Abstract
Aim The Luminex-single antigen bead (SAB) assay is considered the most sensitive method for the identification of anti-HLA antibodies (Ab). However, false negative results can occur due to the prozone effect (PrE) leading to underestimation of some high titer anti-HLA Ab. The aim of this study was to evaluate the ethylenediaminetetraacetic acid-EDTA’s Ca++ chelating effect on the C1 complement in eliminating the PrE in anti-HLA Ab identification by SAB. Method Ten patients with PRAs > 65% were included in the study. Neat and EDTA treated samples were tested using SAB for either HLA class I (n=14) or class II (n = 10) specificities. A mean fluorescence intensity (MFI) ⩾ 2 fold increase in EDTA treated versus neat sera was considered relevant to PrE. The PrE was confirmed with 1:10 dilution and with dithiothreitol treatment of the sera. To confirm the clinical significance of the new identified Ab, sera with defined PrE and MFI value 1000 in neat sera were further tested with AHG-CDC crossmatch against donor cells expressing the relevant HLA. Results PrE was found in 7/10 patients concerning either HLA class I (n = 4) or class II (n = 3) Ab specificities. Using an MFI = 1000 as a cut off, six ‘new’ HLA specificities were identified either HLA class I (HLA-A∗01:01, A∗34:02, A∗11:02) or class II (DQB1∗03:01, DQB1∗03:02, DQB1∗03:03). However, a significant shift of MFI value of HLA Ab class I and II was observed. More precisely, 38 HLA-class I Ab directed against HLA-A (n = 31) and HLA-B (n = 7) showed a significant mean MFI value shift from 5162 (266 lower value) to 20207. Regarding HLA class II Ab, 26 directed against HLA-DQ (n = 23) and HLA-DR (n = 3) showed a significant shift from MFI 4419 (113 lower value) to 23795. New HLA Ab specificities detected after EDTA treatment were confirmed with a strong positive AHG-CDC crossmatch. Conclusion Taking into account that selection of potential recipients pre-Tx as well as the follow up post-Tx is based to HLA Ab identification, these preliminary results highlight the importance of using EDTA treatment of the patient’s sera prior to SAB assay, in order to abolish the prozone effect in HLA antibody detection.
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