(P-BC-24) Development and Validation of a Standardized RBC Biotinylation Protocol

Background/Case Studies: Licensing of novel red blood cell (RBC) products in the United States requires measurement of 24 hour post-transfusion recovery and survival in autologous, healthy volunteers. Current radiolabeling methods using ⁵¹Cr and ^99mTc are becoming prohibitive from a regulatory, financial, and logistical standpoint, due to the unavailability of GMP grade ⁵¹Cr. Biotinylation of RBCs, a promising alternative, allows labeling of RBC populations with different biotin densities that can be detected and tracked by flow cytometry. In a multicenter collaborative effort, Hoxworth Blood Center, Vitalant Research Institute, and the American Red Cross (HOXVITARC) developed a standardized method to biotinylate and detect RBCs to measure post-transfusion pharmacokinetics in regulatory studies of novel or modified RBC products.

Study

Design/Methods: A biobank of frozen RBCs - either unbiotinylated or biotinylated at two different densities (s-NHS-biotin; 6 and 18μg/mL, Thermo Scientific) - was created by pooling 10 O+ leukoreduced RBC units following a common protocol developed following a technical workshop. These samples were distributed to three sites for flow cytometry staining (SA-PE, 0.25 μg/mL, BD Biosciences) and analysis (BD Lyric). Part A: Each site stained three samples per biotin density, diluted them 1:100 (1%) with unlabeled RBCs, and analyzed in triplicate using a standardized protocol. Part B: RBC units (N=5 per site) were collected at each site, split between days 35–42, and sent to the other sites for biotinylation, staining, and flow cytometry after 1% dilution in unlabeled RBCs. Separation Index (SI) between the two biotin densities was calculated as: SI = (MFI₁₈ – MFI₆) / [(85th percentile MFI₆ – MFI₆)/0.995].

Results/Findings: The staining and flow cytometry analysis of biobank cells demonstrated minimal variability in samples run multiple times at all three sites. Coefficients of variation (CVs) for Median Fluorescence Intensity (MFI) were < 1% within sites and across sites < 5%. Part A: SI CVs are summarized in Table 1A. Biotinylation of paired RBC units at multiple sites showed MFI values at one site being higher compared to the other 2 sites, resulting in higher SI values between unlabeled and labeled populations (Table 1B). However, the SI values comparing 6 ug/mL and 18 ug/mL biotin densities were consistent across all sites (10.7±1.9) indicating reliable and reproducible biotinylation ratios.

Conclusions: This study demonstrates that the standardized biotinylation and detection protocol yields reproducible results across multiple institutions, with minimal inter-site variability. Ongoing studies are comparing in vivo performance of biotin-labeled RBCs to traditional ⁵¹Cr and ⁹⁹^ᵐTc labeling methods.