Papanicolaou (PAP) Staining : Introduction, Principle, Procedure and Interpretation

Papanicolaou stain (also Papanicolaou’s stain or PAP stain) is the most important stain utilized in the practice of Cytopathology. It is a polychromatic stain containing multiple dyes to differentially stain various components of the cells.

This technique was developed by George Papanicolaou, the father of Cytopathology. This method is used to differentiate cells in the smear preparation of various gynecological specimens (pap smears), materials containing exfoliative cells and material from fine needle aspiration.

Objectives of Papanicolaou stain

Papanicolaou described three chief objectives for staining of cytological smears:

• Definition of nuclear details : Because of the widespread muclear abnormalities of cancer cells and their diagnostic significance, good staining of the nucleus is of primary importance.
• Transparency of cytoplasm : This is of particular importance because of the varying thickness and the frequent overlapping of cells.
• Differentiation of cells : Differences in the staining reaction such as that between acidophilic and basophilic cells help greatly in the identification of certain cell types found in smears.

Principle of Papanicolaou stain

Papanicolaou stain includes both acidic and basic dyes. Acidic dye stains the basic components of the cell and basic dye stain the acidic components of the cell.

The polychromatic PAP stain involves five dyes in three solutions.

1. Hematoxylin : Natural dye hematoxylin is the nuclear stain which stains cell nuclei blue. It has affinity for chromatin, attaching to sulphate groups on the D.N.A. molecule. Harris’ hematoxylin is the commonest cytologically although Gills’ hematoxylin and Hematoxylin S can be used.
2. Orange Green 6 : This is the first acidic counterstain (cytoplasmic stain) which stains matured and keratinized cells. The target structures are staine d orange in different intensities.
3. Eosin Azure : This is the second counterstain which is a polychrome mixture of eosin Y, light green SF and Bismarck brown. Eosin Y gives a pink colour to cytoplasm of mature squamous cells, nucleoli, cilia and red blood cells. Staining solutions commonly used in cytology are EA 31 and EA 50, while EA 65
4. Light green SF stains blue to cytoplasm of metabolically active cells like parabasal squamous cells, intermediate squamous cells and columnar cells.
5. Bismarck brown Y stains nothing and sometimes it is often omitted.

Composition and preparation of reagents

• Harris’ hematoxylin :
  Hematoxylin = 5g
  Ethanol = 50ml
  Potassium alum = 100g
  Distilled water (50°C) = 1000ml
  Mercuric oxide = 2-5g
  Glacial acetic acid = 40ml
• Orange G 6 :
  Orange G (10% aqueous) = 50ml
  Alcohol = 950ml
  Phosphotungstic acid = 0-15g
• EA 50 :
  0.04 M light green SF = 10ml
  0.3M eosin Y = 20ml
  Phosphotungstic acid = 2g
  Alcohol = 750ml
  Methanol = 250ml
  Glacial acetic acid = 20ml

Filter all stains before use.

Procedure of Papanicolaou staining

Both progressive and regressive nuclear staining techniques can be used in Papanicolaou stain. Before staining, Wet fixation immediately with Cytology spray fixative 96% ethanol for minimum 30 min is required.

Procedure of Progressive Papanicolaou Staining Method

In the progressive method, the nucleus is stained with hematoxylin to a intensity desired. The intensity of the nuclear staining is controlled by the immersion of the slide into a blueing agent. Most commonly used blueing agent is Sott’s tap water (pH 8.02).

2.80% Alcohol2 minutes

Step	Reagent	Time
1.	95% Alcohol (Fixation)	15-30 minutes
3.	60% Alcohol	2 minutes
4.	Distilled Water	5 dips
5.	Distilled Water	5 dips
6.	Hematoxylin stain	3 minutes
7.	Distilled Water	3 minutes
8.	60% Alcohol	2 minutes
9.	80% Alcohol	2 minutes
10.	95% Alcohol	2 minutes
11.	Orange G Stain	3 minutes
12.	95% ALcohol	2 minutes
13.	95% Alcohol	2 minutes
14.	Eosin Azure Stain	3 minutes
15.	95% Alcohol	2 minutes
16.	95% Alcohol	2 minutes
17.	95% Alcohol	2 minutes
18.	95% Alcohol	2 minutes
19.	Absolute Alcohol	2 minutes
20.	Absolute Alcohol	2 minutes
21.	Absolute Alcohol	2 minutes
22.	Absolute Alcohol+Xylene (1:1)	2 minutes
23.	Xylene	2 minutes
24.	Xylene	2 minutes
25.	Xylene	Till clear
26.	Mount in D.P.X

Procedure of Regressive Papanicolaou Staining Method

When using the regressive staining method, the nucleus is deliberately over-stained with a non-acidified haematoxylin. The excess stain is removed with dilute hydrochloric acid solution (acid water).

The decolourising process is then stopped by immersing the slide in running tap water. Timing is crucial in the regressive method as de-staining may lead to a hyperchromatic nucleus becoming hypochromatic.

2.80 % Alcohol 2 minutes 15. Orange G Stain 3 minutes

Step	Reagent	Time
1.	90% Alcohol (Fixation)	15-30 minutes
3.	60% Alcohol	2 minutes
4.	Distilled Water	5 dips
5.	Distilled Water	5 dips
6.	Hematoxylin stain	3 minutes
7.	Distilled Water	10 seconds
8.	1% Acid Alcohol	10 seconds (1 dip)
9.	Distilled Water	10 seconds
10.	Scott’s Tap Water	2-3 minutes
11.	Running Tap Water	2 minutes
12.	60% Alcohol	2 minutes
13.	80% Alcohol	2 minutes
14.	95% Alcohol	2 minutes
16.	95% ALcohol	2 minutes
17.	95% Alcohol	2 minutes
18.	Eosin Azure Stain	3 minutes
19.	95% Alcohol	2 minutes
20.	95% Alcohol	2 minutes
21.	95% Alcohol	2 minutes
22.	95% Alcohol	2 minutes
23.	Absolute Alcohol	2 minutes
24.	Absolute Alcohol	2 minutes
25.	Absolute Alcohol	2 minutes
26.	Absolute Alcohol+Xylene (1:1)	2 minutes
27.	Xylene	2 minutes
28.	Xylene	2 minutes
29.	Xylene	Till clear
30.	Mount in D.P.X

Results and interpretation of Papanicolaou Staining

• Nuclei : Blue
• Acidophilic cells : Red
• Basophilic cells : Blue Green
• Erythrocytes : Orange-red
• Keratin : Orange-red
• Superficial cells : Pink
• Intermediate and Parabasal Cells : Blue Green
• Eosinophil : Orange Red
• Candida : Red
• Trichomonas : Grey green