**HEMATOXYLIN & EOSIN STAINING**


**1. PURPOSE:** (a) To prepare the H & E stains (Harris) as per standard text literature & manufacturer’s guidelines.\\
(b) Lay down procedure for staining the smears prepared from fluids& FNAC in Cytopathology section at NCHLS, Surat.\\
**2. STAFF RESPONSIBLE:** Lab technicians & all the authorized signatories of reports in Cytopathology section.\\
**3. Principle:** Hematoxylin is oxidized to hematin (in situ with lodate) which in acidic conditions binds to lysine residues of nuclear histones by linkage via a metallic ion mordant (aluminium) giving it blue/black color. Eosin Y is acidic colorant which binds with protein in cytoplasm.\\
**4. Reagent:** Hematoxylin& Eosin stain, methanol, acid alcohol, tap water.\\
**5. Performance specification:** NA\\
**6. Sample:** FNAC & body fluids\\
**7. Type of container:** NA\\
**8. Equipment & reagent used:**\\
Hematoxylin\\
|Hematoxylin|2.5 gm|
|Absolute alcohol|50 ml|
|Ammonium/potassium alum|50 gm|
|D.W.|500 ml|
|Mercuric oxide|1.5 gm|

Eosin\\

|Glacial acetic acid|20 ml|
|Eosin w/v yellowish|1 gm|
|D.W.|100 ml|
|Thymol crystal|-|


  *Dissolve the Haematoxylin in the alcohol and the alum in the DW with the gentle heat.\\
  *Mix both the solution in a boiling flask and bring rapidly to boil.\\
  *Add the mercuric oxide slowly and then cool immediately by immersing the flask in cold water.
  *The solution should assume a dark purple color on addition of the mercuric oxide.
  *Transfer to a suitable storage bottle.
  *It is preferable to add glacial acetic acid before use as this gives more precise and selective nuclear staining.


**9. CALIBRATION PROCEDURE:** For weighing of reagents/materials, calibrated weighing balance is used.\\
**10. PROCEDURE (STEPS):**\\ 
  *After preparing the smear immediately fix it in methanol for 10 minutes.(Wet Fixation)
  *Dry the smear.
  *First apply Haematoxylin for 2 minutes.
  *Wash the slide 2 times in tap water.
  *Apply Eosin for 5-10 seconds.
  *Wash with tap water for 2 times.
  *Dry the slides and mount with D.P.X. Label it properly.

**11. QUALITY CONTROL PROCEDURES:** Staining from every new lot (batch) of the reagents (stains) should be done with the in use stain to improve quality.\\
**12. INTERFERENCE:** Precipitate formation on storage. Discard the stains if the staining quality of smears is poor, contaminated alcohol.\\
**13. REFERENCE INTERVAL:**  NA\\
**14. TAT FOR TEST:** 25 min including fixation.\\
**15. LABORATORY INTERPRETATION:** NA\\
**16. ALERT CRITICAL VALUE:** NA\\
**17. SAFETY PRECAUTIONS:** Universal safety precautions (Biosafety manual).\\
**18. VALIDATION OF RESULTS TO BE DONE BY:** By authorized signatories & Assi. Technical manager\\
**19. REVIEW, RECORDS & RECOMMONDETATIONS:** As  per ISO:15189 & NABL 112\\
**20. REFERENCE:** Theory & Practice of Histological Techniques 4th edition 1996, John D Bancroft