Monday, September 27, 2010 2:00 PM - American Society for ...

65-PBETTER DYNAMIC ANTIBODY DETECTION RANGE USING FLOW SINGLE ANTIGENASSAY THAN LUMINEX SINGLE ANTIGEN ASSAY.Chih-Hung Lai 1 , Jacob Okhovat 2 , Geraldine Ong 1 , Qi Wang 1 , Mehrnoush Naim 1 , Kai Cao 1 , Nancy L.Reinsmoen 1 . 1 HLA Lab, Cedars-Sinai Health System, Los Angeles, CA, USA; 2 Biology, California StateUniversity, Northridge, Northridge, CA, USA.Aim: To assess our pooled positive control sera (PPCS) performance comparing antibody detection rangeusing flow single antigen (FSA) and Luminex single antigen (LSA).Methods: The PPCS consisted of serum from 10 highly HLA sensitized patients. Randomly selected 4PPCS data sets of sera were tested using FSA and LSA over a three months period from December 2008 toFebruary 2009. All the tests were carried out with no dilution. Raw Trimmed Mean Fluorescent Intensity(MFI) was used to calculate the Standard Fluorescent Intensity (SFI) through Quantiplex standard curve.FSA bead median value was obtained from BD FACSDiva software. The mean value of each molecularequivalent bead was calculated and compared.Results: The coefficient of variation (CV) from each individual FSA bead using peak-area was from 5.26to 75.51% (average 23%) and using peak-height was 8.47 to 137.47% (55%). Each LSA bead CV was from22.64 to 64.32% (36%) using SFI and 24.39 to 61.71% (38%) using MFI units. Figure 1 shows thecorrelation between FSA bead and corresponding LSA bead matched at molecular level. Figure 2 shows nolinear distortion between raw trimmed MFI and calculated SFI from each SA bead. The equivalencybetween the MFI and SFI through the linear regression is 1:20.Conclusions: FSA has greater a dynamic range than LSA because most Luminex beads reached thesaturation at 300K SFI while the corresponding FSA beads ranged from 15K to 100K on the linear flowscale. The FSA assay may be a better choice for monitoring the progress of desensitization therapy andposttransplant antibody status.[figure1]66-PHLA-SPECIFIC IgM CAN PREVENT POST-TRANSPLANT DETECTION OF DONOR-SPECIFIC IgG.Peter N. Lalli, Lynne L. Klingman, Diane J. Pidwell. Allogen Laboratories, Cleveland Clinic, Cleveland,OH, USA.Aim: To determine if HLA-specific IgM can prevent the detection of donor-specific IgG in post-transplantantibody testing using Luminex single antigen beads.Methods: Six patients that had previously received either a kidney or pancreas allograft were tested forDSA by Luminex single antigen beads to confirm a suspected diagnosis of antibody mediated rejection.When antibody levels to donor antigens were discordant with biopsy results, we hypothesized that IgMmay be interfering with donor-specific IgG detection. Recipient sera were also heat-inactivated to disruptpotential IgM and retested for donor-specific antibodies. Sera were then tested for the presence of donorspecificIgM using the Luminex assay. To confirm the presence of DSA, sera were tested using flowcytometry beads and cellular crossmatches.Results: In the patients tested, there was a significant increase in the detection of DSA when the sera wereheat-treated prior to testing using the Luminex single antigen beads. The heat-inactivation demonstratednearly an 11-fold increase in the donor-specific bead MFI. In all cases, the mismatched donor antigen was aDQA1*0505/DQB1*0301 pair. In 3 of the 6 cases, MFI levels in untreated sera were below the level usedin the lab to identify positive reactivity. Each sera showed the presence of IgM antibody binding to the beadrepresenting the donor antigen. Positive IgG binding was also seen in both single antigen bead and cellularcrossmatches using the flow cytometer.Conclusions: Our data confirms previous studies demonstrating that treatment of sera to disrupt IgM inLuminex testing can reveal IgG that is undetected in untreated sera. In the cases presented here, wedemonstrate that IgM can prevent the detection of DSA in suspected cases of AMR. This suggests thattreatment to diminish IgM interference may allow for more accurate detection of donor specific IgG.67-PANTIBODIES AGAINST HLA ANTIGENS AND KIDNEY BIOPSIES OBTAINED FOR CAUSE.