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PCNA immunopositivity index as a substitute to 3H-thymidine pulse-labeling index (TLI) in methanol-fixed human
P Galand1, G Del Bino, M Morret
1Laboratory of Cytology and Experimental Cancerology, Institute for Interdisciplinary Research, RIBHN School of Medicine, Belgium.
Insights
Optimizing proliferating cell nuclear antigen (PCNA) immunostaining requires specific fixation methods. Methanol-Triton fixation with PC10 antibody offers high sensitivity and specificity for identifying S-phase cells.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Proliferating cell nuclear antigen (PCNA) is a marker for cell proliferation.
- Accurate detection of PCNA is crucial for assessing cell growth fraction.
- Existing immunostaining methods for PCNA have limitations in specificity and sensitivity.
Purpose of the Study:
- To evaluate the efficacy of different fixation and permeabilization procedures for PCNA immunostaining.
- To determine the optimal antibody and protocol for reliable PCNA detection.
- To compare PCNA index with Ki-67 index in hematological diseases.
Main Methods:
- Human lymphocytes and K-562 cells were pulse-labeled with 3H-thymidine.
- Cells underwent various fixation-permeabilization treatments before immunostaining with PCNA antibodies (19A2, 19F4, PC10).
- Specificity and sensitivity scores were calculated by comparing PCNA staining with 3H-thymidine labeling.
Main Results:
- Methanol fixation with Triton X-100 and PC10 antibody demonstrated high sensitivity and specificity for S-phase cells.
- PC10 antibody provided a more intense and readable reaction compared to others.
- PCNA index correlated well with Ki-67 index in hematological diseases (r = 0.79).
Conclusions:
- The combination of methanol-Triton fixation and PC10 antibody is recommended for accurate PCNA detection.
- PCNA immunostaining is a valuable tool for assessing cell proliferation, comparable to Ki-67.
- Further research is needed to refine PCNA's role in measuring growth fraction, especially for G0 cells.
Abstract:
PHA-stimulated human lymphocytes or myelogenous leukemia cells (strain K-562) were pulse labeled with 3H-thymidine and submitted to various fixation-permeabilization procedures. They were then immunostained with the 19A2, 19F4 or PC10 monoclonal antibody against the proliferating cell nuclear antigen (PCNA). The preparations were finally scored for the proportion of unlabeled, double-labeled and single PCNA or 3H-thymidine-labeled nuclei. Unstimulated lymphocytes were immunonegative in all the conditions tested, as also were stimulated lymphocytes checked with an isotype of the primary antibody. A specificity (Sp) and a sensitivity (Se) score was calculated to evaluate the recognition by PCNA staining of the S-phase cells, as defined by the 3H-labeling. The data show that in most instances the three antibodies recognized the 3H-labeled cells with high sensitivity, ie with few false negative, but with low specificity, ie with PCNA positivity extending to variable proportions of non-S-phase cells. By contrast, methanol fixation followed by a brief treatment with the detergent Triton X-100 and immunostaining with either 19F4 or PC10 (but not with 19A2) combined a high sensitivity and specificity scores of the recognition of the 3H-thymidine-labeled cells: PC10 gave a more intense and, hence, more readable reaction. PHA-stimulated lymphocytes that had been preserved at -20 degrees C as cytocentrifuged smears failed to show any immunopositivity for PCNA if not submitted to further fixation prior to the immunocytochemical assay. When methanol-Triton was used for this step, only PC10 gave positive immunoreaction, yet with a lower specificity score (Sp = 76%) than in cells submitted to this fixation-permeabilization procedure without prior cryopreservation (Sp = 91.7%). The PCNA index was measured in cryopreserved, methanol-fixed smears of lymphocytes from patients with various hematological diseases and was compared to the Ki-67 index established independently on a serial sample. A good correlation was found between the two indices (r = 0.79; P < 0.0001) with the PCNA index generally lower than or close to the Ki-67 index. This warrants a note of caution about the use of total (ie stable and labile) PCNA immunostaining to measure the growth fraction (GF), classically defined as the proportion of proliferating cells in a population. However, in the absence of an absolute reference marker for G0 cells, there is no reason to assume that the PCNA index would necessarily be a worse estimate of GF than the Ki-67 index.(ABSTRACT TRUNCATED AT 400 WORDS)
