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

Area of Science:

• Hematology
• Biotechnology
• Flow Cytometry

Background:

• Twenty-four-hour post-transfusion recovery (PTR24) is critical for red blood cell (RBC) quality assessment.
• Traditional ⁵¹Cr radiolabeling methods face supply and facility limitations.
• Biotinylation of RBCs (BioRBCs) presents a non-radioactive flow cytometry alternative.

Purpose of the Study:

• To develop and standardize a biotinylation protocol for RBCs (BioRBCs) across multiple blood centers.
• To assess the reliability and reproducibility of the BioRBC labeling protocol for pharmacokinetic studies.

Main Methods:

• A two-phase multicenter study evaluated flow cytometry variability using RBCs biotinylated at 6 and 18 μg/mL.
• Standardized staining with streptavidin-phycoerythrin (SA-PE) and shared flow cytometry templates were used.
• Inter-site variability of biotinylation was assessed using paired RBC units, with separation index (SI) calculated.

Main Results:

• Minimal inter- and intra-site variability was observed in staining and analysis of BioRBCs (<7.9% within sites, <5.1% across sites).
• Comparable separation indices were achieved for biotinylation ratios (6 and 18 μg/mL), indicating reproducible labeling.
• The developed protocol demonstrated high reliability and reproducibility across different testing sites.

Conclusions:

• The standardized BioRBC protocol provides reliable and reproducible labeling with minimal inter-site variability.
• This method offers a promising non-radioactive alternative for RBC quality assessment.
• Future in vivo PTR24 studies will compare BioRBCs with dual radiolabeling (⁵¹Cr and ^99mTc).