CLINICAL PATHOLOGY LABORATORY 3 Normal values (adult)

USTMED ’07 Sec C - AsM % SI

Myelocytes 0%
Differential Leucocyte Count Juvenile 0 - 1% 0 - 0.01
Stabs 0–5% 0 – 0.05
Preparation of Blood Smears for Differential Count Segmenters 50 – 70% 0.50 – 0.70
Lymphocytes 20 – 40 % 0.20 – 0.40
Procedure: Monocytes 0 – 7% 0 – 0.07
1. Make a finger puncture and wipe of the first drop of Eosinophils 0 – 5% 0 – 0.05
blood
Basophils 0 – 1% 0 – 0.01
2. Touch the slide to the second drop, transferring a
small drop of blood to the slide about ½ inch from
one end or if venipuncture was done, place a drop of
oxalated blood onto a slide
3. Lay the slide on a flat surface and place the end of
the second (spreader) slide on it, held by the
fingertips at an angle no greater than 30 degrees, in
front of the drop of blood
4. Pull the spreader slide back into the drop of blood.
When the blood has spread along the two thirds of
the width, push the spread along the two thirds of Tally counter – used for differential leukocyte count
the width, push the spreader slide forward with a (peripheral smears)
steady even motion. The weight of the slid is the only
pressure applied

Purpose:
- to establish the percentage distribution of the
different leukocytes
Nucleated RBC under the Microscope
Equipment:
Microscope with oil immersion objective Corrected WBC Count
Stained slide
Formula:
Procedure: WBC count x 100
1. Under LPO, slect area where cells are evenly Corrected WBC count =
distributed 100 + number of NRBC counted
2. Place a drop of cedar wood oil and examine under oil
immersion lens.
3. Count 100 leukocytes in the smear. Microscopic view of 1 large square (Area for counting WBCs
under LPO)
Suitable and unsuitable regions for observation of stained
smear

left – (unsuitable) erythrocytes are scattered but their three-dimensional

structure is difficult to observe
middle – (suitable) erythrocytes are uniformly distributed and their
three-dimensional structure is well observed (the central part is bright)
right – (unsuitable) erythrocytes are stacking

Pathway for differential count

 Note: if nucleated RBCs are present, the leukocyte count can
be corrected by using the following formula

Total WBC count x 100

Corrected WBC =
100 + No. of nucleated RBC

Computation:
Cells counted in 4 large squares
WBC/cu mm =
of blood no of squares x depth of counting x dilution***
counted* chamber**

*no. of squares counted = 4

Examples of white blood cells counted in a representative area. **depth of counting chamber = 1/10 or 0.1mm
***dilution = 1/20

Leukocyte Count Normal values

A. Leukocyte count using oxalated blood S.I. 4.5 – 10.0 x 109 / L (5,000-10,000 /mm3)

Principle: Complete Blood Count

- blood is diluted with a fluid that lyses the non- A complete blood count (CBC), a routine hematology
nucleated RBC and not the nucleated WBC screening, includes the following determinations:
• total white blood cell count
Equipment: • total red blood cell count
- WBC pipette • hematocrit
- Counting chamber
• hemoglobin
- Sucking tube
• differential white cell count
- WBC Diluting fluid
o Glacial acetic acid 1% with tinge of 1% With an accurate determination of these values,
approximately 70% to 80% of the hematologic diagnosis can be
gentian violet
made as well as significant amount of information gathered for
the purpose of _____________ the stages of a particular
disease or of diagnosing some disease entities not directly
related to the hematopoetic system.

Small lymphocyte
- small lymphocytes are slightly
larger than erythrocytes (about
11 um in diameter and circular
in shape. Their cytoplasm is
scanty and clear blue, having no
granules. The nucleus stains
deeply due to condensation of
chromatin. In healthy adults,
30-45% of leukocytes are
classed as lymphocytes.

Large Lymphocyte
- large lymphocytes are round
and twice as large as the
erythrocyte (about 15 um in
diameter). The nucleus is large
and the cytoplasm plentiful.
The nuclear chromatin structure
and the coloring of the
cytoplasm are similar to those
of small lymphocytes, except
that a few azurophilic granules
are present in the cytoplasm.

Large Lymphocyte (ulit)

The cell shown here has larger
azurophilic granules scattered
1. Fill a WBC pipette with blood to the 0.5 line
in the cytoplasm. This is called
2. Dilute with diluting fluid to the 11 line above the
a large granular lymphocyte
bulb so that the blood is diluted 1:20
(LGL) and is considered, by
some, to be a natural killer cell.
To classify lymphocytes into T or
B forms or into subclasses,
surface antigen exploration with
monoclonal antibodies is
required.

Polymorphonuclear neutrophil
- the size, cytoplasm and
nuclear chromatin structure of
polymorphonuclear neutrophils
are similar to those of
neutrophilic band granulocytes.
However, the nucleus is
segmented into three lobes by 2
3. shake, discharge 3 drops, then fill the counting
filaments. In healthy adults,a
chamber
bout 50% of leukocytes are
4. count (10x objective) the cells of the 4 large corner
polymorphonuclear neutrophils.
squares (1mm each)
Most polymorphonuclear
5. calculate the leukocyte count on the basis of cells
counted, area counted and the dilution
 neutrophils have tow or three
lobes.
Eosinophil
- eosinophils are slightly larger
than neutrophils. The cytoplasm
contains large orange-red
granules. The nuclear chromatin
structure is similar to that of
neutrophils but the nucleus is
only segmented into two
swollen oval lobes of equal size
by a filiform strand. In the
normal blood, about 3% of
leukocytes are eosinophils. The
level increases in cases of
parasitic diseases, such as
ancylostomiasis, allergic
reactions, such as bronchial
asthma, and chronic
granulocytic leukemia.

Neutrophil band granulocyte

(neutrophil band form)
Neutrophil band granulocytes
have diameters about twice
that of erythrocytes (about 15
um). The cytoplasm contains
fine granules staining lightly
orange-purple. The nuclear
chromatin is condensed,
forming coarse, dark purplish-
red clumps. The nucleus is like
a band of varying widths but the
narrow portions are not filiform
as in the mature neutrophil. In
normal blood, 5-10% of
leukocytes are neutropil band
granulocytes.

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