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

eads. Comparing PS beads and TL beads, we found that 92.5% of the reaction patterns were in agreement.In contrast, only 15.2% of positive sera showed agreement between OL beads and PS beads.Conclusions: PS beads and TL beads generally agreed well. About half of positive sera on OL beads didnot react with PS or TL beads. Most discrepancies were from MICA*019 or MICA*001 beads. Serumexchange and antibody absorption studies could be used to better characterize the antibodies against MICAand their serologic patterns.15-ORA MULTI-SITE STUDY EMPLOYING HIGH RESOLUTION HLA GENOTYPING BY NEXTGENERATION 454 GS FLX SEQUENCING.Cherie L. Holcomb 1 , Bryan Höglund 1 , Matthew W. Anderson 2 , Lisbeth A. Blake 3 , Irena Böhme 4 , MichaelEgholm 3 , Deborah Ferriola 5 , Christian Gabriel 6 , Damian Goodridge 7 , Rolf Klein 8 , Martha Ladner 9 , CurtLind 5 , Dimitri Monos 5 , Marcelo Pando 2 , Johannes Pröll 6 , David C. Sayer 7 , Gudrun Schmitz-Agheguian 10 ,Birgitte B. Simen 3 , Bernhard Thiele 8 , Elizabeth Trachtenberg 9 , Dolly B. Tyan 2 , Ralf Wassmuth 4 , ShanaWhite 9 , Henry A. Erlich 1 . 1 Roche Molecular Systems, Pleasanton, CA, USA; 2 Stanford University,Stanford, CA, USA; 3 454 Life Sciences-A Roche Company, Branford, CT, USA; 4 DKMS Life Sciences,Dresden, Germany; 5 Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 6 Blutzentrale, Linz,Austria; 7 Conexio Genomics, Perth, Australia; 8 Institute of Immunology and Genetics, Kaiserslautern,Germany; 9 Children’s Hospital of Oakland, Oakland, CA, USA; 10 Roche Applied Science, Penzberg,Germany.Aim: The 454 GS FLX massively parallel pyrosequencing system is capable of providing high resolutionHLA genotyping for multiple individuals at multiple loci in a single run using the Conexio Genomics ATFsoftware. The clonal property and long sequence lengths of this system reduce genotype ambiguities. Toassess the reproducibility and accuracy of genotyping we performed a multi-site study.Methods: To achieve high throughput genotyping, we used PCR primers with multiplex identifier (MID)tags to amplify HLA exons from individual samples, thereby allowing pooling of the amplicons generatedfrom different individuals prior to the emulsion PCR step. The MIDs allow the software to assign alleles inthe sequence files to the appropriate individuals. We performed a double blind study in which 8 laboratorysites with varying levels of experience in sequencing on the 454 GS-FLX platform used GS-FLX standardchemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, and DRB3, 4, 5 using the Conexio ATF software. Fourteen primer pairs (exon 2, 3, and 4 for class I, exon 2 forDPB1, DQA1, and the DRB loci and exons 2 and 3 for DQB1) with 11 MID tags were used.Results: The average sequence read length was 250 bp and the average number of sequence reads peramplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered,assignment was possible in 95% of the cases. Failure to assign known genotypes was the result ofprocedural error or presence of a novel allele. Overall concordance with known genotypes, in cases whereassignment was possible, was 97.2%. Overall concordance with known alleles was 98.3%.Conclusions: Genotyping using the GS-FLX system and Conexio ATF was very reproducible andaccurate. Further studies are underway to implement the use of GS-FLX Titanium chemistry and ConexioATF to provide even higher resolution HLA genotyping.16-ORDETECTION AND CHARACTERIZATION OF NON-IgG ANTIBODIES BY FLOWCYTOMETRY ANALYSIS OF MULTIPLEX BEAD ARRAYS.Nancy M. Tcheou, Aaron T. Whiteley, Lee Ann Baxter-Lowe. Surgery, UCSF, San Francisco, CA, USA.Aim: Microparticles with unique internal fluorescence, coated with purified HLA antigens are usuallyinterrogated for bound IgG using a phycoerythrin (R-PE) conjugated antibody. This only allows foranalysis of one antibody isotype per test despite documented prevalence of IgA and IgM. This studydetermined feasibility of using multiple fluorescent parameters to quantify HLA-specific antibodies ofmultiple isotypes.Methods: Sequential sera of sensitized patients (n=173) with previously identified HLA antibodies byLabScreen Mixed Class I and II (One Lambda) were tested by LabScreen Single Antigen (One Lambda) todetermine antibody specificities and quantities according to the manufacturers’ recommended protocol.After analysis by Luminex 100, the remaining Microparticles were divided and stained with FITC