Organs transplanted across donor-specific HLA antibodies (DSA) are associated with a variety of clin. outcomes, including a high risk of acute kidney graft rejection.Unfortunately, the currently available assays to determine DSA characteristics are insufficient to clearly discriminate between potentially harmless and harmful DSA.To further explore the hazard potential of DSA, their concentration and binding strength to their natural target, using soluble HLA, may be informative.There are currently a number of biophys. technologies available that allow the assessment of antibody binding strength.However, these methods require prior knowledge of antibody concentrationsOur objective within this study was to develop a novel approach that combines the determination of DSA-affinity as well as DSA-concentration for patient sample evaluation within one assay.We initially tested the reproducibility of previously reported affinities of human HLA-specific monoclonal antibodies and assessed the technol.-specific precision of the obtained results on multiple platforms, including surface plasmon resonance (SPR), bio-layer interferometry (BLI), Luminex (single antigen beads; SAB), and flow-induced dispersion anal. (FIDA).While the first three (solid-phase) technologies revealed comparable high binding-strengths, suggesting measurement of avidity, the latter (in-solution) approach revealed slightly lower binding-strengths, presumably indicating measurement of affinity.We believe that our newly developed in-solution FIDA-assay is particularly suitable to provide useful clin. information by not just measuring DSA-affinities in patient serum samples but simultaneously delivering a particular DSA-concentrationHere, we investigated DSA from 20 pre-transplant patients, all of whom showed neg. CDC-crossmatch results with donor cells and SAB signals ranging between 571 and 14899 mean fluorescence intensity (MFI).DSA-concentrations were found in the range between 11.2 and 1223 nM (median 81.1 nM), and their measured affinities fall between 0.055 and 24.7 nM (median 5.34 nM; 449-fold difference).In 13 of 20 sera (65%), DSA accounted for more than 0.1% of total serum antibodies, and 4/20 sera (20%) revealed a proportion of DSA even higher than 1%.To conclude, this study strengthens the presumption that pre-transplant patient DSA consists of various concentrations and different net affinities.Validation of these results in a larger patient cohort with clin. outcomes will be essential in a further step to assess the clin. relevance of DSA-concentration and DSA-affinity.