PAP SMAER

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Papanicolaou Stain Reagents

Description: Papanicolaou stain (also Papanicolaou's stain and Pap stain) is a multichromatic staining histological technique developed by George Papanikolaou, the father of cytopathology. Pap staining is used to differentiate cells in smear preparations of various bodily secretions; the specimens can be gynecological smears (Pap smears), sputum, brushings, washings, urine, cerebrospinal fluid, abdominal fluid, pleural fluid, synovial fluid, seminal fluid, fine needle aspiration material, tumor touch samples, or other materials containing cells. Pap staining is a very reliable technique. As such, it is used for cervical cancer screening in gynecology. The entire procedure is known as Pap smear.

The classic form of Pap stain involves five dyes in three solutions:

A nuclear stain, haematoxylin, is used to stain cell nuclei.First OG-6 counterstain. The Orange G is used to stain keratin. Its original role was to stain the small cells of keratinizing squamous cell carcinoma present in sputum.Second EA (Eosin Azure) counterstain, comprising of three dyes; the number denotes the proportion of the dyes, eg. EA-50, EA-65.Eosin Y stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells.Light Green SF yellowish stains the cytoplasm of all other cells. This dye is now quite expensive and difficult to obtain, therefore some manufacturers are switching to Fast Green FCF, however it produces visually different results and is not considered satisfactory by some.Bismarck brown Y stains nothing and in contemporary formulations it is often omitted.

When performed properly, the stained specimen should display hues from the entire spectrum: red, orange, yellow, green, blue, and violet. The chromatin patterns are well visible, the cells from borderline lesions are easier to interpret, the photomicrographs are better, and the stained cells are pretty. The staining results in very transparent cells, so even thicker specimens with overlapping cells can be interpreted.

On a well prepared specimen, the cell nuclei are crisp blue to black. Cells with high content of keratin are yellow, glycogen stains yellow as well. Superficial cells are orange to pink, and intermediate and parabasal cells are turquoise green to blue. Metaplastic cells often stain both green and pink at once.

The principle of pap stain

The principle of pap stain is to clearly distinguish between basophilic and acidophilic cell components and obtain a detailed chromatin pattern. The pap stain has 3 solutions having 6 dyes. Solution 1 has Hematoxylin stain, a basic nuclear stain. Solution 2 has OG-6, an acidic cytoplasmic stain, has orange G with phosphotungstic acid (6 denotes the used concentration of phosphotungstic acid). Orange G is an acid dye and stains keratin with a bright intense orange. Phosphotungstic acid, a mordant, strongly binds to protein and helps to intensify the color achieved. Solution 3 has EA (PH 4.5-5 for maximum results) which is a polychromatic stain having 3 components; light green/fast green, eosin Y and Bismarck brown Y. Light green SF is an acid dye which stains the cytoplasm of metabolically active cells i.e. intermediate squamous cells, parabasal cells, endocervical cells, histiocytes, leukocytes, undifferentiated carcinoma cells and cells from adenocarcinoma as green. Eosin Y is an acid dye which stains cytoplasm of superficial squamous cells, nucleoli, erythrocytes and cilia. Bismarck brown Y precipitates phosphotungstic acid, the ingredients responsible for differential staining by light green and eosin. Fast green sometimes used in modified EA which has a lower solubility in alcohol and makes it less prone to being washed away from cells. Fast green is not as stable as light green. EA 36/50 has 2 times more light green than EA65 and hence former is preferred for gynecologic smears and latter for nongynecologic smears/thick smears. However EA65 differentiates adenocarcinoma of endocervix which takes pink stain from that of endometrium which takes blue stain. A well stained smear shows well stained nuclear chromatin with differential cytoplasmic counterstaining and cytoplasmic transparency Bales, 2006Catherine M Keebler, 2008. The greenish cytoplasmic stain of neutrophils are taken as internal control.