Husmhema Upt Stm Bm2

8/3/2019 Husmhema Upt Stm Bm2

1/4

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA

TITLE :BONE MARROW: MAY-GRUNWALD-

GIEMSA STAIN (MGG)VERSION NO. 1

PROCEDURE NO. HUSM/HEMA-UPT/STM-BM2 VERSION DATE. 24.03.2011

Page 1 of 4 STANDARD TECHNICAL MANUAL

APPROVED BY:

...

ASSOC PROF DR ROSLINE HASSANHEAD OF HAEMATOLOGY DEPARTMENT

CONTROLLED COPY NO : 3

REGISTERED HOLDER

HAEMATOLOGY LABORATORY

RECORD OF REVIEW/AMMENDMENT

DATEVERSION

NO.DETAIL OF AMMENDMENT BY


2/4






PREPARED BY : ANG CHENG YONG

DESIGNATION : TRAINING OFFICER / SCIENTIFIC OFFICER

CHECKED BY : DR SHAFINI MOHAMED YUSOFF

DESIGNATION : HAEMATOLOGIST

AUTHORISED BY : ASSOC PROF DR ROSLINE HASSAN

DESIGNATION : 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 ideallyshould 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 Conical flask 250 ml capacity

4.1.2 Mixer

4.1.3 Filter funnel

4.1.4 Microscope

4.1.5 Microscope slides and cover slips

4.2 REAGENT

4.2.1 May-Grunwalds Stain

May Grunwald 0.3 gMethanol 100 ml

Warm the mixture to 50oC for 30 minutes. Allow the mixture to cool to roomtemperature 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.


3/4






4.2.2 Giemsa StainGiemsa - 1.0 g

Methanol - 100 ml

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

ii) Disodium Hydrogen Phosphate (Na2HPO4)

Disodium Hydrogen Phosphate (Na2HPO4)) - 9.5 gDistilled Water - 1L

iii) Working phosphate Buffer pH6.8Solution (i) - 50.8 mlSolution (ii) - 49.2 ml

4.2.4 Absolute Methanol (Fixative)

4.2.5 DPX (mountant)

4.3 SPECIMEN

Air-dried bone marrow smear

5 PROCEDURE

NO. ACTIVITY RESPONSIBILITY

5.1 Fix two (2) air-dried bone marrow smears (if numbers of smearsare sufficient) with absolute methanol for approx. 20 minutes

Note: Bone marrow should be dry (at least 1 hour after preparation)before fix

MLT/SO

5.2 Mix equal volume of May Grunwald stain with working PhosphateBuffer (pH 6.8) in staining jar MLT/SO

5.3 Pour stain on slide for 8 minutes. MLT/SO5.4 Mix 1 part of Giemsa stain with 9 parts of working Phosphate

Buffer (pH6.8)

MLT/SO5.5 Pour off May Grunwald stain and cover the slide with Giemsa stain

for 12 minutes.

MLT/SO5.6 Wash off the stain and differentiate with working Phosphate Buffer

(pH6.8) for approx. 20-30 seconds.

MLT/SO5.7 Wash the smear with tape water for few seconds MLT/SO5.8 Allow the slides to dry in the air before mount the smear with DPX MLT/SO


4/4






6 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 occupiedby haemopoietic marrow then in children and the proportion of fat cells to cellular marrow

is increased. The marrow undergoes slight to moderate hyperplasiain 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.

7 REFERENCES

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

edition. Churchill Livingstone.

End of Document

Husmhema Upt Stm Bm2

Documents

Transcript of Husmhema Upt Stm Bm2