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 fetal hemoglobin-positive (HbF +) RBCs. High-performance liquid chromatography (HPLC) was also used for measuring HbF and adult hemoglobin protein concentrations for the determination of RCV.Results:Concurrent RCV measurements using BioRBCs (18 and 54 g/ml), Kidd antigen, and HbF flow cytometry were not statistically different compared with RCVs measured using the reference BioRBC density (6 g/ml). By contrast, the HbF-HPLC method overestimated RCV by 45% compared with the reference method. All the methods demonstrated 100% 24-h posttransfusion RBC recovery (PTR 24).Conclusion:Because BioRBC, Kidd antigen, and fetal hemoglobin (HbF) flow cytometry are safe and accurate methods requiring <10 l of patient blood for determining RCV and PTR 24 in preterm infants, they can be useful in clinical and research studies of anemia and other conditions.