Dr Piyush Tailor

1.PURPOSE

1. To prepare MGG stain as per standard text literature & manufacturer’s guidelines
2. Lay down procedure for staining the smears prepared from fluids& FNAC in Cytopathology section at NCHLS, Surat.

2.STAFF RESPONSIBLE Lab technicians & all the authorized signatories of reports in Cytopathology section.

3.Principle

• It is a type of Romanowsky stain which has two components - azure B (trimethylthionine) and eosinY (tetrabromofluorescein).
• Azure B binds to anionic molecules &eosinY binds to cationic site on proteins.
• Acidic grouping of nucleic acids, proteins of cell nuclei and primitive cytoplasm determine the uptake of basic dye azure B.
• Basic grouping of cytoplasm results in its affinity for acidic dye and its staining by eosin.

4.Reagent Giemsa powder, glycerin, methanol

5.Performance specification NA

6.Sample Cytology smear

7.Type of container NA

8.Equipment & reagent used

• Giemsa powder: 5 gm
• Glycerin: 350 ml
• Methanol: 600 ml

• Take 5 gm of Giemsa powder and grinding it in 350 ml of glycerin for one hour. Allow it at least for two days for ripening in the dark place. After two days add 600 ml of methanol to it. Shake well and use it after filtering by doing dilution 1:4.
• May - Grünwald powder: 5 gm
• Methanol: 1000ml

• Take May - Grünwald powder 5gm in 1000 ml methanol dissolve it. Then filter the stain with filter paper. Store it as stock solution. At the time of staining 1:10 dilution of the stain is prepared for use.

PREPARATION OF SORENSON PHOSPHATE BUFFER SOLUTION

MATERIALS REQUIRED

• NaH2PO4.2H2O – 6gm
• K2HPO4 – 4.5 gm
• Distilled water – 1000 ml
• Measuring cylinder
• pH meter/strip
• Container

METHOD OF PREPARATION

• Take 1000 ml of distilled water in glass/plastic container.
• Add 6gm of NaH2PO4.2H2O & 4.5 gm of K2HPO4 in it.
• Mix properly with glass rod.
• Measure pH of prepared solution with pH meter/pH strip.
• Store at room temp.

9. CALIBRATION PROCEDURE For weighing of reagents/materials, calibrated weighing balance is used.

10. PROCEDURE (STEPS)

• After preparing the smears first dry it in air for 5 minutes (Dry Fixation) then fix it in methanol for 10 minutes.
• Dilute the MGG stain with buffer
• Apply the stain over the smear for 5 minute.
• Remove the stain from smear.
• Dilute the Giemsa stain with tap water (1:4).
• Apply the stain over the smears for 15- 20 minutes.
• Wash with tap water.
• Dry the smears and mount with D.P.X. Label it properly.

11. QUALITY CONTROL PROCEDURES Staining from every new lot (batch) of the reagents (stains) should be done with the in use stain to improve quality.

12. INTERFERENCE Precipitate formation on storage. Discard the stains if the staining quality of smears is poor, contaminated alcohol.

14. TAT FOR TEST 40 min including fixation

15. LABORATORY INTERPRETATION NA

16. ALERT CRITICAL VALUE NA

17.SAFETY PRECAUTION Universal safety precaution (Biosafety manual).

18.VALIDATION OF RESULTS TO BE DONE BY By authorized signatories &Assistant Technical manager.

19.REVIEW, RECORDS & RECOMMENDATION As per ISO:15189 Guidelines Clause 4.13 & NABL 112

20.Reference Theory & Practice of Histological Techniques 4th edition 1996, John D Bancroft