Pap Stain: Introduction, Principle, Staining Procedure, Result Interpretation and Keynotes

Pap Stain-Introduction, Principle, Staining Procedure, Result Interpretation and Keynote

Intrdouction of Pap Stain

Pap stain was named after the three letters of the surname of developer George Papanicolaou in 1942 and was routinely used for cytological stain. It is the most common staining of the cellular components in the smear of exfoliated cells, vaginal, cervical, prostate secretion, pleural fluid, spinal fluid, etc. A Pap smear is the standard method for cytological examination of a smear from the female genital tract.

Principle of Pap Stain

The stain contains both basic and acidic dyes, with the basic dye staining acidic cell components and the acidic dyes staining basic cell components. This is based on the ionic charges of the cell’s constituents, as well as the attraction and repulsion of ions and dyes.

Nuclear dye: Haematoxylin- stains blue

Cytoplasmic dye (counterstain): Orange G6, Eosin-alcohol-36

OG6 stain keratin ( keratinized squamous cancers) a bright intense orange. EA36 includes light green SF yellowish and eosin Y. Light green SF yellowish stain green to parabasal cells, columnar cells, intermediate squamous cells, leukocytes, and histocytes. Eosin Y stains the cytoplasm of superficial squamous cells, cilia, nuclei, and RBCs. EA 65 was developed for staining thicker cell samples.

Requirements for Pap Stain

Required reagents and equipment are-

  1. Harris’s alum hematoxylin
  2. 0.5% (v/v) hydrochloric acid in distilled water.
  3. 0.25% (v/v) hydrochloric acid in distilled water
  4. Orange G (OG) and EA- 36 or 50 (Eosin – Azure)
  5. Ethanol
  6. Alcohol
  7. Distilled water
  8. Xylene
  9. Timer
  10. Coverslip
  11. Mounting media
  12. Coplin jars
  13. Compound microscope

Composition of Harris’ hematoxylin

  • Hematoxylin:  2.5g
  • Ethanol: 25ml
  • Potassium alum: 50g
  • Distilled water (50°C): 500ml
  • Mercuric oxide: 1.3g
  • Glacial acetic acid:  20ml

Composition of Orange G 6

  • Orange G (10% aqueous) = 25ml
  • Alcohol = 475ml
  • Phosphotungstic acid = 0.8 g

Composition of EA 50

  • 0.04 M light green SF: 5ml
  • 0.3M eosin Y: 10ml
  • Phosphotungstic acid: 1g
  • Alcohol:  365ml
  • Methanol:  125ml
  • Glacial acetic acid: 10ml

Procedure of PAP stain

  1. Fix the smear immediately in 95% alcohol for 5-15 min.
  2. Take the smear to water (Hydration): 6 dips in each for the following solution of ethanol 80% (v/v) , 70%(v/v)and 50%(v/v) respectively.
  3. Rinse gently in distilled water.
  4. Stain in diluted Harris’s hematoxylin for 2 to 3 minutes.
  5. Rinse in distilled water.
  6. Dip in 0.25% (v/v) hydrochloric acid 6 times while in  in 0.5% (v/v) hydrochloric acid only 3 times.
  7. Place under running tap water for 5 minutes.
  8. Check the slides under the microscope to see if the nuclei are stained properly. ( If over-stained, decolorize in acid-alcohol, and if pale, return to hematoxylin stain).
  9. Dehydrate through distilled water and alcohol solution 50%(v/v),  70% (v/v), 80%(v/v)  and 95% (v/v) by  dipping 6 times in each solution.
  10. Stain in OG-6 for 2 minutes.
  11. Rinse in 95%  (v/v)  alcohol 3 changes and 6 dips each.
  12. Stain in EA-36 (or EA-50) for 2 minutes.
  13. Rinse in 95% (v /v)  alcohol (3 separate changes)
  14.  Dehydrate in absolute alcohol ( 2 changes)
  15. Clear alcohol using xylene (6 dips), followed by 3 changes in xylene (6 dips).
  16. Mount in any satisfactory neutral medium.

Result Interpretation of Pap stain

  1. Nuclei: Blue
  2. Cytoplasm: Varying shades of pink, blue, yellow Green- gray
  3. Acidophilic cells: Red or orange
  4. Superficial cell: Pink
  5. RBCs: Orange
  6. Basophilic cell: Green to blue-green
  7. Intermediate and parabasal cells: Green
  8. Eosinophil: Orange Red
  9. Metaplastic cells: May contain both blue/green and pink
  10. Candida (fungus): Red
  11. Trichomonas (parasite): Grey-green

Keynotes on Pap stain

  1. A Pap smear, often known as a Pap test, is a cervical cancer screening technique. It examines your cervix for the presence of precancerous or cancerous cells. The cervix is the uterus’s opening.
  2. There are two types of Pap staining procedures-a. Regressive method: First over staining and then decolorization with hydrochloric acid for differentiation and it is commonly used. b. Progressive method: Stains are applied in a strict sequence. There is no need for washing or decolorization. It is applicable for the samples that do not adhere well to the glass.
  3. Clinical significances of Pap stain are in Pap smear (Pap test), Screening for cervical cancer, examination of myeloma cancer cells of the liver, screening for thyroid cancer, cell carcinomas, examination and characterization of benign tumors, identification of Candida and Chlamydia trachomatis.
  4. Pap smears are useful only after 21 years of age and the women who are at increased risk for cancer or infection, are HIV-positive and have a weakened immune system from chemotherapy or an organ transplant. Women aged 65 with a history of normal Pap smear results may not be necessary to have the test in the future.
  5. Do not allow smears to dry and the smears may be left in the fixative for 3 days if necessary, but prolonged fixation affects the staining reaction.
  6. The slides should be handled carefully during rinsing and washing. Rough handling may wash off the specimen.
  7. A Good Pap smear must contain endocervical cells.

Further Reading on Pap Stain

  1. Bancroft’s Theory and Practice of Histological Techniques (6th edition)
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