Background: Donor-specific HLA antibodies (DSA) are detected by different crossmatch techniques. Flow cytometric crossmatching (FCXM) is sensitive method, but it is subject to false (+) results caused by auto-, non-HLA- antibodies and anti-CD20 (rituximab) treatment. Although single antigen beads (SAB) assay is widely used for virtual crossmatch (VXM), it differs in diagnostic accuracy from physical crossmatch.FlowDSA-XM (One lambda) is an advanced crossmatch which selectively detect the HLA antigen/antibody complex and can make separate detection HLA class I, class II and HLA-DQ DSAs. We evaluated FlowDSA-XM in comparison with VXM and FCXM.

Methods: Total 19 sera (7 sera contain anti-CD20) and fresh four donors' lymphocytes were used in crossmatches. VXM results were assessed using SAB assay (One lambda) and donor HLA typing results for high-resolution HLA-A, B, C, DR, DQA and DQB. The sum of MFI values of individual DSAs (> 1000 MFI) was considered for VXM positive. FCXM (T cell IgG and B cell IgG) was performed according to the laboratory optimized protocol. For FlowDSA-XM, any bound IgG on the lymphocytes is tagged with PE-anti-human IgG, and capture beads detect the antibody/antigen/PE-anti-IgG complex. Median channel shift (MCS) of fluorescent intensity was calculated and the cut-off values of 100, 50 and 50 were used for HLA-I, -IIa (DQ) and –IIb beads, respectively.

Results: In seven VXM with sera contain rituximab, all B-FCXMs were positive, while FlowDSA-XMs were negative in three DSA (-) XM. Class I DSAs (MFI; 1090-14488) were detected in 100% (8/8) of FlowDSA-XM, while two DSAs with weak MFI (1090, 2272) were negative in T-FCXM. MCS values of FlowDSA-XM were correlated with the DSA class I MFI values (r=0.855, P<0.001). For class II DSAs, FlowDSA-XMs detected in 68.7% (11/16) of VXM(+) and showed negative results for weak HLA-DR DSAs (MFI 1022-3014). Of three VXM (+) cases from class II DSA only (+)/rituximab(-) sera, one case was Flow-DSA-XM(+)/B-FCXM(-). In terms of HLA-DQ DSA (MFI; 7578-21603), flowDSA-XM showed 100%(9/10) sensitivity and 90%(9/9) specificity.

Conclusions: FlowDSA-XM could discriminate HLA class I, II and DQ-specific DSAs and the results correlated well with VXM results. FlowDSA-XM showed better sensitivity than T-FCXM and detected DSA regardless of rituximab treatment. This method may be helpful to detect physical DSA, but the cut-off value needs to be adjusted for best diagnostic performance.

CITATION INFORMATION: Lee J., Ryu J., Choi A., Oh E-.J. Evaluation of FlowDSA Crossmatch for Detection of Donor-Specific HLA Antibodies Am J Transplant. 2017;17 (suppl 3).

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