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.