Inception and development of the papanicolaou stain method

Nikolaos Chantziantoniou, Amber D. Donnelly, Maheswari Mukherjee, Mathilde E. Boon, R. Marshall Austin

Research output: Contribution to journalReview articlepeer-review

24 Scopus citations


Objective: Cytodiagnoses of specific malignancies are enabled through analyses of abnormal nuclear chromatin and cytoplasmic features in stained cells. Aim: The objective of this work was to explore the inception, development, and chemistry of the Pap stain method introduced in 1942 by Dr. G.N. Papanicolaou. Study Design: To achieve this, we carried out a review of the English literature. Results: Between 1914 and 1933, Papanicolaou first analyzed vaginal squamous cells in Guinea pigs and later in human vaginal fluid samples using hematoxylin and eosin with limited color reactions, correlating the cell-type morphology with endocrinology and histology. The 5-dye Pap stain method evolved through 2 salient phases. The first, between 1933 and 1942, saw the introduction of alcohol-ether fixation and aqueous waterblue staining to enhance cellular transparency, aiding the distinction of cervical cancer cells from benign cells, with quantitative and qualitative assessment of squamous cell maturity. The second phase, between 1942 and 1960, saw the introduction and refinement of various alcoholic cytoplasmic counterstaining schemes with orange G and EA (light green, Bismarck brown, eosin) and phosphotungstic acid, allowing wider ranges of polychromasia and further enhancing cellular visualization, facilitating the distinction of cell types and improving diagnostic confidence. Conclusions: Development of the Pap stain method followed specific historical and scientific events. The staining method evolved following incremental improvements in cellular transparency achieved through tailored cellular fixation and cytoplasmic staining using variable dye and pH combinations.

Original languageEnglish (US)
Pages (from-to)266-280
Number of pages15
JournalActa Cytologica
Issue number4-5
StatePublished - Aug 1 2017


  • Cornification
  • Fixation
  • Pap stain
  • Pap test
  • Papanicolaou stain
  • Vaginal fluid smear

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology


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