Document Detail


Immunocytochemical detection of p16INK4a protein in scraped cervical cells.
MedLine Citation:
PMID:  12920756     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Chamsai Pientong; Tipaya Ekalaksananan; Usanee Swadpanich; Bunkerd Kongyingyoes; Onanong Kritpetcharat; Pissamai Yuenyao; Nuannit Ruckait
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Acta cytologica     Volume:  47     ISSN:  0001-5547     ISO Abbreviation:  Acta Cytol.     Publication Date:    2003 Jul-Aug
Date Detail:
Created Date:  2003-08-18     Completed Date:  2003-12-18     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370307     Medline TA:  Acta Cytol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  616-23     Citation Subset:  IM    
Affiliation:
Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand.
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MeSH Terms
Descriptor/Qualifier:
Carcinoma / diagnosis*,  metabolism
Cell Cycle Proteins / metabolism
Cell Division / physiology
Cell Transformation, Neoplastic / metabolism
Cervix Uteri / metabolism*,  pathology
Cyclin-Dependent Kinase Inhibitor p16 / metabolism*
Diagnostic Errors / prevention & control*
Epithelial Cells / metabolism*,  pathology
False Negative Reactions
Female
Humans
Immunohistochemistry / methods,  trends
Observer Variation
Reproducibility of Results
Retinoblastoma Protein / metabolism
Tumor Markers, Biological / analysis*
Uterine Cervical Neoplasms / diagnosis*,  metabolism
Vaginal Smears / methods,  trends
Chemical
Reg. No./Substance:
0/Cell Cycle Proteins; 0/Cyclin-Dependent Kinase Inhibitor p16; 0/Retinoblastoma Protein; 0/Tumor Markers, Biological

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