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

Twenty-four-hour post-transfusion recovery (PTR24) is a key measure of red blood cell (RBC) quality. Traditionally assessed using radioactive chromium-51 (⁵¹Cr), 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.

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.

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.

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 ⁵¹Cr and technetium-99m (^99mTc).