basic techniquies in pathology in hematology.

1. STAINS IN HEMATOLOGY
PRESENTED BY-DR.JUHI SINGH
MODERATOR-DR.PARAMVIR KAUR MAM

3. LEISHMAN STAIN

5. GIEMSA STAIN

6. • Romanowsky stains used universally for
routine staining of blood films.
• Two components: azure B (trimethylthionine)
and eosin Y(tetrabromofluorescein).
• The original Romanowsky combination was
polychrome methylene blue and eosin
• Methylene blue enhance staining of
nucleoli and polychromatic red cells
,

7. • Wright stain gives results similar with
Leishman stain, whereas Wright–Giemsa gives
results similar with May–Grünwald–Giemsa.
• A pH of 6.8 is recommended.
• pH of 7.2- malaria parasites (Schüffner
dots.)
• Jenner - simplest and Giemsa - most complex.
• Leishman stain- Routine staining of blood films

8. • Uniform pH, 50 ml of 66 mmol/l Sörensen phosphate
buffer + each litre of the water.
• Azure B bound to anionic molecules and eosin Y bound to
cationic sites on proteins.

9. • Granules in the cytoplasm of neutrophil
leucocytes are weakly stained by the azure
complexes.
• Eosinophilic granules stains strongly with acidic
component of the dye
• Basophilic granules contain heparin,affinity for
basic component of dye
• (DNA) binds rapidly, (RNA) more slowly, and
haemoglobin more slowly still; hence the need to
have the correct azure B to eosin ratio to avoid
contamination of the dyes and to stain for the
right time.

10. Preparation of solutions of Romanowsky dyes
May–Grünwald stain
1. 0.3 g of the powdered dye in a conical flask of 200–250-ml
capacity.
2.Add 100 ml of methanol and warm the mixture to 50 °C.
3. Allow the flask to cool to c. 20 °C and shake several times
during the day.
4.After letting it stand for 24 h, filter the solution.
5. It is then ready for use, no ‘ripening’ being required.
Jenner stain
Prepare a 5 g/l solution in methanol in exactly the same way as
described earlier for the May–Grünwald stain.

11. Giemsa stain
1. 1 g of the powdered dye in a conical flask of
200–250-ml capacity.
2. Add 100 ml of methanol and warm the mixture
to 50 °C,
3. Keep at this temperature for 15 min with
occasional shaking, then filter the solution.
4. It is then ready for use, but it will improve on
standing for a few hours.

14. Azure B–Eosin Y stock solution
• Azure B, tetrafluoroborate or thiocyanate >80% pure
• Eosin Y >80% pure.
• Dissolve 0.6 g of azure B in 60ml of dimethyl
sulphoxide (DMSO) and 0.2 g of eosin Y in 50ml of
DMSO;
• Preheat the DMSO to 37 °C before adding the dyes.
• Allow the solution to stand at 37 °C, shaking
vigorously for 30 s at 5-min intervals.
• Add the eosin Y solution to the azure B solution and
stir well.
• This stock solution should remain stable for several
months if kept at room temperature in the dark.
DMSO will crystallize below 18 °C.

15. STAINING METHODS
May–Grünwald–Giemsa stain
• Dry the films in the air,
• Fix by immersing in methanol for 5–10min.
• For bone marrow films, allow a longer time to dry then
leave it for 15–20min in the methanol.

16. • Methyl alcohol (methanol) is the fixative of choice,
although ethyl alcohol (‘absolute alcohol’) can also
be used.
• Methylated spirits must not be used because it
contains water.
• Transfer the fixed films to a staining jar containing
May–Grünwald stain freshly diluted with an equal
volume of buffered water.
• After the films have been allowed to stain for about
15min, transfer them without washing to a jar
containing Giemsa stain freshly diluted with 9
volumes of buffered water, pH6.8

17. • After staining for 10–15min, transfer the slides to a jar
containing buffered water, pH6.8, rapidly wash in three
or four changes of water, and finally allow slides to
stand undisturbed in water for a short time (usually 2–
5min) for differentiation to take place.
• The slides should be transferred from one staining
solution to the other without being allowed to dry
• When differentiation is complete, stand the slides
upright to dry.
• This method is designed for staining a number of films
at the same time.
• Single slides may be stained by flooding the slide with a
combined fixative and staining solution.

19. Jenner–Giemsa stain -The stain is used with 4
volumes of buffered water and the films, after
being fixed in methanol, are immersed in the
Jenner stain for approximately 4min before being
transferred to the Giemsa stain. They should be
allowed to stain in the latter solution for 7–10min.
Leishman stain Air-dry the film and flood the slide
with the stain. After 2min, add double the volume
of water and stain the film for 5–7min. Then wash
it in a stream of buffered water until it has
acquired a pinkish tinge (up to 2min). After the
back of the slide has been wiped clean, set it
upright to dry.

20. Rapid staining method
The Field method provide a quick method for staining
thick films for malaria parasites .
Field staining
1. Dip the slide with the dried film on it into Stain
A for 3s. 2. Dip into a jar of tap water for 3s with
gentle agitation. 3. Dip into Stain B for 3s. 4. Wash
gently in tap water for a few seconds until all
excess stain is removed. 5. Drain the slide vertically
and leave to dry. Do not blot.

21. Stains -- Stain A (Polychromed methylene blue)

22. Dissolve the methylene blue and the disodium hydrogen
phosphate in 50ml of water.
Then boil the solution in a water bath almost to dryness
to ‘polychrome’ the dye.
Add the potassium dihydrogen phosphate and 500ml of
freshly boiled water.
After stirring to dissolve the stain, set aside the solution
for 24h before filtering.
Filter again before use.
The pH is 6.6–6.8.
Alternatively, azure B may be added to the methylene
blue in the proportion of 0.5g of azure B to 0.8g of
methylene blue, and the combined dyes are then
dissolved directly in the phosphate buffer solution.

23. Stain B (Eosin)
Dissolve the phosphates in warm freshly boiled water and then
add the dye. Filter the solution after letting it stand for 24h.

24. • Method
• Fix the film for 10–15s in methanol.
• Pour off the methanol and drop on the slide 12
drops of diluted Stain B (1 volume of stain to 4
volumes of water).
• Immediately add 12 drops of Stain A.
• Agitate the slide to mix the stains. After 1min, rinse
the slide in water, then differentiate the film for 5s
in phosphate buffer at pH6.6, wash the slide in
water and then place it on end to drain and dry.
• Two-stage stains of this type are also available
commercially