SREL Reprint #1869

 

 

 

 

Sources of error associated with sample collection and preparation of nucleated blood cells for flow cytometric analysis


S.K. Fisher1, C.E. Dallas1, C. Jagoe2, M.H. Smith2, I.L. Brisbin, Jr2 and R.K. Chesser2
1Department of Pharmacology and Toxicology, College of Pharmacy, University of Georgia, Athens, Georgia;
2Savannah River Ecology Laboratory, University of Georgia, P. 0. Drawer E, Aiken, South Carolina, USA

Abstract

Analysis of cellular DNA content by flow cytometry has been used to detect genetic changes associated with exposure to environmental contaminants. In lower vertebrates, nucleated red blood cells can be collected for analysis without harm to the animal. Because erythrocytes sampled from an individual should have identical amounts of DNA, the coefficient of variation (CV) around the G0/G1 peak should be small. Increases in CV can indicate genetic aberrations, but may also be caused by sample handling and preparation or problems with instrumentation. To increase confidence in associating increases in CV with external causes, artifactual changes in CV due to sample treatment and instrument parameters should be identified and minimized. We assessed the effects of various sampling and handling protocols on the CV of nucleated blood cells collected from largemouth bass (Micropterus salmoides). We also compared the distribution of cells among the G0/G1, S, and G2/M phases of the cell cycle to see whether these were affected by sampling or treatment protocols. Groups of 7 fish were bled on 7 consecutive days, and blood from each fish was analyzed by flow cytometry when freshly collected, and after freezing for I hour or 10 days. The same fish were bled again over a consecutive 7-day period, and the experiment was repeated. 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 increased hemopoiesis in response to blood sampling. Day-to-day variation in the CV occurred in both fresh and frozen samples, probably as the 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 variation when using flow cytometry to screen wild populations in genotoxicological studies.

Keywords: DNA, flow cytometry, genotoxicology, largemouth bass, red blood cells

SREL Reprint #1869

Fisher, S.K., C.E. Dallas, C. Jagoe, M.H. Smith, I.L. Brisbin Jr., and R.K. Chesser. 1994. Sources of error associated with sample collection and preparation of nucleated blood cells for flow cytometric analysis. Cell Biology and Toxicology 10:145-153.

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