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

BONE MARROW: PRUSSIAN BLUE REACTION STAIN (PERLS STAIN) HUSM/HEMA-UPT/STM-BM3 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

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

PREPARED BY DESIGNATION CHECKED BY DESIGNATION AUTHORISED BY DESIGNATION

BONE MARROW: PRUSSIAN BLUE REACTION STAIN (PERLS STAIN) HUSM/HEMA-UPT/STM-BM3

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.

OBJECTIVE
To demonstrate the presence of iron in the bone marrow.

2.

METHOD
Manually

3.

PRINCIPLE
The reaction occurs with the treatment of bone marrow smear in acid solution of ferrocyanides. Any ferric ion (+3) in the smear with the ferrocyanide and results in the formation of a bright blue pigment called Prussian blue or ferric ferrocyanide.

4.

REQUIREMENTS
4.1 EQUIPMENT 4.1.1 4.1.2

Staining Container Microscope slides and cover slips

4.2 REAGENT

4.2.1

2% Potassium Ferrocyanide (K4Fe(CN)6.3H2O) Pottasium Ferrocyanide 2g Distilled Water 100 ml 0.2N HCl (0.2 mol/L HCl) Distilled Water Acid Hydrochloride 0.1% Safranin Safranin Distilled Water Absolute Methanol (Fixative) DPX (mountant)

4.2.2

- 500 ml - 2.74 ml - 1g - 100 ml

4.2.3

4.2.4 4.2.5

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

4.3

BONE MARROW: PRUSSIAN BLUE REACTION STAIN (PERLS STAIN) HUSM/HEMA-UPT/STM-BM3

VERSION NO. VERSION DATE.

1 24.03.2011

QUALITY CONTROL MATERIAL A known bone marrow slide which has iron stores as a positive control

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 absolute methanol for approx. 20 minutes Note: Bone marrow should be dry (at least 1 hour after preparation) before fix Mix equal volume of 2% Potassium Ferrocyanide and 0.2N HCl solution in staining jar and put the mixture solution at temperature 50oC to 56oC Place the slides into the container and stain for 10 minutes Wash the slides thoroughly under running rap water for 20 minutes Counter stain the smears with 0.1 % Safranin for 2 minutes Rinse well the smear with tape water for few seconds Allow the slides to dry in the air before mount the smear with DPX RESPONSIBILITY

MLT/SO

5.2

MLT/SO

5.3 5.4 5.5 5.6 5.7

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

RESULTS
The reaction product is blue

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

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