Dr Piyush Tailor

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

2. STAFF RESPONSIBLE: Lab technician & all the authorized signatories of reports in histopathology section.

3. PRINCIPLE AND METHOD OF PROCEDURE USED FOR EXAMINATION: Hematoxylin is oxidized to hematin (in situ with Iodate) which in acidic conditions binds to lysine residues of nuclear histones by linkage via a metallic ion mordant (aluminum) giving it blue/black color. Eosin Y is acidic colorant which binds with protein in cytoplasm.

4. PERFORMANCE CHARACTERISTICS: NA

5. TYPE OF SAMPLE: Tissue sections.

6. PATIENT PREPARATION: NA

7. TYPE OF CONTAINER AND ADDITIVES: NA

8. REQUIRED EQUIPMENT AND REAGENT: Hematoxylin & Eosin stain, Methanol, acid alcohol, Tap water, Jars & assembly.

9. ENVIRONMENTAL AND SAFETY CONTROL: Universal safety precautions.

10. CALIBRATION PROCEDURE (METROLOGICAL TRACEBILITY): 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.

11. PROCEDURE (STEPS) :

Manual method:

• Dewax sections in xylene giving two changes for 15 minutes.

• Put the section in 85% alcohol for 2 minute.

• Put the section in 80% alcohol for 2 minute.

• Dry the section in air.

• Dip the section in water for 2 times

• Stain with Harris Hematoxylin for 5 minutes. • Wash well in tap water for five Minutes till sections are blue.

• Dip in 1% acid-alcohol: 1-2 dips.

• Wash in sodium bicarbonate for 2 minutes.

• Stain in 1% Eosin for 30 second.

• Wash in tap water for 1-2minutes.

• Dehydrate through ascending grade of Alcohol.

• Clear in Xylene and mount with DPX after drying.

• RESULTS:

• Nuclei – blue/black

• Cytoplasm – varying shades of pink

• Muscle fibers – deep pink/ red

• Red blood cells – orange/red

• Fibrin – deep pink.

12. QUALITY CONTROL PROCEDURE: Discard the stains if the staining quality of sections poor.

13. INTERFERENCE: Contaminated Alcohol or Xylene

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

15. BIOLOGICAL REFERENCE INTERVALS OR CLINICAL DECISION VALUES: NA

16. REPORTABLE INTERVAL OF EXAMINATION RESULTS: NA

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

18. ALERT /CRITICAL VALUE, WHERE APPROPRIATE: NA

19. LABORATORY CLINICAL INTERPRETATION: NA

20. POTENTIAL SOURCE OF VARIATION: NA

21. REFERENCE: Histopathological laboratory techniques by Bancroft, 6th edition