=====8.SOP FOR H&E STAIN PROCEDURE=====

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