1.PURPOSE

1. To prepare PAP stain 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 The classic form of Pap stain involves five dyes in three solutions:

1. A nuclear stain, haematoxylin, is used to stain cell nuclei.
2. The unmordantedhaematein may be responsible for the yellow colour imparted to glycogen.
3. Orange G stains keratin. Its original role was to stain the small cells of keratinizing squamous cell .
4. Eosin Y stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells.

4.Reagent Orange gel B6, Eosin, Azure, Bismark brown, Eosin yellow

5.Performance specification NA

6.Sample Cytology smear

7.Type of container Conical flask

8.Equipment & reagent used Staining reagents 1.OG6 (Orange Gelb):

• OG6 stain powder -5 gm
• Phosphotangstic acid -1.5gm
• 95% Ethanol
• Dissolve OG6 in 50ml dist. Water and add 950ml of absolute ethyl alcohol then add phosphotangstic acid.
• OG6 is stored in brown colored bottle in the refrigerator.

2.EA 36 (Eosin Azure):

• EA 36 containing light green SF, Eosin yellow and bismark brown.

• All Stock solution is prepared by dissolving 2.5gm of this stain powder of 2.5ml dist. Water and by adding 475ml of dist. Absolute ethyl alcohol. This solution is refrigerated.

3)Light green SF:

• Light green SF – 2.5gm
• Dist. Water-25ml
• Dist. Ab. Ethanol -475ml

4) Bismark Brown:

• Bismark brown stain -2.5ml
• Dist. Water - 25ml
• Dist. Ab. Ethanol – 475 ml

5)Eosin Yellow:

• Eosin Yellow powder -2.5ml
• Dist. Water - 25ml
• Dist. Ab. Ethanol – 475ml

Working Solutions

• Light green SF – 450ml
• Eosin yellow – 450ml
• Bismark brown 100 ml
• Phosphotangstic acid – 2gm
• Sat. aq. Lithium carbonate -10drops

9. CALIBRATION PROCEDURE the new batch of stains is usedsimultaneously with the in used batch of stains to determine the stainingquality.

10. PROCEDURE (STEPS)

• Fix the smears in methanol for 10 minutes after preparing the smears. (Wet Fixation).
• Wash with tap water for 30 seconds.
• Apply Hematoxylin for 3 minutes.
• Wash with tap water for 2 times.
• Dehydrate the smears with 90% alcohol for 30 seconds.
  • Dehydrate with 90% alcohol for 30seconds.
  • Apply EA 36 for 6 minutes.
  • Dehydrate with 90% and 95% alcohol for 30 seconds.
  • Dry the smears 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