Papanicolaou (Pap) stain

Principle

• A polychromatic stain giving crisp nuclear detail and transparent cytoplasm, so overlapping cells can be evaluated.
• Hematoxylin stains nuclei; OG-6 (Orange G) stains keratin; EA (eosin azure, e.g. EA-50 or EA-65) contains eosin Y and light green SF.
• Cell maturity is reflected in cytoplasm: mature squamous cells are eosinophilic, less mature cells are cyanophilic.

Specimen

• Cervical/vaginal smears or liquid-based preparations (e.g. ThinPrep, SurePath).
• Non-gynecologic samples: body fluids, urine, sputum, bronchial specimens and fine needle aspirates.
• Smears must be wet-fixed immediately in 95% ethanol (at least 15 minutes) or with a spray fixative.

Reagents and equipment

• 95% and absolute alcohol, hematoxylin (e.g. Harris or Gill), bluing agent, OG-6, EA, xylene, mounting medium.

Procedure

• Hydrate through alcohols to water.
• Stain nuclei with hematoxylin, differentiate if regressive, and blue.
• Dehydrate in 95% alcohol, stain with OG-6, rinse in alcohol.
• Stain with EA, rinse and dehydrate in absolute alcohol.
• Clear in xylene and mount.

Results

• Nuclei: blue to black, with visible chromatin pattern.
• Superficial squamous cells: pink/orange; intermediate and parabasal cells: blue-green.
• Keratinized cells: orange; RBCs: red-orange; mucus: pale blue or pink.

Quality control and pitfalls

• Air-drying before fixation causes enlarged, pale nuclei and poor staining.
• Filter stains and change solutions regularly to prevent cross-contamination of cells between slides (especially from highly cellular samples).
• Water carried into xylene causes cloudy, hazy slides.
• Review staining quality daily with control slides.

Clinical use

• Cervical cancer screening reported using the Bethesda System; also for detecting malignancy and infection in non-gynecologic cytology.