=====17.SOP FOR TOLUIDINE BLUE=====

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.