Papanicolaou Stain (PAP Stain)

Introduction:

The Papanicolaou stain, commonly known as the Pap stain, is a widely used cytological staining technique that is used to detect and diagnose abnormal cells in cervical and other gynecological specimens. The Pap stain is a critical tool in screening for cervical cancer and other gynecological diseases.

Requirements:

• Cervical or other gynecological specimens
• Fixative (e.g., ethanol, methanol)
• Hematoxylin solution
• 1% acid alcohol
• 0.1% Ammonia 
• Eosin Azure 50 (EA 50)
• Orange G 6 (OG 6)
• Phosphotungstic acid solution
• Different grades of alcohol (70%, 95%, and absolute)
• Xylene
• Microscope slides
• Coverslips

Principle:

The Pap stain is a multicolor staining technique that differentiates and stains cellular components in gynecological specimens. The stain involves four main steps: nuclear staining with hematoxylin, cytoplasmic staining with orange G, nuclear counterstaining with phosphotungstic acid, and counterstaining with eosin. The resulting coloration allows the identification of cellular abnormalities and the classification of cells into different categories.

Procedure:

1. Fix the cervical or other gynecological specimen using a suitable fixative.
2. Process the specimen using descending grades of alcohol and rinse in tap water.
3. Stain the specimen with hematoxylin solution for 10-15 minutes.
4. Rinse the specimen in running water for 5-10 minutes.
5. Differentiate in 1% acid alcohol 
6. Blue in ammonia water.
7. Stain the specimen with orange G solution for 5-10 minutes.
8.  Wash in tap water.
9. Rinse the specimen in running water for 5-10 minutes.
10. Stain the specimen with phosphotungstic acid solution for 3-5 minutes.
11. Rinse the specimen in running water for 5-10 minutes.
12. Stain the specimen with EA 50 for 1-2 minutes.
13. Rinse the specimen in running water for 5-10 minutes.
14. Dehydrate the specimen using graded alcohol and clear it in xylene.
15. Mount the specimen with a coverslip using a mounting medium (DPX/Canada balsam).

Results:

The Pap stain produces multicolored cellular staining, allowing for the identification of normal and abnormal cellular structures. Normal cells will be classified into different categories, including superficial, intermediate, and basal cells, while abnormal cells may be identified as atypical or cancerous.

QC:

Quality control is critical in ensuring accurate staining results. The following measures should be taken:

• Ensure that the specimen is properly fixed and processed.
• Use fresh staining solutions.
• Follow the staining protocol and timing.
• Ensure that the coverslips are properly mounted and secured.

Interpretation:

The Pap stain is a critical tool in screening for cervical cancer and other gynecological diseases. The staining results allow for the identification of normal and abnormal cells, which can be classified into different categories. Pathologists can use the results to diagnose and monitor disease progression and treatment.