Preparation and staining of peripheral blood smear

2. Preparation and staining of
peripheral blood smear

3. Preparation of blood film
• Making an idel slide is backbone of
Hematology ……….
• Wedge Technique
• Preparation of blood film is a fundamental
technique in haematology and done for
differential leukocyte count and observing red
cells morphology.

4. Requirements
• Spreader
Select a glass microscope slide or use a cover
slip with at least one smooth end as spreader.
The edge must be wiped carefully and dried
before and after each use, and the slide must
be discarded if the spreading edge becomes
chipped.

5. Requirements
• Clean Slides
 It is essential to use clean, dry, dust free slides.
 Boxes of clean grease-free slides may be available.
 If not available then proceed as follows.
 Leave the slides overnight in a detergent solution.
Then wash thoroughly in running tap-water, dry with
a clean linen cloth. Before use, wipe the surface with
methylated spirits(95% ethanol) or methanol and dry
with a clean cloth.

6. Procedure (Thin Blood Film)
1) Place a drop of blood in the centre line of slide 1 cm from
one end.
2) Place the spreader in front of glass slide at an angle of 30o.
3) Move the spreader back, so that it touches the blood
drop. Blood will spread along the edge of spreader by
Capillary action.
4) Push the spreader forward along the length of slide by
rapid but straight movement.
5) Then allow the smear to dry in the air before staining.
6) When dry, patient’s name, number and other
identifications may be written on the slide with a diamond
marker or pencil.

7. Procedure (Thick Blood Film)
• These are widely used in the diagnosis of
Haemoparasites, particularly malarial parasites.
Generally the blood films should be made about
10 times thickness of normal smears.
1) Place a drop of blood on a clean slide and
spread it with the corner of another slide until
the printed matter is just visible through the
smear.
2) Allow to air-dry and label the slide properly.

8. Sources of Errors
1) Film extending to end of slide: blood drop
too large.
2) Short thin film: blood drop too small.
3) Irregular spread with ridges and long tail:
Edge of spreader dirty or chipped; dusty
slide.
4) Holes in film: Slide contaminated with fat or
grease.

9. Sources of Errors
5) Cellular degenerative changes: delay in fixing,
inadequate fixing time or methanol contaminated
with water.
6) Irregular leukocyte and platelet distribution,
especially in tail: poor film-making technique.
7) Film too short and too thick: spreader held at
incorrect angle
8) Film extends to edge of slide: spreader too wide or
not positioned correctly

12. STAINING OF BLOOD FILM
• After preparation, the slide is stained to
distinguish the cells from each other.
Cytohaematology is the study of the cellular
components of blood cells. Parasites may also
be observed during microscopic examination
of the blood smear.

13. LEISHMAN’S STAIN
• It is named after Sir William Boog Leishman from
Scotland.
• Stain Preparation
Dissolve 0.15 g of leishman’s stain powder in 100
ml of absolute methanol.
The stain is then filtered into stock bottle.
Place at 500C for 15 minutes in water bath.
Again filter into clean brown borosilicate glass
bottle and store in dark at room temperature.

14. LEISHMAN’S STAIN
• Staining Procedure
1) Prepare blood film, air dry and place in a
staining rack.
2) Cover the smear with stain and leave for 2
minutes. The methanol fixes the smear.
3) Pour distilled water on the slide about twice
the amount of stain until a metallic scum
appears. Allow this diluted stain to act for 5-7
minutes. During this time differentiation
takes place.

15. Staining Procedure continued…….
4) Without disturbing the slide, flood with
distilled water and wash until thinner parts of
the film are pinkish red.
5) If distilled water doesn’t give adequate
differentiation, a buffer solution of pH 6.8
can also be used.

16. GIEMSA’S STAIN
• It is named after Dr. Gustav Giemsa from
Germany.
Stain Preparation
Transfer 0.3 g of Giemsa stain powder to a
motor and mix with 25 ml of glycerin
thoroughly. This makes the stock solution.

17. GIEMSA’S STAIN
• Staining Procedure
1) Prepare blood film, air dry and place in a
staining rack.
2) Fix with methanol for 3 minutes.
3) Dilute the stain 1 in 10 with distilled water or
buffer solution of pH 6.8.
4) Cover the blood film with diluted Giemsa
stain for 10 minutes.
5) Wash with distilled water, dry and examine
under microscope.