BLOOD SMEAR
EXAMINATION
I- Preparation of blood smear
There are three types of blood
smears:
1. The cover glass smear.
2. The wedge smear .
3. The spun smear.
The are two additional types of blood
smear used for specific purposes
1. Buffy coat smear for WBCs < 1.0×109/L
2. Thick blood smears for blood parasites .
PROCEDURE
Placing a drop of blood from mixed
sample on a clean glass slide.
Spread the blood in forward direction
using another clean glass slide at 30-40
degree angle.
Control thickness of the smear by
changing the angle of spreader slide.
Allow the blood film to air-dry completely
before staining.
STEPS FOR BLOOD FILM
The thickness of the spread
Notes:
1. If the hematocrit is increased, the angle of
the spreader slide should be decreased.
2. If the hematocrit is decreased, the angle of
the spreader slide should be increased.
high HCT
small angle
low HCT
large angle
CHARACTERISTICS OF A GOOD SMEAR
1. Thick at one end, thinning out to a smooth
rounded feather edge.
2. Should occupy 2/3 of the total slide area.
3. Should not touch any edge of the slide.
4. Should be margin free, except for point of
application.
5. Should be well differentiated in three
parts i.e head, body and tail.
COMMON CAUSES OF A POOR BLOOD
SMEAR
1. Drop of blood too large or too small.
2. Spreader slide pushed across the slide in a jerky manner.
3. Failure to keep the entire edge of the spreader slide against
the slide while making the smear.
4. Failure to keep the spreader slide at a 30° angle with the slide.
5. Failure to push the spreader slide completely across the slide.
6. Irregular spread with ridges and long tail: Edge of spreader
is dirty or chipped; dusty slide
7. Holes in film: Slide contaminated with fat or grease.
Examples of unacceptable smears
BIOLOGIC CAUSES OF A POOR SMEAR
1. Cold agglutinin - RBCs will clump
together. Warm the blood at 37° C for 5
minutes, and then remake the smear.
2. Lipemia - holes will appear in the smear.
There is nothing you can do to correct this.
3. Rouleaux - RBC’s will form into stacks
resembling coins. There is nothing you
can do to correct this
SLIDE FIXATION &
STAINING
LEISHMAN'S STAIN
Fixing the films
To preserve the morphology of the cells, films must be fixed
as soon as possible after they have dried.
It is important to prevent contact with water before fixation
is complete.
Methyl alcohol (methanol) is the choice, although ethyl
alcohol ("absolute alcohol") can be used.
Methylated spirit (95% ethanol) must not be used as it
contains water.
To fix the films, place them in a covered staining jar or tray
containing the alcohol for 2-3 minutes.
Staining the film
Romanowsky staining:
Romanowsky stains are universally employed for staining blood
films and are generally very satisfactory.
There are a number of different combinations of these dyes, which
vary, in their staining characteristics.
1. May-Grunwald-Giemsa is a good method for routine work.
2. Giemsa stain is thought to produce more delicate staining
characteristics.
3. Wright's stain is a simpler method.
4. Leishman's is also a simple method, which is especially suitable
when a stained blood film is required urgently.
5. Field's stain is a rapid stain used primarily on thin films for malarial
parasites.
Staining procedure (Leishman’s stain)
Thin smear are air dried.
Flood the smear with stain.
Stain for 1-5 min. Experience will indicate the
optimum time.
Add an equal amount of buffer solution and
mix the stain by blowing air on the slide using
pipette.
Leave the mixture on the slide for 10-15 min.
Wash off by running water directly to the center
of the slide to prevent a residue of precipitated
stain.
Stand slide on end, and let dry in air.
TOO ACIDIC SUITABLE TOO BASIC
CAUSES & CORRECTION
Too Acid Stain:
1. insufficient staining time
2. prolonged buffering or washing
3. old stain
Correction:
1) lengthen staining time
2) check stain and buffer pH
3) shorten buffering or wash time
Too Alkaline Stain:
1. thick blood smear
2. prolonged staining
3. insufficient washing
4. alkaline pH of stain components
Correction :
1) check pH
2) shorten stain time
3) prolong buffering time
Staining procedure (Giemsa’s stain)
Preparation of Giemsa Stain:
Staining procedure (Giemsa’s stain)
Thin smear are air dried.
Flood the smear with stain (1:9 diluted with
buffer solution).
Wash off by running water directly to the center
of the slide to prevent a residue of precipitated
stain.
Stand slide on end, and let dry in air.
