Abstract 3189: Preservation of immunorecognition by the transfer of cells from 10% neutral buffered formalin to 70% ethanol

Because of schedule limitations, cells and tissues initially fixed in 10% neutral buffered formalin (NBF) may sometimes be transferred and held in 70% ethanol until immunostaining can be performed in order to reduce the decrease in immunorecognition that might occur on prolonged fixation in formaldehyde. The specific parameters as to time of fixation in NBF prior to transfer to ethanol as well as the effects on immunorecognition of transfer to 70% ethanol have not been documented adequately in the literature. The cell lines, DU145 (prostate cancer) and SKOV-3 (ovarian cancer) were used as models by fixing of aliquots of each of these cell lines in 10% NBF for 5 minutes and 12, 15, 18, 36, 108 and 180 hours. Concomitantly, after 12 hours of fixation in 10% NBF, an aliquot of the exact same cells were transferred to 70% ethanol for 3, 6, 24, 96 and 168 hours so that comparable times of fixation (e.g., 180 hours 10% NBF versus 12 hours 10% NBF plus 168 hours ethanol) could be compared as to their effects on immunorecognition of PCNA, Ki67 (MIB-1), cytokeratins (AE1-AE3), and EGFr (membrane and cytoplasmic). The experiment was designed to perform all immunostaining concomitantly. The results for PCNA, Ki67 (MIB-1), and AE1/AE3 indicate that fixation of cells decreases immunorecognition after 12 to 18 hours (statistically significant), but the extent of the decreases in immunorecognition was not large enough to be important in evaluation until between 18 and 36 hours of fixation in 10% NBF. At 108 to 180 hours of fixation in 10% NBF, there was almost complete loss of immunorecognition of PNCA, Ki67 (MIB-1) and AE1/AE3. The transfer of cells to 70% ethanol after 12 hours of fixation in 10% NBF preserved immunorecognition at all points of evaluation (up to 180 hours). The effects on immunorecognition of EGFr were less, but transfer to 70% ethanol maintained cytoplasmic and membranous staining of EGFr. In conclusion, to preserve immunorecognition of many antigens in cells, the specimens should be transferred from 10% NBF to 70% ethanol after about 18 hours of fixation. Immunorecognition will then be stable for at least one week. Of note, results do vary with the antigen-antibody pair being evalutaed. Although the results of the study apply most directly to immunostaining of cells, this approach also is a model to determine if tissues fixed in 10% NBF might have their immunorecognition preserved by transfer and holding tissue in 70% ethanol prior to tissue processi