Quantitative Assessment Shows Loss of Antigenic Epitopes as a Function of Pre-analytic Variables

Pre-analytic variables, specifically cold ischemic time, have been implicated as key variables in the measurement of proteins by immunohistochemistry. To determine the significance and magnitude of antigenic loss due to pre-analytic variables, we have compared protein antigenicity in core needle biopsies, with essentially no cold ischemic time, to that in routinely processed tumor resection specimens. Two cohorts of matched core needle biopsies and tumor resections were collected with 20 matched pairs and 14 matched pairs, respectively. Both series were analyzed by quantitative immunofluorescence using the AQUA® method. Antibodies to phospho-ERK, total ERK, phospho-AKT, total AKT, phospho-S6K1, total S6K1, Estrogen Receptor, Ki67, cytokeratin and GAPDH were measured. Detection levels for all phospho-epitopes were significantly decreased in tumor resections compared to biopsies while no significant change was seen in the corresponding total proteins. Of the other four proteins examined, Estrogen Receptor and cytokeratin showed significant loss of antigenicity. This data suggests that measurement of phospho-protein antigenicity in formalin-fixed tissue by immunological methods is dramatically affected by pre-analytic variables. This study suggests that core needle biopsies are more accurate for assessment of tissue biomarkers.