SOURCES OF VARIATION IN THE ASSESSMENT OF CELL-PROLIFERATION USING PROLIFERATING CELL NUCLEAR ANTIGEN IMMUNOHISTOCHEMISTRY

The reproducibility in quantitation of proliferation activity, determi ned using the monoclonal antibody 19A2 to proliferating cell nuclear a ntigen (PCNA), was tested in visual and computer-assisted analyses of brain tumor material. The PCNA labeling index eons scored using count (PCNA-LI, visual and computer analyses) and area (PCNA-LIa, computer a nalysis) estimates of immunopositivity. The quality of immunostaining was the most important reason for variation in the assessment results. Other significant variation sources in the assessment were experience in selecting microscopic fields and distinguishing immunopositive nuc lei from immunonegative ones. Computer-assisted analysis improved the reproducibility of quantitation between different observers (visual r( PCNA-LI) = 0.624 versus computer assisted r(PCNA-LI) = 0.904). Also, t he use of PCNA-LIa improved the intraobserver and interobserver reprod ucibility in different stainings (observer 1:r(PCNA-LI) = 0.857 versus r(PCNA-LIa) = 0.874; observers 1 and 2: r(PCNA-LI) = 0.904 versus r(P CNA-LIa) = 0.927; observers 3 and 4: r(PCNA-LI) = 0.848 versus r(PCNA- LIa) = 0.906). PCNA-LIa by computerized image analysis improves accura cy in the evaluation of the granularly expressed PCNA level. Furthermo re, the effect of tumor heterogeneity on the assessment results can be diminished with the computerized method because large tissue areas ca n be analyzed faster.