Papanicolaou stain (also Papanicolaou's stain and Pap stain) is a multichromatic (multicolored) cytological staining technique developed by George Papanicolaou in 1942.^[1][2][3] The Papanicolaou stain is one of the most widely used stains in cytology,^[1] where it is used to aid pathologists in making a diagnosis. Although most notable for its use in the detection of cervical cancer in the Pap test or Pap smear, it is also used to stain non-gynecological specimen preparations from a variety of bodily secretions and from small needle biopsies of organs and tissues.^[4][5] Papanicolaou published three formulations of this stain in 1942, 1954, and 1960.^[2]

Pap staining is used to differentiate cells in smear preparations (in which samples are spread or smeared onto a glass microscope slide)^[6] from various bodily secretions and needle biopsies; the specimens may include gynecological smears (Pap smears), sputum, brushings, washings, urine, cerebrospinal fluid,^[4] abdominal fluid, pleural fluid, synovial fluid, seminal fluid,^[7] fine needle aspirations, tumor touch samples, or other materials containing loose cells.^[8][4][9]

The pap stain is not fully standardized and comes in several formulations, differing in the exact dyes used, their ratios, and timing of the process.^[2][1] Pap staining is usually associated with cytopathology in which loose cells are examined, but the stain has also been modified and used on tissue slices.^[9]

The classic form of the Papanicolaou stain involves five stains in three solutions.^[2][11][12]

• The first staining solution contains haematoxylin which stains cell nuclei.^[10][2][12] Papanicolaou used Harris's hematoxylin in all three formulations of the stain he published.^[2]
• The second staining solution (designated OG-6), contains Orange G in 95% ethyl alcohol with a small amount of phosphotungstic acid.^[12][2] In the OG-6, the OG signifies Orange G and the '6' denotes the concentration of phosphotungstic acid added; other variants are OG-5 and OG-8).^[2]
• The third staining solution is composed of three dyes, Eosin Y, Light Green SF yellowish, and Bismarck brown Y in 95% ethyl alcohol with a small amount of phosphotungstic acid and lithium carbonate.^[12][2] This solution, designated EA, followed by a number which denotes the proportion of the dyes, other formulations include EA-36, EA-50, and EA-65.^[2]

The counterstains are dissolved in 95% ethyl alcohol which prevents cells from over staining which would obscure nuclear detail and cell outlines especially in the case when cells are overlapping on the slide.^[3][2] Phosphotungstic acid is added to adjust the pH of counterstains and helps to optimize the color intensity.^[2] The EA counterstain contains Bismarck brown and phosphotungstic acid, which when in combination, cause both to precipitate out of solution, reducing the useful life of the mixture.^[2]

The stain should result in cells that are fairly transparent so even thicker specimens with overlapping cells can be interpreted.^[2] Cell nuclei should be crisp, blue to black on color^[12][13] and the chromatin patterns of the nucleus should be well defined. Cell cytoplasm stains blue-green and keratin stains orange in color.^[13][5]

Eosin Y stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells.^[2] Light Green SF yellowish confers a blue staining for the cytoplasm of active cells such as columnar cells, parabasal squamous cells, and intermediate squamous cells.^[14] Superficial cells are orange to pink, and intermediate and parabasal cells are turquoise green to blue.^[12]

Ultrafast Papanicolaou stain is an alternative for the fine needle aspiration samples, developed to achieve comparable visual clarity in significantly shorter time. The process differs in rehydration of the air-dried smear with saline, use 4% formaldehyde in 65% ethanol fixative, and use of Richard-Allan Hematoxylin-2 and Cyto-Stain, resulting in a 90-second process yielding transparent polychromatic stains.^[15]

• Papanicolaou, George N. "A new procedure for staining vaginal smears." Science 95.2469 (1942): 438–439.

• Papanicolaou, George N. "The cell smear method of diagnosing cancer." American Journal of Public Health and the Nation's Health 38.2 (1948): 202–205.

• Papanicolaou, George N. "Atlas of exfoliative cytology." Published for the commonwealth fund by Harvard University Press. (1954).

• Papanicolaou, George N. "Memorandum on staining." Atlas of exfoliative cytology. Cambridge, MA: Harvard University Press, Supplement II (1960): 12.

• Diff-Quik— Romanowsky staining method commonly used in cytology^[16]