Changes in mitotic rate and cell cycle fractions caused by delayed fixation.

The mitosis frequency and flow cytometric data of malignant neoplasms are important, both for diagnosis and for prognosis. It is unclear to what extent these factors are affected by a delay in the fixation of tumor biopsies. We have thus studied the mitotic activity and DNA content in human soft-tissue sarcoma xenotransplants, fixed for periods of 5 minutes and 3, 6, 9 and 12 hours after biopsy. On average, the mitoses counted by two observers were 13% and 10% below initial values after 3 hours, and decreased by 46% and 39% after 12 hours. The mitosis decrease was related to the degree of mitotic activity of individual tumors, and was minimal in the sarcomas with the lowest mitotic rate. These results were reproducible. However, numerous pyknotic mitotic figures were observed, so the decrease in counts is largely due to their reduced identifiability, and only partly attributable to a completion of the cell cycle. Well-preserved mitotic figures demonstrable after 12 hours appear to indicate that the proliferation activity only gradually decreases in unfixed biopsies. The flow cytometric data did not change substantially; only a slight increase in the G2 + M-phase fraction was observed. General conclusions from the results are limited by the fact that the investigated sarcomas had a higher mitotic activity than most carcinomas. Nevertheless, early fixation of biopsies is desirable to accurately measure mitosis counts for the grading of malignancy.