Red blood cell (RBC) volume can be independently determined in vivo in humans using RBCs labeled at different densities of biotin

Abstract

Background: Anemia is a serious problem in critically ill neonates. To investigate the pathophysiology of anemia and responses to red blood cell (RBC) transfusions and erythropoietin therapy, repeated measurement of red blood cell volume (RCV) and blood volume is useful. To extend our previous sheep study in which RBCs were labeled at four different biotin densities, we assessed the validity of this multidensity method for in vivo measurement of circulating RCV in humans.

Study design and methods: In eight healthy adults, autologous RBCs were biotinylated at each of four biotin densities (6, 18, 54, and 162 µg biotinylation reagent/mL RBC), mixed, and infused intravenously; blood was sampled at 10, 20, and 60 minutes. At each time, RCV was calculated from dilution of individual RBC populations enumerated by flow cytometry. RCV measurements from the population of RBCs biotinylated at 6 µg/mL were chosen as the reference values because this density had been previously validated against the 51Cr method in vitro and in vivo in humans.

Results: Values for RCVs were not significantly different among the four densities of biotinylated RBCs at any of the three time points and did not change over 60 minutes.

Conclusions: These studies provide evidence that four densities of biotinylated RBCs can be used in vivo for simultaneous, independent, accurate measurements of RCV in humans. We speculate that this method will also be useful for repeated measurement of RCV and blood volume in infants and other patient populations in whom radioactive labels should be avoided.

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests

© 2010 American Association of Blood Banks.

Figures

Figure 1

Flow cytometry histogram relating number of RBCs enumerated to log of fluorescent intensity for four populations of biotinylated human RBCs demonstrates separation between each population of biotinylated RBCs and from the population of unlabeled RBCs. “6, 18, 54, and 162 µg/mL” denote the populations of biotinylated RBCs produced by successive three-fold increases in the ratio of the mass of biotinylation reagent to volume of RBCs being biotinylated.

Figure 2

RCV values determined at 10, 20, and 60 minutes from three highest populations of biotinylated RBCs (A: 18 µg/mL, B: 54 µg/mL, and C: 162 µg/mL) plotted against RCV determined from the reference biotin density of 6 µg/mL demonstrated excellent agreement. Two of the three regression lines (18 and 54) were not significantly different from the line of identity.

Figure 2

RCV values determined at 10, 20, and 60 minutes from three highest populations of biotinylated RBCs (A: 18 µg/mL, B: 54 µg/mL, and C: 162 µg/mL) plotted against RCV determined from the reference biotin density of 6 µg/mL demonstrated excellent agreement. Two of the three regression lines (18 and 54) were not significantly different from the line of identity.

Figure 2

RCV values determined at 10, 20, and 60 minutes from three highest populations of biotinylated RBCs (A: 18 µg/mL, B: 54 µg/mL, and C: 162 µg/mL) plotted against RCV determined from the reference biotin density of 6 µg/mL demonstrated excellent agreement. Two of the three regression lines (18 and 54) were not significantly different from the line of identity.

Figure 3

Individual RCV values from RBCs biotinylated at 18 µg/mL (Panel A), 48 µg/mL (Panel B) and 96 µg/mL (Panel C) agreed well with the lowest biotin density (6 µg/mL). Depicted are ratios to the corresponding RCV value from 12 µg/mL for individual RCV values. Values for all of the three timed blood samples and all eight humans are depicted. Mean ratio (solid line) and the 95% limits of agreement (dashed line) are also depicted.