Dr Piyush Tailor

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

Eosin

• 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