RESUMO
High-quality patient samples are required for reliable immunohistochemistry test outcomes that provide a significant benefit for patient care. Among the preanalytic variables in tissue handling, tissue thickness is thought to be easily controlled; however, whether the thickness of the tissue effects the staining intensity for antibody immunohistochemistry has not been quantitatively demonstrated. To investigate, we cut multiblock tissue sections of tonsil, liver, and kidney at 2, 4, 6, and 8 µm thicknesses. Interferometry measurements of the sectioned paraffin showed a <1 µm variation within a preset microtome thickness. Sections were then immunostained with antibodies targeting different cellular localizations; Ki-67 and BCL6 (nuclear), CD7 (membranous), and cytokeratin (cytoplasmic). A pathologist annotated regions of interest for each marker and performed brightfield and whole-slide visual scoring. Then a pixel-wise processing algorithm determined intensity of each pixel in these regions of interest and binned them into predetermined 0, 1+, 2+, or 3+ intensities. Visual scores from brightfield and whole-slide images were highly correlated to the percentage of pixels in each intensity bin. A stepwise increase was observed in pathologist scores and algorithmically defined percentage of pixels in each bin with increasing thickness demonstrating that changes in preset section thickness impacts staining intensity. The use of tissue thickness outside vendors' recommendations might change the intensity including the proportion of positive and negative cells and eventually the overall diagnosis outcome. Therefore, we recommend that tissue be consistently cut within the middle of thickness range specified by the assay manufacturer.
RESUMO
BACKGROUND: The aim of this study was to examine p16(INK4a) protein expression in ThinPrep (Cytyc Corporation, Marlborough, Mass) cervical specimens by using the CINtec p16(INK4a) Cytology Kit (Dako, Glostrup, Denmark). The ability of this assay to accurately identify underlying high-grade lesions was assessed by using follow-up biopsies and comparing these results with Hybrid Capture 2 (Digene, Gaithersburg, Md) high-risk HPV (hc(2)) results. METHODS: Three hundred ninety-eight residual ThinPrep samples were collected, and histological follow-up data were retrieved for abnormal cytology specimens. After preparation of a Papanicolaou-stained slide, a second slide was processed in preparation for p16(INK4a) immunostaining. High-risk human papillomavirus testing (hc(2)) was also performed. RESULTS: Of the 163 cytologically abnormal samples, 6-month biopsy follow-up data were available for 45% of the specimens. At initial blinded evaluation, 21 of the 26 cases with cervical intraepithelial neoplasia (CIN) II/III follow-up were positive for p16(INK4a), yielding an overall diagnostic sensitivity of 81%; 29 of the 47 cases diagnosed as CIN I or less were p16(INK4a) negative, yielding a diagnostic specificity of 62%. In comparison, the hc(2) test results indicated a diagnostic sensitivity of 100% with a diagnostic specificity of 15%. After review of selected cases with CIN II/III follow-up, 25 of 26 slides were deemed to be positive for p16(INK4a), increasing the diagnostic sensitivity to 96%. CONCLUSIONS: The CINtec p16(INK4a) Cytology Kit, in combination with ThinPrep cervical samples, allowed clear evaluation of p16(INK4a) protein overexpression. Diagnostic specificity of the CINtec p16(INK4a) assay was significantly improved relative to hc(2). To increase p16(INK4a) immunostaining in abnormal cells, a modified kit version with improved staining performance has been developed and is currently being evaluated.
