PCNA IMMUNOPOSITIVITY INDEX AS A SUBSTITUTE TO H-3 THYMIDINE PULSE-LABELING INDEX (TLI) IN METHANOL-FIXED HUMAN-LYMPHOCYTES

PHA-stimulated human lymphocytes or myelogenous leukemia cells (strain K-562) were pulse labeled with H-3-thymidine and submitted to various fixation-permeabilization procedures. They were then immunostained wi th the 19A2, 19F4 or PC10 monoclonal antibody against the proliferatin g cell nuclear antigen (PCNA). The preparations were finally scored fo r the proportion of unlabeled, double-labeled and single PCNA or (3)Ht hymidine-labeled nuclei. Unstimulated lymphocytes were immunonegative in all the conditions tested, as also were stimulated lymphocytes chec ked with an isotype of the primary antibody. A specificity (Sp) and a sensitivity (Se) score was calculated to evaluate the recognition by P CNA staining of the S-phase cells, as defined by the H-3-labeling. The data show that in most instances the three antibodies recognized the H-3-labeled cells with high sensitivity, ie with few false negative, b ut with low specificity, ie with PCNA positivity extending to variable proportions of non-S-phase cells. By contrast, methanol fixation foll owed by a brief treatment with the detergent Triton X-100 and immunost aining with either 19F4 or PC10 (but not with 19A2) combined a high se nsitivity and specificity scores of the recognition of the H-3-thymidi ne-labeled cells: PC10 gave a more intense and, hence, more readable r eaction. PHA-stimulated lymphocytes that had been preserved at -20 deg rees C as cytocentrifuged smears failed to show any immunopositivity f or PCNA if not submitted to further fixation prior to the immunecytoch emical assay. When methanol-Triton was used for this step, only PC10 g ave positive immunoreaction, yet with a lower specificity score (Sp = 76%) than in cells submitted to this fixation-permeabilization procedu re without prior cryopreservation (Sp = 91.7%). The PCNA index was mea sured in cryopreserved, methanol-fixed smears of lymphocytes from pati ents with various hematological diseases and was compared to the Ki-67 index established independently on a serial sample. A good correlatio n was found between the two indices (r = 0.79; P < 0.0001) with the PC NA index generally lower than or close to the Ki-67 index. This warran ts a note of caution about the use of total tie stable and labile) PCN A immunostaining to measure the growth fraction (GF), classically defi ned as the proportion of proliferating cells in a population. However, in the absence of an absolute reference marker for G(o) cells, there is no reason to assume that the PCNA index would necessarily be a wors e estimate of GF than the Ki-67 index. We conclude that direct fixatio n with methanol and brief treatment with Triton X-100, followed by imm unoreaction with the PC10 antibody offers a valid substitute to H-3-th ymidine pulse-labeling in human lymphocytes for evaluating the classic al thymidine-labeling index or TLI, so that correlations eventually fo und between the latter and any clinical data would also apply to the P CNA index measured in those conditions. Finally, we suggest that conco mitant immunocytochemical analysis of stable PCNA (after methanol fixa tion), of total PCNA leg after formaldehyde fixation) and of the Ki-67 antigen might improve the performances of each separate criterion as prognostic indicators and help to clarify the biological significance of those three types of immunopositivity.