Optimal results
are obtained by fixing and staining immediately after the blood film is
completely air-dried. Fixation of blood films before staining is recommended,
although many laboratories have a practice of staining immediately after
air-drying of blood films. This procedure
usually yields acceptable results. methanol, is essential for good staining and
presentation of cellular detail.
However, if slides cannot be stained
immediately, fixation in methanol is necessary within 4 hours, but preferably 1
hour after air-drying; otherwise, the plasma will cause gray/blue background
effects. Staining and fixing solutions must be as free of water as possible
(<3%) to prevent morphological artifacts.
If manual
staining procedures are used, it is recommended that slides be immersed in
reagent-filled (Coplin) jars rather than covering slides with staining solution
because formation of precipitate by evaporation may occur. If staining is
performed under extremely hot conditions, care must be taken to prevent
evaporation during staining, eg, by performing staining in a closed jar or in a
closed petri dish. Automated staining devices have their own specific staining
procedures that are provided by the manufacturers. Users may modify these
procedures to satisfy local requirements for cell staining. Staining protocols
vary between laboratories, and no generally accepted routine staining method or
result is available.
ICSH has published a “reference” staining method for blood
films based on purified azure B and eosin Y solutions (ICHS, 2010). As a
general rule for judging the quality of a stained blood film, the laboratory
must ensure that all cell types in a blood film can be identified reliably by
the staining procedure. This rule applies equally to manual and automated
methods. Blood films are typically stained by Romanowsky dyes (consisting of a
variety of thiazines and eosins) (Henry, 1998). A number of methods have been
described and include the following: Wright, Wright-Giemsa, May-Grünwald-Giemsa,
and Leishman., giemsa, as described above.
STAINING
TECHNIQUE USING LEISHMANN STAIN:
1.
Prepare
a thin smear as described earlier. Dry the smear in air
2.
Cover
the smear completely with Leishman stain
3.
Stain
for 1 to 2 minutes
4.
Dilute
the stain with twice its volume of the buffer solution
5.
Wash the slide with the buffer solution. Use
tap water if not too acidic or alkaline.
6.
Drain
and dry in air by keeping it in a staining position.
STAINING
TECHNIQUE USING GIEMSA STAIN:
1.
Prepare
a thin blood film as discussed earlier and dry in air
2.
Fix
in methanol for 3 minute
3.
Dilute
one volume of the solution (Azure II eosin, Azure II, glycerol, methyl alcohol)
with 9 volumes of the buffer solution.
4.
Flood
the slide with the stain and allow to act for 10-15 minutes
5. Wash
and differentiate with the buffer solution. Drain, dry in air and examine
microscopically.
METHODS OF BLOOD FILM READING IN
HAEMATOLOGY
There
are two major methods employed while reading blood films in haematology. They
are:
·
Battlement
Method:
In battlement method, the blood film is examined systematically by being
traversed three fields along the edge, two fields up, two fields along and two
fields down. This sequence is continued until a minimum of 100 white cells have
been counted.
·
Longitudinal
method:
in the longitudinal method, the different types of white cells are counted in
one complete longitudinal strip of the film. If less than 100 cells are
counted, a second strip should be similarly enumerated (Baker et al., 2009).
