CHAPTER THREE

HEMATOLOGY

Basic Hematological
tests
Amsalu M
1 03/16/2022
 Learning Objectives

 After the completion of this chapter, the students will

be able to:
 Describe frequently encountered haematological tests.
 Perform venous and capillary blood collection.
 Perform thick and thin blood film.
 Describe the principle of Romanosky stains.
 State the principle, procedure, reference ranges and
interpretation of diffrent haematological tests.

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 Lecture outline

 Introduction.
 Composition of blood.
 Characteristics of blood.
 Blood sample collection.
 Basic Hematological tests.

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 Introduction
Definition:
 The study of blood cells and coagulation
 Analyses of concentration, structure and function of
cells in blood and their precursors in the bone
marrow.
 Haematological tests -are used to diagnose anaemia,
leukaemia, haematological disorders, infectious and
inflammatory disease.
 The frequently used haematological tests in most
clinical laboratories includes:-
CBC
ESR
BF
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 The role of Hematology Laboratory in Clinical
Medicine

Confirming a physician’s clinical impression of a possible

hematological disorder
Establish a diagnosis or rule out a diagnosis
Detect an unsuspected disorder
Monitor the effect of Medication

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 Blood
Is the only fluid tissue

Constitutes 6-8% of the total body weight

Consists of cells suspended in a fluid called plasma

About 45% cells; 55% plasma

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 Composition of Blood
Plasma
 Part of the extracellular fluid
 A complex solution of:-proteins, salts and numerous
metabolic substances
 Acts as a transport medium carrying its constituents to
specialized organs of the body.

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 Cont…
Plasma Consists of:-
 About 91.5% water
 About 8.5% solutes of which about 7% are proteins
Out of the 7% protein:-
 54% albumin
 38% globulin
 7% fibrinogen

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 Formed Elements
The three main blood cells/formed elements are:
 Red blood cells (erythrocytes)

 White blood cells (leucocytes)

 Platelets (thrombocytes)

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 Characteristics of Blood
1. Temperature
 Roughly 38°C (100.4 °F)
1. Viscosity
 Five times that of H2O due to interactions among
dissolved proteins, formed elements, & surrounding
H2O molecules
 Sticky, cohesive, and resistant to flow
1. Ph
 Ranges from 7.35- 7.45, averaging 7.4
4. Volume
 5-6 liters in adult male
 4-5 liters in adult female
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 Function of Blood
Transportation
 O2 from lung to tissues & CO2 from tissues to lung
 Nutrients from GIT to cells
 Heat and waste products from cells for excretion
 Hormones from endocrine glands to other body cells
Regulation
PH
Temperature
Osmotic pressure (influence water and ion content
of cells)

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 Cont…
Protection
 From bleeding (by the clotting mechanism)
 Immunity (phagocytes, lymphocytes, antibodies,
complement proteins, etc)

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 Blood collection
 Blood is the most common specimen analyzed in
hematology laboratory.
 Blood can be collected from veins, arteries or
capillaries.
 Proper collection and reliable processing of blood
specimens is a vital part of the laboratory diagnostic
process.
 The objective of blood sample collection is to obtain a
representative sample of circulating blood.
Blood sources for hematological tests are:
 Venous blood
 Capillary or peripheral blood.

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 Venous puncture
 Is the procedure of collecting blood sample from
veins.
 Necessary for most test that requires anticoagulant or
large quantities of blood, plasma or serum.

 The collection can be done either by using a syringe

or vaccutainer.

Sites:
 Ante-cubital fossa (Median cubital, cephalic & basilic
veins).
 Veins in the jagular, wrist or ankle may be used.
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 The three main veins in the forearm
1. Median Cubital
First choice, well
anchored and easy to
penetrate
2. Cephalic
On the outside surface
Well anchored
3. Basilic
Not well anchored, tends
to roll, painful and can
cause nerve damage
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 Anticoagulant
 When Blood is collected, it clots after sometime.
 Anticoagulants are the chemical agents which prevent the
clotting of blood when mixed with Blood in proper
proportion.
 The choice of anticoagulant depends on the test purpose.
 Purpose of using anticoagulants:
 For study of various constituents of Blood components.
 Study of coagulation(clotting of Blood).
 Preservation of Blood in Blood in Blood Bank.

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 Anticoagulant…
 Properties of anticoagulant:
It must be soluble in Blood.
It must keep the Blood in fluid condition.
It must not bring haemolysis Blood cells.
It must not change the size of RBC.
It must minimize the aggregation of Platelets.
 The common anticoagulants used in hematology are:
EDTA
Sodium Citrate
Heparin
Oxalate

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 Venous Blood collection methods
Two types of venous blood collection techniques:
2.Syringe method
1.Vacutainer method
1

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 Materials required:
 Gloves
 Vaccutainer tube
 Vaccutainer tube holder and Two-way needle
 Sterile syringe and needle (if the syringe method is
used)
 Tourniquet
 Gauze pads or cotton,
 70% alcohol or suitable skin antiseptic
 Test tubes with or without anticoagulant (for
syringe method)
 Sharp container
 Band Aid (to stop further bleeding)
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 Procedure for venous blood collection
1. Assemble all the things required during blood collection.
2. Read carefully the patients form, identify the patient and
decide patient and decide the total amount of blood
needed for the entire test.
3. Select the blood collection container and label them with
the patients identification number.
4. Introduce your self to the patient. Ask the patient to sit
alongside the table used for taking blood. Lay his arm on
the table, palm upwards.

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Cont…
5. Select the puncture site carefully after inspecting both
arms.
6. Apply the tourniquet before drawing blood.
7. Using the index figure of your left hand, feel for the vein
where you will introduce the needle.
8. Disinfect the site with a swab dipped in methanol or
70% alcohol. Rub the vein-puncture site thoroughly.
9. Remove the syringe from the protective warp.

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 Cont…
10. Puncture the vein, try to enter the skin first and then the
vein , at a 30 to 40 degree angle.

11. Release the tourniquet by pooling on the looped end.

12. Place a swab of cotton wool over the hidden point of the
needle. With draw the needle in one rapid movement from
under the swab.

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 Cont…
13. Ask the patient to firmly on the cotton wool swab for 3
to 5 minutes. This stops bleeding from the wound.
Do not bend the arm , this may cause hematoma.
14. Remove the needle from the syringe and gently expel
the blood in to appropriate container.
15. Mix the blood immediately and thoroughly but gently
with the anti coagulant.
16. Immediately discard the syringe and the needle in
appropriate waste disposal equipment.
17. Before the patient leaves, re-inspect the vein-puncture
site to ascertain that the bleeding has stopped.
 Do not leave the patient until the bleeding stops.
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Blood collection precautions
 Excessive venous stasis by prolonged application of
tourniquet should be avoided.
 The syringe needle and the tube should be moisture
free.
 Expelling blood through the needle into the container
should be avoided.
 Do not shake blood in container to mix with
anticoagulant.
 Prolonged contact of serum or plasma with blood cells
should be avoided to minimize glycolysis and/or shift
of constituents from cells to serum or plasma.
 If the patient is already on an intravenous drip,
withdraw blood from the other arm.
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 Venous Blood collection…
Advantage of Venous Puncture :
 It allows various tests to be repeated for checking
doubt full results & allows additional tests to be
performed.
 Plasma or serum can be frozen for further reference.
 Reduce the possibility of error resulting from dilution
with interstitial fluid.
Disadvantage of Venous Puncture :
 Lengthy in procedure.
 Technical difficulty in children, obese individual .

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 Capillary blood collection
 Used when small amount of blood is required for
e.g. Hemoglobin & HCT determination
WBC count
blood smear preparations.
 It is also used when vein puncture is impractical.
 In neonates
 In case of sever burn
 In patients whose both arm veins are being used
for IV medication.

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 Site of puncture
Adults and children:
Palmer surface of the tip of the ring or middle finger
or free margin of the ear lobe
Infants:
 plantar surface of the big toe or the heel.

Correct area

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 Materials required:

Gloves

Sterile disposable lancet (Automatic lancet)

Gauze pads or cotton,

70% alcohol or suitable skin antiseptic

Sharp container
Steps for capillary blood collection
 Preparing necessary equipments
 Preparing the finger
 Puncturing the finger
 Eliminating the first drop
 Producing a large rounded drop
 Withdrawing the blood
 Preventing further bleeding
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 Advantages of Capillary Blood
 Obtained with ease
The preferred specimen for making peripheral blood
films since no anticoagulant is added that may affect
cell morphology

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 Disadvantages of Capillary Blood
 Only small amounts of blood can be obtained and
repeated examinations require a new specimen
 Platelet count cannot be performed on capillary blood.
 Precision is poorer in capillary than venous blood b/se
of variation in blood flow and dilution with interstitial
fluid.
 Blood in micro tubes frequently hemolyze.

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 Hematological Tests
The frequently encountered hematological tests in
most clinical laboratories include:
Hct
Hgb
CBC
ESR
BF

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 1. Hematocrit (PCV) determination
 It is the volume of erythrocytes expressed as a
percentage of the volume of whole blood in a
sample.

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 Cont…
Packed cell volume (PCV) can be used as:
 Screening test for anemia & polycythemia
 Rough guide to the accuracy of Hgb measurement
since PCV is about 3 times the Hgb value
 In conjunction with Hgb value & RBC count it is
used to calculate the red cell indices.
Two methods:
 Microhematocrit method
 Macrohematocrit method

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 Cont…
Microhematocrit method:
 Capillary tubes (75 mm x 1mm) are used.
 Capillary tubes: plain or heparinized
 Tubes are filled about 2/3rd to 3/4th with blood & then
sealed with a sealant (plasticine, clay, plastic sealant)
 Centrifugation for 3-5 min with 10,000-15,000 RPM
speed
 Measurement
Interpretation:
Men: 45% - 47%
Women: 42% - 44%
Infants: 44% - 62%

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 Cont…
Macrohematocrit method:
 Wintrobe tubes are used.
 It is graduated in from 0-100 mm.
 Tubes are filled until 100 mm mark.
 Centrifugation for 30 min with 3000 RPM speed
 Direct reading!
Interpretation:The same as microhematocrit method

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 2. Hemoglobin determination
Clinical significance of Hgb determination:
Screen anemia
Determine severity of anemia
Follow the response to treatment of anemia
Evaluate polycythemia
Citing changes in Hgb concentration before &
after operation & blood transfusion
 Hgb determination is more important in the
evaluation of anemia than the RBC counting.

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 Cont…
Hgb amount can be expressed in g/dl, g/l, mmol/l etc
Reference intervals:
 Men: 13-18 g/dl
 Women: 11-16 g/dl
 Newborns: 14-23 g/dl
Methods used for Hgb determination:
 Visual comparison method
 CuSO4 specific gravity method
 Spectrophotometric method

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 Cont…
Visual comparison method
A. Sahli-Hellige method
 20 L of blood is mixed in a tube containing
0.1mol/l HCl which lyses the RBC & converts the
hemoglobin to Acid Hematin.
 After 10 minutes (or more), 0.1mol/l HCl or water is
added drop by drop, with mixing, until the color of
the solution matches the color of the glass standard
positioned alongside the dilution tube.
 The concentration of Hemoglobin is read from the
graduated scale on the dilution tube
 It is not recommended because of its unacceptable
imprecision & inaccuracy.

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 Cont…
Disadvantages of Sahli-Hellige method
 Fading of the color glass standard  difficulty in
matching it to the acid hematin solution
 Conversion to acid –hematin is slow
 Acid Hematin is unstable
 HbF is not converted to acid hematin  not suitable
for measuring Hgb in infants < 3 months.
 Difficulty in matching the acid hematin with the
glass standard (color matching is subjective/personal
bias)
 Interpersonal difference in reading the endpoint of
dilution.

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 Cont…

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 Cont…
Procedure for Sahli-Hellige method
Fill the graduated tube to the ''20'' mark of the red
graduation or to the 3g/l mark of the yellow graduation
with 0.1N HCl.
 Draw venous or capillary blood to the 0.02ml mark of
the Sahli pipet. Do not allow air bubbles to enter into
the Sahli pipet.
With EDTA anticoagulated venous blood ensure that
it is well mixed by inverting the tube repeatedly for
about 1 minute immediately before pipetting it.
If using capillary blood, wipe away the first drop of
blood from the finger

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 Cont…
 Wipe the outside of the pipette with absorbent paper.
Check that the blood is still on the “20” mark.
 Blow the blood from the pipet into the graduated
tube containing the 0.1N HCl solution.
 Rinse the pipet by drawing & blowing out the acid
solution 3 times.
 Place the graduated tube in the hemoglobinometer
stand facing a window.
 Allow 10 minutes for RBC lysis & formation of acid
hematin
 Compare the color of the tube containing diluted
blood with the color of the standard

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 Cont…
 If the color of the diluted sample is darker than that
of the reference, continue to dilute by adding 0.1N
HCl or distilled water drop by drop.
 Stir with the glass rod after adding each drop.
 Remove the rod & compare the color of the tube with
the standard columns.
 Stop when the colors match.
 Note the mark reached.
 Depending on the type of hemoglobinometer, this
gives the hemoglobin concentration either in g/dl or
as a percentage of ''normal''.
 To convert percentages to g/dl, multiply the reading
by 0.146.

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 Cont…
B. The WHO color scale
 The method is based on comparing the color of a
drop of blood absorbed on a particular type of
chromatographic paper against a printed scale of
colors corresponding to different levels of Hgb
 Easy & inexpensive method
 Measures hemoglobin between 4-14 g/dl in 2 g/dl
increments
 Provides a rough/reliable indication of presence &
severity of anemia where laboratory based
hemoglobinometry methods are not available
 The accuracy of the scale is high in laboratory-based
studies but deteriorates as studies become more field-
based & ‘real life’, & where prevalence of anemia is
46 low.
AMSALU M 03/16/2022
 Cont…

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 Cont…
CuSO4 specific gravity method
 It is a qualitative method based on the capacity of a
standard solution of CuSO4 (specific gravity 1.018-
1.062) to cause the floatation or sinking of a drop of
blood.
 Reference interval for specific gravity of blood:
1.0506-1.0621
 The measurement of specific gravity of a sample of
blood corresponds to its hemoglobin concentration.
 The method is routinely utilized in some blood
banking laboratories while screening blood donors
for the presence of anemia.

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 Cont…

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 Cont….
Spectrophotometric methods
HemoCue® method
 It applies a dry reagent system.
 The RBCs are lysed & Hgb is converted to
azidemethemoglobin by sodium nitrite & sodium azide.
 The absorbance of the color formed will be determined
photometrically at 570nm.
 This method of Hgb measurement is a widely used
point-of-care test.

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 Cont…
Advantages of HemoCue® method
 No dilution necessary
 The instrument reads the result when it is ready (no
need to let stand for lysing of RBCs) & result is
reported directly eliminating errors in reading from a
calibration chart
 High accuracy
 No expensive instrument needed
Dis-Advantages of HemoCue® method
 Test cuvettes are expensive
 Finger prick technique must be good

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 Cont…

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 Complete Blood Count (CBC)
 Provides important information about the kinds and
number of cells in the blood: RBC,WBC,and platelets
A CBC can be done to:
 Investigate the cause of certain symptoms like fatigue,
weakness, fever, bruising or weight loss
 Detect anemia or determine severity of blood loss

 Diagnose polycythemia, leukemia

 Monitor the response to some types of drugs or

radiation treatment.

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 Investigate
AMSALU M a history of abnormal bleeding. 03/16/2022
 CBC…
The CBC consists of:
 Total WBC count

 Differential white blood cell count

 Red blood cell (RBC) count

 Hematocrit (Packed red cell volume)

 Estimation of Hemoglobin

 Reticulocyte count

 Red blood cell indices (MCV, MCH, MCHC)

 Platelet count
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 White Blood Cell (WBC) Count
 WBCs are heterogeneous group of nucleated cells
that are responsible for body’s defense.
Two main groups:
Granulocytes & Agranulocytes
 It is the number of white cells in 1 liter (L) of whole blood

Significance of WBC count:

 To investigate infections and unexplained fever

 To follow prognosis and

 To monitor treatments, which can cause leukopenia

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 WBC…
Manual Hemocytometry
 It is used for enumerating cells: WBCs, RBCs, platelets
 It uses a Hemocytometer: Improved Neubauer Chamber
 Each hemocytometer side has a counting chamber (two)
 Each counting chamber is 3mm x 3mm (area of 9mm2).
 Each chamber is divided into 9 squares – each large
square is 1mm x 1mm (area of 1mm2)
 Each of the four corner squares are divided into 16
smaller squares.

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 Hemocytometry complete set

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 WBC Counting
Principle:
 Whole blood is diluted 1 in 20 in an acid reagent.
 Mature red cells are hemolysed, leaving the white cells
to be counted
 White cells are counted
 The number of WBCs per liter or per microlitre of
blood is calculated

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 WBC…
Procedure (Thoma pipette dilution)
1. Suck blood up to the 0.5 mark; wipe the outside
with clean gauze without touching the tip & take
diluting fluid up to 11 mark
2. Detach the aspirator from the Thoma pipette by
sealing the open tip by your index finger (hold
horizontally)
3. Mix systematically (like figure of 8)
4. The volume contained between 1 and 11 mark will
be 10 units out of which 0.5 is blood & this will
make a 1:20 dilution.
5. Wait at least 5 min

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 Procedure…Cont…
7. Clean chambers & cover slip with alcohol & dry
well with lint free cloth
8. Place cover slip on hemocytometer (press cover
slip on both corners)
9. Re-mix blood with diluent by inverting several
times before charging on hemocytometer to
ensure even distribution of cells
10. Discard a few drops from the pipette & plate one
dilution on each side of hemocytometer.
11. Fill the chamber smoothly & don't overfill or
under fill it; there should be no bubbles
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 Procedure…Cont…
N.B. if overflow cells will spill into the moat, thus
falsely reducing the cell count. Under filling
also gives a falsely lower cell count
12. Allow cells to settle for 3 minutes

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 Procedure…Cont…
13. Use 10x (low power) objective with low light by
lowering the condenser
14. Check for even distribution of cells
N.B. Difference in the number of cells between two
corner squares should not exceed 10%
15. Count WBCs in the four large corner squares

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 Cont…
Calculation
Total number of WBC/ µL = No. of WBCs counted x VCF x DCF
 VCF= volume correction factor
 DCF= dilution correction factor

Reference intervals
Children at 1y 6.0-18.0 x 109/L
Children 4-7 y 5.0-15.0 x 109/L
Adults 4.0-10.0 x 109/L
Pregnant women up to 15 x 109/L

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 Cont…
Interpreting WBC count
 Leukocytosis  Acute infection, metabolic
disorders, inflammation & tissue necrosis, poisoning,
leukemia, pregnancy, acute hemorrhage,…..
 Leukopenia  production failure, bonemarrow
infiltration, Viral, bacterial, parasitic infections;
drugs, radiation, hypersplenism, etc
Exercise
You have counted 80 cells in the 4 corners of a
hemocytometer. The blood was diluted in a Thomma
pipette (1:20). What will be the number of WBCs/μl &
WBCs/l of blood.

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 RBC counting
 Is the total number of red cells in 1Litre of whole blood
Significance of RBC count:
 Is used to diagnose anemia
 To know the number of RBCs in other pathological
conditions
 During normal physiological conditions
Principle:
 A sample of blood is diluted with a diluent (1% Formal
citrate) that preserves the disc-like shape of the red
cells & prevents agglutination.
 Diluted specimen is loaded in a counting chamber &
the cells are counted.

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 RBC…
Procedure (Thoma pipette dilution)
Dilution & counting techniques are similar to WBC
count (differences are in the dilution fluid, dilution
factor & area of counting)
1. Dilute 1 in 200 using the RBC Thoma pipette (blood
to the 0.5 mark, diluting fluid to the 101 mark)
2. Fill chamber smoothly & don't overfill or under
fill; there should be no bubbles
3. Allow cells to settle for 3 minutes
4. Use 40x (low power) objective
5. Check for even distribution of cells
6. Count RBCs in the 5 small squares in the central 1
mm2 area.
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 RBC…
Calculation
Total number of RBCs/ µL = No. of RBCs counted x VCF x DCF

VCF= volume correction factor

DCF= dilution correction factor
Reference intervals
Newborn 5.0 – 6.5 x 1012/L
Children at 1y 3.5 - 5.1 x 1012/L
Adults male 4.2 – 6.0 x 1012/L
Adults female 3.6 – 5.6 x 1012/L

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 RBC…
Interpreting RBC count
 Erythrocytosis  polycythemia (relative or
absolute), …
 Erythrocytopenia  anemia, secondary to other
disorders, …
Exercise
You have counted 440 cells in the 5 RBC sections of
a hemocytometer. The blood was diluted in a Thomma
pipette (1:200). What will be the number of RBCs/μl
& RBCs/l of blood.

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 Platelet counting
 Is the total number of platelets per liter of whole blood
Clinical significance:
 To investigate bleeding disorders,
Principle:
 Blood is diluted 1:20 in a filtered solution of 1%
ammonium oxalate reagent which lyses the RBCs
 Diluted specimen is loaded in a counting chamber &
the Platelets are counted
 The number of platelets per liter of blood is then
calculated.

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 Platelet…
Procedure:
1. Make a 1:20 dilution (0.2 ml blood and 3.8 ml
diluent; for Thoma pipette, blood to the 0.5 mark
& diluent up to the 11 mark)
2. Mix well & charge the hemocytomter
3. Place the chamber in a petridish with wet cotton &
cover with a lid (to prevent drying)
4. Leave the chamber undisturbed for 15-20 minutes
(allows time for platelets to settle).
5. Using the 10×objective focus the ruling of the grid
& focus the central square of the chamber in to
view

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 Platelet…
Procedure…
6. Change into 40x & focus the small platelets
They will be seen as small bright fragments (refractile)
against a dark background
Dirt & debris are distinguishable because of their
high refractility
 Count the platelets in the 5 small squares marked as P
If PLT count < 100, 000, count all 25 small squares.

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 Platelet…
Calculation
Total number of Platelets/ µL = No. of Platelets counted x VCF x DCF

 VCF= volume correction factor

 DCF= dilution correction factor
If the 5 middle squares are counted, multiply the No.
of platelets by 1000
If the 25 of the middle squares are counted, multiply
the No. of platelets by 200
Reference intervals: 150-400 x 109/L

72 AMSALU M 03/16/2022
 Platelet…
Interpreting platelet count:
Thrombocytosis:
The main causes for an increase in PLT numbers include:
 Chronic myeloproliferative diseases:
 essential thrombocythemia
 polycythaemia vera
 chronic myelogenous leukemia
 Following splenoctomy
 Chronic inflammatory disease, e.g. tuberculosis
 Hemorrhage
 Sickle cell disease associated with a non-functioning
spleen or after splenectomy.
 Iron deficiency anemia, associated with active bleeding
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 Platelet…
Thrombocytopenia
The main causes for a reduction in platelet numbers are:
 Thrombocytopenia purpura
 Aplastic anemia
 Acute leukemia
 Infections, e.g. typhoid and other septicemias
 Deficiency of folate or vitamin B12
 Drugs (e.g. cytotoxic, quinine, aspirin), chemicals (e.g.
benzene), some herbal remedies
 Hereditary thrombocytopenia (rare condition).
 Following chemotherapy and radiation
74 AMSALU M 03/16/2022
 Red cell indices
The red cell indices:
 Are absolute values calculated from:
 The measured hemoglobin,
PCV
RBC count
 Are of considerable clinical importance in the
diagnosis and classification of anemia
 Are dependent upon the accuracy of the various red
cell parameter estimations
 Red cell indices include: MCV, MCH & MCHC

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 Red cell indices…
Mean corpuscular volume (MCV)
 It is the average volume of a red cell expressed in
femto litres (fL)  Femtoliter is 10-15 of a liter.
MCV (fl) = PCV (in decimals)
No. of RBC/L

Reference intervals: 80 - 100 fl

MCV increases in macrocytic anemia 
Megaloblastic anemia, Pernicious anemia
MCV decreases in microcytic anemia  Iron
deficiency anemia
76 AMSALU M 03/16/2022
 Red cell indices
Mean corpuscular hemoglobin (MCH)
 It is the average amount of Hgb per individual red
cell expressed in picograms (pg).
MCH (pg) = Hgb (g/L)
No. of RBC/L

Reference intervals: 27 - 31 pg

MCH increases in macrocytic anemia 

Megaloblastic anemia, Pernicious anemia
MCH decreases in microcytic anemia  Iron
deficiency anemia
77 AMSALU M 03/16/2022
 Red cell indices…
Mean corpuscular hemoglobin concentration(MCHC)
 It is the average amount of Hgb per unit volume of red
cell expressed in g/L.
MCHC (g/L) = Hgb (g/L)
PCV (in decimals)

Reference intervals: 32 - 36 %(320-360) g/L

MCHC increases in some cases of hereditary

spherocytosis
MCHC decreases in Iron deficiency anemia
78 AMSALU M 03/16/2022
 Erythrocyte Sedimentation Rate(ESR)
Definition:
 ESR is the rate of fall (sedimentation) of red cells when
an anticoagulated blood is allowed to stand undisturbed
for a specified period of time, usually 1 hour.
 The rate is expressed in mm/hr.
It is:
 a non specific test
 used as an index of the presence and extent of
inflammation (the so-called 'acute phase response' ) and
its response to treatment, e.g., tuberculosis, rheumatoid
arthritis.

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 ESR…
Clinical Significance:
 Normal ESR can not be taken to exclude the
presence of organic disease.
 Majority of acute or chronic infections and most
neoplastic and degenerative diseases are associated
with changes in the plasma proteins which lead to an
acceleration of the sedimentation rate.
 When inflammation is present in the body, certain
proteins cause red blood cells to stick together and
fall more quickly than normal to the bottom of the
tube.

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 ESR…
Principle:
 The ESR is determined by filling a narrow pipette
of predetermined length and bore, with well mixed
anticoagulated blood and placing it in a vertical
position for a set time at the end of which the
distance from the top of the column to the interface
between the plasma and the sedimented red cells is
recorded and expressed in mm/unit time.

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 ESR…
Stages of ESR
ESR has three stages:
i. An initial period of 10 minutes  rouleaux
formation.
ii. A period of approximately 40 minutes  settling or
sedimentation occurs at a constant rate, and
iii. A slower rate of fall (last 10 minutes) packing of
the sedimented red cell column occurs.
The second stage is the most significant phase.

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 ESR…
There are two methods for the determination of ESR.
1. Westergren
2. Wintrobe
Westergren method
 Westergren ESR tube is a straight pipette 30cm long,
2.5mm internal diameter and calibrated from 0-200.
Normal range
 Women: 0-20 mm/ hr

 Men: 0-15 mm/hr

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 ESR…
 There is a progressive increase
with age as there is decline in
plasma albumin concentration
 ESR is increased in pregnancy
as there is a decrease in plasma
albumin due to:
 Hypovolemia and
 Increase in concentration of 
globulin and fibrinogen.

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 ESR…
The Wintrobe Method
 Uses a tube closed at one end, 11cm long with a

bore of 2.5mm and having a graduated scale from 0-

100mm and a special Wintrobe rack.
 Normal Range
Men: 0-7mm/hr
Women: 0-15mm/hr

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 Reticulocyte Count
 Reticulocytes (Retics) are juvenile red cells
 They contain remnants of the ribosomal RNA
 The most immature reticulocytes are those with the
largest amount of precipitable material
 In the least immature Retics, only a few dots or
strands are seen.

86 AMSALU M 03/16/2022
 Retics…
 Reticulocyte count is the number of reticulocyte
present in a peripheral blood per counted RBC and
expressed in percentage.

Clinical significance:
 To assist physicians in the diagnosis, treatment or
monitoring of patients with various anemia.
 The reticulocyte count is an index of bone marrow
red cell production.
 It measures erythropoietic activity.

87 AMSALU M 03/16/2022
 Retics…
Principle:
 The count is based on the property of ribosomal
RNA to react with basic dyes such as methylen
blue or azure B or brilliant cresol blue to form a
blue precipitate.

 Counting reticulocyte is important because their

number in peripheral blood is fairly accurate
reflection of erytropoietic activity.

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 Retics…
Reference value
 0.5 - 2.5% of total erythrocytes (or 25 - 85 X 109/l)
Increased numbers (Reticulocytosis):
 Indicates hyperactive erythropoiesis in the bone
marrow.
 An increase in reticulocyte number is seen in the
following conditions:
Hemolytic anemia:
Immune hemolytic anemias
Primary RBC membrane defects
Sickle cell disease
RBC enzyme defect
Exposure to toxins
89 AMSALU M 03/16/2022
 Retics…
2. Following hemorrhage
3. Following treatment of anemia where an increase in
the reticulocyte number may be used as an index of the
effectiveness of treatment.
e.g.
After doses of iron in iron deficiency anemia, the
reticulocyte count may exceed 20%
Proportional increase of Retic count when
pernicious anemia is treated by transfusion or
vitamin B12 therapy.

90 AMSALU M 03/16/2022
 Retics…
Decreased levels: means that the bone marrow is not
producing enough erythrocytes.
Conditions associated with low Retic count:
 Iron deficiency anemia
 Aplastic anemia
 Radiation therapy
 Untreated pernicious anemia
 Tumour in marrow

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 Blood film preparation and staining
 Examining peripheral blood smears is an important
procedure performed in the Hematology laboratory.

 It is useful in:
 Providing diagnostic information
 Providing additional data
 Guiding the selection and monitoring of therapy
 Indicating adverse effects of treatment

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 BF…
 The validity/reliability of the information obtained
from blood film evaluation depends heavily on quality
of the films.

There are two kinds of blood films:

Thin blood film
Thick blood film

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 Staining Blood Films
 There is little consistency between laboratories in the
precise stain used to prepare a blood film for
microscopic examination.
 Many stains in use are based on the Romanowsky stain
Principle:
 Acidic dyes such as eosin unite with the basic
components of the cell (cytoplasm)
 Basic stains like methylene blue are attracted to &
combine with the acidic parts of the cell (nucleic
acid & nucleoproteins of the nucleus)
 Other structures stained by combination of the two
94 are Mneutrophilic.
AMSALU 03/16/2022
 Differential Counting
 Is the enumeration of the relative proportions
(percentages) of the various types of white cells as
seen in stained blood films
 Is used to determine the relative numbers of each
type of leukocyte.

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 Differential…
The differential cell count also includes:
An evaluation of RBC morphology,
Platelet morphology and numbers, and
General WBC morphology and estimation

Clinical significance of Diff count

 To correlate the distribution of different leukocytes
present in the circulation with different disease
conditions.

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 Differential…
Principle:
 After taking blood sample, blood film is prepared,
and after staining with Romanowsky stains, 100,
200, 300, etc., cells will be counted; then the percent
distribution of each cell is calculated.

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 Differential…
The following points should be checked:
Erythrocytes:
 size, shape, distribution, and degree of
hemoglobinization
 presence of inclusion bodies
 presence of nucleated red cells, if so, the total
leukocyte count must be corrected.
Platelets:
 Estimation (10-20/HPF)
 Do they look normal?
 are there many giant or bizzare forms?

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 Differential…
Leukocytes:
 Mature? Immature? Atypical?
 Average number of lobes? hypersegmented
neutrophils? Hyposegmentation?
 Hypergranulated ones? vacuolation?
 Toxic granulation, other inclusions
 Estimation
Hemoparasites:
 Plasmodium, Borrelia, Babesia, etc.

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 Differential…
Healthy Adult

Cell Type Absolute Relative

Segmented
2.0-7.0 x 109/L 40.0-75.0%
Neutrophil

Lymphocyte 1.5-4.5 x 109/L 20-45%

Monocyte 0.2-0.8 x109/L 2-10%

Eosinophil 0.04-0.4 x 109/L 0-7%

Basophil 0.02-0.1 x 109/L 0-2%

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 Neutrophil
 Approximately 40-75% in peripheral blood.
 2-5 lobes of nucleus joined by thin filament.
 Faint pink cytoplasm sprinkled with pink or purple
neutrophilic granules.

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 Eusinophil
 Approximately 2-7% in peripheral blood
 Bi-lobed or banded nucleus
 Large, red-orange and distinct granules

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 Lymphocyte
 Approximately 40% in peripheral smear
 Small round nucleus, clumpy, chunk chromatin
pattern.

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 Monocyte
 Approximately 5% in peripheral smear
 Large, convoluted, brainy looking nucleus with lacy
looking chromatin.
 Pale, gray blue, ground glass cytoplasm (numerous
fine azurophilic granules

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 Basophil
 Approximately 0-2% in peripheral blood
 Nucleus is bi-lobed & obscured by granules
 Cytoplasm is pale, washed out & contains intense
large blue black granules.
 Granules are scattered throughout the cell & it is
hard to distinguish any distinct nuclear
characteristics.

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 Interpretation of Differential White Cell Count
Neutrophils:
Neutrophilia
An increase in the number neutrophils above normal (>2.0-7.0 x
109/L)
 Overwhelming infections

 Metabolic disorders: uremia, diabetic acidosis

 Drugs and chemicals: lead, mercury, potassium chlorate

 Physical and emotional stress

 Hematological disorders: myelogenous leukemia

 Tissue destruction or necrosis: burns, surgical operations

106 AMSALU M 03/16/2022

 Cont…
Neutropenia:

 A reduction of the neutrophil count below 2.0 x

109/L
Myeloid hypoplasia

Drugs (chloramphenicol, phenylbutazone)

Ionizing radiation

107 AMSALU M 03/16/2022

 Interpretation…
Eusinophil:
Eosinophilia:
 An eosinophil count above 0.5 x 109/L

 Occurs during:

 Allergic diseases: bronchial asthma, seasonal rhinitis

 Intestinal parasitic infections: e.g. trichinosis, taeniasis

 Skin disorders

 Chronic myelogenous leukemia

108 AMSALU M 03/16/2022
 Interpretation…
Eosinopenia:
 An eosinophil count below 0.04 x 109/L
 Occurs during:
Acute stress due to secretion of adrenal
glucocorticoid and epinephrine
Acute inflammatory states
Basophil:
Basophilia:
A basophil count above 0.2 x 109/L
Rare condition
Occurs during:
Allergic reactions
Chronic myelogenous leukemia
Polycythemia vera
109 AMSALU M 03/16/2022
 Interpretation…
Monocyte:
Monocytopenia:
Monocytosis:
A monocyte count
 A monocyte count above
below 0.2 x 109/l
1.0 x 109/l
Occurs during
 Occurs during
 Treatment with
 Recovery from acute
prednisone
infections
 Hairy cell leukemia
 Tuberculosis

110  Monocytic leukemia

AMSALU M 03/16/2022
 Interpretation…
Lymphocyte:
Lymphocytosis:
 Absolute lymphocyte count above 4.0 x 109/L in adults and
above 8.0 x 109/L in children.
 Seen during
 Infectious lymphocytosis associated with coxackie virus

 Other viral infections: Epstein-Barr virus, cytomegalovirus

 Acute and chronic lymphocytic leukemia

 Toxoplasmosis

111 AMSALU M 03/16/2022

 Interpretation…
Lymphocytopenia:
 A lymphocyte count below 1.0 x 109/l in adults
and below 3.0 x 109/l in children
 Seen in
 Immune deficiency disorders: HIV/AIDS
 Drugs, radiation therapy

112 AMSALU M 03/16/2022