Pap Test: Introduction, Principle, Staining Procedure, Result- Interpretation, and Keynotes

Introduction of Pap Test

Pap test is also known as Pap smear and 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 dye 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 (hematoxylin) stains blue while 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 Test

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

Ingredients	Amount
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

Ingredients	Amount
Orange G (10% aqueous)	25ml
Alcohol	475ml
Phosphotungstic acid	0.8 g

Composition of EA 50

Ingredients	Amount
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 Test

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 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 Test

The Result Interpretation of the Pap Test is as follows-

S. No	Staining Components	Staining Colour
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 Test

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 Test

1. Bancroft’s Theory and Practice of Histological Techniques (6th edition)
2. http://www.tulipgroup.com/MicroExpress/Accumix/PackInsert/Stains/207160540125_207160550125_207160560125_Papanicolaou_Stain.pdf
3. https://www.hopkinsmedicine.org/health/treatment-tests-and-therapies/pap-test
4. https://www.healthline.com/health/pap-smear
5. https://www.mayoclinic.org/tests-procedures/pap-smear/about/pac-20394841
6. https://en.wikipedia.org/wiki/Pap_test