Abstract
Aim High levels of IgM immunoglobulins and complement have been shown to interfere in bead based antibody assays. Hypotonic Dialysis (HD) has proven to effectively remove interfering factors from serum in highly sensitized patients; however, this method requires lengthy serum treatments prior to testing. Laboratories have experimented and found some success in removing IgM by DTT treatment, serum dilutions, and removal of complement by addition of EDTA and heat inactivation. We have tried all of these approaches and continue to find HD to be most effective at removing all interference from patient sera. Recently we have experimented with Melon Columns which have shown promise as a quick and effective alternative to HD. Methods Sera from sensitized patients were tested untreated, titrated and following treatment with DTT and EDTA, Melon Column (MC) and HD prior to testing by OneLambda Single Antigen Beads (SAB). Efficacy of treatment, time and cost (labor and reagent) were factors used to determine the usefulness of each method. Results Removal of interference as measured by MFI intensity was observed in the following order: MC>HD>DTT>EDTA>no treatment. MC treated serum is ready for use in ∼ 20 min; however, processing large numbers increases hands on time. HD requires minimal hands on time, but has lengthy dialysis steps. DTT treated serum removes IgM, but can also degrade IgG. EDTA treated serum removes only complement interference and neither DTT or EDTA are as effective as HD for high CPRA sera. Titrations of untreated serum can be effective but are costly and may not be needed for all sera. Conclusions Sera treated with MC is superior to DTT or EDTA treatment, yields comparable results to HD and is more cost effective than serum titrations. Identification of sera requiring treatment prior to testing is the most effective strategy that a laboratory can take to reduce costs and insure accuracy of HLA antibody testing.
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