PEROXIDASE STAINING FOR bone-marrow smear

PEROXIDASE STAINING

This staining reaction is used to differentiate between the cells of lymphoid series and those of myeloid and monocyte series. The granules in both the latter series of cells contain peroxidase which liberates oxygen from hydrogen peroxide and oxidises benzidine. A blue colour indicates a positive reaction.

Specimen A thin peripheral blood film or a bone-marrow smear.

Reagents 

1. Fixative 

95 % ethyl alcohol 9 parts 

Formaldehyde1 part 

2. Staining solution

Benzidine dihydrochloride 0.3 g 

Methanol 30 ml 

Dissolve benzidine dihidrychloride in methanol 

Add distilled water 70 ml 

3.8 % Zinc sulphate 1.0 ml

Sodium acetate 1.0 g 

I N Sodium hydroxide 1.5 ml 

Add 0.7 ml of 10 vol hydrogen peroxide just before use.

Note

Benzidine is carcinogenic, and therefore, must be handled with utmost care.

Dilute the stock Giemsa solution (commercially available) 1:10 in 0.066 M

phosphate buffer, pH 6.4.

Technique

(i)                Fix the dried smear in the fixative for 60 seconds.

(ii)              Wash under tap water for 15 seconds.Immerse in the benzidine staining solution for 30 seconds.

(iii)            Wash under tap water for 5-10 seconds.

(iv)             Counterstain with Giemsa for 10 minutes.

(v)               Wash, dry and examine under the oilimmersion objective.

Interpretation A positive peroxidase reaction is indicated by the presence of blue-back granules in the cytoplasm. Early granulocyte precursors show a weak, localised reaction, becoming stronger and more generalised with increasing cell maturity, Monocytes may be negative, but sometimes show a weak positive reaction. Normal lymphoid and erythroid cells are invariably negative. Auer bodies

can be demonstrated by peroxidase reaction in myeloblasts and promyelocytes. The peroxidase reaction is most useful for differentiation between leukaemias of myeloid and lymphoid series.