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Clin Path Lab 3

Clin Path Lab 3

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CLINICAL PATHOLOGY LABORATORY 3
USTMED \u201907 Sec C - AsM
Differential Leucocyte Count
Preparation of Blood Smears for Differential Count
Procedure:
1. Make a finger puncture and wipe of the first drop of
blood

2. Touch the slide to the second drop, transferring a
small drop of blood to the slide about \u00bd 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
the width, push the spreader slide forward with a
steady even motion. The weight of the slid is the only
pressure applied

Purpose:
-
to establish the percentage distribution of the
different leukocytes

Equipment:
Microscope with oil immersion objective
Stained slide

Procedure:
1. Under LPO, slect area where cells are evenly
distributed
2. Place a drop of cedar wood oil and examine under oil
immersion lens.
3. Count 100 leukocytes in the smear.
Suitable and unsuitable regions for observation of stained
smear

left \u2013 (unsuitable) erythrocytes are scattered but their three-dimensional
structure is difficult to observe
middle \u2013 (suitable) erythrocytes are uniformly distributed and their
three-dimensional structure is well observed (the central part is bright)
right \u2013 (unsuitable) erythrocytes are stacking

Pathway for differential count
Normal values (adult)
%
SI
Myelocytes
0%
Juvenile
0 - 1%
0 - 0.01
Stabs
0\u20135%
0 \u2013 0.05
Segmenters
50 \u2013 70%
0.50 \u2013 0.70
Lymphocytes
20 \u2013 40 %
0.20 \u2013 0.40
Monocytes
0 \u2013 7%
0 \u2013 0.07
Eosinophils
0 \u2013 5%
0 \u2013 0.05
Basophils
0 \u2013 1%
0 \u2013 0.01
Tally counter \u2013 used for differential leukocyte count
(peripheral smears)
Nucleated RBC under the Microscope
Corrected WBC Count
Formula:
WBC count x 100
Corrected WBC count =
100 + number of NRBC counted
Microscopic view of 1 large square (Area for counting WBCs
under LPO)
Examples of white blood cells counted in a representative area.
Leukocyte Count
A. Leukocyte count using oxalated blood
Principle:
-
blood is diluted with a fluid that lyses the non-
nucleated RBC and not the nucleated WBC
Equipment:
-
WBC pipette
-
Counting chamber
-
Sucking tube
-
WBC Diluting fluid
o
Glacial acetic acid 1% with tinge of 1%
gentian violet
1. Fill a WBC pipette with blood to the 0.5 line
2. Dilute with diluting fluid to the 11 line above the
bulb so that the blood is diluted 1:20
3. shake, discharge 3 drops, then fill the counting
chamber
4. count (10x objective) the cells of the 4 large corner
squares (1mm each)
5. calculate the leukocyte count on the basis of cells
counted, area counted and the dilution
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
**depth of counting chamber = 1/10 or 0.1mm
***dilution = 1/20

Normal values
S.I. 4.5 \u2013 10.0 x 109 / L (5,000-10,000 /mm3)
Complete Blood Count
A complete blood count (CBC), a routine hematology
screening, includes the following determinations:
\u2022
total white blood cell count
\u2022
total red blood cell count
\u2022
hematocrit
\u2022
hemoglobin
\u2022
differential white cell count

With an accurate determination of these values,
approximately 70% to 80% of the hematologic diagnosis can be
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
in the cytoplasm. This is called
a large granular lymphocyte
(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
filaments. In healthy adults,a
bout 50% of leukocytes are
polymorphonuclear neutrophils.
Most polymorphonuclear

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