal)
without first consulting the patient’s
physician.
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➢ Lymph node removal can cause
lymphostasis (obstruction or
stoppage of normal lymph flow).
➢ Impaired lymph flow makes the arm
susceptible to swelling, called
lymphedema, and to infection.
vein by direct fusion (fistula),
resulting in a bulging vein, or
with a piece of vein or tubing
(graft) that creates a loop
under the skin.
✓ typically created to be used
for dialysis, commonly joins
the radial artery and cephalic
vein above the wrist on the
underside of the arm
✓ NEVER APPLY tourniquet or
perform venipuncture on a
fistula

• OBESITY
➢ Veins on obese patients may be deep
and difficult to find
• VASCULAR ACCESS DEVICE (VAD’s AND SITES)
➢ ARTERIAL LINE
✓ is a catheter that is placed in an
artery. It is most commonly placed in
a radial artery and is typically used to
provide accurate and continuous
measurement of a patient’s blood
pressure.
✓ NEVER APPLY tourniquet or perform
venipuncture on an arterial line
➢ ARTERIOVENOUS SHUNT, FISTULA
OR GRAFT
✓ is the permanent surgical
connection of an artery and
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• HEPARIN OR SALINE LOCK

➢ is a catheter or cannula connected to
a stopcock or a cap with a diaphragm
(thin rubber-like cover) that provides
access for administering medication
or drawing blood
➢ IMPORANTANT NOTE: A 5-mL discard
tube should be drawn first when
blood specimens are collected from
either type of device
• INTRAVENOUS SITES
➢ An intravenous line, referred to
simply as an IV, is a catheter inserted
in a vein to administer fluids
➢ It is preferred that blood specimens
not be drawn from an arm with an IV
➢ Collecting blood in an IV LINE: Collect
blood below the IV line; Stop IV line COMPLICATIONS OF VENIPUNTURE
for 2 minutes 1. IMMEDIATE LOCAL COMPLICATION
➢ When both arms are involved in 2. LATE LOCAL COMPLICATION
therapy and the IV cannot be 3. LATE GENERAL COMPLICATION
discontinued for a short time, a site
below the IV line should be sought; IMMEDIATE LOCAL COMPLICATION
the initial sample (5 mL) drawn
• Hemoconcentration
should be discarded. Collection of
➢ is an increase in the number formed
blood below the IV line must be
elements in blood resulting either
written on the laboratory requisition
from a decrease or increase
form to inform the staff in the
(hemodilution) in plasma volume
chemistry section
• Failure of blood to enter the
➢ V FLUID CONTAMINATION: An
syringe/vacutainer tube
increase of infused substances such as
➢ Excessive pull of plunger
glucose, chloride, potassium and
➢ Piercing the other pole of the vein
sodium, with a decrease in urea and
➢ Transfixation of vein (moving of the
creatinine
vein)
➢ Incorrect bevel position (bevel down)
➢ Absence of vacuum
• Syncope (fainting)

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➢ is the transient loss of consciousness
due to lack of oxygen in the brain and
results in an inability to stay in an
upright position
➢ If a seated patient feels faint, the
needle should be removed
immediately, the patient's head
should be lowered between the legs
and the patient should be instructed
to breath deeply

LATE LOCAL COMPLICATION

• Thrombosis
➢ is an abnormal vascular condition in
which thrombus develops within a
blood vessel of the body.
• Thrombophlebitis
➢ is inflammation of a vein often
accompanied by a clot which occurs
as a result of trauma to the vessel wall

LATE GENERAL COMPLICATION

• HEMOLYSIS
➢ Using a needle that is too small
➢ Pulling a syringe plunger back too fast
➢ Expelling the blood vigorously into a
tube
➢ Forcing the blood from a syringe into
an evacuated tube
➢ Shaking or mixing the tubes vigorously
➢ Performing blood collection before
the alcohol has dried at the collection
site
• HEMATOMA
➢ The vein is fragile or too small for the
needle size
➢ The needle penetrates all the way
through the vein
➢ The needle is partly inserted into the
vein
➢ The needle is removed while the
tourniquet is still on.
➢ Excessive probing Pressure is not
adequately applied after venipuncture

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TEACHING AND LEARNING ACTIVITIES

• The students will demonstrate the proper

procedures of red blood cell counting. The
students will answer the guide questions after
the experimentation

PRE-ANALYTICAL PHASE

• The red cell count is the number of red cells in

1 uL or 1 cu.mm. The RBC count is one of the
tests that are used for the diagnosis of anemia
and polycythemia or erythrocytosis and
leukemia.
• Affected in diurnal variation: high in the
morning, low in the evening
RBC DILUTING FLUID
MATERIALS AND EQUIPMENT
❖ Lysis of WBC; ISOTONIC SOLUTION
❖ Anticoagulated blood (EDTA) ❖ TYPES OF DILUTING FLUID:
❖ RBC pipet ➢ Gower’s solution
❖ Diluting fluid (Hayem’s or Gower’s solution) ➢ Hayem’s solution
❖ Tally counter ➢ Toisson’s solution
❖ Counting chamber ➢ NSS or Eagle’s solution
❖ Clean gauze ➢ Bethell’s solution
❖ Test tubes ➢ Strong’s solution
❖ Microscope ➢ Dacie’s/Formol Citrate

RBC PIPETTE WBC PIPETTE

BREAD COLOR RED WHITE

GRADUATIONS Up to 101 mark Up to 11 mark

BULB Larger Smaller

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PROCEDURE:

1. Draw blood up to 0.5 mark using the RBC

pipette.
2. Wipe the outside walls of the pipette with
clean gauze.
3. Dip the pipette into diluting fluid, and then
draw the diluting fluid into the pipette slowly,
until the mixture reaches the 101 mark.
Gently rotate the pipette to ensure a proper
amount of mixing. The dilution is 1:200 (DF:
200)
4. Remove the tubing from the pipette, and then
mix it in a horizontal axis for 5 minutes.
5. Discard the first 3-4 drops of the diluted
sample
6. Charge both sides of the hemacytometer
counting chamber with a drop of the diluted
sample and allow to stand for few minutes.
7. While keeping the hemacytometer in a
horizontal position, place it on the microscope
stage.
8. Using HPO, count the red cells in the 5’R’
squares of the central square.
9. Calculate the number of RBC per liter of each
side of the hemacytometer. Average the
results and report this number.

SQUARE COLOR AREA

RED (LARGE SQUARE) 1mm²
GREEN (WBC SQUARE) 0.0625mm²
YELLOW (RBC QAURE) 0.04 mm²
BLUE (SMALLER RBC SQUARE) 0.0025 mm²

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COMPUTATION
SAMPLE PROBLEM
❖ Adult male patient have an average count of
630 RBCs in both chamber using the standard
RBC squares.
• Calculate for the RBC Count of a newborn:
❖ RBC counted: 800
❖ Squares counted: 5 RBC squares
❖ Aspirated blood up to: 1 mark
general, a 1:20 dilution is made, however
other dilution can be used.
• An increase white blood cell count is called
leukocytosis whereas a decrease in the WBC
count is called leukopenia. An increase or
decreased in the count is associated with
infections and conditions such as leukemia.
Leukocytosis is expected in bacterial
infections such as pneumonia, diphtheria,
meningitis, leukemia and appendicitis.
Leukopenia may be associated with hepatitis,
measles, typhoid fever, disease of lymphatic
system such as Hodgkins disease and Systemic
Lupus Erythematosus.
MATERIALS AND EQUIPMENT
❖ Anticoagulated blood (EDTA)
❖ WBC pipet
❖ Diluting fluid
❖ Tally counter
❖ Counting chamber
❖ Clean gauze
❖ Test tubes
❖ Microscope

CLINICAL SIGNIFICANCE

❖ INCREASED
➢ Polycythemia vera
❖ DECREASED
➢ Anemia
➢ 50 years old and above
➢ Horizontal posture of patient during
extraction
➢ After taking a meal

PRE-ANALYTICAL PHASE

• The white blood cell count represents the

number of white blood cells in 1 uL of whole
blood. Whole blood from a capillary puncture
or anticoagulated blood with EDTA is mixed
with a diluting fluid that contains a weak acid
to lyse the non nucleated red blood cells. In

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ANTICIPATED DILUTION THOMA PIPET
WBC CT
0.1-3 x 109/L 1:10 WBC
3.1-30 x 109/L 1:20 WBC
>30 x 109/ 1:100 RBC
>100 x 109/L 1:200 RBC

WBC DILUTING FLUID

• Lysis of RBC; hypotonic solution (causes RBC

swelling; lysis)
• TYPES OF DILUTING FLUID:
➢ 1-3% glacial acetic acid
➢ 1% hydrochloric acid
➢ 1% ammonium oxalate (also used for
platelet count)
➢ Turks solution

SQUARE COLOR AREA

RBC PIPETTE WBC PIPETTE
RED (LARGE SQUARE) 1mm²
GREEN (WBC SQUARE) 0.0625mm²
BREAD COLOR RED WHITE
YELLOW (RBC QAURE) 0.04 mm²
GRADUATIONS Up to 101 mark Up to 11 mark BLUE (SMALLER RBC SQUARE) 0.0025 mm²
BULB Larger Smaller

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COMPUTATION

SAMPLE PROBLEM

❖ Adult male patient have an average count of

525 WBCs in both chamber using 4 corner
squares. Blood was aspirated up to the 0.5
mark.

• In manual WBC counting, count the cells in

the four corner squares of the • Calculate for the WBC Count:
hemacytometer (area is 4mm2 ) ❖ WBC counted: 200
PROCEDURE: ❖ Squares counted: 4 corner squares
❖ Aspirated blood up to: 1 mark
1. Draw blood up to 0.5 mark using the WBC
pipette.
2. Wipe the outside walls of the pipette with
clean gauze.
3. Dip the pipette into diluting fluid, and then
draw the diluting fluid into the pipette slowly, CORRECTED WBC COUNT
until the mixture reaches the 11 mark. Gently
rotate the pipette to ensure a proper amount ❖ Done due to presence of nRBC’s
of mixing. The dilution is 1:20 (DF: 20) (normoblasts)
4. Remove the tubing from the pipette, and then ❖ CRITERIA:
mix it in a horizontal axis for 5 minutes. ➢ NEWBORNS: >10 NRBC per 100 WBC’S
5. Discard the first 3-4 drops of the diluted ➢ ADULTS: >5 NRBC per 100 WBC’S
sample.
6. Charge both sides of the hemacytometer
counting chamber with a drop of the diluted
sample and allow to stand for few minutes. SAMPLE PROBLEM
7. While keeping the hemacytometer in a
horizontal position, place it on the microscope ❖ Total WBC count (adult): 4.8𝑋109 /𝐿
stage. ❖ Number of nRBC’s: 7
8. Using HPO, count the white bloods cells.
9. Calculate the number of WBC per liter of each
side of the hemacytometer. Average the
results and report this number.

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 TRANS: Hematology
[TRANS] LESSON IV: HEMOGLOBIN
DETERMINATION
PRE-ANALYTICAL PHASE
• Hemoglobin is the main content of the red blood cells
which is responsible for transporting oxygen from the lungs
to the tissues and CO2 from the tissues to the lungs to be
eliminated to the outside.
• Hemoglobin is a conjugate protein that consists of 2
portions, the globin (a simple protein) and heme
(organic compound in iron). Each hemoglobin molecule
consists of the one molecule of globin (four globin
chains) and four molecules of heme.
• Hemoglobin may be found in the blood plasma; such a
condition is referred to as hemoglobinemia. When the free
hemoglobin in the plasma reaches a concentration between
30 and 300 mg/100mL of blood, hemoglobin is detected in
the urine; such a condition is called hemoglobinuria.
• In the normal adult, a hemoglobin concentration is as
follows:
o male: 13.5-17.5 g/dL
o female: 12.0-16.0 g/dL
• Value may vary according to age, sex, and locality.
• Abnormal value as in hyperchromia (increased value) is
found in polycythemia, dehydration and in changing from
low to high altitude.
• Hypochromia (decreased value) is seen in anemia.
METHODS FOR HEMOGLOBIN DETERMINATION
VAN SLYKE METHOD
• principle: gasometric
• determines hemoglobin and oxygen saturation
• hemoglobin concentration is related to oxygen binding
capacity of blood.
• 1 gram of hemoglobin = carries 1.34 mL of oxygen
• uses Van slyke apparatus or Natelson microgasometer
KENNEDY’S AND WONG; ASSENDELT METHOD
• principle: chemical method
• measures iron bounded to hemoglobin
• 1 gram of hemoglobin = 3.47 mg of iron
PHYSICAL METHOD
• applicable only to patients that have normocytic,
normochromic RBCs
• hemoglobin: RBC count is multiplied to 3
• hematocrit: Hemoglobin is multiplied to 3
• +/- 3
• PHYSICAL METHOD (RULE OF THREE)
o RBC COUNT = 5.15 X 1012/ L
o HGB = 15.45 g/dL or 154.5 g/L
o HCT = 46.35% or 0.46 L/L
PORRAS | MT 3 YA-4
COPPER SULFATE METHOD
• principle: gravimetric
• based on the specific gravity of copper sulfate (CuSO4) –
1.053
• semi-quantitative; for mass assays (blood donation
drives/blood-letting)
• drop of blood is placed into the copper sulfate solution and
observed whether it will sink within 15 seconds.
• sink: hemoglobin is the same as the specific gravity of
copper sulfate and has a value of >12.5 g/dl
• float: hemoglobin value is <12.5 g/dl (deferred)
POCT (POINT OF CARE TESTING DEVICES)
• tests that can be done bedside.
DIRECT METHODS (ACID HEMATIN & ALKALI
HEMATIN)
• principle: colorimetric
• uses comparator block with an end color of yellow brown
• acid hematin method: 0.1N HCl (Sahli-Hellige method)
• alkali hematin method: 0.1N NaOH
• ACID HEMATIN METHOD
o hemoglobin is converted to acid hematin with dilute 0.1
N HCl and resulting brownish yellow color produced
upon addition of distilled water is matched with the
color standard in the comparator block.
o materials:
▪ EDTA whole blood specimen
▪ distilled water
▪ 0.1N HCl solution
▪ hemoglobinometer
• comparator block
• Sahli pipette
• Sahli graduated tube
• stirrer
• dropper
o procedure:
1. Introduced 0.1 N HCl solution up to 2 mark of the
Sahli graduated tube.
2. Using the Sahli pipette, draw blood up to 0.02 ml
or 20 uL mark. Observe the same technique as in
the use of WBC or RBC pipette.
3. Dispense the measured volume of the blood into
the graduated tube. Gently draw up and down to
rinse the pipette and also to mix the resulting
colloidal suspension.
4. Thoroughly mix with the use of the stirrer and allow
to stand for 5 minutes.
5. Add distilled water drop by drop, stirring after each
addition and compare with the comparator block.
6. When the resulting solution compares with the
color standard in the comparator block, get the
reading either from the grams or percentage scale.
9
 TRANS: Hematology

7. Write your result. PREPARING THE STANDARD/CALIBRATION

CURVE
INDIRECT METHODS (OXYHEMOGLOBIN METHOD & Hemoglobin
CYANMETHEMOGLOBIN METHOD) blank 5 10 15 20
Concentration (g/dL)
• principle: spectrophotometric Cyanmethemoglobin
0 1.5 3 4.5 6
• oxyhemoglobin method: aqueous ammonium hydroxide Standard (mL)
(540 nm) Cyanmethemoglobin
6 4.5 3 1.5 0
• cyanmethemoglobin method: gold standard for Reagent (mL)
hemoglobin determination
• CYANMETHEMOGLOBIN (HiCN METHOD)
o the cyanmethemoglobin is said to be the method of
choice for hemoglobin determination because:
▪ cyanmethemoglobin is stable in dilutions
▪ cyanmethemoglobin standard are readily
available
▪ all hemoglobin derivatives except
sulfhemoglobin are measured with their
conversion to cyanmethemoglobin
▪ the spectrum of cyanmethemoglobin is such
that other types of spectrophotometers can be
used for reading color intensity produced
o hemoglobin iron is converted from ferrous to ferric state
to form methemoglobin by the action of ferricyanide,
methemoglobin then combines with potassium cyanide
o procedure:
to produce the stable cyanmethemoglobin which is
1. Place 5 cc or 5 mL of Drabkin’s reagent into a test
measured spectrophotometrically at 540 nm.
tube.
o % Transmittance: proportional to hemoglobin
2. Using the Sahli pipette, draw blood to 0.02 cc/mL
concentration
or 20 uL mark. Make sure to wipe the outer wall of
o materials and reagents:
the pipette, and then dispense the blood to the test
▪ EDTA whole blood specimen
tube.
▪ test tubes
3. Gently draw up and down to rinse the pipette and
▪ pipettes
also to mix the resulting colloidal suspension.
▪ spectrophotometer
4. Cover and mix well by inversion. Let it stand for 5
▪ modified Drabkin's reagent
minutes.
▪ cyanmethemoglobin standard
5. Transfer the mixture to a cuvette.
• contains 80 mg/dl of hgb; only 6. Set the spectrophotometer to 100% transmittance
standard used in hematology. at the wavelength of 540 nm, using a
▪ Parafilm cyanmethemoglobin reagent as a blank.
7. Continue reading the patient’s sample, and record
the percentage transmittance.
8. The hemoglobin concentration is obtained from a
calibration curve prepared with the use of
standards. (stable standard cyanmethemoglobin
solution of concentration representing 1:250
dilution of whole blood containing 5, 10, and 15
grams of hemoglobin/mL are available
commercially for calibration purposes and for
periodic check-up on the accuracy of the
colorimeter
o errors in cyanmethemoglobin method:
▪ usually caused by turbidity
Hemoglobin (Fe2+) + K3Fe (CN)6 → methemoglobin (Fe3+) + ▪ Drabkin’s reagent is light sensitive
▪ contains cyanide which is highly toxic
KCN → cyanmethemoglobin
ERROR SOLUTION
COMPONENTS OF THE MODIFIED DRABKIN’S increased WBC
REAGENT count: >20 x 10 9/L • sample must be centrifuged and
COMPONENT USE/FUNCTION increased platelet measure supernatant
Potassium • converts to ferrous (Hgb) to count: >700x 10 9/L
Ferricyanide ferric (Hi) • add 0.01 mL of patient’s plasma
• combines with methemoglobin Lipemia and add to 5 mL of reagent and
Potassium Cyanide use solution as blank
(HiCN)
• replaces potassium carbonate • make 1:2 dilution with distilled
Dihydrogen Potassium Hgb S or C
(orig) for faster reaction (3 water and multiply result from
Phosphate (mod) (resistant to lysis)
mins) the standard curve by 2
• improves cell lysis and • add 0.1 gram of potassium
non-ionic abnormal globulins carbonate (only done in original
decreases turbidity by CHONs
detergents/surfactant Drabkin’s reagent)
(proteins)

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 TRANS: Hematology

[TRANS] LESSON III: HEMATOCRIT PROCEDURE

DETERMINATION 1. Mix the blood
2. With the use of a long stem pipette, fill the Wintrobe
PRE-ANALYTICAL PHASE tube to the 10 mark.
• Hematocrit is often referred to as the packed cell volume 3. NOTE: No air bubbles should be present on the
(PCV), volume of erythrocytes or reading of packed cells surface of the blood, it may be removed by touching it
(pRBC). with a tissue paper or with a pipette. If the bubbles are
• This test measures the proportion of red cells to plasma present in the middle of the tube, aspirate the blood
in the peripheral blood but not in the entire circulation. It and proceed to step no. 2 again.
gives the number of millimeters of packed red blood 4. Centrifuge the blood for 30 minutes.
cells/100 millimeter of blood or simply expressed as volume 5. Read the height of the packed red blood cells on the
percent. scale at the right side of the tube, which is graduated
• reported as: PCV (%) or EVF (Erythrocyte volume fraction) from 0-10 cm from the bottom to top, read upward
(L/L
• normal values: height of packed red cells
o at birth: 45-60% (0.45 – 0.60 L/L) hematocrit (%) = x 100
amount of blood used
o females: 36-48%
o males: 40-55%
• IMPORTANT NOTE:
METHODS FOR HEMATOCRIT DETERMINATION o If the tube has been filled to the tenth mark, the
• macrohematocrit method (manual) 30minutes above calculation maybe eliminated by following
each line or mark on the Wintrobe tube that represents
• microhematocrit method (manual)
as 1% of the hematocrit. Therefore, simply count the
• automated method: hematocrit is computed by (MCV x
number of lines from the bottom of the tube to the level
RBC count)
of the packed red blood cells. The number of lines is
the value of your hematocrit reading.
MACROHEMATOCRIT METHOD
• less commonly used; time consuming. METHOD ANTICOAGULANT
• required increased blood volume; higher amount of trapped
Wintrobe’s double oxalate
plasma.
Van Allen’s sodium oxalate
• centrifugation: 30 minutes at 2,000- 2,300 x g (RCF →
relative centrifugal force) Sanford Magath sodium oxalate
• trapped plasma: Haden’s sodium oxalate
o plasma trapped in the RBC portion after centrifugation Bray’s heparin
o increases hematocrit by: 1-2 or 1-3%
o not affected by automated tests MICROHEMATOCRIT METHOD
• materials: • requires small amount of blood; more commonly used.
o Wintrobe tube with rubber caps • aka: ADAM’s method
o long stem pipette/Pasteur pipette • specimen: whole blood, capillary blood
o centrifuge • sealer/clay: 5-6mm long
o EDTA anticoagulated blood (this procedure needs • centrifugation: 5 mins for 10,000-15,000 x g
about 3.0 mL of whole blood) • materials:
o heparinized capillary tube (red mark/band)
o microhematocrit centrifuge
o microhematocrit reader
o sealant/clay
o lancet

GRADUATION TEST USED

0 top to 10/100 bottom ESR
0 bottom to 10/100 top HCT

WINTROBE TUBE
length 115 mm or 11.5 cm
internal bore 3 mm
external bore
graduation/markings 100 • length: 75 mm/7.5 cm 2/3
• internal bore: 1.2 mm
• can hold up to: 0.05 mL of blood

PORRAS | MT 3 YA-4 7
 TRANS: Hematology

LAYERS OF THE SPUN HEMATOCRIT/CENTRIFUGED

WHOLE BLOOD
PROCEDURE barely visible, increased in
1. After necessary preparations, make a capillary fatty layer (topmost)
lipidemia
puncture and produce a rounded drop of blood. normal: pale yellow
2. In a horizontal position, put one end of capillary tube pink: slight hemolysis
on the drop of blood and fill the tube about 2/3 full or ¾ plasma (second) red: gross hemolysis
full. yellow-brown: increased
▪ NOTE: The tube will be filled by capillary bilirubin/icteric (hemolysis)
action. If using tubes with a colored ring at one <1%
end, fill from opposite end. platelets
3. Seal one end of the capillary tube with the clay by buffy coat (third) leukocytes
placing the dry end of the tube into the clay in a vertical reticulocytes
position. NRBC’s (normoblasts)
4. After sealing with clay, seal it again with wax.
PRBC’S (fourth
5. Assemble the tube in microhematocrit centrifuge in PCV (hematocrit)
layer)
such a way that the unsealed end is nearest the center
of the centrifuge.
6. Spin at 10, 000 to 15, 000 rpm for 5 minutes.
7. Using the microhematocrit reading device, determine
the HCT.
▪ NOTE: Buffy coat should not be included in
the reading (increased hematocrit).
8. The reading on the window of the hematocrit reader
corresponds to the hematocrit value.

ERRORS IN HEMATOCRIT DETERMINATION

INCREASED DECREASED INCREASED DECREASED
• prolonged tourniquet • hemoconcentration
• excessive
application (plasma • dehydration
anticoagulant
leakage, • abnormal RBC • blood loss
(EDTA) (RBC
hemoconcentration) shape (plasma
shrinkage)
• insufficient mixing (RBC • trapped plasma: replaced
TECHNICAL • insufficient PHYSIOLOGIC
first) o Sickle cell faster than
mixing (plasma
• inadequate centrifugation anemia RBC’s)
first)
• prolong standing of the o Spherocytosis • tissue fluid
• improper
tube (processed within 10 o Thalassemia • hemolysis
flushing of IV
mins after centrifugation) o Macrocytic
catheter
• reading of the buffy coat anemias

PORRAS | MT 3 YA-4 8