High-Throughput Amplification and Detection of Human Erythrocyte Antigen (HEA) Nucleotide Polymorphisms From Leukocyte Reduced Red Blood Cell (RBC) Units: Implications for Minor RBC Antigen Inventory Management by Hospital Transfusion Services

Technologies have recently been developed for rapid determination of extended human erythrocyte antigen (xHEA) phenotypes. For example, a semi-automated method using allele-specific oligonucleotides targeted against 32 clinically significant minor RBC antigens has been used to determine donor xHEA phenotypes from whole blood samples. This approach is currently used by blood collection centers and medical centers with blood collection facilities (both sites have access to linked donor whole blood samples). Broader access to xHEA information closer to the point-of-care (e.g. Transfusion Services at a Medical Center without a blood collection facility) may provide an opportunity to enhance patient care by more quickly and broadly providing units with xHEA phenotypes (Klapper et al., 2010.) However, transfusion services would need to use integrally attached segments for testing, and with leukoreduced (LR) RBC units these segments have very low numbers of white blood cells (WBC) (and therefore DNA), potentially limiting analysis. This study was performed to determine whether a HEA-elongation mediated multiplex assay in solution (HEA-eMAP-S) (Xin et al., 2010) could accurately genotype segments from LR-RBC units for 32 clinically significant minor RBC antigens.

Segments from pre-storage LR-RBC units (American Red Cross), < 14 days old, were obtained from a large tertiary care Children's Hospital in the Southeastern US and residual WBC were quantified by flow cytometry. DNA was extracted using an extraction method developed at BioArray SolutionS (BAS) using commercial reagents (Qiagen, Inc., Valencia, CA), and then amplified with the Universal Beadchip™ package (HEA LR-eMAP-S Beadchip™ Kits) which contains allele specific oligonucleotides directed to 32 clinically significant blood group antigens (c, C, e, E, V, VS, K, k, Kp^a, Kp^a, Js^a, Js^b, Jk^a, Jk^b, Fy^a, Fy^b, M, N, S, s, Lu^a, Lu^b, Di^a, Di^b, Co^a, Co^b, Do^a, Do^b, Jo^a, Hy, Yt^a, Yt^b mutation for hemoglobin S). DNA analysis results were correlated with RBC storage solution, WBC filter type, and serologic minor RBC antigen phenotypes of the units.

102 LR-RBC units from whole blood donations were studied, 74 /102 (73 %) stored in AS-1 and 28 /103 (27 %) in CPDA-1 solution. All AS-1 units were pre-storage LR with Fenwal Sepacell Flex Excel Filters and all CPDA-1 units were pre-storage LR with Whole Blood Fenwal Filters (Fenwal Inc. Lake Zurich, IL). All units demonstrated < 5 × 10⁶ WBC/unit with 47 % having < 4 × 10⁴ WBC/unit, which is at or below the limit of flow cytometric detection. Complete genotyping data was obtained from all samples. Ten samples showing initial indeterminate results on Diego and one for N antigens produced complete results after repeat testing. Fifty-four percent of units were serologically phenotyped for 1–8 antigens by the blood collection center; there was 100% correlation between predicted phenotype from DNA analysis and serology for these units.

The HEA LR-eMAP-S DNA analysis can be applied to optimally pre-storage LR-RBC units yielding > 99 % accuracy for all minor red blood cell antigens tested. The ability to perform this type of testing in a hospital transfusion service opens up new possibilities for transfusion services to select from their existing inventory and more efficiently allocate units to recipients with specific phenotypic requirements for RBC units.

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