Isovolumetric sphering of erythrocytes for more accurate and precise cell volume measurement by flow cytometry.

Typical mammalian erythrocytes are biconcave disks. Flow cytometric light-scattering measurements of red cell volume show large, orientation-dependent variations in signal size. To eliminate errors caused by shape factors, we sphere erythrocytes isovolumetrically on an Auto-Analyzer manifold (Technicon Instruments Corp., Tarrytown, N. Y.) using a carefully controlled ratio of concentrations of a salt of long chain sulfated fatty alcohol (e.g.), sodium dodecyl sulfate (SDS) to albumin in neutrally buffered isotonic saline, producing a suspension with at least 2 1/2 hours of stability with respect to cell volume, cell count and retention of hemoglobin. A two-step manifold modification of this procedure is preferred. It uses the endogeneous serum protein and a first SDS-saline reagent without added albumin at a low dilution, and further dilution with a second SDS-saline containing a very low concentration of glutaraldehyde. This bypasses a serious shelf life problem with the albumin-SDS reagent. Microhematocrit and Coulter electronic impedance measurements were correlated with light-scattering measurements on our own flow cytometer system of unsphered, sphered, and sphered and fixed samples. These reveal marked superiority over unsphered samples (e.g., reduced coefficients of variation of cell volume and improved correlation with microhematocrit) of the sphered and of the sphered and lightly fixed cells. The resulting tighter unimodal cell volume distributions also provide the following advantages: (1) The separation of signals of large platelets from that of the smallest red cells is substantially increased permitting better resolution of microcytic red cells in some abnormal bloods, and (2) bimodality of red cell volume distribution in abnormal bloods is more easily resolved.