Accurate determination of mean cell volume by isotope dilution in erythrocyte populations with variable deformability

Variations in erythrocyte deformability and morphology lead to artifacts in electronic determinations of mean cellular volume (MCV) by the aperture-impedance method. The micropipette-aspiration technique loses accuracy when applied to severely aberrant cells such as dense sickle cells. A new light-scattering technique requires that the cells be capable of undergoing isovolumetric sphering. In contrast, the isotope-dilution (ID) method measures absolute mean volume and is free of artifacts associated with abnormal deformability or morphology. It does not depend on any algorithms or correction factors and does not subject the cells to any stringent processing, not even centrifugation. The ID method can be used to determine the mean volume of red cells in hypo- or hypertonic media or in the presence of pharmacologic agents. It requires no more than a 1-ml aliquot of suspended cells at a hematocrit of at least 30%. The cells can be readily recovered, washed, and reused. Using EDTA labeled with 57Co as an extracellular space marker we have used ID to determine the MCV of fractionated normal human red blood cells (RBC), unfractionated RBC containing SS hemoglobin, and RBC from four other mammalian species. In the case of human RBC obtained from eight normal donors, we obtained mean MCV values (+/- SD) of 83.6 +/- 3.0, 87.5 +/- 3.9, and 76.5 +/- 5.3 fl for unfractionated and top and bottom 10% density fractions, respectively. The value 83.6 is significantly lower than the generally accepted range of 89-91 indicated by electronic analyzers calibrated against spun microhematocrits. The discrepancy of about 7% can account for the difference between mean cell hemoglobin concentration (MCHC) data determined by a calibrated Coulter Counter and corresponding data obtained with paired samples using a cyanmethemoglobin procedure specified in NCCLS Standard H15-A and corrected for trapped plasma.