Dr Piyush Tailor

1. PURPOSE OF EXAMINATION: To lay down the procedure for Masson’s trichrome stain in Histo-pathology section at NCHLS Surat.

2. PRINCIPLE AND METHOD OF PROCEDURE USED FOR EXAMINATION: The structure and density of protein is directly related to the staining of tissue components. Smaller dye molecule will penetrate a tissue element. When a large dye molecule penetrates the same element, the smaller molecules will be replaced by it. RBCs are stained 1st with smaller molecules of Orange-G followed by Acid Fuschin treatment which is a larger particle sized dye. This enters collagen and muscle. This is followed by Phosphotungstic acid differentiation which allows displacement of Acid Fuschin particle from collagen. Muscle retains acid fuscin. Lastly, Light Green treatment is given which stains collagen as green color.

3. STAFF RESPONSIBLE: lab. Technician under supervision of resident doctor/tutor.

4. PERFORMANCE CHARACTERISTICS: NA

5. TYPE OF SAMPLE: Histopathology section slide

6. PATIENT PREPARATION: NA

7. TYPE OF CONTAINER AND ADDITIVES:

8. POSITIVE CONTROL: Skeletal Muscle tissue, cirrhosis liver.

9. REQUIRED EQUIPMENT AND REAGENTS:

EQUIPMENTS: Glasswares Cleaned with distilled water, Coplin jars.

REAGENTS: 0.5 % periodic acid, Schiff’s regent, Glacial acetic acid, Methyl blue, Celestine blue, Phosphomolybdic acid, Ferric ammonium sulphate, Glycerin, Harris hematoxylin, absolute alcohol, Acetic acid, xylene, DPX

10. ENVIRONMENTAL AND SAFETY CONTROLS:

11. CALIBRATION PROCEDURE (METROLOGICAL TRACEBILITY):NA

12. PROCEDURE(STEPS):

Solutions preparation:

(1) Solution A

• Acid Fuchsin 0.5gm

• Glacial acetic acid 0.5ml

• D.W. 100ml

(2) Solution B

• Phosphomolybdic acid 1gm

• D.W 100ml

(3) Solution C

• Methyl blue 2gm

• Glacial acetic acid 2.5ml

• D.W. 100ml

(4) Celestin blue solution

• Celestin blue B 2.5

• Ferric ammonium sulphate 25gm

• Glycerin 70ml

• D.W. 500ml

(5) Acid alcohol 1%

(6) Acetic acid 1% (1gm in 100 ml DW)

Preparation of Celestin Blue Solution:

 The ferric ammonium sulphate is dissolved in the cold DW with stirring. The Celestin blue is added to this solution, and the mixture is boiled for a few minutes.

 After cooling the stain is filtered and glycerin is added.

 The final stain should be usable for over 5 months.

 Filter before use.

Staining Procedure:

 Deparaffinize sections by keeping in oven for 5 minute; Clear it with xylene 2 changes for 15 minute each.

 Wash in tap water.

 Stain nuclei by the Hematoxylin for 5 minute.

 Differentiate with 1% acid alcohol.

 Wash well in tap water.

 Stain in acid Fuschin solution for 5 minutes.

 Rinse in DW.

 Treat with Phosphomolybdic acid solution for 5 minutes.

 Drain.

 Stain with methyl blue solution for 2 minutes.

 Rinse in DW.

 Treat with 1% acetic acid for 2 minutes.

 Dehydrate through alcohols.

 Clear in xylene and mount in DPX.

Result:

• Nuclei: Blue black

• Cytoplasm, muscle erythrocytes: Red

• Collagen: Blue

13. QUALITY CONTROL PROCEDURE: The newer batch of stain is tested before being put in to use by checking their efficiency with the batch of stain already in use.

14. INTERFERENCE: Inadequate quality/improper dilution

15. PRINCIPLE OF PROCEDURE FOR CALCULATING RESULTS INCLUDING, WHERE RELEVENT, THE MEASUREMENT OF UNCERTAINITY OF MEASURED QUANTITY VALUES:NA

16. BIOLOGICAL REFERENCE INTERVALS OR CLINICAL DECISION VALUES:NA

17. REPORTABLE INTERVAL OF EXAMINATION RESULTS: NA

18. INSTRUCTION FOR DETERMINING QUANTITATIVE RESULTS WHEN RESULTS IS NOT WITHIN THE MEASUREMENT INTERVAL: NA

19. ALERT /CRITICAL VALUE, WHERE APPROPRIATE: NA

20. LABORATORY CLINICAL INTERPRETATION: interpret test result after true positivity of control slide.

21. POTENTIAL SOURCE OF VARIATION: NA

22. REFERENCE: Histopathological laboratory techniques by Culling’s.