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

BONE MARROW: DUAL ESTERASE STAIN HUSM/HEMA-UPT/STM-BM5 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

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DATE

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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 detect the monocytic and granulocytic components in bone marrow aspiration. 2. 3. METHOD Manually PRINCIPLE These are a group of hydrolases with a wide range of pH activity which vary in their localization in cells of different bone marrow lineages. It is best to consider separately the results obtained with the various substrates used for their cytochemical demonstration. Those currently in use are -naphthol acetate, -naphthol butyrate, naphthol AS (or AS-D) acetate and naphthol AS-D chloracetate. Li et al described nine estrate isoenzymes in leucocytes:1,2,7,8 and 9 represents the specific esterase of granulocytes which can demonstrated by means of naphthol AS-D chloracetate; 3,4,5 and 6 represent the so-callednon-specific esterases which are sensitive to sodium fluoride (NaF) anf are found in monocytes, megakaryocytes and platelet, and are demonstrated by -naphthol esterase acetate and butrate. Naphthol AS (or AS-D) reacts with most isoenzymes but is inhibited by NaF in its reaction with the nonspecific esterases.

BONE MARROW: DUAL ESTERASE STAIN HUSM/HEMA-UPT/STM-BM5

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

4.

REQUIREMENTS
4.1

EQUIPMENT
4.1.1 4.1.2

Staining Container Microscope slides and cover slips

4.2

REAGENT 4.2.1 Buffered Formalin/Aceton Na2HPO4 KH2PO4 Formaldehyde Acetone Distilled water Keep in 4oC, can last for few months. KH2PO4 Solution
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0.02 g 0.10 g 25 ml 45 ml 30 ml

4.2.2
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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO. BONE MARROW: DUAL ESTERASE STAIN HUSM/HEMA-UPT/STM-BM5
KH2PO4 Distilled water 4.2.3 Na2HPO4 Solution Na2HPO4 Distilled water 4.2.4 Phosphate Buffer pH 8.0 KH2PO4 Solution (refer to 4.2.2) Na2HPO4 Solution (refer to 4.2.3) 4.2.3 Fast Blue BBsalt solution a. Fast blue BBsalt (BDH:34117) b. Phosphate buffer pH8.0 (refer to 4.2.4) Mix a and b till dissolve Napthol AS-D Chloroacetate Solution a. Napthol AS-D Chloroacetate (Sigma: N0758) b. Acetone -napthyl butyrate Solution b. -napthyl butyrate (Sigma:8000) c. Aceton Dual Esterase working Solution a. Fast Blue BBsalt solution (refer to 4.2.3) b. Napthol AS-D Chloroacetate Solution (refer to 4.2.4_ c. -napthyl butyrate Solution (refer to 4.2.5) Mix well and filter before use Mayers Hematoxylin (Sigma:MHS16)

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1 24.03.2011
6.9 g 500 ml 7.2 g 500 ml 2.75 ml 47.25 ml 0.1 g 50 ml

4.2.4

0.08 g 1 ml 40 l 1 ml 50 ml 1 ml 1 ml

4.2.5

4.2.6

4.2.7

4.2 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 4.3 SPECIMEN Air-dried bone marrow smear 5 PROCEDURE NO. 5.1 ACTIVITY Fix both of the air-dried patient and positive control bone marrow smears with Buffered Formalin/Aceton for approx. 2 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 Prepare Dual Esterase working Solution (refer to 4.2.6) in staining
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RESPONSIBILITY

MLT/SO

5.2 5.3

MLT/SO MLT/SO

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO. BONE MARROW: DUAL ESTERASE STAIN HUSM/HEMA-UPT/STM-BM5 VERSION NO. VERSION DATE. 1 24.03.2011

container and incubate the slides into it for approx. 60 minutes in dark condition 5.4 5.5 5.6 5.7 Wash in running tap water for approx. 30 min. Pour Mayers Hematoxylin on the slides and incubate for approx. 5 min Rinse with tap water for few seconds Air dry the slides MLT/SO MLT/SO MLT/SO MLT/SO

RESULTS/INTERPRETATION The Chloroacetate Estrase (CAE) reaction product is bright blue (granulocytes) and the Butyrate Esterase (ANBE) product is dark brown (monocytes). In myelomonocytic leukemic, cells staining with both esterase maybe be present. REFERENCE 7.1 S. M. Lewis, B. J. Bain & I. Bates. (2006) Dacie and Lewis Practical Haematology, 10th edition. Churchill Livingstone. End of Document

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