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Cytochemistry
Cytochemistry
Cytochemistry
ENZYMATIC NON-ENZYMATIC
1. Myeloperoxidase 4. Sudan black B
2. Esterase 5. Periodic acid Schiff
a) specific 6. Toluidine blue
b) non specific 7. Perl’ stain
3. Phosphatase
a) leucocyte alkaline phosphatase
b)acid phosphatase
MYELOPEROXIDASE (MPO)
❖ It is an enzyme present in primary and secondary granules of neutrophils
and their precursors.
❖ Also present in eosinophil granules and in azurophillic granules of
monocytes.
Purpose
❖ Differentiate between myelogenous and monocytic leukaemia from acute
lymphoblastic leukaemia(ALL).
❖ Diagnose congenital deficiency of neutrophil MPO.
Principle:-
Myeloperoxidase splits H2O2 in the presence of chromogenic
electron donor (e.g. DAB) and forms an insoluble reaction product.
The reaction product is stable, insoluble and non diffusible.
Method:-
Pseudo peroxidase or Lepehre reaction
Reagents:-
• Fixative:- Buffered Formal acetone (BFE)
Acetone - 40 ml
Buffer - 30 ml
Formalin- 25 ml
• Substrate:- 3,3’- DAB (Diaminobenzidine)
• Buffer:- Sorensen's phosphate buffer, PH-7.3
• Hydrogen peroxide(H2O2, 30% w/v)
• Counterstain:- Aqueous haematoxylin.
Procedure:-
1) Fix air dried smear in cold buffered acetone for 30 sec.
2) Rinse in running tap water and dry.
3) Incubate for 10 min. in working substrate solution.
Results:-
The reaction product is brown and granular.
All nuclei are blue.
Interpretation:-
• Early myeloblasts are negative, with granular positivity appearing
progressively as they mature.
• Dark brown granules in the cytoplasm of granulocytes and monocytes.
• Monocytes exhibit weaker and more scattered staining properties than
granulocytes.
• RBC’S will stain diffusely brown because haemoglobin has pseudo-
peroxidase activity. Hence, act as internal control.
• Eosinophil granules stain strongly and they are cyanide resistant MPO
positive.
• Auer rods stain well with DAB.
• Plasma cells and lymphoblast are negative.
• Peroxidase activity is present in basophil but not demonstrable by DAB.
red brown precipitate
MPO stains reveal strong granular cytoplasmic staining in many leukemic
blasts. MPO positive Auer rods are present .(Intensified Stain )
• MPO Stained positive •Myeloperoxidase staining of
neutrophil at left. The blast is negative.
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 which MPO can not be performed.
Principle:-
The SBB is a lipophilic dye binds irreversibly to an unidentified /
undefined granule in granulocytes and eosinophils.
Method:- SHEEHAN AND STOREY
Reagents:-
• Fixative:- 40% formaldehyde vapours
• Stain:- 0.3% SBB in absolute alcohol
• Phenolic buffer:- 16gm crystalline phenol in 30ml of absolute
ethanol and final volume up to 100ml with buffer.
• Working stain solution:- add 40ml phenolic buffer to 60ml SBB
solution.
• Counter stain :- Leishman stain
Procedure:-
1) Fix air dried smear in in formalin vapours for 5-10 min.
2) Then air wash for 15 min.
3) Now stain with working solution of SBB stain solution for 1 hour.
4) Now give 3 washings of ethanol for 30 sec each.
5) Wash with water and air dry.
6) Now counter stain with Leishman stain.
Results:-
The reaction product is black and granular.
All nuclei are blue.
Interpretation:-
• The results are similar to MPO staining
both in normal and leukemic cells.
• The differences are:-
The eosinophil granules are SBB negative
• In rare cases (1-2%) of ALL shows non
granular smudgy positivity not seen in
MPO staining e.g. in Burkett's lymphoma.
• Lipids are present in azurophilic and
secondary granules of myeloblast so they
give positive result
• Lymphoblast have no lipids so they give
Sudan Black B
• Positive sudan black B (SBB)
stain in a patient with AML ,
• Not the black staining
cytoplasmic granules in the
myeloblasts
4+
2+
Principle:-
Esterase enzyme present in leucocyte hydrolyses the substrate esters
and the product formed react with a diazonium salt and form
brightly formed coloured compound.
Purpose:-
• Useful in differentiating myelocytic series from monocytic series.
• To distinguish between normal and leukemic cells of both series.
1) Naphthol AS-D chloroacetate esterase
useful as a marker of cytoplasmic maturation in myeloid
leukaemia.
Reagents:-
• Fixative :- buffered formal acetone
• Buffer :- 66mmol/l phosphate buffer,ph7.4
• Substrate :- Naphthol AS-D chloroacrtate in buffer
• Coupling reagent :- Hexazotized new fuchsin in 4% sodium nitrate
solution.
• Couterstain :- Aqueous haematoxylin
Procedure:-
1) Fix air dried smears in cold buffered formal acetone for 30sec.
2) Rinse gently in running tap water and air dry.
3) Treat the slides with substrate solution for 5- 10 min.
4) Rinse in running tap water and air dry.
5) Couterstain with aqueous haematoxylin for 1 min.
6) Blue in running tap water and air dry.
Results :-
• The reaction product is bright red.
Interpretation :-
• The positivity is confined to the neutrophil series and mast cells.
• Positivity in myeloblast but mature atages stains strongly.
• Auer rods are positive.