Introduction
- The Papanicolaou stain is one of the most important cytological staining techniques used in diagnostic pathology.
- Dr. George Papanicolaou developed it and is primarily employed in the screening and diagnosing cervical cancer through Pap smear tests.
- This stain enables the differentiation of cells based on their nuclear and cytoplasmic features, providing a clear visualization of cellular morphology.
- It is also widely applied to other cytological specimens, such as pleural fluid, urine, sputum, and fine-needle aspiration (FNA) samples, aiding in detecting malignancies, infections, and other pathological conditions.
Principle
The PAP stain is a multichromatic technique that uses various dyes to stain cellular components differentially:
- Nuclei: Stained with a basic dye, hematoxylin, which binds to acidic nuclear material (DNA), producing a blue or purple color.
- Cytoplasm: Stained with counterstains (OG-6 and EA series), differentiating cell types based on their keratinization, maturity, and metabolic activity.
- Orange G (OG-6): Stains keratinized cells orange.
- Eosin Azure (EA): A mixture of eosin (acidic dye) and light green SF or fast green (basic dye), used for cytoplasmic differentiation.
Combining these dyes highlights nuclear and cytoplasmic details, aiding in classifying normal, inflammatory, and malignant cells.
Requirements
Equipment
- Clean glass slides
- Coplin jars or staining jars
- Slide rack
- Light microscope
- Timer or stopwatch
Materials
- Buffered water (pH 6.8)
- Immersion oil (for microscopy)
- Fixative (e.g., 95% ethanol or spray fixative)
Reagents
- Hematoxylin (Gill’s, Harris, or Mayer’s):
- Stains nuclei blue or purple by binding to chromatin.
- Orange G (OG-6):
- An acidic dye that stains keratinized cells orange.
- Eosin Azure (EA-36, EA-50, or EA-65):
- A polychromatic stain containing eosin Y, light green SF, and sometimes bismarck brown.
- Eosin Y stains mature cells pink.
- Light green SF stains immature and metabolically active cells blue-green.
- Ethanol (95%):
- Used as a fixative and in dehydration steps.
- Xylene:
- Used for clearing during the final steps before mounting.
- Mounting medium:
- A synthetic resin to mount coverslips on stained slides.
Procedure
1. Fixation
- Fix the smear immediately with 95% ethanol or a commercial spray fixative to preserve cellular morphology and prevent air-drying artifacts.
2. Hematoxylin Staining
- Immerse the fixed slide in hematoxylin for 3–5 minutes.
- Rinse with distilled water to remove excess stain.
- Differentiate by dipping the slide in a weak acid-alcohol solution (if required).
- Wash thoroughly with tap water and dip in Scott’s tap water substitute or another blueing agent to enhance nuclear clarity.
3. Orange G (OG-6) Staining
- Stain the slide in OG-6 solution for 2–3 minutes.
- Rinse briefly with 95% ethanol to remove excess stain.
4. Eosin Azure (EA) Staining
- Stain the slide in EA solution (e.g., EA-36, EA-50, or EA-65) for 3–5 minutes.
- Rinse briefly with 95% ethanol to remove excess stain.
5. Dehydration
- Dehydrate the slide through graded alcohols (95% and 100% ethanol).
- Ensure no water remains in the sample to prevent artifacts.
6. Clearing
- Clear the slide by immersing it in xylene to make the specimen transparent.
7. Mounting
- Apply a drop of mounting medium on the slide and place a coverslip to preserve the stained smear.
Results
- Nuclei: Stain blue to purple, allowing detailed chromatin patterns and nuclear-cytoplasmic ratio evaluation.
- Keratinized cells: Stain orange with OG-6.
- Mature squamous cells: Stain pink with eosin (from EA).
- Immature or metabolically active cells: Stain green or blue-green with light green SF (from EA).
- Cytoplasm: Shades of pink, green, or blue depending on cell type and metabolic activity.
Advantages
- Excellent cellular detail: Provides sharp nuclear and cytoplasmic contrast for reliable interpretation.
- Diagnostic versatility: Applicable to various cytological specimens beyond Pap smears, including sputum, urine, and FNAs.
- Identification of cell maturity: Differentiates keratinized, non-keratinized, and metabolically active cells.
- Standardized method: Globally recognized and standardized for cervical cancer screening.
- Long-lasting slides: Well-stained slides can be stored for extended periods without fading.
Disadvantages
- Time-consuming: The multistep procedure takes longer than simpler stains like Diff-Quik.
- Requires skill: Proper interpretation of PAP-stained slides requires training and expertise.
- Cost: Reagents like EA series and OG-6 can be expensive compared to simpler stains.
- pH sensitivity: Staining results can vary with fluctuations in pH during reagent preparation or rinsing.
- Limited microbial detection: While useful for cellular morphology, PAP staining is less effective for detecting intracellular microorganisms than special stains like Gram or Ziehl-Neelsen.