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

BONE MARROW: MAY-GRUNWALDGIEMSA STAIN (MGG) HUSM/HEMA-UPT/STM-BM2 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

Page 1 of 4

STANDARD TECHNICAL MANUAL

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

PREPARED BY DESIGNATION CHECKED BY DESIGNATION AUTHORISED BY DESIGNATION

BONE MARROW: MAY-GRUNWALDGIEMSA STAIN (MGG) HUSM/HEMA-UPT/STM-BM2

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 study the morphology of cells in the bone marrow aspirate

2.

METHOD
Manually

3.

PRINCIPLE The neutral dyes combining the basic dye methylene blue and the acid dye eosin, give a wide color range when staining. The pH of the staining solution is critical and ideally should be adjusted for different fixatives. More acid and pH levels give more selective chromatin staining and less cytoplasmic basophilic; less acid pH levels give denser nuclei and increased cytoplasmic basophilic. The pH range should be between 6.4 and 6.9.

4.

REQUIREMENTS
4.1 EQUIPMENT 4.1.1 4.1.2 4.1.3 4.1.4 4.1.5

Conical flask 250 ml capacity Mixer Filter funnel Microscope Microscope slides and cover slips

4.2 REAGENT

4.2.1

May-Grunwalds Stain May Grunwald 0.3 g Methanol 100 ml Warm the mixture to 50oC for 30 minutes. Allow the mixture to cool to room temperature and shake several times during the day. After standing for 24 hours, filter the solution. It is then ready for use, no ripening being required.

Page 2 of 4

STANDARD TECHNICAL MANUAL

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

4.2.2

BONE MARROW: MAY-GRUNWALDGIEMSA STAIN (MGG) HUSM/HEMA-UPT/STM-BM2

Giemsa Stain Giemsa - 1.0 g Methanol - 100 ml

VERSION NO. VERSION DATE.

1 24.03.2011

Warm the mixture to 50oC for 15 minutes with occasional shaking. Filter the solution. It is then ready for use. 4.2.3 Phosphate Buffer pH 6.8 i) 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) - 9.5 g - 1L - 50.8 ml - 49.2 ml

ii)

iii)

4.2.4 4.2.5

Absolute Methanol (Fixative) DPX (mountant)

4.3 SPECIMEN Air-dried bone marrow smear 5 PROCEDURE NO. 5.1 ACTIVITY Fix two (2) air-dried bone marrow smears (if numbers of smears are sufficient) 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 May Grunwald stain with working Phosphate Buffer (pH 6.8) in staining jar Pour stain on slide for 8 minutes. Mix 1 part of Giemsa stain with 9 parts of working Phosphate Buffer (pH6.8) Pour off May Grunwald stain and cover the slide with Giemsa stain for 12 minutes. Wash off the stain and differentiate with working Phosphate Buffer (pH6.8) for approx. 20-30 seconds. Wash 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 5.3 5.4 5.5 5.6 5.7 5.8

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

Page 3 of 4

STANDARD TECHNICAL MANUAL

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE : PROCEDURE NO. BONE MARROW: MAY-GRUNWALDGIEMSA STAIN (MGG) HUSM/HEMA-UPT/STM-BM2 VERSION NO. VERSION DATE. 1 24.03.2011

RESULTS/INTERPRETATION
6.1 Quantitative cell counts The degree of marrow cellularity can be assessed within broad limits as increased, normal or reduced by inspection of a stained film containing marrow particles As a rough guide i) If less than 25% of the particle is occupied by haemopoietic cells hypocellular ii) If more than 75-80% is occupied hypercellular Note: physiological variation in cell content has to be taken into account. The cellularity of marrow is affected by age. In adults, a smaller proportion of the marrow cavity is occupied by haemopoietic marrow then in children and the proportion of fat cells to cellular marrow is increased. The marrow undergoes slight to moderate hyperplasia in pregnancy. 6.2 Differential cell counts Is necessary to document the proportion of every stage of each cell type on the marrow slide. 6.3 Morphology The morphology of the abnormal cells should be described in details.

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

End of Document

Page 4 of 4

STANDARD TECHNICAL MANUAL