Dr Piyush Tailor

1.PURPOSE

1. To prepare the H & E stains (Harris) as per standard text literature & manufacturer’s guidelines
2. 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