Routine hematology

examination
Rahajuningsih D Setiabudy
Department of Clinical Pathology
Faculty of Medicine
University of Indonesia

Content

Definition
Indication
Specimen
Method
Effect of storage
Interpretation

Routine hematology tests

Before the era of automatic blood cell
counter routine hematology tests consist of
Hemoglobin level
Leukocyte count
Differential count
Erythrocyte sedimentation rate (ESR)

Now complete blood count (CBC) consist

of
Hb, Ht, RBC, MCV, MCH,MCHC, WBC, diff.
count, Platelet + ESR (manual)

Indication
The purpose of hematology examination :
To detect abnormalities of RBC (anemia,
polisitemia)
To detect abnormalities of WBC (leukocytosis,
leukopenia)
To detect abnormalities of platelet
(thrombocytopenia, thrombocytosis)
To detect abnormalities in leukocyte differential
count
To detect abnormality of ESR (inflammation)

Specimen
Blood is mixed with ethylene diamine
tetra acetic acid / EDTA blood
(K3EDTA or K2EDTA)
Purple top vacuum tube
Venous blood or capillary blood (infant
from heel prick)
ESR : 4 volume of blood are mixed with
1 volume of sodium citrate (1.09 M)

Measurement of
Hemoglobin
1. Sahli / acid hematin method
less accurate, color develops slowly,
unstable
2. Haemiglobincyanide (HiCN)
Internationally recommended method
stable and reliable reference preparation
available, KCN potential hazard sodium
azide or sodium lauryl sulphate
3. Oxyhemoglobin
Standard solution unstable

Haematocrit or PCV
Microhematocrit method: accuracy
and precision adequate for clinical
utility
K2EDTA is recommended because
K3EDTA shrinking of RBC
reducing PCV by 2%
Excess of anticoagulant cell
shrinkage falsely low PCV
PCV gradually with storage

Blood cell counting

Manual method:
Diluting the blood with specific
diluent, (Turk solution for WBC)
Fill the counting chamber
(Improved Neubauer)
Under the microscope count the
cell in a certain area (volume)
Automatic blood cell counter

Automatic blood cell

counter

Principles :

Impedance
blood cells are poor conductors of electricity
while certain diluents are good conductors
Light-scattering technology
Cells pass in single file in front of a light
source.
Light is scattered by the cells passing
through light beam. Scattered light is detected
by photodiode. The amount of light scattered
is proportional to the volume of cell

Impedance method

resistance

direct current
(approx. 100 V)
external electrode

vacuum

external
electrod
e

internal
electrode

internal electrode

vacuum

orifice
blood cells

orifice

sample
sample beaker

Light Scattering system

Erythrocyte indices
Mean Cell Volume
In automated system, MCV is measured
directly
In manual system MCV = (PCV x 10): RBC

Mean Cell Hemoglobin

MCH = (Hb x 10): RBC

Mean Cell Hemoglobin Concentration

MCHC= (Hb x 100) : PCV

RBC indices are needed to classify

anemia based on morphology

Automatic blood cell

counter
Advantages
CV < manual
More rapid
Less volume of
blood is needed
More efficient
(multiparameter,
less human
resources)

Disadvantages
Machine and
reagent are
expensive
Cumbersome
maintenance
Inaccurate in some
conditions

Precision of cell counting

Parameter

RBC

CV of
manual
method
11%

CV of
automatic
method
3%

WBC

16%

4%

Platelet

22%

7.5%

Wintrobe Clinical Hematology 1974

Sources of inaccuracy in
automatic cell counting
Agglutination : RBC MCV , MCH MCHC
Microcytosis or many fragmented red cells:
RBC , Platelet
Hemolysis: RBC and PCV
Lipemic blood: Hb , MCH , MCHC
Two population of RBC: MCV normal, RDW
Giant platelet, clumping, satellitism: Plt
Nucleated red blood cell: WBC

Erythrocyte sedimentation
rate

The rate of sinking RBC is influenced by:

The difference of specific gravity
between RBC and plasma
The capacity of RBC to form rouleaux
The ratio of RBC to plasma
The plasma viscosity
The vertically of sedimentation tube
The bore of the tube
Dilution

Erythrocyte sedimentation
rate
ESR is not specific test
Accelerated ESR can be found in :
Acute and chronic infection due to
acute-phase protein (tbc,
rheumatoid arthritis, AMI)
Multiple myeloma due to
immunoglobulin
Anemia

Low ESR
ESR is low (0-1 mm) can be found
in :
Polycythemia
Hypofibrinogenemia
Abnormalities of red cells difficult
to form rouleaux : poikilocytosis,
spherocytosis, sickle cell

Effect of storage on the

blood count
RBC, WBC, platelet count , and red cell
indices stable for 8 hours. Although RBC
start to swell
Ht and MCV start
At 4 C the effect on blood count insignificant
for up to 24 hours.
It is best to count WBC and especially platelet
within 2 hours
Reticulocyte count begin within 6 hours at
room temp.

Stability of blood count

Parameter

Room
temperat
ure

WBC, RBC,
8 hours
Platelet
Reticulocyte 6 hours
Hemoglobin Up to 2
concentratio days
n

Refrigerat
or
up to 24
hours
24 hours
days

Dacie and Lewis Practical Haematology 2006

Effect of storage on
blood cell morphology
Striking changes after 12 18 hours at room
temperature
Nucleus stain more homogenously
Nuclear lobe separated,
Vacuoles appear in cytoplasm
Cytoplasmic margin ragged
> 6 hours : RBC crenation and sphering
Importance of making blood film as soon
as possible after blood collection

INTERPRETATION OF
HEMATOLOGIC TEST

Hematological values for normal

adults

MCV men and women

5.0 0.5 million/uL

4.3 0.5 million/uL
15 2 g/dL
13.5 1.5 g/dL
5 %
41 5 %
92 9 fl

MCH men and women

29.5 2.5 pg

MCHC men and women

WBC

33 1.5 g/dL
4000 10.000/uL

Platelet

150 000 400 000/uL

RBC men
RBC women
Hb men
Hb women
Ht men
Ht women

Dacie and Lewis Practical Haematology 10th ed 2006

Hemoglobin level
Hb anemia
Red cell indices are needed to classify
anemia: microcytic hypochromic,
normocytoc normochromic,
macrocytic
Hb polycythemia :
Polycythemia vera
Secondary polycythemia
Relative polycythemia

WBC count
WBC leukopenia : viral infection
aplastic anemia
SLE
WBC leukocytosis : physiologic
(epinephrine)
pathologic : infection
inflammation
tissue injury
malignancy

Platelet count
Platelet count : thrombocytopenia
decreased production
increased destruction
increased consumption
dilution
pooling
Platelet count : thrombocytosis
Reactive/secondary
Primary

Abnormalities in differential
count
Neutrophilia : bacterial infection,

inflammation, intoxication, acute bleeding

Neutropenia : viral infection, typhoid,
chemicals and drug (benzene, antithyroid )
Lymphocytosis : chronic infection,
pertussis, tbc, CLL, mumps
Monocytosis: tbc, lymphoma, malignancy
Eosinophilia: allergic disorder, parasitic
infection
Basophilia: CML, PV, ulcerative colitis

Thank you