Comparison of multiple red cell volume methods performed concurrently in premature infants following allogeneic transfusion

Abstract

Background: Study of the pathophysiology and treatment of anemia of prematurity is facilitated by direct measurement of red cell volume (RCV) utilizing microliter quantities of blood samples. Our objective was to compare concurrent measurements of multiple direct RCV methods in infants. Methods: Eighteen preterm infants receiving clinically-indicated transfusions had concurrent flow cytometric determinations of RCV and 24 h red blood cell (RBC) recovery based on donor-recipient differences of biotin–labeled RBCs (BioRBCs), Kidd antigen mismatched RBCs, and HbF positive (HbF+) RBCs. HPLC was also used to measure HbF and HbA protein concentrations for RCV determination. Results: Concurrent RCV measurements using BioRBCs (18 and 54 μg/ml), Kidd antigen, and HbF flow cytometry were not statistically different compared to RCVs measured using the reference BioRBC density (6 μg/ml). In contrast, the HbF HPLC method over estimated RCV by 45% compared to the reference method. All methods demonstrated 100% 24 h post-transfusion RBC recovery (PTR24). Conclusions: Because BioRBC, Kidd antigen, and HbF flow cytometry are safe and accurate methods requiring <10 μl of patient blood to determine RCV and PTR24 in preterm infants, they can be useful in clinical and research studies of anemia and other conditions.

Measurement of Posttransfusion Red Cell Survival With the Biotin Label

“…The method described below was developed jointly by the Iowa, Arkansas, and Cincinnati groups and is being applied in all three locations with minor variations [5, 6, 49, 50]. All are adaptations of the method of Cavill et al who reported the first human application of the biotin RBC labeling method to measure RCV in adults [51].…”

“…As long as the peaks representing the fluorescent-labeled BioRBC population(s) remains separate from the unlabeled RBC and from each other, enumeration of the proportion of BioRBC can be accurately determined. Consequently, loss of label does not limit accuracy in determining RCV [5, 6, 55] or RCS under conditions of either normal survival [53] or accelerated removal [50]. …”

“…For unstored RBC, immediate recovery is tacitly assumed to be 100% including all of the studies that used the biotin method. At least three lines of evidence indicate that immediate loss of biotin labeled RBC is negligible: a) In normal individuals and sickle cell disease patients, circulating RCV calculated from times within 10 min of infusion agreed with RCV calculated from 51 Cr [2, 5]; b) survival of autologous RBC labeled at single density exhibited no detectable RBC loss based on post-transfusion samples at 5, 10, and 20 min [5]; little or no loss of labeled RBC between 5 and 20 min indirectly suggests that there was little immediate loss of BioRBC; and c) survival of allogeneic RBC labeled at multiple densities exhibited no detectable RBC loss in infants based on samples drawn at 20 min [6] and the circulating RCV calculated from these time points agreed with circulating red cell volume calculated from minor antigen mismatch, which presumably has no immediate loss because none of the RBC are labeled. Thus, while absolute proof is difficult, we conclude that the preponderance of the evidence indicates that immediate post-transfusion BioRBC label loss is likely negligible in comparison to the inherent accuracy of the method.…”

Changes in circulating red cell volume during the first 6 weeks of life in very-low-birth-weight infants

“…Nevertheless, the use of radioactive substances for research in infants would be considered ethically unacceptable in the 21st century. Fortunately, biotin has been developed as a safe, nonradioactive RBC label for studies of RCV and RBC survival in infants (10,11,14,24,25), allowing continued research in this important area.…”

“…Others have subsequently reported their experiences measuring RCV directly using chromium-51 ( 51 Cr) (2-6), chromium-50 ( 50 Cr) (7), technitium-99 ( 99 Tc) (8), and biotin (9)(10)(11) as red blood cell (RBC) labels. In addition, several other methods have been used to calculate RCV based on changes in fetal hemoglobin concentration (12)(13)(14) or mismatched RBC antigens (14,15) following a transfusion of packed RBCs, or by calculation from measured plasma volume and hematocrit (16).…”