Papanicolaou stain (alsoPapanicolaou's stain andPap stain) is a multichromatic (multicolored)cytologicalstaining technique developed byGeorge Papanicolaou in 1942.^[1][2][3] The Papanicolaou stain is one of the most widely used stains incytology,^[1] where it is used to aidpathologists in making a diagnosis. Although most notable for its use in the detection ofcervical cancer in thePap test or Pap smear, it is also used to stain non-gynecological specimen preparations from a variety ofbodily secretions and fromsmall 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 variousbodily secretions andneedle 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 the timing of the process.^[2][1] Pap staining is usually associated withcytopathology in which loose cells are examined, but the stain has also been modified and used on tissue slices.^[9]

Pap staining is used in thePap smear (or Pap test) and is a reliable technique incervical cancerscreening ingynecology.^[10]

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

• The first staining solution containshaematoxylin which stainscell nuclei.^[10][2][12] Papanicolaou usedHarris's hematoxylin in all three formulations of the stain he published.^[2]
• The second staining solution (designated OG-6), containsOrange G in 95%ethyl alcohol with a small amount ofphosphotungstic acid.^[12][2] In the OG-6, the OG signifies Orange G, and the '6' denotes the concentration ofphosphotungstic 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, andBismarck brown Y in 95% ethyl alcohol with a small amount of phosphotungstic acid andlithium carbonate.^[12][2] This solution, designated EA, followed by a number that 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 thepH 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 toprecipitate 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 in color^[12][13] and thechromatin patterns of the nucleus should be well defined. Cellcytoplasm stains blue-green andkeratin stains orange in color.^[13][5]

Eosin Y stains the superficial epithelialsquamous cells,nucleoli,cilia, andred blood cells.^[2]Light Green SF yellowish confers a blue staining for thecytoplasm 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 a significantly shorter time. The process differs in rehydration of the air-dried smear withsaline, use 4%formaldehyde in 65%ethanolfixative, and use of Richard-AllanHematoxylin-2 andCyto-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]

• Staining
• Cytopathology
• Microbiology techniques
• Laboratory techniques
• Histopathology
• Staining dyes
• Cervical cancer

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