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

BONE MARROW: PERIODIC ACID SCHIFF STAIN (PAS STAIN) HUSM/HEMA-UPT/STM-BM6 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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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.

PREPARED BY DESIGNATION CHECKED BY DESIGNATION AUTHORISED BY DESIGNATION 1. 2. 3. OBJECTIVE To detect the presence of glycogen in the hematopoietic cells METHOD Manually PRINCIPLE The periodic acid oxidizes the carbon bond forming aldehydes which react to fuchsinsulfurous acid which form the magenta color 4. REQUIREMENTS
4.1

BONE MARROW: PERIODIC ACID SCHIFF STAIN (PAS STAIN) HUSM/HEMA-UPT/STM-BM6

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

EQUIPMENT
4.1.1 4.1.2

Staining Container Microscope slides and cover slips 10% Formal Ethanol Formaldehyde Ethanol 1% Periodic Acid
Periodic Acid (Sigma: P 7875)

4.2

REAGENT 4.2.1 1 ml 9 ml 1g 100 ml

4.2.2

Distilled water Store in refrigerator, it stable for 6 months

4.2.3 Schiff Reagent Distilled water 768 ml Concentrated HCl 32 ml Sodium Sulfite 20 g Pararosaniline (Sigma: P 1528) 8g Mix the above solution/compounds in amber bottle. Stir the mixture for at least 2 hours till the solution turn to clear dark brown colour. Add in 10 g of activated charcoal powder and stir for at least 1 min. Filter and store the solution in refrigerator, it stable for 6 months. 4.2.4 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)
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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO.

4.2.3

BONE MARROW: PERIODIC ACID SCHIFF STAIN (PAS STAIN) HUSM/HEMA-UPT/STM-BM6

DPX (mountant)

VERSION NO. VERSION DATE.

1 24.03.2011

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 5 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

RESPONSIBILITY MLT/SO MLT/SO MLT/SO MLT/SO MLT/SO MLT/SO MLT/SO MLT/SO MLT/SO

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 5.9 Wash slides in running tap water for approx. 20 minutes and air dry the slides Incubate the slides into 1 % Periodic Acid solution for approx. 20 minutes Rinse in running tap water for 3 to 4 times Pour Schiff Reagent 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 Air dry the slides and mount with DPX

RESULTS/INTERPRETATION The reaction product is red/magenta. Lymphocytes show granular to block positivity in the cytoplasm.

7 7.1

REFERENCE 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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