Protocol for immunocytochemical detection of SIL in cervical smears using MIB-1 antibody to Ki-67 [corrected]

Ki-67 nuclear antigen is expressed in upper epithelial levels of intraepithelial neoplasia of the cervix and vulva, variably in condyloma, and in basal and parabasal cells of normal squamous mucosa in histologic preparations. The application of antibodies to Ki-67 as a marker of squamous intraepithelial lesions in cervical smears was explored using either air-dried, acetone-fixed cervical smears obtained from 106 consenting patients or a single slide from archival two-slide cases of squamous intraepithelial lesions MIB-1 monoclonal antibody to Ki-67 was tested using two immunocytochemical techniques. In one set of smears, avidin-biotin peroxidase was used for detection and diaminobenzidine with H2O2 as the chromogen. Some specimens were incubated with 0.3% H2O2 and phosphate buffered saline for blockade of endogenous peroxidase. Alternatively, other air-dried smears were stained using alkaline phosphatase antialkaline phosphatase for detection and new fuchsin as the chromogen. Nuclear staining in squamous intraepithelial lesions was identified in air-dried smears using all of the above methods. Slides stained with avidin-biotin peroxidase and blocked with 0.3% H2O2 and phosphate buffered saline showed less background staining from neutrophils and erythrocytes compared with those without blocking. Slides stained using alkaline phosphatase antialkaline phosphatase showed excessive cytoplasmic staining of endocervical cells, making intraepithelial difficult. No nuclear staining of squamous intraepithelial lesions was observed in destained archival smears. Air-dried smears blocked with 0.3% H2O2 and phosphate buffered saline, incubated with MIB-1, and stained using avidin-biotin peroxidase gave the best results for identification of Ki-67 expression in squamous intraepithelial lesions.