Multi‐center development and validation of a standardized protocol for biotinylated red blood cell analysis

Abstract Background Twenty‐four‐hour post‐transfusion recovery (PTR24) is a key measure of red blood cell (RBC) quality. Traditionally assessed using radioactive chromium‐51 ( 51 Cr), supply and facility limitations have prompted the need for alternatives. Biotinylation of RBCs (BioRBCs) offers a non‐radioactive method capable of distinguishing multiple RBC populations by flow cytometry. Researchers at three major blood centers developed a standardized BioRBC labeling protocol for pharmacokinetic studies. Study Design and Methods The multicenter development of the standardized protocol followed a two‐phase design. First, inter‐ and intra‐site flow cytometry variability was assessed using RBCs biotinylated at 6 and 18 μg/mL. Samples were stained with streptavidin‐phycoerythrin (SA‐PE), diluted, and analyzed in triplicates using shared flow cytometry templates. Second, inter‐site variability of biotinylation was tested using paired RBC units split and processed simultaneously at each site. Separation index (SI) was calculated as: SI = (median BioRBC − median unlabeled)/(84th percentile unlabeled − median unlabeled)/0.995. Results Staining and analysis of BioRBCs demonstrated minimal inter‐ and intra‐site variability when using identical protocols. Coefficients of variation for median fluorescence intensity were <7.9% within sites and <5.1% across sites. Biotinylation of paired RBC with 6 and 18 μg/mL of biotin resulted in comparable separation indices, suggesting reliable and reproducible biotinylation ratios. Discussion The standardized BioRBC protocol produces reliable, reproducible labeling with minimal inter‐site variability. In vivo performance will be assessed in upcoming PTR24 studies comparing BioRBCs with dual radiolabeling using 51 Cr and technetium‐99m ( 99m Tc).