Hematology

Blood Cell Count

Function of the count

A RBC count is used to evaluate any type
of decrease or increase in red blood cells.
These changes must be interpreted in
conjunction with other parameters, such
as hemoglobin and/or hematocrit.
Normal Value RBC:
1. adult female: 4.2 to 5.4 million/mm
3

2. adult male: 4.6 to 6.2 million/mm
3

Below N
may indicate anemia, bleeding, kidney disease,
bone marrow failure
Above N
may indicate congenital heart disease,
dehydration, obstructive lung disease, or bone
marrow over-production , beta thalassemia trait
, deficiencies of iron, folate, vitamin B12, and
vitamin B6.



Leukocyte
Cell contain nucleus
with various
morphology and
various size
Leukocyte
Function of counting
The WBC count is used to suggest the presence of an infection or
leukemia. It is also used to help monitor the bodys response to
various treatments and to monitor bone marrow function.

Normal Value WBC: adult: 4,500 to 11,000/mL

Below N (leukopenia). It can result from many different situations,
such as chemotherapy, radiation therapy, or diseases of the immune
system.

Above N (leukocytosis). This can result from bacterial infections,
inflammation, leukemia, trauma, or stress.

Function of counting
Bleeding disorders or other bone marrow
diseases, such as leukemia, require the
determination of the number of platelets
present and/or their ability to function
correctly.

Methods of Cell Counting
Principle :

manual
Blood diluted count

automatic
Dilution
Diluting pipet :
- Thoma for erythrocyte
(scale : 0,5; 1 -101 )

- Thoma for leukocyte (scale: 0,5 ;1- 11)

- Unopette



Red Cell Counting Pipette (Thoma)
0
,
5

1

1
0
1

101 vol = Total capacity of pipet







0,5 vol of blood goes into bulb first
101 1 = 100 vol of diluted blood,
of which 0,5 vol is undiluted blood.
Dilution of blood is 0,5 : 100 or 1:200
1 vol diluting fluid remains in the stem
and does not dilute blood
Leukocyte Cell Counting Pipette (Thoma)
0
,
5

1

1
1

11 vol = Total capacity of pipet








0,5 vol of blood goes into bulb first
11 1 = 10 vol of diluted blood,
of which 0,5 vol is undiluted blood.
Dilution of blood is 0,5 : 10 or 1:20
1 vol diluting fluid remains in the stem
and does not dilute blood
Diluting solution

Erythrocyte : Hayem solution
(HgCl
2
,Na
2
SO
4,
NaCl)
Leukocyte: Turk solution
(glacial, gentian violet)
Platelet : ammonium oxalat 1%

Manual counting
Tool : Haemacytometer
Negative :
tedious
Time consuming
Coefficient of variation 8-20%


R R
R R
R


1 mm


1 mm


1 mm

1

m
m


1

m
m


1

m
m

0,2 mm
1 mm
2

0,0025 mm
2

R
0,04 mm
2

Automatic COUNTING
Positive :
speed
precision
Automated calculation of Ht and hematologic
constants
printouts
Coefficient of variation 2-4%
Example procedure to counting leukocyte with Thoma pipette:
draw EDTA blood to exactly 0,5
wipe the tip of pipet (without touching the opening capillary)
immerse pipet in the diluting fluid
suction by mouth gently until exactly 101 (beyond 101 discard; air
bubbles discard)
rotate the pipet, shake about 1 min
discard the first 3 or 4 drops of diluted blood from pipet
fill the counting chamber via capillary attraction
if overfilling (presence of fluid in the moat surrounding) clean & refill
if insufficient adding dilution
if air bubbles are present (indicate moisture / dirty hemocytometer)
clean & refill
making the count

Manual Procedure
No. of cells/mm
3
undiluted blood = counted cell x 10000

No. of cells/mm
3
undiluted blood =
total no. of counted cells in 5 squares
surface area counted x height of counting chamber x dilution of blood

~ counted cells
(0,2x0,2)x5 x
1
/
10
x
1
/
200



area which erythrocyte can be counted : A, B, C, D, E
counting begin in the left upper corner to the right, drop down to the
second row and count to the left.
- do not count cells those are touching lower and right hands lines
Formula for erythrocyte count
Dihitung

Tidak dihitung
Formula for leukocyte count
Leukocytes/mm
3
blood =
cells counted in 4 large squarer
area counted x height of chamber x dilution

counted cells = counted cells x 50
(1x1)x 4 x
1
/
10
x
1
/
20



Hematocrit (packed cell volume)
Hct : the percentage of red blood cells volume to the
whole blood volume
A measure of both the number and the size of red blood
cell
Hct reflects the concentration of red blood cell, not the
total of red blood cell mass
Hct value :
Male 40-48
Female 37-43
Hematocrit
Hct test is used to evaluate :
Anemia
Polycythemia
Response to treatment of
anemia and polycythemia
Decision to undergo blood
transfusion and the
effectiveness of those
transfusions
Principle of Hct test
Blood + anticoagulant

Centrifuge

Blood cells will precipitate


Erythrocyte platelet+
(bottom) leukocyte


Technique to do Hct test
Macromethod of wintrobe
Venous blood +anticoagulant

Wintrobe tube

centrifuge 3000 rpm 30

Read the scale from bottom
Hct = L1/L2
L1 = the height of erythrocyte column, exluded buffy coat
L2 = the height of the whole blood specimen
Can be done after ESR test
Interpretation of Hct result
Increased hematocrit :
Dehydration resolved with adequate hydration
Polycythemia vera could be due to problem with bone
marrow
Early indication of dengue shock syndrome
Polycythemia secondary to chronic hipoxia i.e COPD
Living in high altitude
Interpretation of Hct result
Decreased hematocrit :
Indication of anemia
Further testing may be necessary to determine the exact
cause of anemia ( i.e deficiency of iron, folate, vit B12)
Recent bleeding
Liver cirrhosis
Bone marrow suppressant effect of drugs
Sickle cell anemia
Hemolysis
Pregnancy due to extra fluid in the blood
Erythrocyte Sedimentation Rate (ESR)
The rate at which erythrocytes settle to the bottom of a
test tube through the forces of gravity in a certain period
of time, usually 60 minutes
It is directly proportional to the weight of cell aggregates
and inversely to the surface area and plasma viscosity
(microcyte < macrocyte)
ESR is a sensitive but nonspecific (the earliest indicator
when other chemical and physical signs are normal)
Function of ESR test :
monitor inflammatory or malignant disease
Aid detection and diagnosis of occult disease i.e tb,
tissue necrosis, chronic infection

ESR is governed by the balance between pro-
sedimentation factors,mainly plasma protein
(fibrinogen, IgG, CRP) and factors resisteng
sedimentation (negative charge of erythrocyte called
zeta potential and ratio of surface area to volume)

When an inflammatory process occurs, the high
proportion of plasma proteins causes erythrocyte to
stick to each other to form roleaux

Roleaux will settle faster

sedimentation
Methods to determine ESR
Principle :
Anticoagulated blood is
placed in vertical tube

Erythrocytes will tend to fall
toward the bottom

The length of fall of the
column of erythrocytes in
60 minutes =ESR
ESR test
Westergren Wintrobe
Comparison of ESR method
Westergren Wintrobe
Anticoagulant Na sitrat 3.8%
(0.4 ml/1.6 ml blood)
Balanced oxalate
Dried EDTA
Dilution 5/4 -
Length of equipment 300 mm, pipet 110 mm, tube
Diameter 2.5 mm 2.5 mm
Scale 0-200 mm 0-100 mm
Normal value
Male
Female

0-10 mm/hr
0-15 mm/hr

0-10 mm/hr
0-20 mm/hr

Advantage easy Reliable
disadvantage If ESR>50 mm/hr, the
result is unreliable
More difficult
Westergren wintrobe
Peripheral Blood Smear
(Blood Film)
Blood Film : Overview
A blood test that gives information about the number and
shape of blood cells
It is still the best method for definitely evaluating and
identifying immature and abnormal cells (if examined by
trained eyes)
The results of blood film evaluation are not always
diagnostic and more often indicate the presence of an
underlying condition
further diagnostic testing
Sample : capillary blood
venous blood + EDTA/heparin (not oxalate!!!)
Film Staining
Staining : technique used in microscopy to
enhance contrast in the microscopic image
Principle procedure
Fixation

Staining

Mounting
Fixation
To preserve the shape of
cells/tissues due to difference
in tonicity
Saline concentration inside
cells > staining fluid
hypotonic shock
Fixation will hinder inflation of
cells when stainning
Fixation can be done by
immersing the film in a jar
containing fixative solution or
covering horizontally
supported film
Fixative solution :
formaldehyde, methanol,
ethanol, citric acid


Staining
To observe and recognize
different kind of cells by using
certain dyes


Mounting
To keep the film for a long
time
After drying the slide, place a
drop of Canada balsam or
another medium mountant on
the smear, then mount the
coverslip.

Acidophilic : structures that take up only acid dye
Basophilic : structures that take up only basic dye
Neutrophilic : structures stained by combination of acid and basic dye
Dyes
Acid
i.e eosin
Basic
i.e methylene blue and its
analogues (azure A.B,C)
Romanowsky principle
A staining technique based on electrostatic interaction between dye
and target molecules
Acid dyes carry negative charges and bind to basic component of
cells i.e RBC and granules of eosinophil
Basic dyes carry positive charges and bind to acidic component of
cells i.e nuclei of WBC, granules of basophil, RNA molecule of WBC
cytoplasm
Some staining methods following romanowsky principle :
Wright stain
Giemsa Stain
Leishmann Stain
Jenner Stain
Wright Stain
It is used to stain peripheral blood film and bone marrow
aspirates
Dye : methyl alcoholic solution of eosin and a complex
mixture of thiazine (methylene blue 50-75% and azure B
10-25%)
Function of wright stain
to distinguish blood cell types
to perform differential WBC count
to stain chromosome to facilitate diagnosing of diseases
Procedure of Wright Stain

Slide is exposed to undiluted stain solution for 2 minutes.
An equal amount of buffer (pH 6.4) is added and mixed
gently for 10 minutes
The stain is flushed gently to coarsely from the slide with
aquadest
Air dry the slide in a tilted position

Cell Figure with Wright Stain
(films stained well with Wright stain have a pink color when viewed with naked eye)
Basophils has dark
purple granules
(which contain
histamines) in their
cytoplasm

Eosinophils has red-
orange granules and
each distinctly
discernible
Neutrophils (50-70% WBC)
Diameter 12 m
The cytoplasm is pale and
often contains small pink to
purple granule
Erythrocytes are pink
platelet
erythrocyte
Monocytes (6% WBC)
The largest WBC (12-20 m)
Characterized by blue-grey
cytoplasm and have a folded
nucleus( a great reniform
or horseshoe-shaped nucleus,
in some cases even bi-lobed.)


Lymphocytes
Accounts for 20-40%
WBC
Round in shape
Nucleus is generally large
in relation to cytoplasm
Cytoplasm appears pale
blue and generally
doesnt contain any
granules
Type B and T cannot be
distinguished