Papanicolaou (PAP) Stain

 


1. Objective

The objective of this test was to use the Papanicolaou stain to examine exfoliated cells, particularly from the cervix, for cytological abnormalities, including precancerous and cancerous changes.

2. Principle

The PAP stain was a multichromatic staining technique that used nuclear and cytoplasmic stains to differentiate cells based on their morphology and maturity. The method highlighted subtle changes in nuclear detail, cytoplasmic transparency, and keratinization, critical for detecting cervical neoplasia.

3. Materials

  • Exfoliated cell smear on glass slide (e.g., cervical/vaginal smear)

  • Fixative (95% ethanol)

  • Stains:

    • Hematoxylin (for nuclei)

    • Orange G (keratinized cells)

    • EA (Eosin Azure) containing Eosin Y, Light Green SF, and Bismarck Brown

  • Reagents: alcohol series, xylene, water

  • Coverslips, mounting medium

  • Slide holder, staining jars

  • Light microscope

4. Procedure

  • Smears were fixed in 95% ethanol.

  • Stained with Harris hematoxylin for nuclear detail.

  • Rinsed in water, then differentiated in acid alcohol.

  • Counterstained with Orange G (for keratinized cytoplasm).

  • Stained with EA solution (for cytoplasmic staining of non-keratinized cells).

  • Dehydrated through alcohol, cleared in xylene, and mounted.

5. Result (Example)

Cell Component Stain Color
Nuclei Dark blue/purple
Superficial squamous Pink/orange
Intermediate squamous Green-blue
Keratinized cells Orange
Cytoplasm (non-ker.) Green to blue-green

  • Example: A smear showed high nuclear-to-cytoplasmic ratio, nuclear hyperchromasia, and irregular membranes — suggestive of high-grade squamous intraepithelial lesion (HSIL).

6. Uses

  • Cervical cancer screening (Pap smear)

  • Identified HPV-related dysplasia

  • Evaluated vaginal, endocervical, and endometrial cells

  • Assisted in detecting infection, inflammation, or hormonal status

7. Conclusion

The Papanicolaou stain was a cornerstone technique in cytopathology, enabling early detection of cervical precancerous and cancerous lesions, significantly reducing cervical cancer mortality.

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