P274 Discrepancy between DSA and HLA flow cross match associated with high frequency of prozone phenomenon in solid phase assay in organ transplantation

Abstract

Aim Human Leukocyte Antigen (HLA) antibody identification solid phase assays play a key role in defining Donor Specific Antibody (DSA) which can determine the compatibility between the recipient and donor in cross match results. Luminex bead array test becomes high acceptance and wide reach in HLA antibody detection. However, HLA antibodies with high titer lead to activation of complement and might remove C1 complex on the bead. Subsequently, Interference of HLA antibodies and the secondary IgG antibodies might be occurred. This may lead to false-negative or weak antibody results, which is known as prozone phenomena. Technical methods, like treatment with EDTA, dithiothreitol (DTT), heat inactivation and dilution with normal saline can detect this phenomenon. Methods HLA Typing was performed for all recipients and donors before renal transplantation using One Lambda Sequence specific oligonucleotide revers and sequence specific primers (SSOr/SSP). HLA Antibody Identification, Single Antigen Class I/II and C1q test, was performed by One Lambda. T and B cell IgG XM is performed by FACSCanto II flow cytometer, where the cut-off for positive XM is determined based on normal human studies. Native serum samples of 11 recipients were diluted 1:10 with normal saline when clear discrepancy was found between DSA and HLA Flow XM (DSA 300 MCS). Results All cases (N = 11) showed strong elevating of DSA after 1:10 dilution, often >15000 MFI, which was correlated with strong positive HLA flow cross match results. Conclusions: Our results demonstrate the large incidence of prozone effect in solid phase assay. It is therefore strong recommended to treat the serum -in our cases dilution- for prozone effect always and whenever there is no correlation between strong positive FXM and weak or negative DSA.