====SOP of Hematoxytin & Eosin stain====

**1.PURPOSE**
  -To prepare the H & E stains (Harris) as per standard text literature & manufacturer’s guidelines
  -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**
  *Linearity
  *Precision & Accuracy
  *Sensitivity/Specificity

**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.



**14. TAT FOR TEST**
25 min including fixation

**15. LABORATORY INTERPRETATION**
 NA

**16. ALERT CRITICAL VALUE**
 NA

**17.SAFETY PRECAUTION**
 Universal safety precaution (Biosafety manual).

**18.VALIDATION OF RESULTS TO BE DONE BY**
 By authorized signatories &Assistant  Technical manager.

**19.REVIEW, RECORDS & RECOMMENDATION**
 As per ISO:15189 Guidelines Clause 4.13 & NABL 112

**20.Reference**
Theory & Practice of Histological Techniques 4th edition 1996, John D Bancroft