Evaluation of p16/Ki-67 dual-stain cytology performed on self-collected vaginal and clinician-collected cervical specimens for the detection of cervical pre-cancer

Clin Microbiol Infect. 2020 Jun;26(6):748-752. doi: 10.1016/j.cmi.2019.10.020. Epub 2019 Oct 22.


Objectives: To compare the performance of dual immunostaining of p16INK4a and Ki-67 proteins performed on self-collected vaginal specimens and clinician-collected cervical specimens, and to evaluate the performance of this technique in predicting high-grade disease.

Methods: Women aged 30-59 years (n = 1005) were recruited at two well-women clinics in Papua New Guinea. Each woman provided both cervical and vaginal specimens that were tested for high-risk human papillomavirus (hrHPV) DNA using the Xpert HPV Test (Cepheid) at point of care. A subset of paired cervical and vaginal specimens (n = 243) were selected to undergo CINTec® PLUS (Roche) p16/Ki-67 dual-stain cytology and liquid-based cytology (LBC).

Results: Fifty-five pairs (22%) were excluded from further analysis because the smears were not assessable. Of the 189 remaining paired specimens, 74 pairs (39.1%) were positive for one or more hrHPV genotypes. When comparing results of the dual stain, the overall percent agreement, positive and negative percent agreements and κ value between the cervical and vaginal specimens were 87.8% (CI 82.3-92.1%), 64.6% (CI 49.5-77.8%), 95.7% (CI 91.0-98.0%) and 0.65 (CI 0.51-0.79%) respectively. The sensitivity of the dual stain performed on the cervical specimen to predict high-grade disease, determined by LBC, was superior to that of the dual stain performed on the vaginal specimen: 100% (CI 84.6-100%) versus 68.2% (CI 45.1-86.1%).

Conclusion: Although further evaluation may be warranted, these findings indicate that dual-stain testing of vaginal specimens cannot be advocated as part of cervical screening programmes in low- and middle-income countries. However, dual-stain cytology performed on cervical specimens may have a role in quality assurance in such settings.

Keywords: Cervical cancer; Cytology; Dual stain; HPV; Screening; p16/Ki-67.

Publication types

  • Comparative Study

MeSH terms

  • Adult
  • Cervix Uteri / virology*
  • Cyclin-Dependent Kinase Inhibitor p16 / analysis
  • Cytological Techniques*
  • Early Detection of Cancer / methods*
  • Female
  • Humans
  • Ki-67 Antigen / analysis
  • Middle Aged
  • Papillomavirus Infections / diagnosis
  • Papua New Guinea
  • Self-Testing*
  • Sensitivity and Specificity
  • Specimen Handling / methods*
  • Staining and Labeling / methods
  • Uterine Cervical Dysplasia / diagnosis
  • Uterine Cervical Neoplasms / diagnosis*
  • Uterine Cervical Neoplasms / genetics
  • Vagina / virology


  • CDKN2A protein, human
  • Cyclin-Dependent Kinase Inhibitor p16
  • Ki-67 Antigen