Cytochemistry ii

1. Rabab Salama Clinical and Chemical Pathology Consultant,
HCQM specialist

2. Why are the special stains special?

3. Objectives of Special Staining
To provide a definitive diagnosis.
 In a differential diagnosis of
hematological conditions.

4. • Stains for collagen
• Stains for muscle
• Stains for elastic tissue
• Stains for reticulin fibers
• Stains for carbohydrates
• Stains for amyloid
• Stains for lipid
• Stains for pigments & minerals
• Stains for nerve tissue
• Stains for microorganisms
• Stains for decalcified bone
Broadly classifying the special stains under
following category…..

5. Sudan Black B (SBB)
Sudan black B is a lipophilic dye that stains intra cellular
phospholipids and other lipids.
Purpose:
 Same significant as MPO i.e. to differentiate between ALL and
AML.
 Done in old smears in witch MPO can not be performed.
Principle:
 The SBB is a lipophilic dye binds irreversibly to an unidentified
/ undefined granule in granulocytes and eosinophils.
Results:
 The reaction product is black and granular.
 All nuclei are blue.

6. Reagents
 Fixatives
 40% formaldehyde solution
Stain
 0.3% SBB in absolute alcohol
Phenol buffer
 16 gm crystalline phenol in 30 ml of absolute
ethanol and final volume up to 100 ml with
buffer.
 Counterstain
 Leishman stain.
Sudan Black B (SBB)

7. Results and interpretation
• Reaction product is black and granular
• Results are essentially similar to MPO staining
• MPO negative neutrophils are also SBB negative
• The only notable difference is in eosinophil granules
which have a clear core when stained with SBB
• Rare cases of ALL show non-granular smudge
positivity not seen with MPO

8.  Positive sudan black B (SBB)
stain in a patient with AML
 Note the black staining
cytoplasmic granules in the
myeloblasts
Sudan Black B (SBB)

9.  Positive sudan black B (SBB) stain in a patient
with AML ,
 Not the black staining cytoplasmic granules in
the myeloblasts
SBB positive prominent Auer rods in
bone marrow smear
Sudan Black B (SBB)

10. Periodic Acid Schiff (PAS)
Purpose:
 Differentiate between AML (diffuse positivity) and ALL (block
positivity).
 Useful in AML and MDS to identify abnormal erythroblast an
dysplastic megakaryocytes.
 Confirm diagnosis of acute promyelocytic leukaemia.
Principle:
 Periodic acid specifically oxidizes 1-2 glycol groups of
carbohydrates to produce stable di aldehydes. These di aldehydes
give a red reaction product when exposed to Schiff’s reagent.

11. Reagents
 Fixatives
 Methanol
Stain
 1% periodic acid (HIO4)
 Schiff’s reagent
 Counterstain
 Aqueous hematoxylin
Periodic Acid Schiff (PAS)

12. Results:
 The reaction product is red.
 Intensity ranging from pink to bright red.
 cytoplasmic positivity may be diffuse or granular.
Interpretation:
 In hemopoietic cells, the main source of positive reaction is glycogen.
 Granular precursors shows diffuse weak positivity with neutrophils
showing intense granular positivity and act as internal positive controls.
 Eosinophil granules are negative with diffuse cytoplasmic positivity.
 Basophils are negative.
Periodic Acid Schiff (PAS)

13. Periodic Acid Schiff (PAS)

14. Positive PAS stain acute
megakaryocytic leukemia AML, M7.
Positive PAS stain in ALL
PAS positivity in M6. Not the
intense staining of the large
abnormal erythroblast.

15. Diffuse PAS positivity Block PAS positivity
Periodic Acid Schiff (PAS)

16. Toluidine Blue
It is a basic thiazine metachromatic dye with high affinity to acidic tissue
components.
Purpose:
 Useful for the enumeration of basophils and mast cells

17. Reagents:
 1% toluidine blue in methanol (w/v)
Results and interpretation:
 The granules of basophils and mast cells stain a bright red and
purple.
 Nuclei stain blue and cells with abundant RNA may show a blue tint to
the cytoplasm.
Toluidine Blue

18. Perl’s Iron stain
(Prussian Blue Reaction)
Principle:
 Sidrotic granules are found in the cytoplasm of developing
cells in [BM] in the form of Ferric [Fe+3].
 Perls' reagent is formed of (Potassium Ferricyanide + HCL)
 Sidrotic granules are found in nRBCs & some reticulocytes

19. Reagents
 Fixatives
 Methanol
Stain
 Potassium ferricyanide
 HCl
 Counterstain
 Aqueous neutral red or eosin
Perl’s Iron stain
(Prussian Blue Reaction)

20. Notes
Care must be taken to avoid contamination by
iron which may have been present on the slides.
Prepare glassware by soaking in HCl before
washing.
For QC, a +ve bone marrow film should always be
stained together with the test film.
Perl’s stain can be applied to films which have
previously been stained by Romanowsky dyes,
even after years of storage (It's advisable to let
the films stand in methanol overnight to remove
most of the stain).

21. Pappenheimer bodies:
These represent iron
deposits which as dense blue,
irregular granules in wright
stain.
Found in:
 Hemolytic anemia
 Splenectomy
 Sideroblastic anemia
 Thalassemia.

22. Perl’s Iron stain
(Prussian Blue Reaction)

23. Reticular fibres
 fine, delicate, branching fibres
 bulk of supporting framework of liver, spleen and
lymph nodes.
Dyes  Not reliable
 Demonstration
Metal impregnation  best
Gomori’s
 Metal impregnation
Gordhon and Sweets’
Reticulin Stain

24. Principle:
Silver from ammonical silver solution gets
selectively deposited on reticular fibers. On
treatment with a reducing agent, silver gets reduced
to metallic silver allowing mineralization of reticulin
fibers.
Results:
Reticulin fibers – Black color
Reticulin Stain

25. Reticulin Stain

26. Reticulin Stain

27. Reticulin Stain

28. Reticulin Stain

29. Reticulin Stain

30. Reticulin Stain

31. Reticulin Stain

32. Disadvantages
i) Strongly alkaline solution tends to lift the sections  mounting on
gelatin coated slides.
ii) Ammonical silver salt is highly explosive
 DO NOT expose to sunlight
 Unused reagent  add dilute HCl.
iii) Excess of ammonia in solution  Loss of sensitivity  add less of
ammonia
Other Precautions :
 Clean glassware
 Fresh reagent
 Accurate measurement
 Fresh ammonia
Reticulin Stain

33. Conclusion

34. Stain Structure stained Use
Periodic assay Schiff (PAS) Carbohydrates, principally
glycogen
In AML and MDS to identify
abnormal erythroblasts and
dysplastic megakaryocytes
To confirm the diagnosis of
acute promyelocytic leukaemia
Perl’s Prussian Blue Reaction Iron For demonstration of ring
sideroblasts and Pappenheimer
bodies
Leucocyte alkaline phosphatase
(LAP)
Neutrophil alkaline phosphatase Differentiate chronic
myelogenous leukemia from
leukemoid reactions and other
myeloproliferative disorders

35. Stain Structure stained Use
Myeloperoxidase Myeloperoxidase in neutrophils,
monocytes and eosinophils
To differentiate a myelogenous or
monocytic leukaemia from acute
lymphocytic leukaemia.
To visualize auer rods
Sudan Black B Intracellular lipid Similar to MPO
Toluidine Blue Basophils and mast cells To identify dysplastic basophils in
myeloproliferative diseases
Specific esterases Neutrophil series and mast cells Marker of cytoplasmic maturation
in myeloid leukaemias
Non- Specific esterases Monocytes Monocytic component in AML
Tartarate resistant acid
phosphatase
T-cells and granulocytes Diagnosis of T-cell ALL and hairy
cell leukaemia