CHAPTER -2

BASIC HAEMATOLOGICAL TESTS

Learning objectives

 At the end of the session, you are expected to;

-Discus different types of blood sample collection techniques

-Describe the principle of basic haematological tests

-Interpret values of tests

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 Hematological test
Are a panel of tests that yield information on

The qualitative and quantitative composition of the

cellular components of the blood.

Can evaluate d/t conditions involving blood and its

components.
 Used to diagnose different diseases related to:-
 Inflammation, anemia, infection, hemophilia,
 Blood-clotting disorders, leukemia,
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 Used to check response to chemotherapy or drugs
 Hematologic Tests
Common hematologic tests include:
1. Complete Blood Count (CBC),
2. Differential WBC Count
3. Erythrocyte Sedimentation Rate ( ESR) ,
4. Blood Film (BF),
5. peripheral morphology examination
6. Coagulation test
 Bleeding time
 Prothrombin time
 Thrombin time
 Activated partial prothrombin time
 INR
 Fibrinogen assay…etc
7. Cytochemical test
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SBB, LAP score, MPO, specific and non-specfic esters test…..ectc
Blood Sample collection
There are different types of blood collection techniques
Venus blood collection

Capillary blood collection

Arterial blood collection

 Specimen : Blood

 Proper collection & reliable processing of blood specimens is a

vital part of the laboratory diagnosis process in hematology as

well as laboratory disciplines.

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Blood must be collected with care & adequate safety

precautions to ensure that:

Test results are reliable

To avoided

contamination of test sample

Contamination of staff

Contamination of environment

From blood transmissible pathogens

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 Materials for blood collection

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Principle
Assemble all materials, reagents & patient identification
before collection.
Collect the specimen from appropriate site.
For all Hematological & Serological tests, use EDTA
anticoagulated test tube except blood coagulation test.
Blood sample for serum preparation should be refrigerated in
order to prevent hemolysis which interfere with the result.
Clinical significance
To diagnose febrile disease
To diagnose renal & liver diseases
To diagnose, and to differentiate types of Anemia, Leukemia
To diagnosis coagulation disorder
7 Cardiac disorder…..etc
 The major blood sources for hematological tests are:
1. Capillary &
2. Venous blood
1. Capillary Blood collection (Skin Puncture)
 also called Micro-blood samples or Dermal Puncture.
 is collected using lancet.
 is mainly used:
 when the patient is an infant or young child and
 when the small volume (amount) of blood is required
 to measure Haemoglobin,
 perform a WBC count, and
 to make thick & thin blood films.
 It is also used when vein puncture is impractical in:-
 neonates
 case of sever burn patients whose arm veins are being used for
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IV medication
Sites of Skin Puncture

Adults & Children

 lateral surface of either tip of the
‘ring’ or ‘middle’ finger as shown
in Fig. 8.1.
 Don’t stick the thumb/index finger as
these are the most sensitive.
 Free margin of earlobe
Infants
 lateral surface of big toe & heel.
 The heel of an infant up to one year
old.
 Care must be taken not to damage the
heel by sticking it too near the edge or
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The areas of the foot of a baby or
infant that are suitable for
obtaining capillary blood

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Technique for collecting capillary blood
Materials-cotton, 70% alcohol(Ethanol), Automatic lancet
Procedure:
1. Make sure the puncture area is warm to allow the blood to flow
freely. On cold days soak the hand or foot of an infant in warm water
prior to collecting a sample.
2. Rub the site vigorously with cotton moistened with 70% alcohol to
remove dirt & increase circulation to the area. Allow the area to dry.
3. Using a sterile pricker or lancet, make a rapid & firm puncture,
sufficiently deep (2-3mm) to allow the free flow of blood.
4. Wipe away the first drop of blood with a dry piece of cotton wool and
use the next few freely flowing blood drops for the test. Do not
squeeze too hard because this will result in an unreliable test result.
5. When sufficient blood has been collected, press a piece of dry cotton

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wool over the puncture area until bleeding stops. 11/28/2024
 Advantage of skin puncture
It can be obtained with ease

It’s preferred specimen for making peripheral blood films because

no anticoagulant is used, cell morphology is not affected.

In district laboratories, capillary blood is also used to monitor

anemia during pregnancy.

Thick BF for malaria parasites are best made from capillary blood
(anticoagulated blood is more easily washed from slides during
staining).

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Disadvantages in using capillary blood for blood tests include:
1. Capillary blood can be used for only a few tests.
2. Greater possibility of sampling errors particularly when the
blood is not free-flowing. E.g. dilution of the sample with tissue
juice can occur if the puncture area is squeezed excessively.
3. Difficulty in obtaining sufficient blood particularly when it is
required for more than one test. Meaning, tests cannot be
repeated immediately or further tests can not be performed
when results are unexpected or seriously abnormal.
4. Rapid clotting of blood in a pipette is common, particularly in
tropical temperatures.
5. It is not possible to estimate platelets in blood films made from
capillary blood (platelets clump). Platelet adherence in the
puncture site
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 Venous blood collection(VBC) (Venipuncture) (Also
called Phlebotomy)

 Procedure of collecting blood sample from veins necessary when:

 test require anticoagulant or
 larger quantities of blood, plasma or serum are required.

 Site of puncture:
Three main veins used for venipuncture are:
 Median Cubital Veins
 Basilic Veins
 Cephalic Veins
 Veins in the wrist or ankle may be used
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 The three main veins in the forearm

1. Medial 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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Venous blood collection in infants and children
presents special problems because of:

small size of the veins

difficulty in controlling the patient; needs excellent

interpersonal skill
Require experience

 Areas of Veni-Puncture in infants & children are:

External jugular vein in the neck region.

Femoral vein in the inguinal.

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 There are two types of venous blood collection
 Vacutainer venous blood collection
 syringe venous blood collection

Materials for venous blood collection

 Gloves, Vacutainer tube,

 Vacutainer tube holder & two-way needle,

 sterile syringe & needle

 tourniquet, cotton

 70% alcohol,

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 Vacutainer technique of Venous Blood collection
1. Assemble necessary materials and equipment
2. Identify right patient
3. Patient preparation (allow proper sitting)
4. Reassure patient
5. Apply tourniquet 7.5-10 cm above venipuncture site.
6. Swab the area with 70% alcohol moistened cotton
7. Grasp back of patients arm elbow
8. Insert needle properly into vein
9. Point of needle is advanced 0.5-1cm into subcutaneous tissue at an
angle of 45 degree & pushed forward at lesser angle to pierce
vein wall.
10. When needle is properly in vein, vacuum tube pushed into needle &
blood flow into tube.
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 11. Tourniquet should be released at moment blood starts to flow
12. After blood drawn, apply cotton ball & withdraw needle
13. Instruct patient to press on the cotton
14. Remove tube from Vacutainer holder & if tube is with
anticoagulant-gently invert 8-10 times for EDTA tubes
15. Label tube with patient’s site to ascertain bleeding stopped.

Vacutainer tube Vacutainer tube

holder

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Procedure for Syringe Method of Venous Blood collection
1. Remove syringe from its protective wrapper & needle from cepad &
assemble them.
2. Attach the needle-bevel faces into the same direction as graduation on
syringe
3. Check that: needle is sharp, moves smoothly & there is no air left in barrel
4. Identify the right patient & let him/her sit
5. Reassure the patient
6. Apply tourniquet & swab the site with 70% alcohol moistened cotton
7. Grasp back of patients arm elbow & anchor selected vein enter skin & then
vein
8. The plunger is drown back to create secretion pressure to draw the blood

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 Advantage of Venous Blood Collection
1. Performing additional tests, allows various tests to be repeated
for checking doubtful results.
2. Plasma or serum can be frozen for further reference.
3. Reduce possibility of error resulting from dilution with interstitial
fluid.
 Disadvantage of Venous Blood Collection
1. Lengthy in procedure.
2. Technical difficulty in children, and obese individual.
3. Occurrence of Hemolysis
4. Hematoma

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 Arterial blood collection
 An ideal specimen for many analyses because its
composition is consistent throughout the body
 whereas venous blood varies relative to the metabolic

needs of the areas of the body it serves

 Not used for routine tests

More invasive
Technically difficult
Used for blood gas analyses(metabolic syndrome)

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1. Complete Blood Count (CBC)
 To measure total cell count to assess changes from normal values
 Provides important information about kinds & No. of cells in blood
CBC can be done to:
 investigate the cause of certain symptoms
 detect anemia or determine severity of blood loss
 diagnose polycythemia , leukemia
 monitor the response to some types of drugs
 To diagnosis abnormal bleeding
CBC Consists of:
 Total WBC count, Differential WBC count, RBC count, RBC indices,
Hematocrit, Hemoglobin determination, Platelet count and platelet
parameters

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Principle:
 CBC is run by automation & separates cells based on their Cellular Granularity & Size.

 Clinical significance
 To know individual/patient blood or blood products level
 Result
 Cells are counted & displayed by machine reported per mm3 for
RBC,WBC,PLT
 Interpretation
 Since machine did all at the same time, the correct recording at the moment
 Limitation
 Sometimes the machine fails to work & the test is not performed if the
machine is non-functional.

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 The White Blood Cell count

 Heterogeneous group of nucleated cells responsible for body’s

defense.

 divided into two main groups.

o Granulocytes

 Neutrophil,

 Eosinophil

 Basophiles

o Agranulocytes

 Lymphocyte

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 Monocyte
 Normal values for WBC count
 Children at 1 year-------------6.0-18X103/mm3
 Children 4-7 years ------------5.0- 15.0X103/mm3
 Adults ---------------------------4.0-10X10 3/mm3
 Pregnant women --------------up to15X103/mm3
 Significance of the test
 a total WBC count + differential WBC count
 to differentiate whether infectious agent is bacterial or viral

 Total WBC count is increased during:

 Infection, inflammation,
 Damage to the body tissue,
 Disease such as in some cancer types.
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 A low WBC count may occur:
late in pernicious anemia

after cancer chemotherapy, radiation therapy

bone marrow failure( Aplastic anemia)

viral infection, Malaria

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3. The Differential WBC Count
There are 5 major kinds of WBC:

 The differential WBC count is the enumeration of the relative

proportion of various types of WBCs as seen in stained examinations.

 Differential Count(DC) is performed in thin Smear stained

by Wright’s Stain Solution or Giemsa’s stain solution

when the Smear is Fixed by Methanol.

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 NEUTROPHIL LYMPHOCYTE

BASOPHIL EOSINOPHIL

MONOCYTE

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 Cells Relative value Absolute value

Neutrophil 60-70% 3000-7000/ul

Eosinophil 1-4% 50-400/ul

0.5-1% 25-100/ul
Basophile

Lymphocyte 20-40% 1000-4000/ul

Monocyte 2-6% 100-600/ul

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 Neutrophils
Also referred to as polymorph nuclear (many shaped nucleus)
neutrophils has 2-5 clear lobes, separated by chromatin
threads.
Neutrophilia / Neutrophilic leucocytosis
 is a absolute increase in the number of circulating
neutrophils.
 the conditions associated with this include:
 infections or tissue damage
 metabolic disorders (uraemia, diabetic acidosis)
 drugs & chemicals (Pb, Hg, Potassium chlorate)
 physical and emotional stress

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Neutrophilia An absolute increase in neutrophils can be found in:
● Acute bacterial infections (often with left shift), e.g.
abscesses, wound infections, meningitis, pneumonia,
gonorrhoea, UTIs
● Tissue damage, e.g. burns, trauma
● Snake envenomation
● Acute myocardial infarction
● Acute hemorrhage
● Malignant diseases
● Myeloid leukemia
● Reactions to some drugs e.g. steroid therapy, and
chemicals
● Metabolic disorders
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 Neutropenia
 a reduction of the absolute neutrophil count below 2.0 x 109/l.

 Common causes of a reduced neutrophil count are:

 Myeloid hypoplasia

 Drugs(chloramphenicol, phenylbutazone)

 Ionizing radiation

 Viral infections, e.g. HIV disease, hepatitis, influenza

 Bacterial infections, e.g. typhoid fever, brucellosis,

 Megaloblastic anaemia

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 Eosinophils
Eosinophilia
 is an absolute increase Eosinophil count above 0.5 x 109/l
 conditions associated with this include:
 Allergic conditions, e.g. asthma, food & drug allergies
 diseases (bronchial asthma),
 Skin disorders,
 chronic myelogenous leukemia
 Helminth parasitic infections
 Pulmonary eosinophilia
Eosinopenia
 is a decrease in Eosinophil count below 0.04 x109/l
 conditions associated with this include:
 Acute stress due to secretion of Adrenal Glucocorticoid & Epinephrine
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 Acute inflammatory states
Basophils
Basophilia
 is an increase in Basophil count above 0. 2 x109/l
 conditions associated with this include:
 Allergic rxns,
 Chronic myelogenous leukemia, &
 Polycythemia.

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Monocytes
Monocytosis
 is an increase in monocyte count above 1.0 x109/l .
 conditions associated with Monocytosis include:
 Recovery from acute infections,
 Chronic bacterial infections, e.g. tuberculosis, brucellosis, typhoid,
bacterial endocarditis
 Protozoal infections, e.g. malaria, trypanosomiasis
 Chronic myelomonocytic leukaemia

Monocytopenia
 is a decrease in monocyte count below 0.2 x109/l.
 conditions associated with Monocytopenia include:
 anti-inflammatory drug rx of Arthritis
 Hairy cell leukemia.
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 Lymphocytosis
 is an increase in absolute lymphocyte count
 Conditions associated with Lymphocytosis includes:

 Infectious lymphocytosis associated with Coxackie virus, other viral

infections (EBV, Cytomegalo virus....etc

 Acute and chronic lymphocytic leukemia, lymphomas

 Protozoal infections, e.g. malaria, toxoplasmosis

 Infections in children, e.g. whooping cough,

 Bacterial infections, e.g. brucellosis, typhoid fever, chronic

tuberculosis, syphilis
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 Lymphocytopenia
a decrease in lymphocyte count below normal range
Common causes of a reduced lymphocyte count are:
Immune deficiency disorders (HIV/AIDS),
drugs and radiation therapy
Severe bone marrow failure
Some acute viral infections

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.RED CELLS (Erythrocytes) count
 form the main cellular component of blood, i.e. about 45% of

total blood volume in an adult, giving blood its red colour.

 Each liter of blood contains about 3.5- 6x1012 red cells, the exact

number varying with age, gender, & state of health.

 Production of red cells

 Red cells are produced in the bone marrow. Tissue hypoxia (lack

of oxygen) leads to the release of the hormone erythropoietin

which stimulates progenitor cells to develop into erythrocyte
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Normal breakdown of red cells
At the end of their life-span of about 120 days, degenerate

red cells are removed from the circulation by

reticuloendothelial cells, mainly in the spleen
The haem is split from the globin. Most of the globins are

hydrolyzed and the constituents returned to the amino acid

pool..
 The iron component is either reused to produce new red

cells or stored in the reticuloendothelial cells in as

41 ferritin or haemosiderin. 11/28/2024
The remaining porphyrin of the haem is metabolized to bilirubin

which binds to albumin in the plasma.

In the liver, the bilirubin is conjugated to glucuronic acid,

forming water soluble conjugated bilirubin which passes into

the intestine.
It is metabolized to urobilinogen and mostly excreted as

stercobilin and stercobilinogen in the faeces.

Some of the urobilinogen is reabsorbed from the intestine

and excreted in the urine as urobilin and urobilinogen.

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 Disorders of red cells
The main disorders of red cells are:

 Anaemia

 Haemoglobinopathies (thalassemia, abnormal haemoglobins)

 Disorders due to red cell enzyme defects, e.g. G6PD deficiency

 Disorders due to red cell membrane defects,

e.g. hereditary spherocytosis

 Polycythaemia

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ANAEMIA
is the commonest red cell disorder.

It occurs when the concentration of haemoglobin falls below the

normal for a person’s age, gender, and environment, resulting in

the oxygen carrying capacity of the blood being reduced.
Haemoglobin values are lower in women than men, probably due

to menstrual loss and the influence of hormones on erythropoiesis.

Hemoglobin levels fall in normal pregnancy due to an increase in
plasma volume. Dilution effect

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 Anaemia can develop:

 following blood loss,

 when the normal production of red cells is reduced,

 when red cells are destroyed (haemolyzed) prematurely.

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  Red blood cell (RBC) Count.
 Normal value in adult-Women=4.0 - 5.5X106/mm3
-Men = 4.5 - 6.2 X 106/mm3
Significance of the test
 With hematocrit & hemoglobin, it is used to calculate red
cell indices which is used to classify anemia.
parameters of RBCs
 Packed cell volume/Hematocrit

 Hgb

 and red cell indices(MCV, MCH, MCHC, RDW)

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5. Packed cell volume(PCV) or Hematocrit
 Ratio of volume of Erythrocytes to Whole Blood.

 expressed as percentage or preferably as decimal.

 Hematocrit can be used as:

• screening test for Anemia, when it is not possible to measure

hemoglobulin
• Rough guide for hemoglobin estimation (%Hct=3x Hgb )

Used to calculate red cell indices. These are:

 Mean cell volume (MCV),
Mean cell Hb concentration (MCHC)
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 Methods

 Two methods are available:

a. Micro hematocrit method

wintrobe method

b. Macro hematocrit method

Adams Microhaematocrit method

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 Erythrocyte Sedimentation Rate(ESR)
 Erythrocyte sedimentation rate 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 for 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

normal ESR can not be taken to exclude the presence of organic

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Erythrocyte Sedimentation Rate(ESR)
Principle-
 ESR determined by filling a narrow pipette of predetermined length
& bore size with well mixed anti-coagulated blood & placing in
vertical position for set of time about 1hr & read result.

Result-expressed as ESR= X mm/hr

Interpretation-normal ESR can’t be taken to exclude presence of

disease as it’s non-specific test.

Clinical significance-used as index of presence & absence of inflammation

Reference range- Men: 0-15mm/hr

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 Several factors affect ESR positively or negatively.
I. Plasma proteins
Fibrinogen -----------------accelerate ESR
 Albumin and lecithin-----retard ESR
II . Red cell factors
Anemia increase ESR
Microcytes sediment slower than Macrocytes
Stages in ESR
1. Initial period of 10 minute for Roulaex formation
2. Approximately 40 minute for settling (Sedimentation)
3. Last 10 minute for packing of red cell column

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 Rouleaux formation

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 There are 2 methods for ESR determination.
1. Western Green Method
2. Wintrobe‘s Method

1. Western green Method

 The Westerngreen ESR tube is a straight
pipette 30cm long , 2.5mm internal
diameter & calibrated from 0-200.
 Normal range:
• Women: 0-20 mm/ hr
• Men : 0-15 mm/hr

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 Some conditions with very
Some conditions with low ESR:
high ESR

Polycythemia
Multiple myeloma
Severe Leukocytosis
Connective tissue disorders
Sickle cell disease (anemia)
- SLE, RA and other
autoimmune diseases Hereditary spherocytosis
Tuberculosis
Malignancies
Severe anemia

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  Platelet count
Platelets:
 are Cytoplasmic fragments of megakaryocytic mother cells
are produced in bone marrow.
the smallest of formed elements
 are non-nucleated, round,oval,flattened disk shaped structures.
Normal value 150 - 450X 103/mm3
 Platelet count may be requested
 to investigate abnormal bleeding which can occur when the
platelet count is very low
 when patients are being treated with cytotoxic drugs / other
drugs that can cause Thrombocytopenia

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