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 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 µg/ml), Kidd antigen, and HbF flow cytometry were not statistically different compared with RCVs measured using the reference BioRBC density (6 µg/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 (PTR24).

Conclusion: Because BioRBC, Kidd antigen, and fetal hemoglobin (HbF) flow cytometry are safe and accurate methods requiring <10 µl of patient blood for determining RCV and PTR24 in preterm infants, they can be useful in clinical and research studies of anemia and other conditions.

Figure 1

Flow cytometry histograms of pre- and post-transfusion samples showing number of RBCs enumerated (y-axis) to log of florescent intensity (x-axis) showing distinct RBC populations detected by different methods. Dashed lines indicate pre-transfusion samples and solid lines indicate post-transfusion sample results. (a) Subject 15, BioRBCs detected using Alexa-streptavidin; (b) Subject 14 with no previous transfusion, Jkb⁺ RBCs detected using anti-Jkb (infant RBCs are Jkb₋ and donor RBCs are Jkb⁺); (c) Subject 4 with a previous transfusion, Jka⁺ cells detected using anti-Jka (Infant RBCs are Jka⁺ and donor RBCs are Jka₋); (d) Subject 12 with no previous transfusion, HbF⁺RBCs detected using anti-HbF; and (e) Subject 15 with a previous transfusion, HbF⁺RBCs detected using anti-HbF.

Figure 2

Agreement of RCV measurements determined at 20 min using BioRBC densities (a) 18 μg/ml, (b) 54 μg/ml, (c) 162 μg/ml, (d) Kidd antigen (Jkb), (e) HbF flow cytometry methods plotted versus RCV measured from the reference BioRBC density, (f) RCVs determined by HbF HPLC method were overestimated compare to the reference BioRBC density. The line of identity is indicated by the thin diagonal gray line. The triangles in panels (e)-(f) represent the outlier data point.

Figure 3

Bland-Altman plots of RCV relative to the reference BioRBC density compared to: (a) BioRBC 18 μg/ml, (b) BioRBC 54 μg/ml, (c) BioRBC 162 μg/ml, (d) Kidd-Jkb, (e) HbF flow cytometry, and (f) HbF HPLC. The mean ratio of RCVs (solid line), 95% CI (dashed line), and ratio = 1.0 (dotted line) are shown. With the exception of (f) in which HbF HPLC significantly overestimated the limits of agreement for RCV (i.e., the area between the two dashed horizontal lines), all other plots demonstrated good agreement relative to the reference BioRBC density. The triangle data points in panels (d)-(f) indicate the single outlier data point.

Figure 4

Results of mean (± SD) RCV and BV determinations by each method. (a) Pretransfusion RCV in ml/kg (white bars) and posttransfusion RCV in ml/kg (gray bars); (b) posttransfusion BV in ml/kg.