HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.

BONE MARROW: PEROXIDASE STAIN HUSM/HEMA-UPT/STM-BM4 VERSION NO. VERSION DATE. 1 24.03.2011

APPROVED BY:

... ASSOC PROF DR ROSLINE HASSAN HEAD OF HAEMATOLOGY DEPARTMENT CONTROLLED COPY NO : 3 REGISTERED HOLDER HAEMATOLOGY LABORATORY

RECORD OF REVIEW/AMMENDMENT

DATE

VERSION NO.

DETAIL OF AMMENDMENT

BY

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.

PREPARED BY DESIGNATION CHECKED BY DESIGNATION AUTHORISED BY DESIGNATION

BONE MARROW: PEROXIDASE STAIN HUSM/HEMA-UPT/STM-BM4

VERSION NO. VERSION DATE.

1 24.03.2011

: ANG CHENG YONG : TRAINING OFFICER / SCIENTIFIC OFFICER : DR SHAFINI MOHAMED YUSOFF : HAEMATOLOGIST : ASSOC PROF DR ROSLINE HASSAN : HAEMATOLOGIST/LAB DIRECTOR

1. 2. 3.

OBJECTIVE
To detects the presence of the enzyme peroxidase in cells.

METHOD
Manually

PRINCIPLE
The members of the granulocytic series contain an enzyme proxidase, which liberates the oxygen from hydrogen peroxide. This enzyme is more prominent in mature forms. A benzidine derivative is used as an indicator of peroxidase activity; the indicator is oxidased and precipitates in the form of brown to blue granules. This stain is used to help differentiate leukemias.
peroxidase

DAB + H2O2 4.

----------------> oxidized DAB (brown pigment)

REQUIREMENTS
4.1

EQUIPMENT
4.1.1 4.1.2

Staining Container Microscope slides and cover slips 10% Formal Ethanol 40% Formaldehyde 90% Absolute Ethanol Peroxide Solution 30% H2O2 Distilled Water -

4.2

REAGENT 4.2.1 10 ml 90 ml

4.2.2

0.2 ml 50 ml

4.2.3

DAB Stock 30% Ethanol 3,3 Diaminhobenzide (Sigma: D8001) ZnSO4.7H2O (38g/L or 1.9 g/50ml)) Sodium Acetate (NaC2H3O2.3H2O)

100 ml 0.3 g 1.0 ml 1.0 g

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO. BONE MARROW: PEROXIDASE STAIN HUSM/HEMA-UPT/STM-BM4 VERSION NO. VERSION DATE. 1 24.03.2011

Mix the reagents and adjust the pH to 6 with 0.8 ml of 2N HCl. Filter the solution and keep it in 4oC in amber bottle. Add in 0.006% of H2O2 (final concentration) before use. 4.2.4 i) Phosphate Buffer pH 6.8 Potassium Dihydrogen Orthophosphate (KH2PO4) Potassium Dihydrogen Orthophosphate (KH2PO4) - 9.1 g Distilled Water - 1L Disodium Hydrogen Phosphate (Na2HPO4) Disodium Hydrogen Phosphate (Na2HPO4)) Distilled Water Working phosphate Buffer pH6.8 Solution (i) Solution (ii) Giemsa stain DPX (mountant) - 9.5 g - 1L - 50.8 ml - 49.2 ml

ii)

iii)

4.2.5 4.2.6 4.3

QUALITY CONTROL MATERIAL Smear from labour rooms patient with high white blood cell count range from 11 x 103/l to 18 x 103/l

4.4

SPECIMEN Air-dried bone marrow smear

PROCEDURE
NO. 5.1 ACTIVITY Fix both of the air-dried patient and positive control bone marrow smears with 10% of Formal Ethanol for approx. 10 minutes Note: Bone marrow should be dry (at least 1 hour after preparation) before fix Wash slides in running tap water and air dry the slides Mix 19 ml of DAB stock with 1.0 ml of Peroxide Solution and incubate the slide into it for approx. 10 minutes Wash in running tap water and air dry. Mix 1 part of Giemsa in 9 parts of Phosphate Buffer pH6.8and incubate the slides in the stain for approx. for 10 minutes Wash with running tap water for few seconds Air dry the slide and mount the smear with DPX RESPONSIBILITY

MLT/SO

5.2 5.3 5.4 5.5 5.6 5.7

MLT/SO MLT/SO MLT/SO MLT/SO MLT/SO MLT/SO

6.

RESULTS/INTERPRETATION
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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO. BONE MARROW: PEROXIDASE STAIN HUSM/HEMA-UPT/STM-BM4 VERSION NO. VERSION DATE. 1 24.03.2011

The reaction product is brown and granular. Red cells and erythroid precursors show diffuse brown cytoplasmic staining. The most primitive myeloblasts are negative with granular positivity appearing progressively as they mature toward the promyelocyte stage. Promyelocytes and myelocytes are the most strongly staining cells. Metamyelocytes and neutrophilis have progressively fewer positive granular. eosinophil granules stain strongly and large specific eosinophil granular are easily distinguished from neutrophil granules . Monoblast and monocytes may be negative or positive. The granules are smaller than in neutrophils and diffuse scattered throughout the cytoplasmic if the reaction present considered as positive. 7. REFERENCES
6.1 S. M. Lewis, B. J. Bain & I. Bates. (2006) Dacie and Lewis Practical Haematology, 10
th

edition. Churchill Livingstone.

End of Document

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