Exp : 16

PERIPHERAL SMEAR STUDY USING LEISHMAN’S STAIN

Preparation of blood films:
(1) Place the clean glass slide on a flat table.
(2) Touch the end of a slide keep one drop of blood sample.
(3) Spread the drop with a spreader or second slide.
(4) As soon as the blood has spread, push the spreader towards the other end of the slide (the
spreader must be held at an angle of about 30 – 45 degrees).
(5) The blood film should not be too thin or thick and allow it to dry.

Staining technique:
(1) Place slides on the staining rack with the blood film facing up.
(2) Cover the slide with Leishman’s stain for 2 to 3 minutes (this also allows fixation of
smears)
(3) Then add double the quantity of distilled water or buffer solution. The stain and distilled
water should be mixed evenly.
(4) Allow it stain for 6 – 8 minutes.
(5) The stain is then washed off with fresh distilled water.
(6) Dry the smear in air and examine microscopically.

DETERMINATION OF DIFFERENTIAL COUNT

AIM:
To determine differential count in the given blood sample.
PROCEDURE:
Differential count is done by one of the three methods.
(1) Going back and forth lengthwise on the smear including both the body and tail.
(2) Going back and forth sidewise on the smear including both the edges and center.
(3) Battlement method: In this a few cells in 1 – 2 strips longitudinally along the edge are
counted and then go across the smear and 1 – 2 strips along the opposite side are counted.
Once again the entire width of the smear is covered counting all seen cells come back to
the same edge.
 The different types of white cells seen in a normal peripheral blood film may be divided
into 3 main groups.
(1) Granulocytes
(2) Agranulocytes
(3) Monocytes
(1) Granulocytes :
Specific granules present in the cytoplasm of the cells are called granulocytes. There are
3 types of granulocytes.
(a) Neutrophils or polymorphs
(b) Eosinophils
(c) Basophils
(a) Neutrophils :
Granules are present and are uniformly distributed in the cytoplasm. The nucleus is usually
divided into 1 – 4 lobes. Sometimes more and the number of lobes gives an indication of the age
of the cells (the greater the number the older the cell).
Diameter -- 10 – 12 microns
(b) Eosinophils :
The cytoplasm contains large oval or round orange red granules which usually fill the
cytoplasm completely. The nucleus is divided into 2 lobes, connected by a thin strand of
chromatin and also called “spectacle” arrangement. The granules show affinity towards the acid
such as eosin.
Diameter -- 10 – 12 microns
(c) Basophils or mast cells :
The most prominent feature is the presence of large, round, deep purple to black granules
which vary considerably in size. They are fewer in number than eosinophilic granules and most
often are found overlying the nucleus. Therefore the nucleus is not clearly seen.
Diameter -- 8 – 10 microns
(2) Agranulocytes : (Lymphocytes)
The cell does not have any granules and hence it is called as agranulocytes. They has
relatively more cytoplasm and large round or slightly indented nucleus.
Diameter -- 10 – 12 microns
(3) Monocytes :
The cytoplasm is more abundant in relation to the nucleus. The nucleus is kidney shaped
or horse shoe shaped. If the cell is very large, vacuolated cytoplasm it is some times called
histocyte.
Diameter -- 16 – 22 microns
Result:
The differential count of the given blood sample is --------------

Normal range:
Neutrophils : 40 – 75 %
Eosinophils : 1 – 6 %
Basophils: 0 – 1 %
Lymphocytes: 20 – 50 %
Monocytes : 2 – 10 %