HEM

311

Introduction to

Hematology
Ma. Christy V. Gonzales, RMT, MPH
College of Medical Laboratory Science
Our Lady of Fatima University- Valenzuela
Hematology 1 (HEM 311)
 HEM
311

Leaning
objective At the end of this session, the students will be able to:
s 1. Describethe characteristics and components of
blood
2. Differentiate the laboratory procedures in
hematology section
3. Explain the specimen collection performed
in hematology
4. Relate the principle of quality assurance to concepts
in hematology

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Hematology

• study of blood cellsby staining, counting analyzing and

recording the appearance, phenotype and genotype of all types of cell
• to predict, detect and diagnose blood diseases and many systemic diseases
that affect blood cells
• to select and monitor therapy for diseases

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Blood
• functions:

• transports O2 from lungs to tissues

• clears CO2 from tissue

• transports biomolecules (proteins,

glucose, fats)

• delivers waste to liver and kidneys

• provides coagulation enzymes

• protect vessels from trauma and

hemostasis

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Blood
• average of 5L
• composition

• plasma

• transports and nourishes

blood cells

• cells

• RBC (erythrocytes)

• WBC (leukocytes)

• platelets (thrombocytes)

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Histor
y
• Athanasius Kircher (1657)
• described “worm” in blood
• Anton van Leeuwenhoek
(1674)
• gave account to
RBC
• Giulio Bizzozero (1880’s)
• described platelets
as “petites plaques”
• James Homer Wright
(1902)
• developed Wright
stain (Wright’s Romanowsky-
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type stain)
Terminologies (prefixes)
PREFIX MEANING
a-/ an- lack, without, absent decreased
aniso- unequal, dissimilar
cyte- cell
dys- abnormal, difficult, bad
erythro- red
ferr- iron
hemo-/ hemato- blood
hypo- beneath, under, deficient, decreased
hyper- above, beyond, extreme
iso- equal, alike, same

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Terminologies (prefixes)
PREFIX MEANING
leuko- white
macro- large, long
mega- large, giant
meta- after, next, change
micro- small
myel/ myelo- from BM or spinal cord
pan- all, overall, all-inclusive
phleb- vein
phago- eat, ingest
poikilo- varied, irregular

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Terminologies (prefixes)
PREFIX MEANING
poly- many
schis- split
scler- hard
splen- spleen
thromb/ thrombo- clot, thrombus
xanth- yellow

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Terminologies (suffixes)
SUFFIXES MEANING
-cyte cell
-emia blood
-itis inflammation
-lysis destruction. dissolving
-oma swelling, tumor
-opathy disease
-penia deficiency
-phil/ -philic attracted to, affinity for
-plasia/ -plastic cell production or repair
-poiesis cell production, formation & development
-poietin stimulates production
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Hematologic Procedures
I. Complete Blood Count
• can be performed by automation
and manual method

• enumeration of cellular elements,

quantitation of hemoglobin and
describes cell appearance

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 Red Blood Cells
• anucleate, biconcave, discoid cells filled with a reddish protein
• site of production:
• appear pink to red cell that measure 6 to 8 um in diameter with a zone of pallor which
occupies 1/3 of their center reflecting its biconcavity
• measurement of volume detects the presence of anemia or polycythemia

• Anemia

• Polycythemia
• RBC count in cells per microliter (uL, mcL, mm3, cc, L)
• first visual RBC counting (1900) but inaccurate
• Joseph and Wallace Coulter of Chicago, Illinois (1953)

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 Red Blood Cells
Hemoglobin
• protein inside the RBC
• reagents: potassium cyanide and potassium ferricyanide
• hgb - stable cyanmethemoglobin
• color intensity of the solution is measured in a spectrophotometer at 540nm
• compared with a known standard and mathematically converted to hemoglobin
concentration
• replace it with ionic surfactant sodium lauryl sulfate

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 Red Blood Cells
Hematocrit
• ratio of the volume of packed RBC’s to the volume of whole blood
• also known as PCV determined by transferring blood to a graduated plastic tube with
a uniform bore
• after centrifugation, measure the column of RBC’s and dividing by the total length of
the column of RBC’s plus plasma.
Buffy coat
• light colored layer between the RBC and plasma
• contains the WBC’s and platelets
• excluded in Hct determination

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 Red Blood Cells
RBC indices
• MCV (Mean Cell Volume)
• reflects RBC diameter
• expressed in fL
• MCHC (Mean Cell Hemoglobin
Concentration)
• reflects RBC staining intensity
and the amount of central pallor
• expressed in g/dL
RBC indices
• MCH (Mean Cell Hemoglobin)
• reflects the mass of
hemoglobin
• expressed in pg
• RDW (RBC distribution width)
• expressed the degree of variation
in RBC volume
• Anisocytosis
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 White Blood Cells
• leukocytes (granulocytes and agranulocytes)
• cells for protection from infection and injury
• site of production: bone marrow or lymphoid tissue
• colorless in unstained cell suspension, hence “white blood cell”
• normal count: 4,500-11,500 cells/uL of blood
Leukopenia

Leukocytosis

Leukemia

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 White Blood Cells
Granulocytes
1. Neutrophil
- neut’s, segmented neutrophil, segs, polymorphonuclear neutrophils, PMN
- phagocytic cells which engulf and destroy microorganisms and foreign
materials
- “segmented” refers to the multilobed nuclei
Neutropenia

Neutrophilia

- “bands”
- cytoplasm of the cell contains submicroscopic, pink or lavender-staining
granules (bacterial secretions)
- left shift
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 White Blood Cells
Granulocytes
2. Eosinophil
- eo’s
- cells with bright orange-red, regular cytoplasmic granules
Eosinophilia

Eosinopenia
3. Basophil
- baso’s
- cells with dark purple, irregular cytoplasmic granules which
obscure nucleus
- granules contain histamine and various proteins
Eosophilia

Eosopenia 18
 White Blood Cells
Agranulocytes
1. Lymphocyte
- “lymph’s”
- complex system of cells that provide host immunity
- part of humoral and cell-mediated responses
- cell: round, slightly larger than RBCs
- nuclei: round featureless and has a thin rim of nongranular
cytoplasm
Lymphocytosis

Lymphocytopenia

Leukemia

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 White Blood Cells
Agranulocytes
2. Monocyte
- mono’s
- immature macrophage passing through blood from its site of
production
- macrophages
- comprise the most abundant cell type of the body
- comprise minor component of peripheral blood WBCs
- phagocytosed foreign particle
- assist in assembly and presentation of immunogenic epitopes to
the lymphocytes
Monocytosis
Monocytopenia

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 Platelets
• “thrombocytes”
• true blood cells that maintain blood vessel integrity by initiating vessel repair
• adhere to the damaged surfaces, form aggregates to plug the BV and secrete proteins
and small molecules that trigger clot formation or thrombosis
• control hemostasis
• 2-4 um in diameter, round or oval, anucleated and slightly granular
Thrombocytosis
Thrombocytopenia
Essential thrombocythemia

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Hematologic Procedures
II. Blood film examination
- “wedge prep” blood film on a glass microscope slide
- allows it to dry, fixes and stains it with Wright or Wright-
Giemsa
- examines for abnormalities in shape, diameter, color or
inclusion bodies using OIO
- estimate WBC count and platelet count for comparison with
automation
- WBC differential count

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Hematologic Procedures
III. Other procedures
a. Coagulation
b. BM examination
c. Flow cytometry immunophenotyping
d. Cytogenetic analysis
e. Molecular diagnosis assay

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Specimen Collection
Equipment for Venipuncture

1. Tourniquet
• barrier against venous blood flow to locate a vein
• disposable elastic strap, Velco strap, blood pressure cuff
• 3-4 in above the venipuncture site
• left no longer than 1 min before venipuncture is performed

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Specimen Collection
Equipment for Venipuncture

2. Collection tubes
• plastic or glass (OSHA recommends plastic tubes whenever possible)
• plastic tubes are covered with silicone (help decrease possibility of hemolysis and
prevent blood from adhering to the sides)
• additives

 Clot activators

 Anticoagulants

 Antiglycolytic agent

 Separator Gel 25
Specimen Collection
Equipment for Venipuncture

2. Collection tubes
a. Clot activators
** blood specimens for serum testing: allowed to clot for 30-60
minutes prior to centrifugation and removal of serum
- accelerates clotting process and decreases specimen
preparation time
- glass or silica particles
- thrombin

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Specimen Collection
Equipment for Venipuncture

2. Collection tubes
b. Anticoagulants
• prevents blood from clotting
• EDTA, sodium citrate, heparin

• remove calcium needed for clotting by forming insoluble

calcium salts
• Heparin
• binds to antithrombin in plasma and inhibiting thrombin
and activated factor X
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Specimen Collection
Equipment for Venipuncture

2. Collection tubes
b. Anticoagulants
EDTA
- EDTA + calcium= insoluble calcium salt
- dry additives (K2 EDTA or Na2 EDTA)
- liquid additive (K3 EDTA)
- coagulant of choice for cell counting and sizing
- less shrinkage of RBCs and less of an increase in cell volume on
standing

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Specimen Collection
Equipment for Venipuncture

2. Collection tubes
b. Anticoagulants
Sodium Citrate
- EDTA + calcium= insoluble calcium salt
- 3.2% sodium citrate
- 3.8% sodium citrate
- anticoagulant for aPTT, PT testing and Westergren ESR
- due to dilution of anticoagulant to blood, sodium citrate is
generally unacceptable for most other hematology tests

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Specimen Collection
Equipment for Venipuncture

2. Collection tubes
b. Anticoagulants
Heparin
- inactivates the blood-clotting factor thrombin & factor
Xa
- in vitro and in vivo anticoagulant
- coat capillary blood collection tubes
- inappropriate anticoagulant for many hematology tests
(Wright-stained blood smears)

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Specimen Collection
Equipment for Venipuncture

2. Collection tubes
b. Anticoagulants
Oxalate
- distorts the cell morphology
- RBCs become crenated
- vacuoles appear in the granulocytes
- bizarre forms of lymphocytes and monocytes appear
rapidly

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Specimen Collection
Equipment for Venipuncture

2. Collection tubes
c. Antiglycolytic agent
- inhibits metabolism of glucose by blood cells
- sodium fluoride

** anticoagulated plasma can be immediately centrifuged to

obtain plasma

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Specimen Collection
Equipment for Venipuncture

2. Collection tubes
d. Separator Gel
- inert material that undergoes a temporary change in viscosity
during centrifugation process
- separation barrier between liquid and cells
- cannot be used with certain instruments or for blood bank
procedures

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Specimen Collection
Equipment for Venipuncture

3. Needles
- sterile and available in variety of lengths and gauges
- inserted into the vein and has a point with slanted side
- gauge number is inversely related to the bore diameter
- 19-23 gauge: bore for blood extraction
- 21 gauge with 1 in length: most common needle size for adults
- 1 in length: provides better control during venipuncture

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Specimen Collection
Equipment for Venipuncture

4. Needle holders
• designed to comply with OSHA’s revised
• Occupational Exposure to Bloodborne Pathogens Standards
• needle holders are made to fit specific manufacturer’s needle
and should not be interchanged
• Vacutainer Eclipse Blood Collection System (BD Medical,
Franklin Lakes, NY) with BD Eclipse needle
• Jelco multisample blood collection needle with Venipuncture
Needle-Pro Device (Smith Medical ASD, Norwell, MA)
• Greiner Bio-One (Monroe, NC VACUETTE QUICK SHIELD
with VACUETTE Visio PLUS multisample needle

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Specimen Collection
Equipment for Venipuncture
4. Winged Blood Collection Set (Butterfly)
- short needle with plastic wings connected to thin tubing
- other end of the tubing can be connected to a needle holder for an
evacuated tube, syringe or a blood culture bottle
- useful for children or other patient from whom it is difficult to
collect blood
5. Syringes
- consist of barrel, graduated in mm and a plunger
- have a point at one end and an open hub at the other end that attaches to
the barrel
- pediatric, geriatric and other patients with fragile, tiny or rolling veins (that
would not be able to withstand the vacuum pressure from evacuated
tubes
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Specimen Collection
Blood Collection Method

1. Evacuated Tube System

- most commonly used
- vacutainer two-way needle, plastic tube holder, evacuated tube
- vacuum tube set should be assembled before applying
tourniquet
- provides closed sterile system for specimen
collection
- follows “order of draw” recommended by CLSI
- avoids cross-contamination

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Specimen Collection
Blood Collection Method

1. Evacuated Tube System

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Specimen Collection
Blood Collection Method

1. Evacuated Tube System

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Specimen Collection
Blood Collection Method

1. Evacuated Tube System

- factors affecting quality of tubes:
a. ambient temperature
low:
high:
b. altitude
high
(>5,
000
ft):
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Specimen Collection
Blood Collection Method

1. Evacuated Tube System

- factors affecting quality of tubes:
a. humidity
low: hasten the escape of water vapor from a tube
containing a wet additive
high: migration of water vapor inside a tube
b. light
CTAD

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Specimen Collection
Blood Collection Method

2. Syringe Method
- used less often, still method of choice for some instances
- barrel and plunger
- small, fragile and damaged vein (may collapse under vacuum
pressure)

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Specimen Collection
Blood Collection Method

3. Butterfly Infusion Set

- needle with plastic wings, plastic tubing, adapter
- useful for children or other patient from whom it is difficult to
collect blood
- costly

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Specimen Collection
Blood Collection Method

4. Capillary blood
- microhematocrit tubes
- small tube may be heparinized or plain
- mylar layer
- keep the pieces intact and safely contained
- not interfere with the accuracy for the user’s visual
inspection of the sample

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Quality Control and Quality Assurance
Quality control
• implies the ability to provide accurate, reproducible/ reliable assay results that offer
clinically useful information

Quality assurance
• broader concept
• preanalytical, analytical, and postanalytical variables

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Examples of Components of Quality Assurance

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Quality Control and Quality Assurance
Internal Quality Control
Controls
• prepared or purchased assay control
• provide known values and are sampled alongside
patient specimens to accomplish within-run assay
validation
• normal, low or high
• run at least once per shift
• within +/-2 SD.
• when run is rejected, the cause should be found and
corrected
Calibration verification
• detects systematic errors caused by deterioration of the
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calibrator or a change in the analytical process
Quality Control and Quality Assurance
Internal Quality Control

Steps to correct out-of-control run

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Quality Control and Quality Assurance
Internal Quality Control
Delta check
compares a current analyte result with the result from the
most recent previous analysis for the same patient
20% deviation
• investigated for an intervention

• analytical error or mislabeled specimen

MCV, red cell distribution width (RDW), hemoglobin (HGB),
platelet count (PLT), PT, INR, and PTT

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Quality Control and Quality Assurance
External Quality Assessment
• validates the accuracy of hematology and hemostasis assays by comparing results
from identical aliquots of specimens distributed at regular intervals among
laboratories nationwide or worldwide
• survey or proficiency testing specimens

• aliquots
• target values for the test specimens are established in-house by their manufacturer
or distributor
• then further validated by preliminary distribution to a handful of “expert”
laboratories
NEQAS
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• requiring laboratories to participate as a condition of licensure.
Quality Control and Quality Assurance
External Quality Assessment

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References:
• Lotspeich-Steininger e.t al; Clinical hematology principles,
procedures, correlations, Lippincott Company, 1992
• Turgeon, Clinical Hematology: Theory and Procedures 5ht
ed., Lippincott Williams & Wilkins, 2012
• Keohane et. al, Rodak’s Hematology: Clincal Principles 6th
ed., Elsevier, 2020
• DOH Dept Circ 2017-0173 Schedule of EQAS Application

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 Thanks!
Any questions?
You can find me at:
mvgonzales@fatima.edu.ph

HEM
311

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