1. PURPOSE OF EXAMINATION: To lay down the procedure for PAS stain in Histo-pathology section at NCHLS Surat.
2. PRINCIPLE AND METHOD OF PROCEDURE USED FOR EXAMINATION: Certain tissue containing 1-2 glycol groups are oxidized by 1% per iodic acid to form dialdehyde which combine with Schiff’s reagent to form insoluble magenta color complex by restoration of quinoid chromophoric group.
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: Appendix.
9. REQUIRED EQUIPMENT AND REAGENTS:
EQUIPMENTS: Coplin jars, Brown bottle.
REAGENT: 0.5 % periodic acid, Schiff’s reagent, Harris hematoxylin, absolute alcohol, xylene, DPX
10. ENVIRONMENTAL AND SAFETY CONTROL: Universal safety precaution
11. CALIBRATION PROCEDURE (METROLOGICAL TRACEBILITY):NA
12. PROCEDURE(STEPS):
Preparation of Schiff’s reagent:
• Dissolve 1 gm basic Fuschin in 200 ml of boiling distilled water, removing flask from the burner just before adding basic fuscin. This will avoid premature renovation of the laboratory in deep magenta color.
• Allow the solution to cool to 50.C and add 2 gm potassium metabisulfite with mixing. Cool it room temperature, then add 2 ml concentrated hydrochloric acid, mix, add 2 gm activated charcoal and leave overnight in dark place at room temperature. Filter through whatman no. 4 paper when solution should be either clear or pale yellow color. Store in a dark container at 4C.
Staining Procedure:
• Take test and positive control slides.
• Deparaffinize section by keeping in oven for 5 min and clear by 2 changes of xylene for 15 min each.
• Dehydration with graded alcohol 2 change for 2 min.
• Bring section to distilled water.
• Place slide in jar containing 0.5% Periodic acid for 5 min.
• Wash well in several changes of distilled water.
• Stain with Schiff’s reagent for 10-15 min.
• Tap water washes for 5-10 min.
• Counter stain with Harris Hematoxylin for 1 min.
• Wash in running tap water
• Differentiate in 1% acid alcohol for 1-2 sec.
• Wash well in running tap water.
• Dehydrate in alcohol, clear in xylene, mount with DPX.
Result
• Nuclei : blue
• Glycogen, mucin, fungi: purple red or magenta color.
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.