A new method for cell volume measurement based on volume exclusion of a fluorescent dye

Several methods currently in use for measuring mean corpuscular volume include: centrifuged packed cell volume, electronic impedance, and light scattering methods. Although these techniques are widely used and accepted, there are problems inherent to each method which may produce systematic errors that are difficult to estimate. This paper describes a new flow cytometric method of cell volume determination, based on the principle of volume exclusion, which may overcome the systematic errors of the methods currently in use. This method requires that the cells be suspended in a fluorescent dye which is unable to penetrate the cell membrane. The level of fluorescence which is produced when a narrow stream of the cell suspension is excited by a focused laser beam will remain constant until a cell arrives in the illuminated region thereby causing a decrease in fluorescence which is directly proportional to the cell's volume. The volume exclusion method is shown to give an estimate of mean red cell volume which correlates well with existing methods.