Red blood cell (RBC) survival determined in humans using RBCs labeled at multiple biotin densities

Abstract

Background: Safe, accurate methods permitting simultaneous and/or repeated measurement of red blood cell (RBC) survival (RCS) are important to investigate pathophysiology and therapy of anemia. Methods using chromium 51 ((51) Cr)-labeled RBCs are unacceptable for infants, children, and pregnant women. We report RCS measured in vivo using RBCs labeled with several densities of biotin (BioRBCs).

Study design and methods: Aliquots of autologous RBCs from eight healthy adult subjects were labeled separately at four discrete biotin densities, mixed, and infused. The proportion of each population of BioRBCs circulating was determined serially by flow cytometry over 20 weeks. For each population, RCS was assessed by the following: 1) posttransfusion BioRBC recovery at 24 hours (PTR(24) ); 2) time to decrease to 50% of the enrichment at 24 hours (T(50) ); and 3) mean potential lifespan (MPL).

Results: Among the four BioRBC densities, no significant differences in PTR(24) were observed. T(50) and MPL were similar for the two lowest BioRBC densities. In contrast, the two highest BioRBC densities demonstrated progressively decreased T(50) and MPL.

Conclusions: RBCs labeled at four biotin densities can be used to independently and accurately measure PTR(24 ) and two lowest biotin densities can accurately quantitate long-term RCS. This method provides a tool for investigating anemia in infants, fetuses, and pregnant women with the following advantages over the standard (51) Cr method: 1) study subjects are not exposed to radiation; 2) small blood volumes (e.g., 20 µL) are required; and 3) multiple independent RCS measurements can be made simultaneously in the same individual.

Fig. 1

Flow cytometry histogram (Subject 1, Day 3 of survival) relating number of RBCs enumerated to log of fluorescent intensity for four populations of biotinylated human RBCs demonstrates the complete separation observed between each biotinylated RBC population and from the unlabeled RBC peak. “6, 18, 54, and 162 μg/mL” denote the populations of biotinylated RBCs produced by successive three-fold incremental increased of the biotinylation reagent, sulfo-succinimido-biotin.

Fig. 2

RBC survival for Subjects 1–4. RCS determined independently and simultaneously from the four populations of autologous BioRBC densities is depicted for Subjects 1–4.

Fig. 3

RBC survival for Subjects 5–8. RCS determined independently and simultaneously as per Fig. 2.

Fig. 4

Mean T₅₀ and MPL for each density of BioRBCs. Mean T₅₀ values (A) for the four populations of autologous BioRBCs agree for the two lowest densities but are progressively shorter for the two highest densities. Mean MPL values (B) for the four populations of autologous BioRBCs agree for the two lowest densities but are progressively shorter for the two highest densities.

Fig. 4

Mean T₅₀ and MPL for each density of BioRBCs. Mean T₅₀ values (A) for the four populations of autologous BioRBCs agree for the two lowest densities but are progressively shorter for the two highest densities. Mean MPL values (B) for the four populations of autologous BioRBCs agree for the two lowest densities but are progressively shorter for the two highest densities.

Fig. 5

Effect of Length of Observation on Precision of Estimates of Long-Term RCS. For each the three lowest densities of autologous BioRBCs, mean ± 1 standard deviation of the difference in T₅₀ (A) and in MPL (B) from the value obtained from the full data set is plotted against the interval of observation.

Fig. 5

Effect of Length of Observation on Precision of Estimates of Long-Term RCS. For each the three lowest densities of autologous BioRBCs, mean ± 1 standard deviation of the difference in T₅₀ (A) and in MPL (B) from the value obtained from the full data set is plotted against the interval of observation.