P204 The complement interference effect in HLA antibody detection in hematopoietic stem cell transplant patients: A validation study of EDTA treatment in LabScreen PRA and single antigen assay

Aim The complement interfering (CI) effect has been shown to cause falsely decreased or negative result in the Luminex LabScreen assay in solid organ transplantation. Complement bound by anti-HLA antibodies blocks the access of reporting antibody to anti-HLA antibodies. Pretreatment of serum with EDTA can abolish the CI effect by chelating calcium cations required for complement complex association. Only a few studies demonstrated the CI effect in hematopoietic stem cell transplant (HSCT) setting despite the fact that the presence of donor specific antibody (DSA), especially complement binding DSA, is associated with inferior engraftment and overall survival in HSCT recipients. We validated the EDTA pretreatment of serum to improve the quality of HLA antibody testing in both PRA and SAB tests using the samples from HSCT patients. Methods 21 and 20 specimens were selected and underwent PRA or SAB test with and without EDTA pretreatment, respectively. Dilution studies (1:8) were performed on all samples to verify the CI effect or other prozone effect by an alternative method. 9 SAB samples with high PRA were tested additionally with C1q test. The positive effect was defined as MFI (EDTA or Dilution) – MFI (Neat) × 1.2 ⩾ 400. Results In both SAB and PRA tests, 99–100% of the samples/beads detected with CI effect using the dilution method were confirmed by the EDTA method. In contrast, only 50–60% CI positive samples by EDTA method remained positive using Dilution method. In SAB assay, many antibodies tested negative with dilution (MFI  r 2  = 0.91 vs 0.85, respectively). A lower regression slope is seen with the Dilution method (0.663) compared to that with EDTA method (1.093) due to the dilutional effect. Moreover, no significant prozone effect was observed in the non-complement binding antibodies suggested by negative C1q test. Conclusions High prevalence of CI effect was seen in the HSCT setting. Most if not all antibodies demonstrating the prozone effect were complement fixing. Compared to dilution method, EDTA treatment provides a better resolution in identifying CI effect and a single dilution is not sufficient to reveal all the CI effects.