Leukoreduction (LR) is a process by which leukocytes (WBC) are filtered from blood or blood component, as the leukocytes may increase the incidence of alloimmunization to human leukocyte antigens and may cause febrile nonhemolytic adverse transfusion reactions. Current transfusion practice also requires that, at minimum, blood selected for transfusion to a patient be checked (phenotyped) to be antigen negative to the existing alloantibodies in the patient's serum. Human Erythrocyte Antigen (HEA) Beadchip™ (Hashmi, G. et al. Transfusion, 47, April 2007, 736–747) has been adopted by blood centers and transfusion services for routine antigen-negative screening. A recent prospective study (Klapper, E. et al. Transfusion, 50, March 2010, 536–546) conducted in four large hospital transfusion services concluded that it is theoretically feasible to establish an inventory of DNA tested donor components from existing hospital inventories to result in the provision of more extensively matched RBC components than is the current standard of practice. Here described is a HEA LR eMAP-S BeadChip™ platform developed based on a combination of a novel DNA extraction protocol and a novel proprietary assay platform for Human Erythrocyte Antigens determination from leukoreduced blood samples.
The BioArray Solutions HEA LR eMAP-S BeadChip Kit uses the novel Elongation Mediated Multiplexed Analysis of Polymorphisms in Solution (eMAP-S) technology (Lin, X. et al., 2010). The multiplex PCR amplifies 21 DNA fragments covering 24 allele variants associated with 32 human erythrocyte antigens plus one mutation for hemoglobin S. The PCR, is then followed by clean-up, multiplex allele specific primer extension (ASPE), and on-Beadchip™ detection and read-out by using the AIS-400 Array Imaging System. Verification studies was performed using 203 leukoreduced blood segments collected from various blood centers and transfusion services in US. A set of 30 blood samples was also obtained from before leukoreduction (whole blood, WBC count: 2308–6052 cells/ μL) and after leukoreduction (LR blood, WBC count:0.36-2.63 cells/μL), DNAs from LR blood samples were extracted by a novel DNA extraction method (BAS) using commercial reagents ((Qiagen, Inc., Valencia, CA).
To evaluate the performance of HEA LR eMAP-S Beadchip™ Kit, the extracted DNA samples from before and after LR were analyzed by both HEA LR eMAP-S Beadchip™ and commercially available HEA 1.2 Beadchip™ Kit along with 88 whole blood DNA samples and positive controls with known HEA calls and analyzed for 10 red blood group systems (Rh, K, JK, FY, MNS, DO, Lu, Yt, Di, Co); and one mutation associated with hemoglobinopathies. Phenotype results obtained from HEA LR eMAP-S BeadChip™ Kit during the studies were compared to the data from the HEA 1.2 BeadChip™ Kit.
DNA was successfully extracted from 30 donor DNA samples before (DNA ng/ul: 20–56) and after LR (DNA ng/ul: 8–20) and analyzed using HEA LR eMAP-S Beadchip™ Kit with 100% concordance in HEA phenotype results. The WB DNAs were further analyzed using HEA 1.2 Beadchip™ kit and the results showed 100% concordance with LR DNA results. In addition, results from positive controls (HEA Ref-pA, HEA Ref-pB and cell line DNAs) all produced correct calls, and HEA LR eMAP-S assay and HEA 1.2 assay testing results on 88 donor whole-blood DNAs showed 100% concordance.
HEA LR eMAP-S Beadchip™ Kit could be used for reliable determination of human erythrocyte antigens for leukoreduced blood samples.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.