Abstract

Microsphere-based Luminex antibody detection and specification assays of different levels of resolution have for years been used as standard tools to determine anti-HLA antibody specificities.The so-called prozone-effect, an inhibitory mechanism that leads to reduction or complete loss of fluorescent signals (MFI-values), was first described several years ago.This interference is attributed to the deposition products of the complement cascade, which finally block the binding of the fluorescent dye-conjugated secondary antibodies.As the EFI-guidelines defined a pretreatment of the sera under investigation, the most common way has been the use of EDTA (10mM) merely to avoid the activation of the classical complement pathway.It was our aim to systematically investigate and validate the EDTAtreatment using ten selected patients' sera, combining 734 antibody specificities.The MFI-values of these definable specificities with and without EDTA-treatment were subsequently analysed using our routine Luminex-based Single Antigen System (LSA).Comparative measurements of these specificities showed that 568 out of 734 (77%) led to reduction of their MFIvalues after EDTA addition, 291 (40%) exhibited a notable reduction of their MFI-values between 20% and 80%.77 specificities (10%) were no longer classifiable as positive at a given cut-off value of MFI 750 thus representing positive-to-negative conversions.In contrast negative-to-positive conversions were not observed, even if 166 specificities (23%) showed an increasement of their MFI-values.Due to the majority of specificities (77%), which are characterized by signal reductions, we conclude that EDTA-treatment does not provide any benefit.Quite in contrast, the portion of 10% of initially positive specificities, which after EDTA-treatment are no longer classifiable as positive, rather indicates a possible risk of this procedure.Consequently, the postulated correction of the prozone-effect by EDTA-treatment was not at all demonstrable.Our data hold true for the LSA of the manufacturer Werfen.Possible differences regarding the observed outcomes of the corresponding LSA of OneLambda may be explainable by variations in sera/bead ratios apparently differing by a factor of 16 and speculatively leading to the postulated prozone-effect in that system.