1. PURPOSE OF EXAMINATION: To lay down the procedure Toluidine blue staining in Histo-pathology section at NCHLS Surat.

2. PRINCIPLE AND METHOD OF PROCEDURE USED FOR EXAMINATION: when tissue is stained with Toluidine blue stain it gives rise orthochromatic blue color to amyloid deposits.

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

5. TYPE OF SAMPLE: Histopathology section slide

6. PATIENTS PREPARATIONS: NA

7. TYPE OF CONTAINERS AND ADDITIVES:

8. POSITIVE CONTROL: Amyloidosis.

9. REQUIRED EQUIPMENTS AND REAGENTS:

EQUIPMENTS: Clean labelled containers, Coplin jars.

REAGENT: 1% Toluidine blue, 50% isopropyle alcohol, absolute alcohol, Xylene, DPX.

10. ENVIRONMENTAL AND SAFETY CONTROLS: Universal safety precautions

11. CALIBRATION PROCEDURE (METROLOGICAL TRACEBILITY):NA

12. PROCEDURE(STEPS):

REAGENT preparation-

1gm Toluidine blue in 100ml of 50% isopropanol.

Staining procedure:

• Take test slide on which staining has to be performed along with positive control.

• Deparaffinize section by keeping in oven for 5min and clear it with 2 changes of xylene each for 15 minute.

• Bring section to water.

• Stain in freshly prepared 1% Toluidine blue in 50% isopropyle alcohol for 30 minute at 37C.

• Blot with tissue paper and place in a isopropyle alcohol for 1 minute.

• Dehydrate in alcohol, clear in xylene, mount with DPX

Result

• Amyloid: orthochromatic 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 VALUES, 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.