HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA

TITLE : PROCEDURE NO. BONE MARROW: ACID PHOSPHATE STAIN HUSM/HEMA-UPT/STM-BM7 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 1. OBJECTIVE
To demonstrate acid phosphatase enzyme is ubiquitous in haemopoietic cells, T-lymphocytes and lymphoblasts have a characteristic "dot-like" focus of intense positivity, whereas the activity in most other cells is diffuse.

BONE MARROW: ACID PHOSPHATE STAIN HUSM/HEMA-UPT/STM-BM7

VERSION NO. VERSION DATE.

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24.03.2011

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

2. 3.

METHOD Manually PRINCIPLE

Acid phospahate in the cells hydrolyzes the substrate, napthol AS-BI phosphoric acid. The naphthol released is insoluble and couples with hexazotized pararosaniline. The reddish colored precipitate in the cytoplasm of the cells indicates acid phosphatase activity.

4.

REQUIREMENTS
4.1

EQUIPMENT
4.1.1 4.1.2 4.1.3

Staining Container Water bath Microscope slides and cover slips

4.2

REAGENT 4.2.1 0.03 M Citric Buffer pH5.4 0.03M Citric Acid (6.30 g/L) 0.03 M Trisodium Citrate (8.823 g/L) 4.2.2 160 ml 340ml

Fixative solution Methanol 1.6 ml Acetone 9ml 0.03M Citric Buffer pH5.4 6 ml Keep the mixture solution in freezer for approx. 5 minutes 0.2 M Glacial acetic acid Glacial acetic acid Distilled water 0.2 M Sodium Acetate Sodium Acetate Distilled water 0.2 M Acetate Buffer pH 5.0 0.2 M Glacial Acetate acid (refer to 4.2.3) 1.2 ml 98.8ml 2.7 g 100 ml 7.4 ml

4.2.3

4.2.4 4.2.5
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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA

TITLE : PROCEDURE NO. BONE MARROW: ACID PHOSPHATE STAIN HUSM/HEMA-UPT/STM-BM7 0.2 M Sodium Acetate (refer to 4.2.4) Distilled water 4.2.6 VERSION NO. VERSION DATE. -

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24.03.2011 17.6 ml 25 ml

Napthol AS-BI Phosphate Acid & N-N Dimethyl Formamide Solution Napthol AS-BI Phosphate Acid 0.02 g N-N Dimethyl Formamide 1 ml 4 % Sodium Nitrate Sodium Nitrate 4g Distilled water 100 ml Aliquot into 1 ml of 4 % Sodium Nitrate into test tube and keep in freezer 1 ml 1 ml

4.2.7

4.2.8 Sodium Nitrate & Pararosanaline Solution 4 % Sodium Nitrate Pararosanaline Mix the solution for at least 2 minutes 4.2.9 Acid Phosphate Working Solution 0.2 M Acetate Buffer pH 5.0 (refer to 4.2.5) Napthol AS-BI Phosphate Acid & N-N Dimethyl Formamide Solution (refer to 4.2.6) Sodium Nitrate & Pararosanaline Solution (refer to 4.2.8) 0.1 N NaOH

25 ml 1 ml 1 ml 0.4 ml 0.4 g 100 ml

4.2.10 0.1 N NaOH Sodium Hydroxide Distilled Water

4.2.11 2 % Methyl Green Methyl Green 2g Distilled water 100 ml Dissolve the Methyl Green in distilled water. Then add in 150 ml of Chloroform and leave it for 48 hours in dark condition. Discard the bottom part solution (clear solution) 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
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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA

TITLE : PROCEDURE NO. 5 PROCEDURE NO. 5.1 ACTIVITY
Fix both of the air-dried patient and positive control bone marrow smears with Fixative solution (refer to 4.2.2) for approx. 5 minutes

BONE MARROW: ACID PHOSPHATE STAIN HUSM/HEMA-UPT/STM-BM7

VERSION NO. VERSION DATE.

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24.03.2011

RESPONSIBILITY

Note: Bone marrow should be dry (at least 1 hour after preparation) before fix 5.2 5.3 5.4 5.5 5.6 5.7 5.8 Wash slides in running tap water for few seconds and air dry the slides Incubate the slides into Acid Phosphate Working Solution (refer to 4.2.9) for at least 2 hours Rinse in running tap water for few seconds Pour 2 % Methyl Green (refer to 4.2. on the slides and incubate for approx. 20 minutes Wash in running tap water for approx. 15 minutes Pour 2 % Methyl Green on the slides and incubate for approx. 10 minutes Rinse in running tap water and air dry the slides

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

RESULTS/INTERPRETATION The reaction product is red with a mixture of granular and diffuse positivity. In T-cells, acid phosphatase is an early differentiation feature. Almost all acute and chronic T-lineage leukaemias show strong activity. In T-lineage ALL, the activity is usually highly localized (polar). Granulocytes are strongly positive. Monocytes, eosionophils, and platelets show variable positivity. In the bone marrow, macrophages, plasma cells, and megakaryocytes are strongly positive.

REFERENCE 7.1 S. M. Lewis, B. J. Bain & I. Bates. (2006) Dacie and Lewis Practical Haematology, 10th edition. Churchill Livingstone.

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