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Aim: In seeking a method of reducing the background on the control beads in order to clarify the results of the Gen-Probe™ Luminex Single Antigen (LSA) assays, we hypothesized that heat- treating sera would be of great benefit. Methods: When initially processed in the laboratory, five hundred microliter aliquots of sera are frozen at −80°C for a minimum of 10minutes. The sample is then thawed to room temperature (22°C), heated in a 63°C water bath for 12minutes then ultra-centrifuged for 10minutes at 13,200rpm. Results: We have seen no noticeable change in the negative or positive control beads. Heat treating appeared to intensify our positive results. We see an increase in Mean Fluorescence Intensity (MFI) of the positively-reacting Luminex beads while also noting a decrease of the MFI in the negatively-reacting beads. We are uncovering antibodies that were obscured before. However, there has been an unexpected negative side effect of heat treatment. In approximately 30% of our population we are finding the sera are unfit for testing due to coagulation. We found that after ultracentrifuging for 10minutes at 13,200 rpm, 75% of the coagulated samples are suitable for testing. Conclusions: The heat treating process appears to rid the sample of a heat-labile inhibitor, presumably IgM. We believe that through the heat treating process we are able to better detect antibodies in patient sera. In an effort to streamline our testing algorithm, we now heat treat all sera that will be used for single antigen testing. [Copyright &y& Elsevier]

Aim: LIFECODES Class II IDv2 (LM2Q) is a bead-based immunoassay for the detection of class II panel reactive antibodies (IgG). Purified Class II antigens are covalently attached to Luminex beads to create a target for antibodies that may be found in sera. Compared to its predecessor product (LM2), the number of beads found in LM2Q was increased to include beads conjugated with purified antigens from the DQ locus. The aim of the current study was to compare the detection of antibodies when using the expanded panel (LM2Q) vs. the original panel alone (LM2). A second aim of the study was to compare the detection of DR-reactive antibodies when using LM2Q vs. LM2. The third and final aim of the study was to compare the detection of antibodies reactive to DQ antigens when using the expanded panel (LM2Q) vs. the pool of DQ antigens conjugated to a single bead population in LIFECODES LifeScreen Deluxe (LMX). Methods: Six hundred (600) serum samples were identified from libraries of excess samples. The sera were tested with LM2Q, LM2, and LMX. Briefly, and separately with each product, an aliquot of beads was diluted into wash buffer to which the serum was then added. Following an incubation and a series of wash steps, the treated beads were then exposed to a anti-human IgG-conjugated to phycoerythrin. After an additional incubation, the bead/conjugate blend was diluted and the fluorescence measured in a Luminex instrument. The observed Median Fluorescence Intensity (MFI) values of each reaction were analyzed to determine a reportable result of positive or negative. An individual bead reaction was considered positive when the observed MFI (normalized to the observed background) was greater than the lot-specific cutoff value and negative when the normalized MFI value was less than the lot-specific value. The presence of positive bead reactions which created a pattern of antibody reactivity was deemed to be a positive reportable result. The final reportable results were analyzed with 2x2 table analysis to determine the % co-positivity (sensitivity), % co-negativity (specificity), and % agreement between LM2Q, LM2, and LMX. Results: In the comparison of the detection of class II reactive antibody, the LIFECODES Class II IDv2 kit showed 99.7% co-positivity (98.3-99.9% [95%CI]), 72.2% co-negativity (66.6-77.1% [95%CI]) and 87.2% agreement (84.3-89.6% [95%CI]) when compared to results obtained with LIFECODES Class II ID. In the comparison of the detection of DR-reactive antibody, the LIFECODES Class II IDv2 kit showed 97.5% co-positivity (95.2-98.7% [95%CI]), 98.2% co-negativity (95.9-99.2% [95%CI]) and 97.8% agreement (96.3-98.7% [95%CI]) when compared to results obtained with LIFECODES Class II ID. In the comparison of the detection of DQ-reactive antibody, the LIFECODES Class II IDv2 kit showed 99.6% co-positivity (98.0-99.9% [95%CI]), 81.9% co-negativity (77.3-85.8%[95%CI]) and 90.3% agreement (87.7-92.4% [95%CI]) when compared to results obtained with LIFECODES LifeScreen Deluxe (LMX). The results of the study indicated that the expanded panel of DQ antigen-conjugated beads found in LIFECODES Class II IDv2 was associated with an increase in antibody detection; that the presence of the DQ-conjugated beads did not alter the ability to detect DR-reactive antibodies; and that the panel of DQ-antigen-conjugated beads was associated with an increase in antibody detection beyond that which was detected using a pool of DQ antigens conjugated to a single bead population. [Copyright &y& Elsevier]