SOURCES OF ERROR ASSOCIATED WITH SAMPLE COLLECTION AND PREPARATION OFNUCLEATED BLOOD-CELLS FOR FLOW CYTOMETRIC ANALYSIS

Analysis of cellular DNA content by flow cytometry has been used to de tect genetic changes associated with exposure to environmental contami nants. In lower vertebrates, nucleated red blood cells can be collecte d for analysis without harm to the animal. Because erythrocytes sample d from an individual should have identical amounts of DNA, the coeffic ient of variation (CV) around the G0/G1 peak should be small. Increase s in CV can indicate genetic aberrations, but may also be caused by sa mple handling and preparation or problems with instrumentation. To inc rease confidence in associating increases in CV with external causes, artifactual changes in CV due to sample treatment and instrument param eters should be identified and minimized. We assessed the effects of v arious sampling and handling protocols on the CV of nucleated blood ce lls collected from largemouth bass (Micropterus salmoides). We also co mpared the distribution of cells among the G0/G1, S, and G2/M phases o f the cell cycle to see whether these were affected by sampling or tre atment protocols. Groups of 7 fish were bled on 7 consecutive days, an d blood from each fish was analyzed by flow cytometry when freshly col lected, and after freezing for 1 hour or 10 days. The same fish were b led again over a consecutive 7-day period, and the experiment was repe ated. CV and cell cycle distribution were not affected by our freezing protocol. Repeat sampling from the same individual did not affect CV, but altered the distribution of cells in the cell cycle, suggesting i ncreased hemopoiesis in response to blood sampling. Day-to-day variati on in the CV occurred in both fresh and frozen samples, probably as th e result of small variations in instrument adjustments. These results demonstrate the suitability of this freezing protocol for these blood samples, and illustrate the importance of assessing sources of variati on when using flow cytometry to screen wild populations in genotoxicol ogical studies.