Comparative Cytometric and Microscopic Analyses of Oyster Hemocytes

Abstract

Automated techniques for counting molluscan hemocytes and measuring certain of their characteristics are becoming more common, but there has not been a concerted attempt to compare results with more traditional microscopy. We compared electrical resistance (Coulter counter) and light-scatter (flow-cytometry) measurements of eastern oyster, Crassostrea virginica, hemocytes with those from phase-contrast light microscopy. Our findings showed that the clarity of results from the two automated measurements was inversely related to the complexity, in type and size, of the cell population being analyzed. Microscopy produced the most detailed information, but flow-cytometric measurements, even when relying on light scatter alone, clearly surpassed the discriminatory ability of the Coulter counter. Microscopic measurements documented considerable size overlap among morphologically different types of hemocytes. Because it measures size only, the Coulter counter is relatively insensitive to these differences. The flow cytometer consistently described three subpopulations: two granular and one agranular. Because of the wide range and overlap in hemocyte size and intracellular contents (the two criteria measured by light scatter) within individuals, however, we found only a loose correlation between subpopulation counts from microscopy and from flow cytometry. By combining light scatter with the flow cytometer's ability to detect specific fluorescent dyes and its capacity to physically sort cells according to defined parameters, however, we expect to greatly increase its usefulness for studying molluscan hemocytes.