Red blood cell volume can be independently determined in vitro using sheep and human red blood cells labeled at different densities of biotin

Abstract

Background: The development of valid methods for repeatedly measuring red blood cell (RBC) volume (RCV) in the same individual would be useful in furthering understanding of the physiology and pathophysiology of the pregnant woman, fetus, and infant under a variety of conditions.

Study design and methods: Small volumes (5 to 100 mL) of either sheep or human blood were used to test the hypothesis that there is no significant difference in RCV and blood volume determined in vitro using as many as five populations of RBCs labeled at distinct biotin densities. By varying the mass of biotinylating reagent, the density of biotin on the surface of RBCs was incrementally increased to produce discrete populations as assessed by flow cytometric enumeration. Calculation of RCV for each biotin-labeled RBC population was based on the dilution principle.

Results: All biotin densities, except the most densely labeled, where variance was the greatest, accurately quantitated the in vitro blood volume to within 10 percent of the correct value. There was no bias of either overestimation or underestimation in the determination of the blood volume using either sheep or human RBCs.

Conclusion: These in vitro results provide evidence that the multidensity biotin labeling method is sufficiently accurate to utilize in vivo for repeated determination of circulating RCV and blood volume.

Figures

Fig. 1

Flow cytometric results from a representative in vitro mixture of human unlabeled and biotinylated RBCs from one study subject (Human Study 2 from Table 1). (A) Enumeration of RBCs. Selected region includes unlabeled and labeled RBCs. (B) Histogram of unlabeled and labeled RBCs complexed with FITC-AV.Markers delineate the different densities of biotinylated RBCs and the total events enumerated for each population. Marker 1 includes all the RBCs; Marker 2 through 6 denote individual populations labeled at 5.8, 23, 70, 212, and 634 µg per mL RBCs, respectively.

Fig. 2

Effect of increasing concentrations of biotinylation reagent on fluorescent intensity of RBCs from human (■) and sheep (○). (A) Saturation of biotin binding sites. A study of RBCs from one human and one sheep shows evidence of saturation. (B) Relationship of biotinylation reagent in µg per mL RBCs to fluorescent intensity per RBC. There is a linear relationship between mass of sulfo-NHS-biotin and the fluorescent intensity of the biotinylated RBC for masses of 200 µg per mL or less for humans and 500 µg per mL or less for sheep. Data are depicted as mean ± 1SD.

Fig. 3

Representative family of standard curves for an in vitro sheep study. The measured enrichment determined by flow cytometry versus the known enrichment determined gravimetrically yields slopes between 0.98 and 1.09 with correlation coefficients of 0.999 or more. A standard curve for each separate density of biotinylated RBCs was prepared using enrichments of biotinylated RBCs that encompassed the expected enrichment of that label in the total blood volume.