title: Immunocytochemical detection of p16INK4a protein in scraped cervical cells.
abstract: OBJECTIVE
  To develop an immunocytochemical technique for p16INK4a protein detection in scraped cervical cells for cancer screening.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand.
authors.forename: Chamsai
authors.initials: C
authors.lastname: Pientong
authors.forename: Tipaya
authors.initials: T
authors.lastname: Ekalaksananan
authors.forename: Usanee
authors.initials: U
authors.lastname: Swadpanich
authors.forename: Bunkerd
authors.initials: B
authors.lastname: Kongyingyoes
authors.forename: Onanong
authors.initials: O
authors.lastname: Kritpetcharat
authors.forename: Pissamai
authors.initials: P
authors.lastname: Yuenyao
authors.forename: Nuannit
authors.initials: N
authors.lastname: Ruckait
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D002277:Carcinoma / Q000175:diagnosis* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D002471:Cell Transformation, Neoplastic / Q000378:metabolism; D002584:Cervix Uteri / Q000378:metabolism* / Q000473:pathology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D003951:Diagnostic Errors / Q000517:prevention & control*; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D005188:False Negative Reactions; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods / Q000639:trends; D015588:Observer Variation; D065006:Papanicolaou Test; D015203:Reproducibility of Results; D016160:Retinoblastoma Protein / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis* / Q000378:metabolism; D014626:Vaginal Smears / Q000379:methods / Q000639:trends
chemical_list: D014408:Biomarkers, Tumor; D018797:Cell Cycle Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D016160:Retinoblastoma Protein