COMPLETE

BLOOD
COUNTS
 Dr Nadeem Ikam
MBBS;M.Phil;FCPS;CPHE
Professor of Pathology
Akhatar Saeed Medical College
& Frooq Hsopital,
Rawalpindi
 LEARNING OBJECTIVES
Significance of Blood Counts Determination

Methods Employed for blood counts determination

Normal Blood Counts

Variations in Blood Counts and their significance

 Complete Blood Count- What it
Signifies ?

A complete blood count is a series of tests

used to evaluate the composition and
characteristics of the cellular components of
blood .

It tests for disorders and abnormalities of the

blood and gives an indication of diseases
present in other organs also
Blood Cells – Evaluated in
Complete Blood Counts
Leucocytes soldiers of the
(white blood body!
cells)

Erythrocytes- food for the

red blood cells body!

Platelets bleeding
control .
Complete Blood Counts - Report generation
 Collection and Handling of Blood

Blood can be collected form three sources:

1.Venous Blood: venous blood is used for most
hematological examinations.

2.Arterial Blood: This sample is occasionally required

for estimation of blood gases.

3.Capillary Blood: Obtained form heel or finger pulp. A

free flow of blood is essential. All counts are slightly
higher in capillary blood, except the platelet counts that
appears to be higher in venous blood.
 Anticoagulant used for Hematological Studies

1.EDTA:It removes calcium from blood (CALCIUM

CHELATION) which is essential for coagulation. It is used for
blood CP.
2.Sodium Citrate: It removes calcium from blood which is
essential for coagulation. It is used for coagulation profile
3.Heparin: It inhibits coagulation by neutralizing thrombin.
It is used for sample collection of chromosomal studies, blood
gases analysis and osmotic fragility test.
4.Calcium Oxalate.
5.Acid Citrate Dextrose (ACD) or Citrate Phosphate Dextrose
(CPD) or Citrate Phosphate Dextrose with Adenine & Inosine
(CPD-AI): Used in blood transfusion collection bags. They
ensure the vitality and viability of blood cells during the
period of their shelf life.
 Vacutainers for Blood Samples Collection

 VACUTAINER:Tube used for collection of

blood sample for different tests
These tubes have a preformed vacuum which
facilitates the suction of required blood volume
Contian a proper additive (anticoagulant, etc)
for the test required
For each test the tube stopper is of specific
colour
 Vacutainers for Blood Samples Collection

(i)Lavender stopper: EDTA tube for blood C/P.

(ii)Green stopper: Heparin tube for blood gases analysis,
chromosomal analysis and osmotic fragility test.
(iii)Grey stopper: Fluoride/oxalate tubes for blood glucose
estimation (fluoride inhibits glycoysis) and blood alcohol
level.
(iv)Light blue stopper: Tri sodium citrate tubes for PT, APTT,
Fibrinogen, D-D dimmer; factor assay and Lupus
Anticoagulant.
(v)Black stopper: For ESR determination.
(vi)Golden stopper/Gel Tubes: Serum separation tubes
containing a polyoner gel and silica clot activator that speeds
up clotting process.
Vacutainers for Blood Samples Collection
 EDTA Vacutainer for Blood C.P

ETHYLENDYAMINE TETRA ACIDIC

ACID (EDTA) Vacutainer for Blood C.P
EDTA removes calcium (Calcium Chelation) required for
blood clotting. Hence EDTA ensures that blood will not
clot
 METHODS EMPLOYED

Blood evaluation requires

quantification of blood components by
either manual or automated methods
First Step: Evaluation of blood counts
through automated haematology analyzer
Second Step: Microscopic evaluation of
a stained blood film to complement
haematology analyzer data
.
 Working of Automated Haematology Analyzer
Automated Haematology Analyzer

-Haematocrit/PCV
-White Blood Cells Counts -MCV
Haemoglobin
-Red Blood Cells Counts -MCH
Estimation
-Platelets Counts -MCHC
-RDW

Spectrophotometrically -Electrical Impedance

by Cyanmet -Derived parameters
-Lazar based
haemoglobin method measured by claculations
-Flow Cytometry
 Haemoglobin Estimation

Haemoglobin is an intensely coloured protein

allowing it’s measurement by spectrophotometric
techniques.
Haemoglobin is measured by
Cyanmethaemoglobin method .
Cynamethaemoglobin Method: Drabkin’s
Reagent lyses the red blood cells , which release
haemoglobin . Coloured produced is then
measured photometrically and colour produced is
proportionate to the concentration of haemoglobin
in given blood
Cell Counts by Automated Haemotology Analyzer

Automated haematology analyzers for the

counting of Total Leucocytes Count (TLC) Or
White Blood Cells (WBC) , Differential Leucocytes
Count (DLC), Red Blood Cells (RBC) Counts and
Platelets Counts use several distinct technical
approaches like:
Techniques Used in Automated Haematology Analyzer
Electrical Impedance

Differential Light Scatter

Optical Flow Cytometric, using Laser Beam

Fluorescence Based Flow Cytometry

 Blood Cells Counts

Blood Cells Counts

Red Blood Cells

Non-Nucleated Cells in Platelets
Peripheral Blood
Neutrophils
Lymphocytes
Nucleated Cells in
Monocytes
Peripheral Blood
Eosinophils
Basophilis
 WBC, RBC & platelet count:
Provide results obtained by electrical and light
signal generated when blood cells pass through
the sensing zone of the machine.

Data is recorded

Results are generated numerically and

graphically

Distribution curves of white blood cell, red

bland cells and platelets can provide important
information when viewed in context of entire
histogram
 Total Leucocytes Count (TLC) and
Differential Leucocytes Count (DLC)

Leucocytes are counted after

dilution of blood in a diluent
(usually acidic) that lyses red blood
cells .
Electrical Impedance
 Electrical Impedance
(Electrical Resistance Principle)

Counting chamber consists of:

-Beaker
- Two Electrodes with a direct current
- An Orifice with specified dimensions
Principle: Blood cells are not conductive to electricity
While passing through electrical field they will increase
electrical resistance .
When suspended cell passes through the aperture
It will lead to an increase in electrical
impedance(resistance) between the two electrodes
A pulse will be generated
Sum of Pluses= Cells Count
Height of Pulses= indicates cell volume
 Optical Flow Cytometric Type of
Automated Haematology Analyzer

A .

A: As incident light beam hits the

cells, different parameters such as B:Schematic showing the particles
extinct, scatter, and fluorescence are lined in a single stream using
measured and the interaction sheath fluid as they interact with
provides information on optical the laser light which gets collected
properties and composition of the by detector
cell
 Optical Flow Cytometric Type of Automated Haematology Analyzer
Suspension of cells is passed through flow chamber

Focused into a single cell sample stream

Cells pass through a chamber

Interact with a laser light beam

Scatter of the laser light beam at different signals is recorded

Signals are generated

Converted to electronic signals

Electronic signals give information about cells number, size, internal strength and granularity
 DIFFERENTIAL LEUCOCYTES COUNT

In WBC three peaks relate directly to three specific types of

cell population, i.e., Neutrophils, Lymphocytes and Mixed
cells
 Red Blood Cells and Platelets Counts

Each time a cell passes through the aperture, a pulse is produced. The
pulse height is proportional to the cell volume. The distribution
curves for the volume are seperated from each other with a moving
discriminator . The platelets with a volume of 8-12 fl and the Red
Blood Cells with a volume of 80-100 fl are detected from 40-250 fl
 EXAMINATION OF PERIPHEAL BLOOD
SMEAR
Key Features in the Blood Film
RED Size
BLOOD Shape
CELLS Pattern of staining (reflecting
hemoglobin concentration)
Inclusions
Associated findings: Red cells
aggregates, rouleaux formation
WHITE BLOOD CELLS Relative number -decreased,
normal or increased
Morphology
PLATELETS Relative number -decreased,
normal or increased
Morphology: normal or
enlarged.
CELLS IN PERIPHERAL BLOOD FILM

Non-Nucleated Cells
-Red Blood Cells
-Platelets
Nucleated Cells(Leucocytes)
-Neutrophils
-Lymphocytes
-Monocytes
-Eosinophils
-Basophils
 Red Blood Cells
Normal range 4.2-
5.5 million per mm3
in adults.

Biconcave shape.

Diameter 7μ.

Life span 120 days.

 Platelets
Smallest cells in the blood.

Normal range 150,000-

450,000.

Size : 1-4 µm, usually

rounded, light blue and
contains purple-reddish
granules

Active role in coagulation

and hemostasis.
 DIFFERENTIAL LEUCOCYTES COUNT

DLC is the relative number of each type of Leucocyte in blood, expressed as

percentage. The differential leucocytes count is done on a well spread and well
stained blood film. It requires an even distribution of white cells. If the edge of the
spreader is rough, then many leucocytes especially neutrophils may accumulate at
the tail end.
 Leukocytes
Normal range 4 - 11
thousand per mm3 in
adults.

Involved in fighting
infection, combating
allergic reactions,
and immune
responses.
 Neutrophil

The nucleus is lobulated having two to

five lobes
Lobes are connected by thin chromatin
strands.
Cytoplasm is pink and contains numerous
fine purplish granules.
 LYMPHOCYTE

 In mature lymphocyte cytoplasm is scanty

and stain blue.
Nucleus is rounded or slightly indented
Nuclear chromatin shows a clumped
pattern with uneven distribution.
 EOSINOPHIL

Mature eosinophil is slightly larger than

mature neutrophil.
The nucleus usually has two lobes.
Cytoplasm is packed with relatively large
granules which do not overlap the nucleus
Granules stain reddish-orange.
 BASOPHIL

Mature basophil has a lighter staining nucleus

than the nentrophil.
Seldom contains more than two lobes.
Cytoplasm is pink and contains a number of
large oval or round, deeply staining basophilic
granules.
These granules characteristically overlie the
nucleus.
NORMAL LEUCOCYTES VALUES

Neutrophils = 40-75%
Lymphocytes = 20-45%
Monocytes = 2-8%
Eosinophils = 2-10%
Basophils = <1%
 Blood
Counts
Variations
COMPLETE BLOOD COUNTS VARIATIONS

COMPONENT HIGH LOW

HAEMOGLOBIN Polycythemia Anaemia
PLATELELTS Thrombocytosis Thrombocytopenia
(Thromobocytes)
NEUTROPHILS Neutrophilia Neutropenia
LYMPHOCYTES Lymhpocytosis Lymphopenia
EOSINOPHILS Eosinophilia
MONOCYTES Monocytosis Monocytopenia
BASOPHILS Basophilia
 NORMAL BLOOD PARAMETERS

NORMAL BLOOD PARAMETERS

Hemoglobin Females: 12-15

concentration (Hb) g/dl
Males: 14-17.5
g/dl
Leucocytes Count 4-11 x 109/l (4-11 x 103/
l)
Platelet count (Plt) 150 – 450 x 103/l (150-
450 x 109/l)
 ANAEMIA
Definition:“Reduction in the
concentration of haemoglobin in
the blood below the lower limit of
normal for a particular age and sex
of an individual in a particular
environment
 ANAEMIA

Can Be Classified
On the basis of Red Blood Cells Morphology
On the Basis of Cause/Etiology of Anaemia(Kinetic
Classification)
 COMMON CAUSES OF ANAEMIA
Iron Deficiency Anaemia (World Over
Commonest Cause of Anaemia)

Vitamin B12 and Folate Deficiency

Anaemia (Megaloblastic Anaemia

Mixed Deficiency Anaemia

(Iron+B12+ Folate Deficiency)

Congenital Haemolytic Anaemia

(Beta Thalassaemia Major)
CAUSES OF NEUTROPHILIC
LEUCOCYTOSIS (NEUTROPHILIA)
CAUSES OF NEUTROPHILIA
Bacterial Infections (especially pyogenic
bacterial
Inflammation and Tissue necrosis (Vasculitis,
Infarct, Trauma)
Metabolic Disorders (e.g., Uremai, Eclampsia,
acidosis
Pregnancy
Acute Haemorrhage or Haemolysis
Drugs (Corticosteroids therapy, etc)
Myeloproliferative Neoplasms
 CAUSES OF LYMPHOCYTOSIS

CAUSES OF LYMPHOCYTOSIS
INFECTIONS:
Acute: Infectious Mononucleaosis ;
Rubella;Pertusis; Mumps; Infectious Hepatitits;
Cytomegalovirus; HIV; Herpez Simplex or
Zooster
Chronic: Tuberculosis ; Toxoplasmosis;
Brucellosis; Syphilis
Lymphoid Malignancies: Lymphoid Leukaemias;
Lymphomas
CAUSES OF MONOCYTOSIS

CAUSES OF MONOCYTOSIS
CHRONIC BACTERIAL INFECTIONS:
Tuberculosis; Brucellosis;
Connective Tissue Diseases: SLE; Temporal
Arteritis
Protozoan Infections
Chronic Neutropenia
Monocytic Leukaemias
 CAUSES OF EOSINOPHILIA

CAUSES OF EOSINOPHILIA
ALLERGIC DISEASES: Especially
Hypersensitivity of the atopic type (e.g., Bronchial
asthma; Hay Fever; Urticaria, etc)
PARASITIC DISEASES: Amoebiasis; Hookworm;
Ascariasis; Tapeworm; Filariasis; Schistosomiasis;
Trichoriasis
Certain Skin Diseases: Psoraisis; Pemphigus; Urticaria,etc
Hiodgkin’s Lymphoma
Hypereosinophilic Syndrome
Eosinophilic Pulmonary Syndromes
Eosinophilic Leukaemias
 PANCYTOPENIA

Anaemia
Leucopenia
Thrombocytopenia
Blood Complete Picture tests for
disorders and abnormalities of the
blood and gives an indirect- indication
of diseases present in other organs also
In Automated Haematology
Analyzer, Haemoglobin s estimated
spectrophotometrically, Cell Counts
are estimated with Electrical
Impedance A , Laser Based Technology
or Flow Cytometrically. Red cells
Indices (derived parameters) are
calculated
Anaemia is defined as reduction
in the concentration of
haemoglobin in the blood below
the lower limit of normal for a
particular age and sex of an
individual in a particular
environment.
Worldwide Iron Deficiency
is the commonest cause of
anaemia .
Beta Thalassaemia Major is
the commonest inherited
haemolytic anaemia in
Pakistan
Acute Bacterial Infections
are the commonest cause of
Neutrophilic Leucocytosis .
Viral Infections are
the commonest cause of
Lymphocytosis .
Allergic Disorders and
Worm Infestations are the
commonest cause of
eosinophilia.
 -

MULTIPLE
CHOICE
QUESTIONS
(MCQs)
 A house officer sent an attendant to
laboratory to bring vacutainer for
complete blood counts with purple cap,
which contains EDTA

EDTA which prevents blood clotting by:

A.Removing Calcium (Calcium Chelation)

B. By Inhibiting Anti Thrombin
C. By Fibrinogen inhibition
D.By blocking arachidonic acid
metabolism pathways
 (A) EDTA , by removing calcium stops
steps required in coagulation

LEVEL OF COGNITION
C2
 A child from rural background presented
with lethargy, progressive pallor abdominal
pain and habit of clay eating. Treating
physician ordered his complete blood counts
and deworm the child

Which findings are likely to be seen in his

peripheral blood film?

A. Megaloblastic Anaemia
B. Anaemia with Eosinophilia
C. Thrombocytopenia
D. Normal serum Ferritin
 (B) Intestinal parasites will lead to
bleeding and abdominal pain. Child
will have anaemia and on blood film
examination there will be eisinophilia

LEVEL OF COGNITION
C3
Allergic Disorders and
Worm Infestations are the
commonest cause of
eosinophilia.