Immunocytochemical detection of p16INK4a protein in scraped cervical cells

Acta Cytol. Jul-Aug 2003;47(4):616-23. doi: 10.1159/000326578.

Abstract

Objective: To develop an immunocytochemical technique for p16INK4a protein detection in scraped cervical cells for cancer screening.

Study design: We took duplicate cervical scrapes from each participant, the first for a Pap smear and the second for p16INK4a protein detection. From a 50-microL cell suspension prepared from the scrape rinsing, a 10-microL aliquot was dropped in a 5-mm-diameter circle on a glass slide, air dried and fixed in 0.1% formal saline (1 hour) and in 95% ethanol (10 minutes). Using the immunocytochemical technique, slides from 30 samples of each Pap diagnosis class were stained sequentially with mouse monoclonal anti-p16INK4a (primary antibody), biotinylated goat antimouse IgG (secondary antibody), horse-radish peroxidase-labelled streptavidin and 3,3'-diaminobenzidine and mixed hydrogen peroxide, then counterstained with hematoxylin. A positive sample had to contain > or = 3 immunoreactive cells. Results were confirmed by western blot analysis of lysates from the remaining 40 microL of each cervical cell suspension.

Results: Samples were grouped as control (normal cervical cells), mild dysplasia (ASCUS, LSIL) and high abnormality (HSIL, SCC). Using the immunocytochemical technique, > 95% of the positive (SiHa cells) but 0% of the negative controls (human embryonic lung fibroblast cells) showed immunoreactive cells. All slides displayed a clear background without mucus, and positive cells were stained in both the cytoplasm and nucleus. p16INK4a Protein was detected in 17 of 30 (56.67%) ASCUS and 10 of 30 (33.33%) LSIL and increased with the degree of abnormality to 93.33% (28 of 30) and 96.67% (29 of 30) in the HSIL and SCC group, respectively. Normal cervical cells and degenerated malignant cells were nonimmunoreactive. Western blot analysis confirmed similar positive samples in the low-abnormality group, while the whole high-abnormality group was immunoreactive. A sampling error might have caused the 2 HSIL and 1 SCC sample to be negative using our immunocytochemical technique.

Conclusion: p16INK4a Protein detection in scraped cervical cells using the immunocytochemical technique correlated with western blot analysis and was nontraumatic and precise. It offers a significant diagnostic adjunct to the Pap test for cervical cancer screening.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers, Tumor / analysis*
  • Carcinoma / diagnosis*
  • Carcinoma / metabolism
  • Cell Cycle Proteins / metabolism
  • Cell Division / physiology
  • Cell Transformation, Neoplastic / metabolism
  • Cervix Uteri / metabolism*
  • Cervix Uteri / pathology
  • Cyclin-Dependent Kinase Inhibitor p16 / metabolism*
  • Diagnostic Errors / prevention & control*
  • Epithelial Cells / metabolism*
  • Epithelial Cells / pathology
  • False Negative Reactions
  • Female
  • Humans
  • Immunohistochemistry / methods
  • Immunohistochemistry / trends
  • Observer Variation
  • Papanicolaou Test
  • Reproducibility of Results
  • Retinoblastoma Protein / metabolism
  • Uterine Cervical Neoplasms / diagnosis*
  • Uterine Cervical Neoplasms / metabolism
  • Vaginal Smears / methods
  • Vaginal Smears / trends

Substances

  • Biomarkers, Tumor
  • Cell Cycle Proteins
  • Cyclin-Dependent Kinase Inhibitor p16
  • Retinoblastoma Protein