Volumetric capillary cytometry: A new method for absolute cell enumeration

This article describes a novel cytometry system that is capable of imaging, identifying, and counting populations of fluorescently labeled cells within a precisely known volume of unlysed whole blood contained in a precision capillary. The whole blood is reacted with dye‐labeled antibodies directed at cell surface antigens. A red excitation source (helium‐neon laser) is used to minimize auto‐fluorescence and interference by blood components. A two‐color fluorescence detection system is described, using the dyes Cy5 and Cy5.5, allowing the simultaneous detection and quantitation of two different types of surface antigens. Image processing techniques are used to identify cellular events in the image and to quantitate the fluorescent intensities in both color planes. The utility of this technique is demonstrated by performing absolute CD3+/CD4+ lymphocyte counts in whole blood, with minimal sample preparation, and with the blood sample completely contained in a disposable cartridge. A correlation study comparing this technique to flow cytometry gives a correlation coefficient of 0.98. In addition, the ability of this system to monitor reaction kinetics is demonstrated with a measurement of the rate of association of the anti‐CD4 antibody onto the surface of CD4+ lymphocytes. © 1996 Wiley‐Liss, Inc.