HLA Antibody testing- it’s not a one stop assay

Abstract

HLA laboratories rely on single antigen (SA) assays to determine unacceptable antigens and perform virtual crossmatches (vXM) that can immunologically guide clinicians in the identification of an appropriate donor. Although solid phase antibody detection assays are sensitive, specific and robust-instances arise when further testing is needed to verify results or resolve inconsistencies between these tests. Patient S.T. is a 50 year old male diagnosed with heart failure. Routine work-up in our laboratory is to evaluate % FlowPRA and compare those results to solid phase assays on a Luminex platform which measures the breadth and strength of antibodies in a patient’s sera. Patient S.T. had two samples tested three months apart with a class I FlowPRA of 58% and 62%, respectively. However, The Luminex SA results consistently demonstrated only 5 class I (all B15) antibodies in the positive MFI range leading to a cPRA of about 15%. Moreover, the internal negative control values were > 2500 MFI leading us to believe that there may be additional specificities masked by the high background. How should we deal with these contradictory test results? Can we rely on a vXM to accept a heart for this patient? To reconcile these seemingly contradictory results and to determine if the antibodies detected by Luminex assays were relevant, surrogate FCXM were performed. Surrogate Donor 1 (B15 positive) was FCXM positive. Surrogate donor 2 (B15 negative) was FCXM negative indicating that non-specific binding/false-positivity due to high background should not affect FCXM interpretation, Importantly, testing using phenotype beads were not affected by high background and further confirmed the presence of B15 antibodies. Due to the presence of HLA antibodies and the high background exhibited by this patient, a prospective FCXM must be performed prior to accepting an organ. In conclusion, this is an example in which different tests provide contradictory (or non-consistent) results - and our approach to resolve these ambiguities to support successful organ transplantation.