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

Monday, September 27, 20102:00 PM - 3:30 PMWorkshop 1: Case Studies in Solid Organ Transplantation1-ORUNRECOGNIZED ALLOANTIBODY AGAINST B*14:01 AND B*14:02 FROM A B*14:12PATIENT.Bethany L. Dale, Brian Inglehart, Paul Sikorski, Andrea A. Zachary, Mary S. Leffell. Medicine, JohnsHopkins University, School of Medicine, Baltimore, MD, USA.Aim: To investigate antibody to an apparent self antigen. Antibody (Ab) reactive with B64 and B65 wasobserved in a B64+ patient. The patient typed as B*14:01 by rSSOP and was positive in a CDC crossmatchagainst a B*14:02 donor for which the patient had no other donor specific Ab.Methods: Typing was performed by rSSOP (LabType, One Lambda) and by sequence based typing on anApplied Biosystems 3100 Genetic Analyzer. Ab analyses were performed using both phenotype panels andsingle antigen beads (SAB) (Lifecodes, One Lambda).Results: The patient’s phenotype was verified by repeat rSSOP and was further confirmed by serologictyping as a B14. Reactivity, consistent with a positive CDC crossmatch, was noted forB*14:01(MFI=10480, by SAB) and B*14:02(MFI=9879). To resolve this reactivity, the allele wassequenced and shown to be B*14:12. B*14:12 is a relatively new allele that differs from B*14:01:01 byone amino acid (Arg157→Gly157) and from B*14:02:01 by two amino acids (Ala11→Ser11 and Arg157→Gly157.)Conclusions: Codon 157 is located in the α2 domain of the class I heavy chain, and the substitution resultsin a change from the hydrophilic, charged arginine residue to the hydrophobic glycine. An epitope sharedamong B8, B64 and B65 is partially defined by codon 158. It is therefore possible that the substitution inB*14:12 at codon 157 alters the conformation sufficiently to effect a new epitope. In summary, theB*14:12 allele was not defined by intermediate level rSSOP typing and was also not distinguishable fromB*14:01 or *14:02 by serologic typing. Although this is the third known description of B*14:12, it is thefirst report defining its serologic reactivity and has further provided additional intronic and exon 4 sequencedata. Importantly, the B*14:12 allele may be more common than previously recognized and could result infailure to recognize when other B14 alleles are contraindicatory to transplantation.2-ORHLA-B44. IS IT TIME TO SPLIT INTO TWO?Nabil Mohsin, Isabelle Wood, Indira Guleria, Edgar Milford. Tissue Typing Lab, Brigham and Women’sHospital, Boston, MA, USA.Aim: HLA-B44 is currently a single WHO serological specificity. However, several reports suggestserological differences between B*44:02 and B*44:03. The purpose of our study was to examine patternsof serological reactivity against B*44:02 and B*44:03.Methods: We tested 1466 sera from 1024 transplant candidates for anti-HLA class I antibodies by singleantigen bead assay (One Lambda, Luminex). We classified each serum for reactivity against B*44:02 andB*44:03 based on Luminex Mean Fluorescence Intensity (MFI). Sera were classified positive for anti-B44when the MFI was at least 1000. Sera were considered positive for B*44:02 and negative for B*44:03 (+/-)when the ratio of the MFI of B*44:02/B*44:03 was more than 2. They were classified as negative forB*44:02 and positive for B*44:03(-/+), when the MFI ratio of B*44:02 /B*44:03was less than 0.5.Results: We found that 500 (34.1%) sera were positive for B*44:02, B*44:03 or both. While 96% ofsamples showed excellent correlation between B*44:02 and B*44:03 (r-square=0.98, linear slope 0.96),10.6% of the positive sera reacted with B*44:02 but not B*44:03.[figure1]Only 0.4% reacted selectivelywith B*44:03.[table1]Conclusions: Our results suggest that B*44:02 and B*44:03 are distinct serologic entities. The clinicalsignificance of the selective reactivity to HLA-B4402 is yet to be determined. Further testing on nativeantigens on the cell surface is needed for confirmation.

3-ORIgM ANTIBODIES SPECIFIC TO DONOR HLA-B61 ARE ASSOCIATED WITH REJECTIONOF A KIDNEY TRANSPLANT.Qingyong Xu 1,2 , Terry Akister 1 , Donna Rich-Sperling 1 , Elly Johnson 1 , Rajni Chibbar 2 , Ahmed Shoker 3 .1 Lab Medicine, St. Paul’s Hospital, Saskatoon, SK, Canada; 2 Pathology and Lab Medicine, University ofSaskatchewan, Saskatoon, SK, Canada; 3 Saskatchewan Transplant Program, St. Paul’s Hospital,Saskatoon, SK, Canada.Aim: To investigate causes of antibody-mediated rejection (AMR) in kidney transplant without any anti-HLA IgG antibodies.Methods: To detect IgM antibodies, sera were screened with LABScreen® beads with PE-conjugated antihuman IgM as 2 nd reagent. DTT-treated, IgM-inactivated sera were used as negative control.Results: A 45 years old male received a living unrelated kidney transplant. Patient’s pre-transplantation(Tx) PRA were negative with both ELISA and Flow methods. T-AHG XM and T/B FCXM were negative,while T/B extended long incubation CDC XM were positive. Auto antibodies were excluded with negativeauto XM by CDC and flow. Tx was proceeded based on the fact that there is no IgG antibody to donorHLA pre-Tx. Eighteen to 24 hours after Tx, urine volume dropped precipitously, serum creatinine rose bythree fold. A biopsy confirmed severe AMR. The patient was treated with thymoglobulin, MMF,immunoadsorption, IVIG, and achieved excellent graft function. We hypothesize that IgM anti-HLAantibodies cause positive extended CDC XM and are associated with AMR. T/B extended CDC XM arepositive with serum collected when patient was rejecting the transplant. T-AHG XM is negative and thereare no anti-HLA IgG antibodies by ELISA and Luminex methods. The positive T/B extended CDC XMbecome negative when serum IgM is heat-inactivated. This indicates that IgM antibodies may cause thepositive CDC XM. IgM anti HLA class I antibodies were found in the pre-Tx serum with LABScreenmixed antigen beads. With IgM SAB, antibodies to B7, B27, B60, B61, B73, and B81 are found in theserum pre-Tx and when the transplant was rejected. IgM donor specific antibodies to B61 is present pre-Tx(MFI=1564) and is elevated when the transplant is rejected (MFI=2439), but is very low when the kidneytransplant functions well (MFI=359) (MFI=359).Conclusions: Donor-specific anti-HLA IgM antibodies are able to cause AMR in kidney transplant.4-ORPOSITIVE VIRTUAL CROSSMATCH (VXM), NEGATIVE FLOW CROSSMATCH (FXM): ACASE OF ANTIBODY (Abs) TO DENATURED ANTIGEN (dAg).Manish J. Gandhi, Nancy A. Ploeger, Steven R. DeGoey. Mayo Clinic, Rochester, MN, USA.Aim: Donor specific antibodies(DSA) identified by solid phase assay(SpA) are used to screen donors byperforming VXM as a surrogate for XM. We present a case of positive VXM and negative FXM.Methods: 29 yr patient for combined heart/lung transplant (Tx) had positve VXM as SpA demonstratedDSA to HLA-B44 (MFI=13,486). Retrospective T & B-FXM were negative. Possible reasons andinvestigation for the same are: 1. HLA-Abs is allele specific Donor typing confirmed DSA. 2.Error inperforming FXM Repeat testing with historical sample & spleenic cells: negative 3.Error in performingSpA Unlikely as historical Abs profile similar to current result 4.Non-HLA Abs directed to beads/reagents.Unlikely as Abs profile similar using a different product from same manufacturer and a differentmanufacturer.Results: As the SpA have some proportion of dAg, Abs to the dAg was considered.SpA beads were acidtreated(AT) to denature antigens. AT beads with positive control demonstrated a significant decrease inMFI values:[figure1]AT and untreated beads with patient demonstrated similar Abs profiles:[figure2]Datademonstrate Abs specificity is to dAg.Conclusions: Clinical significance of positive VXM and negative FXM is unknown. Post-Tx patient wasstable and biopsies in the past 13 months are normal.

5-ORTWO CASES OF DONOR SPECIFIC ANTIBODIES (DSA): ONE PATIENT’S DSA HAD IgGSUBCLASS 1 & 3 WITH GRAFT FAILURE, AND THE OTHER PATIENT HAD DSA IgGSUBCLASS 4 AND THE GRAFT IS FUNCTIONING WELL.James Cicciarelli 1,2 , Robert Adamson 2 , Steven Steinberg 2 , Barry Browne 2 , Benny Pitpitan 1 , BruceWilliams 1 , Nori Kasahara 1 . 1 Immunogenetics Lab, MNIT, Los Angeles, CA, USA; 2 Heart and RenalTransplantation, Sharp Memorial Medical Center, San Diego, CA, USA.Aim: We hypothesize that patients with DSAs have differing outcomes based on the IgG subclass of theDSAs.Methods: A standard single antigen luminex assay was used with IgG subclass specific murine monoclonalantibodies & goat anti-murine F(ab)2 PE. One Lambda software was used to interpret binding andnormalized fluorescence intensity (FI) is reported. Two patient sera were analyzed for IgG subclassesretrospectively, following high levels of IgG DSAs shown by single antigen luminex.Results: The first case was a renal transplant recipient who had strong DSAs up to 17,000 five days posttransplant.[table1]Thepatient was transplanted with negative crossmatches including pronase, but had fourweak DSAs. The patient diuresed, but became anuric at POD 6 and a nephrectomy was performed on POD34. Our second patient was a heart transplant recipient with a strong DSA toDQ2 (16000), but doingwell.[table2]Conclusions: Graft loss was clearly associated with DSA to IgG subclasses 1 & 3. Albeit, the pattern ofDSA IgG subclasses was somewhat complicated in the heart transplant recipient, IgG subclass 4 seems tobe a blocking or protective DSA.6-ORUNDEFINED TRANSIENT ANTIBODY IN A RENAL PATIENT.Sarah N. Schumacher, D. Phelan, T. Mohanakumar. HLA Laboratory, Barnes-Jewish Hospital;Department of Surgery, Washington University School of Medicine, St. Louis, MO, USA.Aim: Patient CF received a LURD renal transplant in May 2006. In March 2009, acute antibody-mediatedrejection was diagnosed. The goal of this study was to find a crossmatch (XM) compatible donor for retransplant.Methods: Luminex Single Antigen Assay (LSA); Four Wash Amos (FWA), Antiglobulin Augmented(AHG), and Flow XM.Results: Luminex SA was negative pre-transplant. Post-transplant, only 4 antibodies were detected: allweak, none donor specific. In August and September 2009, serologic (FWA and AHG) and flow XM wereperformed with 7 living donors. Five were positive serologically, all were positive by flow. Auto wasnegative. In October 2009, patient serum was adsorbed with T cells, and both T and B cells, from a positivedonor (DP). Sera were diluted and flow XM with T and B cells from DP. Mean channel shift (MCS)increased with dilution in all sera.[table1]Adsorption of serum in plastic did not result in negative XM.With serum from November 2009, 61 unrelated donors were flow XM. Of these, 58 were positive and 3negative. Two donors were HLA identical, and one was positive, the other negative. Five endothelial celllines were flow XM with patient serum: all 5 were positive. In January and February 2010, serologic andflow XM with 2 previously positive donors were negative, as were flow XM with endothelial cell lines.Conclusions: Despite all attempts, antibody specificity could not be defined. Antibody was not HLA,shown by negative LSA in August, September, and November 2009. We conclude that the antibodydeveloped against non-HLA antigens was short-lived, although strong, resulting in reluctance to retransplant.

7-ORTHE IMPACT OF A POSITIVE ENDOTHELIAL CELL CROSSMATCH (ECXM) IN ANINCOMPATIBLE RENAL TRANSPLANT RECIPIENT.Annette M. Jackson 1 , Robert A. Montgomery 2 , Lorraine C. Racusen 3 , Edward S. Kraus 1 . 1 Medicine, JohnsHopkins University, Baltimore, MD, USA; 2 Surgery, Johns Hopkins University, Baltimore, MD, USA;3 Pathology, Johns Hopkins University, Baltimore, MD, USA.Aim: To investigate the impact of non-HLA antibodies following an ABO and HLA incompatibletransplant (tx).Methods: Flow cytometric crossmatches were performed using donor lymphocytes (FCXM) andendothelial cell precursor cells (ECXM) as targets. ECs were isolated using magnetic beads coated withanti-Tie2 antibodies. Donor specific HLA antibodies (DSA) were detected using solid phaseimmunoassays. Allograft rejection was defined using Banff criteria.Results: A 60 year old male with a cPRA of 55% received a kidney tx via paired kidney donation. Thedonor was ABO incompatible (blood group B to O) but provided a reduced HLA barrier (FCXM+) whencompared to his original donor (CDC+, titer 32). Positive T and B cell FCXMs were consistent with DSAto HLA-A11 and A31.The patient was desensitized using plasmapheresis (PP), low dose IVIg, anti-CD25,and anti-CD20 antibodies. The patient’s anti-B titer fell from 256 to 8 and FCXMs were negative by day oftx. The patient was discharged with a serum creatinine (SCr) of 1.3 mg/dl and an anti-B titer of 4. A biopsy,24 days post-tx, showed 2A cellular rejection with a component of AMR with no rise in DSA or anti-Btiter. Following treatment with a T cell depleting antibody and PP, his SCr returned to baseline. Biopsiesperformed at 3 and 4 months post-tx showed persistent AMR with no significant change in DSA or ABOantibodies; the patient was treated with high dose IVIg. ECXM tests were performed and showed a positiveECXM pre-tx in the presence of HLA-A11 and A31 DSA, a negative post-tx ECXM followingdesensitization, and positive ECXMs at 4 and 7 month post-tx (pre and post high dose IVIg treatment).Conclusions: Non-HLA antibodies were detected concomitant with AMR in a patient following anincompatible tx. These antibodies were reduced following PP and low dose IVIg treatments but weobserved no reduction following treatment with high dose IVIg.8-ORARE HLA Cw DONOR SPECIFIC ANTIBODIES AS CLINICALLY SIGNIFICANT IN AMR INRENAL TRANSPLANTATION AS ARE OTHER HLA ANTIBODIES? A CASE STUDY.Luz Stamm 1 , Mauricio Monroy-Cuadros 2 , Kevin McLaughlin 2 , Noureddine Berka 1 . 1 Tissue Typing,Calgary Laboratory Services, Calgary, AB, Canada; 2 Southern Alberta Transplant Program, AlbertaHealth Services, Calgary, AB, Canada.Aim: A 35 year old female with Goodpasture’s disease was worked up for an unrelated living donorkidney.[table1]The AHG-CDC crossmatch was negative, Flow T cell crossmatch was positive. SingleAntigen (SA) by Luminex showed antibodies with specificity to Cw antigens with the strongest bead (MFI5000) directed to donor specific antibody (DSA) Cw9 (3).[table2]Methods: Patient received 360 mg of Mycophenolate Acid a week prior to the transplant and was inducedwith 75mg of ATG. A triple therapy immunosuppression was started with FK-506, Mychophenolate acidand steroids.Results: Creatinine level came down and reached her baseline of 85 µmol/L after the first week. The titerand specificity of DSA is being monitored closely in this patient and up to date DSA remain the same asinitially tested.Conclusions: Little information can be found in the literature as to the implication and relevance of HLACw donor specific antibodies in the risk of acute antibody mediated rejection in renal transplantation. Inthis unique case, this patient’s antibodies are specific to HLA Cw making it an interesting case for followup. Since the cell surface expression of the C locus antigens has been estimated at 10% in relation to A andB antigen, its relevancy in the importance of rejection is not known. Studies have been focusing on theimportance of the HLA epitopes sites to which the antibodies bind. With the development of SAtechnology, the identification of these epitopes has proven very useful. This case may help to prove theclinical importance in the future analysis of HLA Cw antibodies.

Monday, September 27, 20102:00 PM - 3:30 PMAbstract Session 1: New Technologies and Assays9-ORMICROARRAY-BASED HLA TYPING, ON UNPURIFED DNA SAMPLES FROM BLOOD ANDBUCCAL SWABS.Gina Lopez 1 , Andrew Abalos 1 , Kevin Keisler 1 , Melissa May 2 , Rick Eggers 1 , Kevin Obrien 1 , Paul Dunn 3 ,Krishna Jayaraman 1 , Michael Hogan 1 . 1 Research & Development, Genomics USA, Tucson, AZ, USA;2 Arizona Cancer Center, University of Arizona, Tucson, AZ, USA; 3 HLA Laboratory, New Zealand BloodService, Auckland, Epsom, New Zealand.Aim: Current technologies for high resolution HLA typing were originally developed to support organ andmarrow transplantation in the clinic, where large amounts of genomic DNA are readily available from avenous blood draw, and where the medical procedure is valuable enough to justify lengthy, relativelyexpensive, sample preparation. However, if HLA typing is to be applied to population-scale screening,sample size and the allowable test cost must be reduced, thereby necessitating a search for HLA typingtechnologies that are less expensive and, ideally, performed on unprocessed blood or cheek swabs.Methods: HLA typing of this study was performed on raw venous blood, stored frozen until use, and uponcheek swabs, collected with a cotton swab, stored dry until rehydration and use. The raw samples were thensubmitted to tandem PCR, then microarray analysis on chips specific for HLA A, B & DRB21 loci.Results: We present preliminary validation of a low-cost microarray approach to HLA typing, that isoptimized so that testing can be performed directly on a microliter of raw blood or 2 microliters of a raw,rehydrated cheek swab sample without DNA extraction. These data show that, beginning with raw blood orcheek swabs, HLA types are obtained for HLA-A, HLA-B and HLA-DRB1 that are identical, withinexperimental accuracy, to those obtained with extracted DNA as the sample input.Conclusions: We demonstrate that HLA-typing can be performed in a relatively simple microarray format,with little specialized equipment, other than a slide imager, and with no special knowledge of thecomplexity of HLA allele structure, on DNA samples that are equivalent to 1/50th of a drop of blood or1/50th of a raw cheek swab. We propose that because of its technical simplicity and very modest samplerequirements, this simplified HLA microarray technology may enable routine HLA typing as part ofpopulation screening and clinical research.Lopez: Genomics USA: Employee; Stockholder. Abalos: Genomics USA: Employee; Stockholder. Keisler:Genomics USA: Employee; Stockholder. Eggers: Genomics USA: Employee; Stockholder. Obrien:Genomics USA: Employee; Stockholder. Dunn: Genomics USA: Consultant; Science Med Advisor.Jayaraman: Genomics USA: Employee; Stockholder. Hogan: Genomics USA: Employee; Stockholder.10-ORDOUBLE CORD TRANSPLANT SCREENING AND MONITORING ENABLED BY RUOQUANTITATIVE PCR AND CUSTOM SOFTWARE.Douglas A. Bost, Ian J. McLaughlin, Steve Beckert. Celera, Alameda, CA, USA.Aim: STR-PCR is limited in its utility for screening and monitoring of donors and recipients in double cordtransplants, given its inherent lack of sensitivity and the presence of stutter artifacts which obscurepotentially informative alleles. If markers are identified for each individual in a double cord transplant, themanual execution of the algorithm for quantification of individuals is quite time-consuming. To simplythese analyses, we designed a system of RUO assays and software enabling instantaneous markeridentification and quantification post-PCR.Methods: We employ a panel of 31 quantitative PCR assays to bi-allelic indels in which the probability offinding at least one informative marker is >99.9% in unrelated individuals (Caucasian, African, Japanese,Amerindian populations). The probability of finding at least one informative marker in siblings is > 99%,98%, 98% and 97% for European Caucasian, Japanese, Amerindian and African populations, respectively.Results: Each assay was tested using simulated DNA mixtures at 0.05%, 0.1%, 0.2% and 0.4% minorcomponent for 250 ng of input genomic DNA. Each assay detected the 0.05% minor component mixture.The limitation at this low sensitivity is input copy number. In addition, each of the assays in the panel was

able to detect, with statistical significance (p20>100 ng/ml) & high(>125 ng/ml) levels of the molecule. The sHLA class Ilevels were not different & most of them didn’t fluctuate Post-Tx. Carriers of the HLA-G II genotype hadlow levels of sHLA-G(

Results: PHA-induced mRNA expression was compared before and after CI treatment in pre-transplantpatients. PHA-induced interleukin-2 (IL2) mRNA expression was significantly (p

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

conjugated IgM (BD Biosciences), IgA1, or IgA2 (both Southern Biotech). The microparticles wereacquired on a FACS Canto II and classified for their corresponding antigen. Correction for non-specificbinding was analogous to the HLA Fusion (One Lambda) formula. The positive thresholds for themicroparticles were determined at 5 times the average standard deviation of no fewer than 2 negativecontrol sera for each isotype.Results: Using a very conservative threshold, the prevalence of HLA class I antibodies was 7%, 22%, and46% of sensitized patients for IgA1, IgA2, and IgM, respectively. The prevalence of Class II was 36%,11%, 21% for IgA1, IgA2 and IgM respectively.Conclusions: Using this method, it is possible to measure HLA specific non-IgG antibodies by usingmultiple fluorescent parameters to interrogate microparticles. By characterizing multiple antibody isotypessimultaneously we can increase access to data on non-IgG antibodies and better understand their impact onsolid organ transplantation.Monday, September 27, 20102:00 PM - 3:30 PMAbstract Session 2: Innate Immunity and Regulation17-ORHUMAN SEMAPHORIN 5A ACTS AS POTENT ACTIVATOR OF NK CELLS.Christiane Gras, Yarua Jaimes, Stephan Immenschuh, Rainer Blasczyk, Constanca Figueiredo. Institute forTransfusion Medicine, Hannover Medical School, Hannover, Lower Saxony, Germany.Aim: Semaphorins are a wide family of signaling molecules. Semaphorin 5A (Sema5A) and Sema7A areknown to regulate axon guidance. Sema7A has been shown to modulate the T and NK cell-mediatedimmune response. The role of Sema5A in the immune system is unknown. Here, we investigated theinfluence of Sema5A on NK cell function.Methods: The extracellular domain of Sema5A was expressed as soluble His-tagged protein in HEK cellsand purified by immobilized-metal affinity chromatography. NK cells were isolated from peripheral wholeblood of healthy donors and stimulated with Sema5A at a concentration of 10mg/ml during 48h. Sema5Astimulated and non-stimulated NK cells were analysed for phenotype changes in the surface expression ofKIR2DL1, KIR2DL2, NKp30, NKp44, NKp46, NKG2A, NKG2D, CD25 and CD69 receptors using flowcytometry. The cytotoxic potential of NK cells was analysed by measuring granzyme B production by realtime PCR and killing rates of K562 target cells by FACS. For all assays heat-denatured Sema5A was usedas negative control.Results: Sema5A stimulation induced an increase of the frequency of NKp44+ NK cells by up to 94%. Inaddition, the NKG2A+ and NKG2D+ NK cell population increased by up to 44% and 7% respectively. Thefrequency of the early activation markers CD25 and CD69 increased by up to 2.4 fold and 4.3 fold,respectively. Interestingly, the upregulation of the NK cell receptors by Sema5A was decreased or evenabolished in presence of IL-2 and IL-15. This effect can be explained by the downregulation of theSema5A-receptor Plexin-B3 by up to 95% observed upon IL2 and IL15 stimulation. Furthermore, uponstimulation with Sema5A, granzyme B mRNA levels were increased by up to 70% and the cytotoxicactivity of NK cells was increased by up to 20%.Conclusions: These results show for the first time that Sema5A is a potent activator of NK cells in vitroand demonstrate that it may play an important role in immune cell regulation.18-ORG-CSF: A STRONG INHIBITOR OF NK CELL FUNCTION.Laura Schlahsa, Yarua Jaimes, Rainer Blasczyk, Constanca Figueiredo. Institute for Transfusion, HannoverMedical School, Hannover, Lower Saxony, Germany.Aim: The human cytokine granulocyte-colony stimulatory factor (G-CSF) has found widespreadapplication in the medical treatment of neutropenia and to mobilise haematopoietic stem cells (HSC) usedfor transplantation purposes. So far, the effect of G-CSF on Natural Killer (NK) cells, which play a pivotalrole in haploidentical HSC transplantation, has not been fully investigated.

Methods: For this purpose, we accessed the effect of G-CSF on the phenotype, cytokine secretion profileand cytotoxicity of NK cells incubated in vitro for 48h in presence of G-CSF as well as of NK cells isolatedfrom peripheral blood of G-CSF mobilised stem cell donors (in vivo).Results: In vitro, G-CSF caused a strongly altered phenotype in NK cells with 49% downregulation ofNKp44 frequency. Furthermore, the expression of the activating receptors NKp46 and NKG2D decreased40% and 64%, respectively. The expression of two inhibitory receptors, KIR2DL1 and KIR2DL2,decreased by 46% each. In cytotoxicity assays, the lytic capacity of G-CSF exposed NK cells againstallogeneic target cells is reduced by up to 68% in vitro and up to 83% in vivo. Accordingly, the levels ofgranzyme B of in vivo G-CSF-exposed NK cells were reduced by up to 87% in comparison to nonstimulatedNK cells. Cytokine production of in vitro and in vivo incubated NK cells was strongly decreasedfor IFN-gamma, TNF-alpha, GM-CSF as well as IL-6 and IL-8. Furthermore, we observed a reduction inproliferation and a positive feedback loop that increased the expression of the G-CSF receptor (G-CSFR3).Conclusions: In conclusion, G-CSF demonstrated to be a strong inhibitor of NK cells activity and mayprevent GVL effect post-transplantation.19-ORPHENOTYPING STUDY OF KIR EXPRESSION ON NK AND NKT CELLS.Mei Han 1 , Lin Fan 2 , Lama Hussein 2 , Peter Stastny 1,2 . 1 Internal Medicine - Transplant Immunology, UTSouthwestern Medical Center, Dallas, TX, USA; 2 Pathology, UT Southwestern Medical Center, Dallas, TX,USA.Aim: The function of NK cells is regulated in large part by the interaction of inhibitory receptors and theirHLA ligands. Expression of KIR on the cell surface is clonally regulated and the frequency of NK cellsexpressing single or multiple KIR may vary from person to person. Using multicolor flow cytometry it ispossible to analyze the expression of KIR and NKG2A on the surface of single cells and to obtain robustphenotypic data.Methods: We have analyzed NK cells by flow cytometry from 19 Caucasoids and 18 Asians and stainedthem for CD3, CD56, KIR2DL1, KIR2DL2/L3, KIR3DL1, and NKG2A. The KIR expression pattern wasanalyzed on gated CD3 - CD56 + NK cells as well as CD3 + CD56 + NKT cells using a 6-color flow cytometer.Each person was also genotyped for KIR and HLA class I by PCR-SSP.Results: Widely different frequencies were observed. One KIR could be found in 8% of NK cells, anotherKIR in 90% of NK cells in the same person. Differences of this magnitude were also observed betweenindividuals. NK cells expressing KIR2DL1 ranged from 8% to 46%, KIR2DL2/L3 from 21% to 91% andKIR3DL1 from 6% to 77% of NK cells. The expression of KIR2DL2/L3 was more frequent in Asians thanin Caucasoids (p

Methods: Peripheral blood and endomyocardial biopsy specimens were tested by real-time PCR andimmunohistochemistry (IHC) stains for identification of TLR-2, -4, and AIF-1 markers and analyzedagainst clinical ISHLT rejection grades. The group differences for mRNA transcript levels between therejection grades were determined by one-way ANOVA.Results: The mRNA expression levels were significantly varied for TLR-2 in monocytes from the groupswith different ISHLT grades (p1x10 6 /kg on day +5 and at 3, 6 & 9 months. Tacrolimus (TAC) and mycophenolate (MMF)

immunosuppression (IS) for 3 months is converted to sirolimus (SRL) and MMF followed by ISwithdrawal, first MMF and then SRL at 12-30 months post-op. Serial PBMC flow immunophenotyping(IP) and sequential Treg-MLR inhibition/recruitment assays are performed. In the Treg-MLR,cryopreserved recipient pre-RT PBMC (CFSE labeled) respond to irradiated donor or third partystimulating PBMC in the presence of (cryopreserved) pre- vs post- RT recipient PBMC as modulators.Then, lymphoproliferation and newly generated (recruited) CD4 + CD25 high FOXP3 + responding cells aremeasured by flow.Results: The first 4 patients are now between 18 and 30 months post-op with IS (SRL monotherapy)dosing ranging from none to 1 mg QOD with no rejection (clean 1 & 2 year biopsies and no cell growth inbiopsy cultures). Immunophenotyping of PBMC indicated that the (CD127 - ) CD4 + CD25 high FOXP3 +(natural) Tregs increased >3X in recipient PBMC up to 30 months post-RT. In the Treg-MLR, recipientPBMC from 1 year post-RT inhibited anti-donor cell proliferation and increased the proportion(recruitment) of newly developed donor-specific recipient responder CD4 + CD25 high FOXp3 + cells by 10-fold (n=4). Chimerism transiently reached 3% in some recipients at 3 to 6 months (real-time quantitativePCR assay).Conclusions: DHSC in AL treated HLA-identical RT recipients converted from TAC & MMF to SRL andthen IS withdrawn, is associated with high PBMC Tregs, and in the Treg-MLR with donor-specificinhibition and Treg recruitment.23-ORKIR3DS1-2DS1-2DS5, RECEPTORS IMPLICATED IN NK CELL ACTIVATION PROVIDE ARISK OF DEVELOPING ADVANCED STAGE BREAST CANCER.Elham Ashouri 1 , Abbas Ghaderi 1 , Raja Rajalingam 2 . 1 Shiraz Institute for Cancer Research, ShirazUniversity of Medical Sciences, Shiraz, Islamic Republic of Iran; 2 UCLA Immunogenetics Center,Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Universityof California at Los Angeles, Los Angeles, CA, USA.Aim: Polymorphic KIRs are the key receptors of human NK cells that trigger early immune responseagainst infection and tumors. Here we investigated if certain KIR genes are associated with breast cancer.Methods: DNA from 167 women with breast cancer diagnosed at the multidisciplinary breast cancer unitof Shiraz Institute for Cancer Research, Iran, and 278 healthy controls from the same geographical areawere typed for 16 KIR genes using a duplex-PCR typing system.Results: The frequency of Bx genotypes that possess 2-6 activating KIR genes were predominant in thepatients compared to the controls (84.4% vs. 72.6%, P = 0.005; 95% CI, 1.24-3.34, OR = 2.04). ActivatingKIR genes, 3DS1 (46.7% vs. 34.1%, P = 0.009; 95% CI, 1.14-2.5, OR = 1.68), 2DS1 (55.6% vs. 36.3%, P= 0.00007; 95% CI, 1.48-3.25, OR = 2.2) and 2DS5 (42.5% vs. 25.5%, P = 0.0002; 95% CI, 1.43-3.24, OR= 2.15) were significantly increased in breast cancer compared to the controls. Particularly, 36.4% of thepatients carried all these three activating KIR genes compared to only 21.4% controls (P = 0.00067; 95%CI, 1.37-3.2, OR = 2.09). Notably, 41.8% of the patients with advanced stage III cancer carried all thesethree KIRs.Conclusions: These findings contrast the classical view that activating NK cell receptors mediatespontaneous lysis of tumor transformed cells. NK cells expressing 3DS1-2DS1-2DS5 may trigger alocalized hyperresponsiveness exacerbating cancer growth. Consistent with this findings, a detrimental rolefor NK cells against cancer and non-viral pathogens has been suggested from the following studies:increased 3DS1 in patients with cervical neoplasia (J Exp Med 2005, 201:1069), depletion of NK cellsincreased the ability of the host to control L.Monocytogenes (J Immunol 1994, 152:1873); host NK cells arenecessary for the growth of human filarial parasite B.Malayi (J Immunol 1998, 161:1428).24-OREFFECT OF KILLER CELL IMMUNOGLOBULIN LIKE RECEPTOR (KIR) AND HUMANLEUKOCYTE ANTIGEN (HLA) LIGAND INCOMPATIBILITY ON HUMAN RENALTRANSPLANTATION: ASSOCIATION WITH ARTERIAL THICKENING AND TUBULITIS.Faisal M. Khan 1,2 , Jagdeep Doulla 2 , Meena Assad 2 , Aylin Sar 2 , Ipek I. Gonul 2 , Serdar Yilmaz 2 , NoureddineBerka 1,2 . 1 Calgary Laboratory Services, Calgary, AB, Canada; 2 University of Calgary, Calgary, AB,Canada.

Aim: NK cells have an established role in defense against tumors, viral infection and outcomes of HCT buttheir importance in solid organ transplantation is restricted to the reports showing increased numbers ofcirculating and graft interstitium infiltrating NK cells during acute rejection of renal allograft. Theactivation and inhibition of NK cells depend on the interaction of killer immunoglobulin like receptors(KIRs) with HLA class I molecules expressed on target cells. Here we assessed the impact of recipient KIRand donor HLA incompatibility on occurrence of acute kidney allograft rejection and histological scoreindicative of allograft failure.Methods: A total of 135 kidney transplant recipients were genotyped for 16 KIR genes by a multiplex KIRgenotyping assay using Genprobe KIR genotyping kit. Incidence of acute rejection and eleven individualhistological parameters scored in 6-12-months post-transplant surveillance biopsies were utilized as clinicalend points in the analysis.Results: Recipient KIR – donor HLA incompatibility was found associated with high scores for arterialthickening and tubulitis, but not with acute rejection. Recipient KIR2DL3 pos and donor HLAC1 negincompatibility was associated with high scores for arterial thickening (p=0.026) and recipient KIR3DL2 posand donor HLA-A3/A11 neg incompatibility was correlated with high scores for tubulitis (p=0.04).Conclusions: The allogeneic interaction between recipient KIR and donor HLA antigens may constitute arisk of kidney allograft failure. NK cell mediated response may represent another potent mechanism ofallo-immunity in solid organ transplantation though not as critical as T cell mediated allo-immunity.Monday, September 27, 20105:30 PM - 7:00 PMPoster Session1-PCONSIDERATIONS IN INTERPRETING SOLID PHASE ANTIBODY DATA.Jessica L. Badders, Julie A. Houp, Jeffrey T. Sholander, Mary S. Leffell, Andrea A. Zachary. Medicine,Johns Hopkins University, Baltimore, MD, USA.Aim: Solid phase immunoassays provide rapid detection of HLA-specific antibodies with unprecedentedsensitivity and specificity. However, interference in the assays may be caused by extrinsic factors such astherapeutic agents and intrinsic factors such as non-specific binding of IgM and immune complexes. Theaim of this study was to evaluate factors affecting the results of solid phase immunoassays.Methods: Sera, untreated and treated to eliminate IgM, were tested by solid phase assay on the Luminexplatform. Results were analyzed by two or more experienced individuals.Results: Examples of the effect of these are shown in the table.[table1]Reactivity of the controls and testserum can be affected appreciably by the treatment used to eliminate intrinsic factors and we have reportedsignificant differences in the effects of DTT treatment and hypotonic dialysis. Daily variation in reactivityof pooled antigen assays can vary up to 30% and we have observed that sensitivity between lots of the sameproduct can vary two-fold.Conclusions: Accurate and meaningful interpretation of these assays requires recognition of these issuesand their resolution whenever possible. We will present examples of various causes of test interference andpossible solutions.2-PHLA EXPRESSION VARIES ON SUBSETS OF CD3+ AND CD19+ CELLS AND CANINFLUENCE FLOW CYTOMETRY CROSSMATCHS.Aaron T. Whiteley, Mary M. Garrison, Lee Ann Baxter-Lowe. Surgery, University of California, SanFrancisco, San Francisco, CA, USA.Aim: The flow cytometery crossmatch (FCXM) evaluates the quantity of donor specific antibodies (DSA)that bind to CD3+ T cells and/or CD19+ B cells. The resulting histograms representing bound human IgGare at times bi-nodal or irregular indicating cells with different FCXM median channel shifts (MCS). Thisstudy examines HLA expression level on different cell subsets and how expression level affects the MCS.Methods: FCXM were performed according to previously described methods. CD3+ cells were classifiedusing CD4, CD8, CD25, and CD56. CD19+ cells were classified using CD27, CD25, CD38, CD22,

CD138, HLA-DR, IgM, and IgD. The contribution of Fc receptors was assessed by CD23, CD32, andCD16. Cellular activation was analyzed by CD69. HLA expression level was quantified by monoclonalantibodies to monomorphic epitopes on either class I or II (G46-2.6 or Tü39).Results: On CD3+ cell subsets CD4+, CD4+/CD25+, CD8+CD56-, CD8+CD56+ HLA class I expressionlevel correlated linearly to MCS in serum with class I DSA. In serum with class II DSA, CD19+ cellsshowed a high degree of correlation between HLA-DR expression and MCS. For CD3+ cell subsets, theCD69+ activated form of each subset had elevated HLA expression. While HLA class I and II expressionlevel varied on CD19+ cells, no one marker correlated with higher or lower HLA expression. However onCD19+ cells the level of Fc receptors (FcR) CD32 and CD23 correlated with bound IgG in sera withoutDSA. CD56+/CD16+ cells also showed a high degree of bound antibody in serum without DSA thatcorrelated with CD16 expression level.Conclusions: Different subsets of T and B cells have variable levels of HLA expression, which affect theMCS in and FCXM. This suggests that variation in relative proportions of these subsets could influenceFCXM results. Similarly, variation in FcR expression influences nonspecific antibody binding in a densitydependent manner.3-PPREDICTIVE VALUE OF USING LabScreen MIXED BEADS TO DETERMINE DILUTIONSNEEDED FOR QUANTITATIVE ANALYSIS ON LabScreen SINGLE ANTIGEN BEADS.P. Brailey, E. Portwood, A.L. Girnita. Transplantation Immunology Division, University of Cincinnati,Cincinnati, OH, USA.Aim: When analyzing solid organ transplant patients for HLA antibodies, the LabScreen Single AntigenBeads can be saturated with antibody. There are numerous consequences of bead saturation, with theinability to observe patient treatment response being one of the most significant. This inability requires theuse of dilution studies which are time and reagent intensive, and expensive. The aim of this study was todetermine if LabScreen Mixed Bead reagents could be used to determine the dilutions needed for SingleAntigen Bead analysis.Methods: LabScreen Mixed Beads (One Lambda, Inc.) were run against 4 renal patients at serial dilutionsfrom Neat to 1:1024 for both HLA Class I and Class II. The serum samples were first treated with DTT anddiluted using phosphate buffered saline (PBS). MFI values for either an immuno-dominant or donorspecific antibody were plotted on a graph. The graph was analyzed for the dilution values that were at thelower end of the linear portion of the graph. These values were used for dilutions of the LabScreen SingleAntigen Beads (One Lambda, Inc.) for that patient.Results: Mixed Bead dilution studies predicted that 3 of the 4 patients were diluted at 1:256 or 1:1024.LabScreen Single Antigen Bead studies at this dilution were successful for monitoring the patient’s therapyresponse. Mixed Bead dilution studies predicated for 1 patient successful dilution at 1:64, however thispatient actually required LabScreen Single Antigen Bead dilution at 1:2048.Conclusions: LabScreen Mixed Beads can be used to predict the dilutions needed for quantitative analysison LabScreen Single Antigen Beads for many patients. Care must be taken to observe that dilution studiesaccount for two linear curves on patients with extremely high levels of HLA antibodies.4-PUSE OF QUANTIPLEX BETWEEN INSTRUMENTS: WORTH THE BOTHER?Megan Brown, Anne Halpin, Luis Hidalgo, Patricia Campbell. Histocompatibility Laboratory, AlbertaHealth Services, Edmonton, AB, Canada.Aim: Many HLA laboratories use Luminex methodologies to identify HLA antibodies. QuantiplexBeads (One Lambda Inc) are reference beads for standardization of fluorescent signal. They are acquiredwith single antigen beads (LabScreen® products, One Lambda Inc) used to identify antibodies to HLAclass I (LSA1 beads). Quantiplex beads are intended to correct differences in instrument performance.Luminex output is measured as median fluorescence intensity (MFI); Quantiplex beads convert MFI tostandard fluorescence intensity (SFI) by creating a linear standard curve from Quantiplex bead values.We investigated whether Quantiplex beads provide standardization for the same samples run on twoLuminex instruments.

Methods: Sera (n=22) were tested for Class I antibodies using LSA1 beads (cat# LS1A04) as per themanufacturer’s product insert; the vacuum wash method was employed. The beads were acquired on twoLuminex instruments in 2 runs on 2 different days. Quantiplex beads (cat# LXQNTPLX) were acquiredwith each run. Raw data for all positive results (n=14) were exported to Microsoft Excel and analyzed.Spearman correlations were calculated using GraphPad Prism 5.Results: MFI and SFI have a correlation of 1.0 on the same instrument. The MFI to MFI and SFI to SFIcorrelations between instruments are 0.98. When the MFI vs SFI values are plotted on a line graph, thelines are almost identical, although the SFI values are higher. Slight differences in MFI and SFI output areobserved between instruments. One run shows slightly higher output on one instrument vs another and theopposite is seen in the second run. No observed instrument variation is corrected by SFI.Conclusions: Quantiplex TM beads are inexpensive but create additional work such as inventory, QC andadditional Fusion® import. Due to our inability to see a clear benefit with the use of Quantiplex beads,we conclude that they not worth the bother and we will discontinue their use.5-P“NATURAL” HLA ANTIBODIES IN THE PATIENTS ON UNOS KIDNEY OR PANCREASWAIT LIST.Heather A. Casey, Lorie H. Kumer, Carrie L. Mowery, Lori A. Malec, Margaret A. Maybach, Jennifer L.Tyler, Dorothy K. Felt, Jean A. Hess, Justine L. Gaspari, Ronald E. Domen, Hiroko Shike. HLALaboratory, Penn State Hershey Medical Center, Hershey, PA, USA.Aim: “Natural” HLA antibodies in nonalloimmunized healthy males was published by Morales-Buenrostro, et al (2008), referring to naturally occurring non-HLA IgG antibody cross-reactive possibly tostructurally altered HLA antigens on the Luminex beads. The reactivity is considered clinicallyinsignificant and should not be assigned as HLA antibody specificity to patients. Recognition criteria areneeded.Methods: One Lambda LABScreen PRA Class I and II and LABScreen Single Antigen (SAB) were testedby Luminex for patients on wait lists in last 3 years. Specificity assigned by SAB (MFI ≥1000 MFI) wastested by flow crossmatch (XM) of selected serum against donors with only one corresponding HLAantigen.Results: SAB, PRA, and XM results of 52 samples (n=38 for class I; n=14 for class II) were correlated togenerate criteria that may identify non-HLA SAB reactivity. The specificity was supported by clustering(pattern observation on HLA Fusion software) of PRA beads in 39 samples. Of the 39 samples, XM waspositive in 20/22 samples with corresponding PRA beads ≥1000 MFI, and 10/17 samples withcorresponding PRA beads

Methods: HLA antibody specificities were determined by Luminex SAB. A calculated panel antibodyreactivity (cPRA) was obtained for antibodies detected at MFI≥1000, 2000, 3000, and 4000 by using theUNOS cPRA calculator. Fifty sensitized patients who were tested for antibody specificity in the past yearwere calculated for cPRA and only patients (n=32) with HLA class I cPRA≥30% at MFI≥1000 wereincluded in the final study.Results: The average cPRA was 77% at MFI≥1000, 68% at MFI≥2000, 62% at MFI≥3000, and 54% atMFI≥4000. The average probability of having a donor selected for a FCXM would increase from 23% atMFI≥1000 to 46% at MFI≥4000, which would double the size of the donor pool for sensitized patients. Forthe highly sensitized patients (cPRA≥80%, n=18), the average probability would increase from 5% atMFI≥1000 to 21% at MFI≥4000 resulting in an increase of >4X donor pool for highly sensitized patients.Conclusions: The increase cutoff from MFI≥1000 to ≥4000 may at least double the donor pool forsensitized patients. A final FCXM may be used for the definitive decision of transplant. Aggressive posttransplantmanagement may be considered for recipients of kidney allografts with unacceptable antigen(s)detected at a lower cutoff. The increase of the positive FCXM rate caused by increasing the MFI cutoff canbe investigated retrospectively.7-PHEART TRANSPLANTATION OF SENSITIZED PATIENTS.Dong-Feng Chen 1 , Nancy L. Reinsmoen 2 , Adella Clark 1 , Barbara O. Burgess 1 , Gregory F. Egnaczyk 1 ,Chetan B. Patel 1 , Carmelo Milano 1 , Joseph G. Rogers 1 . 1 Duke University Health System, Durham, NC,USA; 2 Cedars-Sinai Health Systems, Los Angeles, CA, USA.Aim: For sensitized heart transplant recipient, our center uses the virtual crossmatch (vXM) to selectcompatible donors. The unacceptable HLA antigens (UAG) were determined based on the specificity andtiters of HLA antibodies (Ab) detected by solid phase technologies. The aim of the study was to evaluatethe accuracy of vXM and its impact on the success of heart transplantation in sensitized recipients.Methods: Total 222 patients received heart transplants at our center between 2005 and 2009 were included.Sensitization was defined by presence of positive flow PRA and defined specificity of HLA Abs. Usingthese criteria, 62 (28%) were pre-transplant sensitized. For sensitized recipients needing emergenttransplant, titration test was performed and anti-HLA Abs not detected at a dilution of 1:16 were considered“low titer” and were not considered in donor selection. Utilizing the principle of vXM, only donors withoutUAGs were used for non-emergent sensitized patients.Results: Among the 62 sensitized patients, 16 patients received organs with UAGs as determined by Absdetected in undiluted serum samples. 10 of these 16 patients had positive HLA XM by flow cytometry. Ofthese 10 positive XMs, 8 were expected due to the presence of weak Abs detected in evaluation seracollected prior to time of listing. In addition, there were 3 positive XMs which most likely were caused bynon-HLA Abs because no DSA were determined. Among the 62 sensitized recipients, 52 had an at leastone-year follow-up. The one-year survival rate for these 52 sensitized recipients was 94%, and for the 16recipients who received organs with UAGs was 93%.Conclusions: Based on these data, we conclude that the vXM used for heart donor selection in sensitizedpatients had a 95% (5 with unexpected positive XM among 62 sensitized) predictive validity. The one-yearoverall survival rate for patients received organs with low-titer UAGs was comparable to those receivedorgan without UAGs.8-PVARIABLE EXPRESSION OF C LOCUS ANTIGENS IN RELATIONSHIP TO FLOWCROSSMATCHING AND SAB TESTING.Steven R. De Goey, Deborah K. Falbo, Nancy A. Ploeger, Gandhi J. Manish. Laboratory Medicine andPathology, Mayo Clinic, Rochester, MN, USA.Aim: Identifying donor specific anibody (DSA) to HLA, using single antigen beads (SAB), is used inperforming a virtual crossmatch (VXM) as a surrogate for an actual crossmatch (XM). DSA strength isdefined by bead fluorescence (MFI) and usually a single value is used as a cutoff. However, this does nottake into consideration the variable expression of HLA antigens on lymphocytes. As HLA-C has a variable

expression on lymphocytes, we hypothesize that HLA-C DSA with a high MFI value can result in anegative or weakly positive Flow Cytometric XM (FXM).Methods: Seven donor-recipient pairs were identified with HLA-C DSA. Correlation between the meanchannel shift (MCS) value of the FXM assay (cutoff: T-cell=52) and HLA-C DSA MFI values wereanalyzed. HLA typing was performed by low to medium resolution SSO/SSP.Results: In our laboratory, DSA MFI>4000 strongly correlates with a positive FXM and DSA MFI values4000 as a cutoff, 6/7 cases had a positive VXM. However, 3 of those 6 cases(50%) were clearly FXM negative and the remaining 3 cases (50%) resulted in an actual (although weak inour hands) positive FXM. One case S.J., had a DSA MFI of less than 4000 however, the FXM MCS wasonly 75.[table1]Conclusions: SAB MFI does not account for the variable expression of different HLA antigens onlymphocytes. However; because of what appears to be a variable expression of C locus antigens onlymphocytes, additional caution must be exercised when predicting a FXM outcome, when utilizing asingle cutoff for VXM.9-PANTIBODY ANALYSIS IN A PATIENT ON THE RENAL TRANSPLANT WAITING LISTUNDERGOING DESENSITIZATION TREATMENT.S. Dilioglou, G. Land. Pathology, Methodist Hospital, Houston, TX, USA.Aim: Desensitization protocols are commonly used in solid organ transplant candidates in order to decreasedonor specific antibodies. We evaluated the antibody status in a 41 year old female patient with ESRD dueto pregnancy-induced hypertension undergoing desensitization treatment as a means of receiving apotential cadaveric kidney.Methods: LABScreen® Single Antigen class I and II, One Lambda Inc. were used for antibody testing.The patient received one-1000 mg dosage IV infusion of Rituxan®, Genentech Inc., and three cycles of 1.3mg/m 2 per dose of Velcade®, Millennium Inc.Results: Table 1 shows the patient’s antibody profile before treatment. Following desensitization, therewas a significant decrease of class I antibodies (p 3000 and MFI > 2000 for first-time andre-grafts respectively. FXMs were performed at Gift of Life Michigan and results were reported according

to Mean Channel shift (MCS) of fluorescence intensity. Cut-off value used for positive FXM was 60 and75 for T and B cells respectively. We examined 262 crossmatches that were performed during the period ofNovember-March 2010.Results: A summary of FXM results and DSA is shown in Table 1. Of the 262 FXM, 8.8% were positivewith MCS>100 and had anti-HLA Abs; 35% of these appear to be due to anti-DP or DRB3 allele-specificantibodies and the remaining were due to weak DSA (MFI: 1000-3000). 44% (18/41) of positive FXM withMCS>100 were not associated with DSA (MFI cut-off value500), FCXMs were performed when donor cells were available (n=35). Outcomes werecompared overall and between FCXM- vs unsensitized groups.Results: Table 1[table1]Conclusions: When DSA is present, patients are more likely to experience AMR than unsensitizedpatients. Collectively, the DSA+/FCXM+ patients have more AMR and lower CrCL than DSA+/FCXMpatients.This latter group still has significant AMR compared to unsensitized patients. Thus,DSA+/FCXM- recipients represent a higher risk transplant than DSA- recipients. Nonetheless, ∼50% ofDSA+ recipients (whether FCXM + or -) were rejection free, meaning that certain donor:recipientincompatibilities are acceptable. Further studies should address which elements dictate good outcome.12-PA NOVEL HLA CLASS I SINGLE ANTIGEN BEAD PREPARATION ELIMINATES FALSEPOSITIVE REACTIONS ATTRIBUTED TO NATURAL ANTIBODIES – IN THE SERA OFNORMAL MALES AND PRE-TRANSPLANT PATIENTS.Nadim R. El-Awar 1 , Paul I. Terasaki 2 , Ali Hajeer 3 , Afzal Nikaein 4 , Matthew Averly 1 , Judy Hopfield 1 , AnhNguyen 1 . 1 Research II, One Lambda Inc., Canoga Park, CA, USA; 2 Terasaki Foundation Laboratory, LosAngeles, CA, USA; 3 King Fahad National Guard Hospital, Riyadh, Saudi Arabia; 4 Texas MedicalSpecialty, Inc., Dallas, TX, USA.Aim: Single antigen (SA) beads have been shown to produce “false” irrelevant reactions in the sera of nonimmunizednormal males. We tested a new bead preparation produced to eliminate these reactions.Methods: 30 normal sera and 46 sera from pre-transplant (pre-Tx) patients were tested with SA beads, EBtreated beads and Clean SA Beads (patent pending) - free of denatured class I antigens (One Lambda Inc.).All MFI values >1000 of the adjusted and normalized trimmed mean were considered positive.Results: Examples of HLA class I specificities of normal male (A) and pre-Tx (B) sera are shown.Reactions of the Clean Beads become negative as compared to the reactions with SA beads and reactionswith the EB treated beads are positive indicating the antibodies target cryptic epitopes. For example.,B0702 (A) has 5,345 MFI with SA beads, 352 MFI with the clean beads, and 7,625 MFI with the EB beads.2/13 (15%), 20/24 (83%), and 5/6 (83%) of the normal sera specificities for the A, B, and C locusrespectively are negative with the clean beads.[figure1]

Conclusions: Clean SA beads can eliminate “extra” reactions attributed to natural anti-HLA antibodies intransplant sera. Positive reactions with the SA beads become negative with the clean beads. Reactions ofnatural antibodies against exposed epitopes are not eliminated. Since their specificities are rare, have lowMFI, and share distinct epitopes, most can be identified. Clean beads have no effect on the reactivity ofalloantibodies and can potentially simplify sera analysis.El-Awar: One Lambda Inc.: Employee; Stockholder. Terasaki: One Lambda Inc.: Stockholder. Averly: OneLambda Inc.: Employee. Hopfield: One Lambda Inc.: Employee. Nguyen: One Lambda Inc.: Employee.13-PIMMUNOGENICITY OF HLA ANTIGENS.Hooi S. Eng, Inessa Kaplan, Mary S. Leffell, Andrea A. Zachary. Medicine, Johns Hopkins University,Baltimore, MD, USA.Aim: Solid phase immunoassays permit prediction of crossmatches (XMs) at different sensitivity levels.Knowledge about differing immunogenicity of HLA antigens could aid donor selection for transplantation.We examined antibodies predicted to yield positive XMs in 54 sensitized patients to those expected basedon equal antigen immunogenicity.Methods: Expected Ab frequencies were determined by: freq. of Ag in the donor population X (1- freq. ofAg in the patient population). Donor Ag frequencies were from the UNOS data base and recipient Ag freq.were calculated from the patient phenotypes.Results: Ten patients had antibodies to class I only (19%); 10 patients to class II only (19%) and 34patients had antibodies to both cI & cII (62%). Antibodies to DR were the most common (41/54, 76%),followed by B (37/54, 69%), A (35/54, 65%) and DQ (31/54, 57%). Anti-C antibodies were found in 2patients only and were excluded from further analysis. Of specificities observed, differences >10% betweenobserved and expected frequencies occurred in 9/11 (82%) A, 20/28 (71%) B, 5/10 (50%) DRB1, 1/3(33%) DRB345 and 2/4 (50%) DQ antigens. A10 (56%), A19 (56%), B15 (75%) and DR9 (36%) had thegreatest frequency differences. The specificities with 70% incidence) present at only the flowcytometric XM level.Conclusions: While differences in frequency may be due to immunogenicity, differences in antibodystrength may reflect repeated exposure to the more common antigens.14-PASSOCIATION BETWEEN DONOR SPECIFIC ANTIBODY (DSA) DETECTED BY SOLIDPHASE ASSAY (SpA) AND FLOW CYTOMETRIC CROSSMATCH (FXM).Manish J. Gandhi, Sandra Bryant, Rebekah M. Knauer, Lisa M. Hallaway, Steven R. DeGoey. MayoClinic, Rochester, MN, USA.Aim: Recipient DSA by SpA is used to screen donors as a surrogate for crossmatch as it is thought thatthere is good correlation between them. Association between 436 FXM and DSA from 273 unique pairswas studied.Methods: SpA was performed by single antigen beads. Correlation between FXM mean channelshift(MCS) and DSA MFI were analyzed using MCS as the dependent variable and MFI as the independentvariable for 3 scenarios: 1.highest DSA(hDSA) 2.sum of DSA 3.each HLA DSA separately Generalizedestimating equations were used to assess these associations to properly adjust for the correlation structurewithin each pair, using an AR1 correlation structure within pairs. Models with the highest quasi-likelihoodunder the independence criterion(QIC) will provide the best-fitting models for this data. p-values

15-PSOLID PHASE ANTIBODY (SpA) IDENTIFICATION: EFFECT OF INTERFERINGSUBSTANCES OR PROZONE PHENOMENON.Manish J. Gandhi, Nancy A. Ploeger, Deborah K. Falbo, Steven R. DeGoey. Division of TransfusionMedicine, Mayo Clinic, Rochester, MN, USA.Aim: HLA antibodies (HLA-Abs), especially donor specific antibodies (DSA) identified by SpA are usedpre-transplant (Tx) to screen donors and for post Tx monitoring. However, there are questions regarding itstechnical limitation.Methods: Results from 11 recipients with low to high levels of HLA-Abs were compared after performingSpA by: 1.manufacturer protocol 2.after treatment at 56C for 30 minutes (inactivate complement) 3.aftertreatment at 63C for 13 minutes (inactivate IgM) 4.after 1:8 dilution.Results: Heat treatment resulted in increased mean flouresence intensity (MFI) of the negative control beadas shown below:[table1]Heat treatment resulted in increased number of allele specificities with MFI >500:untreated=626, 56C=702, 63C=661, diluted=633. However, dilution demonstrated higher number of allelespecificities with MFI >6000 as shown below:[table2]Significantly, for BP and WK, untreated DSA MFIwas 50,000 and correlated with B-flow crossmatchchannel shift >500.Conclusions: HLA-Abs detection by SpA can be effected by the steric interference from complement orIgM. Heat treating the sample helps however, there is increased background. Some cases with high level ofantibodies also demonstrate a prozone phenomenon as demonstrated by significantly higher number ofalleles with MFI >6000.16-PGOOD ANTIBODY OR BAD ANTIBODY?Ralph Graff, Huiling Xiao, Mark Schnitzler, Janet Tuttle-Newhall, Krista Lentine. Saint Louis University,St. Louis, MO, USA.Aim: We examined national registry data to characterize the associations of complement-dependentcytotoxicity crossmatch (CDCXM) results with graft survival among transplants performed incontemporary practice.Methods: OPTN registry data for deceased-donor (DD) and live donor (LD) kidney transplants performedafter CDCXM testing in 1995-2009 were examined. We estimated the association of CDCXM results (+ vs-) with allograft survival over 5 yrs result by the Kaplan-Meier method in the full samples and indemographic and clinical subgroups.Results: Among 24,607 and 32,279 LD and DD transplants performed after CDCXM testing, 1155 DD and1019 LD recipients received CDCXM+ transplants in the study period. Overall, compared to CDCXMtransplants,CDCXM+ was associated with absolute reductions in 1yr LD and DD graft survival of 2.2%and 3.8%, respectively (p50%), white or 18-64 yo.Notably, CDCXM+ vs CDCXM- was not associated with increased risk of graft loss in re-transplants or inblack, 64 yr old recipients (Table 1).[figure1]Conclusions: Among transplants performed in contemporary practice, the benefit of avoiding CDCXM+appears to be an 2-4% absolute benefit in 5yr graft survival. Given the moderate magnitude of risk,mitigating factors that resulted in the decision to transplant despite CDCXM+ were well-considered. Thesedata support that CDCXM+ should not be considered a universal contraindication to transplantation.17-PSINGLE ANTIGEN ANTIBODY ASSAY RUN TO RUN VARIATION: HOW QUANTITATIVEARE WE REALLY?Anne Halpin, Megan Brown, Luis Hidalgo, Patricia Campbell. Histocompatibility Laboratory, Universityof Alberta Hospital, Alberta Health Services, Edmonton, AB, Canada.Aim: Our aim is to evaluate the run to run variation of our single antigen antibody assay. The antibody(Ab) strength is measured by the median fluorescence intensity (MFI) for a given antigen. A secondary

goal is to re-examine our previous findings that the vaccum wash (VW) method is at least as effective asthe centrifugation wash (CW) method.Methods: We tested 10 sera with multiple Ab (5 Class I and 5 Class II) using LabScreen® Single Antigenbeads (LS1A04 and LS2A01, One Lambda Inc.); beads will be referred to as LSA1 (class I) and LSA2(class II). Sera were tested as per product insert using VW and the CW with dry vortex methods. Most serawere tested twice by each VW and CW but 3 were tested once by VW. Two lot# of LSA2 beads were usedbetween wash techniques thus these results are limited to INTRA-method comparison. Raw data wereexported to Excel and analysed.Results: There is variability within VW and CW runs as measured by the mean delta value of the MFIdifference. There is less variation in the VW runs than the CW. The mean baseline MFI to known donorantigens in LSA1 (7 Ab) is higher in the VW method than the CW method (5700 vs 4131 MFI). Althoughthe beads appeared in the same order in almost every case, the MFI of most Ab were higher in the FWresults. The LSA1 mean NC value is 97 vs 37 and the mean PC value is 6690 vs 4709 for the VW and CWrespectively. The LSA2 mean NC value is 117 vs 25 and the mean PC value is 6368 vs 4585 for the VWand CW respectively. The VW method saves at least 30 minutes of bench time and eliminates the risk ofcreating biohazardous aerosols.Conclusions: Regardless of wash technique used, there is clear run to run variation, especially for strongantibodies. For this reason, we will continue our practice of re-testing a previously tested serum in parallelwith new sera samples for patients being monitored for Ab strength. We will continue to use the filter platewash method.18-PAN HLA-DP ANTIBODY DILEMMA – CASE STUDY.Jean L. Heneghan, Karen A. Sullivan. Medicine, Tulane University, New Orleans, LA, USA.Aim: In an on-going effort to improve our ability to perform virtual crossmatching, we retrospectivelyinvestigate all positive donor crossmatches. While our prediction rate has improved over the years due toimproved methodology, there are still some cases which will require adding HLA-DP results to the UNOSdatabase system.Methods: FT is a 32BM who received a 0 Ag MM DCD kidney in 2001. It failed in 2003 likely due toFSGF. No donor-specific HLA Ab was detected post transplant by ELISA methodology. However, FT wassensitized to other HLA antigens due to blood transfusions. FT was re-wait listed. He soon received another0 Ag MM DCD kidney offer. He was not transplanted due to positive flow B cell crossmatches. The patientand donor were typed for HLA-DP antigens using SSO methodology. Detection of HLA-DP antibodies wasperformed using LUMINEX.Results: Retrospective studies indicated the crossmatches were likely positive due to donor-specific HLA-DP antibody. FT has received several additional well matched kidney offers. Each respective donor B cellflow crossmatch was positive, and antibody to donor-specific HLA-DP antigens determined to be the likelycause. In each case FT was not transplanted, as our program does not usually transplant across a positivecrossmatch which is likely due to any donor-specific HLA antibody.Conclusions: Until HLA-DP typing results and unacceptable antigens (and Cw antigens as well) can beadded to the UNOS patient database and become incorporated into the match algorhythm, patients andtransplant programs will continue to incur the unnecessary travel, expense and time of bringing in thesepatients for final crossmatching. Perhaps if HLA-DP results were incorporated into the UNOS system,global data analysis could answer the question of whether such antibodies are even a contraindication totransplantation.19-PHLA ANTIBODY IDENTIFICATION USING LUMINEX-BASED ASSAYS – ARE WE ON THESAME PAGE? THE MICHIGAN EXPERIENCE.Chak-Sum Ho 1 , Mary A. Jackowski 1 , Dorothy Levis 1 , Omar R. Fagoaga 2 , John A. Gerlach 3 , MalekKamoun 4 , Gabriel N. Maine 5 , Jerry C. Rosenberg 1 , A. Bradley Eisenbrey 1,6 . 1 Gift of Life Michigan, AnnArbor, MI, USA; 2 Detroit Medical Center, Detroit, MI, USA; 3 Michigan State University, East Lansing, MI,USA; 4 University of Michigan, Ann Arbor, MI, USA; 5 William Beaumont Hospital, Royal Oak, MI, USA;6 Henry Ford Hospital, Detroit, MI, USA.

Aim: Solid-phase antibody (Ab) detection assays are among the most important tools for determiningunacceptable antigens (UA) currently used in HLA labs. We report here a comparison of Ab identificationamong the Michigan HLA labs aiming to evaluate the extent of inter-laboratory variability in the solidphaseplatform.Methods: Four sera were tested by six labs using the Luminex-based bead assays. Data collected includedtest performed, reagent vendor and lot used, Ab specificities identified and the MFI values, UA assigned,and thresholds used for reporting.Results: While strong Ab with a mean MFI of >6000 were concordantly reported as UAs by all labs, muchvariability was observed in the identification of moderate (MFI 2000-6000) and weak (MFI

Aim: There is increasing evidence that allosensitization represents an important risk factor in hearttransplantation. Progress in antibody detection and desensitization of patients with DSA has resulted inbetter patient and kidney allograft survival, yet its impact on heart transplantation has not been analyzed indepth. We have analyzed the impact of anti-HLA antibodies developed prior and/or followingtransplantation by patients from a cohort of 950 adult recipients of heart allografts performed at ourInstitution between January 1,1995 and December 31, 2009.Methods: Sera were tested for anti-HLA antibodies using the standard complement-dependentmicrolymphocytotoxicity method. Antibody specificities were assigned based on correlation coefficients≥0.70 between serum reactivity and expression of distinct HLA antigen(s) on target panel cells. Sera wereconsidered positive for anti-HLA antibodies when they reacted with more than 10% of the HLA referencepanel.Results: Allosensitization reflected in PRA ≥10% and/or in antibodies with distinct anti-HLA specificitieswas present prior to transplantation in 16% (151/950) of the patient population. The actuarial graft survivalafter 15 years was not significantly different in allo-sensitized compared to non-sensitized recipients.However, post-transplant monitoring of PRA demonstrated that there was a significant difference (p500, as determined by singleantigen bead assays, with presumably minimal interferences from the class II antibodies (class II DSA ofMFI

patients with antibody specific to intact CI-HLA have worse graft survival than those with antibody todenatured CI-HLA; results were dependent upon testing sera with two distinct bead sets to identify intactCI-HLA specific antibodies.Methods: A total of 442 heart and kidney transplant patient sera from four centers are tested with a betaproduct from One Lambda Inc., provisionally designated as “Clean” beads. These beads have the samespecificities as SAB, but have high levels of intact CI-HLA and negligible levels of denatured antigen asverified by monoclonal antibodies.Results: Results indicate that antibodies in some patient sera bind to denatured HLA on class I singleantigen beads, but not to clean beads. Patients with no HLA antibody (n=324) had a 10 year graft survivalof 77% antibody. Patients with antibody to intact CI-HLA had a significantly lower (p

esult in poor graft survival. The goal of this study is to determine the strength of Ab to DP and DQAwhich results in a positive crossmatch.Methods: Luminex Single Antigen (SA) is used to define Ab specificity. XM are performed bycytotoxicity methods, Four Wash Amos (FWA) and Antiglobulin Augmented (AHG) techniques. DTT XMwere tested to determine Ab isotype. Additional XM were done by Flow Cytometry. Antigen typing wasdetermined by SSP methods.Results: Table 1. 17% of our listed renal patients have DP and DQA Abs.[table1]Table 2. Preliminarystudies demonstrate that weak Abs to DP, DR, and DQ do not always result in a positive XM and cantherefore be transplanted across. Patients 1 & 2 have weak antibodies to DP and DQ yet have negative Bcell XM.[table2]Conclusions: With the increase in definition of anti-DP and -DQA antibodies in solid organ transplantrecipients, typing for these loci will be very beneficial in determining antigens to avoid pre-transplant andthe presence of donor specific antibodies post-transplant. Further crossmatching is required to define theMFI/ratio which results in a positive crossmatch to contraindicate transplantation.26-PFREQUENCY OF DONOR SPECIFIC HLA (DSA) ANTIBODY IN A TRANSPLANTPOPULATION AND THEIR CLINICAL IMPACT.Ronald H. Kerman, Jacqueline A. Lappin, Stephen M. Katz, Jerome G. Saltarrelli, Charles T. Van Buren,Phillip Erice, Luis Rodriguez, Jenna Mitchell, Nicholas Woolley, Eva McKissick, Noriel Acorda, KristahMiller, Alfred J. Eaton, Craig Adkins, Esther Kelley. Surgery-Organ Transplantation, University of TexasHealth Science Center-Houston, Houston, TX, USA.Aim: Sera from transplant (Tx) candidates are evaluated for HLA antibody (Ab) and specificity.Methods: We tested the pre-Tx sera from 720 Tx recipients (recips) for HLA Ab and specificitiesincluding DSAs. We also evaluated the clinical outcomes of recips transplanted across donor-specific HLAAbs but who had negative donor specific flow cytometery crosmatches (FCXM).Results: The frequency of pre-Tx (+) DSAs for recips with 0% Flow PRA was 0.6% class I with 1 ACR,POD 14; and 1 AbMR POD 14 with no graft losses and 0.75% for class II with 1 ACR, POD 10 and nograft losses. The frequency of pre-Tx (+) DSAs for recips with 1 -10% Flow PRA was 3.2% for class I withno rejections, and no graft losses and 5.2% for class II with no rejections, and no graft losses. Thefrequency of pre-Tx (+) DSAs for recips with 11-25% Flow PRA was 6.9% for class I with 2 AbMR onPOD 21 and 1 graft loss at 4 mos and 5% for class II with no rejections, and no graft losses. Finally, thefrequency of (+) DSAs for recips with greater than 25% Flow PRA is 14% for class I with 1 ACR on POD41, 2 AbMR on POD 8, POD 19 and 2 graft losses at 2 and 8 mos and 16% for class II with 3 AbMR onPOD 7, 10, 16 and 1 graft loss at 14 mos. The mean frequency of (+) DSAs was 5.2% (38/720). In theseDSA(+)/FCXM(-) recips we experienced yearly frequencies of 2.0% cellular rejection, 8.8% antibodymediated rejection and graft loss of 4% for these high risk recips. These rejections and graft losses areequal to and/or less than patients transplanted with (-) FCXMs and no DSAs.Conclusions: Therefore, the presence of HLA Ab identified by binding to third party surrogate HLAantigen,that apparently is donor HLA antigen directed, may not be a clinical risk if the donor specific flowcytometery crossmatch is negative. These patients should not be excluded from consideration to transplantbased upon a presumptuous virtual crossmatch.27-PTRANSPLANTATION OF DONOR SPECIFIC HLA ANTIBODY POSITIVE, FCXM NEGATIVERENAL ALLOGRAFT RECIPIENTS.Ronald H. Kerman, Jacqueline A. Lappin, Jose B. Abraham, Jerome G. Saltarrelli, Charles T. Van Buren,Esther Kelley, Eva McKissick, Noriel Acorda, Kristah Miller, Nicholas Woolley, Alfred J. Eaton, CraigAdkins, Jenna Mitchell, Phillip Erice, Luis Rodriguez. Surgery-Organ Transplantation, University of TexasHealth Science Center-Houston, Houston, TX, USA.Aim: Donor specific (DSA) HLA antibody (Ab) is considered a transplant (Tx) risk and may be acontraindication to Tx. We investigated the credibility of this with negative donor specific flow cytometerycrossmatches (FCXM) when testing high PRA historical sera, current and pre-Tx sera.

Methods: From January, 2008 we treated 13 DSA(+)/FCXM(-) recips with Thymoglobulin,plasmaphereses (PP) Rituxan and/or IVIG. We investigated 12 month (mos) serum creatinines (SCr mg/dl),patient and graft survivals and the influence on clinical outcome of the class I, class II DSA, the meanfluorescence intensity (MFI) of the DSA and the DSA titer. Surveillance DSA titers and clinically indicatedbiopsies were pursued.Results: At a mean follow up of 12 mos post-Tx (range 4 - 16 mos) the patient and graft survivals are100% respectively, with a mean SCr of 1.68 mg/dl for 12 of the recips and a SCr of 4.7 mg/dl for one recip.There were 4 early rejections (1 cell and 3 Ab mediated) occurring 7 - 21 days post-Tx and 1 delayedmixed rejection occurring 12 mos post-Tx. There were no graft losses and no CMV or de novo BK-virusinfections. The pre-Tx immune studies revealed 8 recips with HLA class I DSAs with DSA titers from 1:2 -1:128 and DSA specific MFIs from 1,500 - 15,000. There were 5 recips with pre-Tx HLA class II DSAswith DSA titers from 1:1 - 1:128 and DSA specific MFIs of 4,762 - 14,986. The pre-Tx class of HLAantibody, DSA titer or DSA - MFI were not predictive of rejection or graft loss.Conclusions: These results suggest that patients with surrogate HLA antigen-bead identified Ab may besuccessfully transplanted as long as the donor-specific FCXMs are negative and the patients receiveaggressive immunosuppression. Furthermore, these patients should not be excluded from transplantation(because of virtual crossmatch considerations) and, in addition, may not need pre-Tx desensitization.28-PPOSITIVE FLOW CYTOMETERY CROSSMATCHES AND DONOR-SPECIFIC HLAANTIBODIES MAY NOT BE CONTRAINDICATIONS TO HEART TRANSPLANTATION.Ronald H. Kerman 1 , Rajko Radovancevic 2 , Paul Allison 2 , Eva McKissick 1 , Jerome G. Saltarrelli 1 , RobertaBogaev 2 , Igor Gregoric 2 , Arthur Bracey 2 , O.H. Frazier 2 , Noriel Acorda 1 , Kristah Miller 1 , NicholasWoolley 1 , Alfred J. Eaton 1 , Craig Adkins 1 , Jenna Mitchell 1 , Phillip Erice 1 , Luis Rodriguez 1 , Esther Kelley 1 .1 Surgery-Organ Transplantation, University of Texas Health Science Center-Houston, Houston, TX, USA;2 Texas Heart Institute, St. Luke’s Episcopal Hospital, Houston, TX, USA.Aim: Flow cytometry and Luminex-based single HLA antigen assays are sensitive methods to determinethe presence of HLA antibodies (Ab), donor specifically directed HLA Abs (DSA) and pre-Tx crossmatch(FCXM) outcomes for potential heart Tx (HTX) recipients (recips). We evaluated the effect of sensitizationfrom pre-Tx blood transfusions (BT) received before HTx on HLA Ab (PRA), DSA, FCXM results andclinical outcomes.Methods: Between January 2004 and May 2009, 182 adult patients underwent HTx (171 - 1º and 11 - Re-Txs). We reviewed their UNOS status, pre-Tx BT, pregnancies, Flow PRA, pre-Tx recip/donor FCXMs,HLA Abs, specificity and specificity titers, induction therapy (OKT3 or ATG), and patient and graftsurvivals of the 171 - 1 recips.Results: Of the 171 first HTx recips 167 (98%) received BTs before HTx. Pre-Tx PRAs were 0% in 108(63%); 1-10% in 21 (12%), 11-25% in 15 (9%) and >25% in 27 (16%) patients. There were no differencesin pre-Tx BT exposures. One year survivals were 90%, 86%, 100% and 96% respectively for each BTgroup. All patients were pre-Tx AHG-XM negative. Nine of the 171 patients were pre-Tx FCXM positive(3 with 0% PRA; 1 with 11-25% PRA and 5 with >25% PRA). All 9 were DSA positive, however, only 3of these 9 recips expired within the first 12 months post-Tx. These three recips were not immunologicallydifferent from the remaining 6 DSA positive, FCXM positive recips in regard to PRA, DSA or DSA titer.Conclusions: Therefore, these data suggest that heart Tx recips, with positive pre-Tx FCXMs and DSAsmay be successfully transplanted. Some recips with positive FCXMs and DSAs may result in patient/graftloss. We do not yet know how to identify the immune factors relevant to good or bad patient outcomes.29-PPRONASE-INDUCED FALSE POSITIVE T-CELL FLOW XM OBSERVED IN CERTAINPATIENT-DONOR COMBINATIONS.Lorie H. Kumer, Carrie L. Mowery, Carolyn L. Fisher, Lori A. Malec, Thomas L. Bement, Justine L.Gaspari, Ronald E. Domen, Hiroko Shike. HLA Laboratory, Penn State Hershey Medical center, Hershey,PA, USA.

Aim: Pronase treatment eliminates irrelevant IgG binding via FcR and reduces background in B cell XM.However, we experienced two patients’ sera with unexpected positive T-cell XM attributed to pronasetreatment. Our experience may be helpful to other laboratories who may encounter similar challenges.Methods: Lymphocytes were treated with pronase (2mg/mL) for 15min at 37°C. XM used F(ab) 2 antihumanIgG-FITC, CD3-PE, CD19-PerCP, and Facscan. MESF units were determined by standard beads(Bangs Lab). XM threshold is 6,000 (T) and 4,500 (B) defined by 1.5-2x average NHS MESF from 50donor cells. Patients on wait list were tested by Luminex-based One Lambda LABScreen PRA Class I andII and LABScreen Single Antigen every 2 and 6 months, respectively. Unexpected XM was investigated byflow XM with additional random donors.Results: Patient A on pancreas list (PRA I and II=0% since 2008) and Patient B on kidney list (PRA I=0%and II=97% since 2008, multiple DPs and DR11) had positive T-XM with different deceased donors towhich other patients were compatible. T-MESF values of additional XMs were greatly variable withdifferent donors but similar between different serum dates. Patient A and B had positive T-XM with 4/6donors (MESF range: 6,794-12,582) and 6/7 donors (MESF range: 6,939-70,370), respectively. Correlationwith donor HLA types failed to identify cause of positive T-XM. Finally, when XM was repeated withoutpronase, T-XM was negative (MESF with/without pronase: 7,886/2,556 in a XM of Patient A vs. XDS489;70,370/5,492 in a XM of Patient B vs. XDU124). In retrospective review of the 13 XMs, all controls are inrange and no consistent sign of false reactivity were found except T-cell histogram patterns; broad-based T(6 XM) and a slightly double-peaked T (1 XM).Conclusions: Pronase-induced false positive T-flow XM is infrequent but should be considered as a causeof unexpected T-flow XM.30-PTECHNICAL VARIATION IN HLA ANTIBODY LEVELS MEASURED BY SINGLE ANTIGENLUMINEX ASSAY IS DIRECTLY PROPORTIONAL TO TOTAL BOUND ANTIBODY.Christian P. Larsen, Phillip J. Summers, Aaron T. Whiteley, Lee Ann Baxter-Lowe. Surgery, UCSF, SanFrancisco, CA, USA.Aim: Specific HLA molecule bound microparticles are utilized to quantify antibody concentration in seraby measuring the associated mean fluorescence intensity (MFI). Variation of MFI plays a critical role inpatient management, however the contributions from technical variability are not always considered. Thisstudy characterizes the technical variation of one serum over a period of 5 months utilizing the same lot ofLabscreen reagents.Methods: Average adjusted MFI (Labscreen, OneLambda) for repeated tests performed on a single serum(n=65) was determined for each HLA molecule. Adjusted MFI was calculated for each assay, as well as themean, standard deviation (STD), and coefficient of variance (CV) for each HLA molecule.Results: Average adjusted MFI demonstrated a strong linear correlation with average microparticle STD(R2=0.97). An STD of greater than 1000 MFI and greater than 100 MFI occurred in 9% and 27% ofmicroparticles, respectively. The average STD and CV values for clinically relevant MFI categories arenoted in Table 1.[table1]Conclusions: This study demonstrates that the standard deviation of MFI in the Luminex single antigen(SA) assay is directly proportional to the level of bound antibody. As expected, STD increased withincreasing MFI. Conversely, the CV decreased with increasing MFI, indicative of more predictable rangeof variability among higher MFI microparticles. These data suggest that interpretation of SA data inlongitudinal studies of sera should consider contributions from technical variation.31-PIMMUNOGENICITY ANALYSIS AMONG DIFFERENT HLA ANTIGENS.Min Ling, Enver Akalin, Rosanne Scandaliato, Ludner Malary, XiaMei Liu, Amy Lu, Milan Kinkhabwala.Transplant Center, Montefiore Medical Center, New York, NY, USA.Aim: HLA immunogenicity is important for graft survival. This study will focus on analyzing the HLAimmunogenicity from sensitized patients waiting for kidney transplant.Methods: This study includes 186 sensitized patients with HLA antibody’s MFI over 5000. Luminexsingle antigen was performed. Statistical analysis: Fisher test.

Results: Analysis of HLA A/B and Cw immunogenicity. Table 1 shows that ratio of anti-HLA A/B ismuch higher than anti-Cw (p=0.0002). The previous transplant has higher chance to generate anti-A/B(p=0.014) and has no difference to generate anti-Cw (P = 0.2821).[table1]Analysis of HLA DR/DQ and DPimmunogenicity. Table 2 shows that DP is a weaker stimulator than DR/DQ (P = 0.0001). Previoustransplant has higher chance to generate anti- DR/DQ (P = 0.0001). Anti-DP is associated with the previoustransplant (P = 0.025).[table2]Conclusions: Based on this study, HLA A/B and DR/DQ have higher immunogenicity than Cw and DP.The previous transplant is a stronger stimulator to generate anti -A/B and DR/DQ antibody. Anti-Cw is notassociated with previous transplant but Anti-DP is associated with previous transplant.32-PDO WE REALLY KNOW EVERYTHING ABOUT CROSS MATCH (OR DOES IgG SUBTYPEMATTER)?Andrew L. Lobashevsky, Kevin Rosner, William Goggins, Nancy G. Higgins. HLA Laboratory, ClarianHealth Transplant Center, Indianapolis, IN, USA; Medicine, Histocompatibility Laboratory, IndianaUniversity, Indianapolis, IN, USA; Department of Surgery, Indiana University, Indianapolis, IN, USA.Aim: Preexisting donor-specific antibodies (DSA) play a critical role in the success of solid-organtransplantation. Strong anti-HLA class II DSA in three transplant candidates (TC) constantly producedpositive B cell Flow Cytometry (FC) cross match (CM) (MCS > 380), whereas CDC CM tests were alwaysnegative. This study was carried out to investigate this paradox.Methods: SAB LUMINEX technology was used to determine the IgG class II antibody subclasses. Thehuman IgG-specific secondary antibodies conjugated to PE were replaced with antibodies specific forhuman IgG1, IgG2, IgG3, and IgG4 subclasses conjugated to the same fluorochrome.Results: IgG solid-phase subtype analysis showed that 26%-86% of DSA were represented bynoncomplement binding IgG2/IgG4 subtypes.[table1]Furthermore, the MFI values of DSA of IgG1/IgG3subtypes were always below the cut off producing positive CDC CM. These findings account for antibodymediated rejection free post-transplant course in these TCs, despite the high level of DSA.Conclusions: Routine application of SAB IgG subtype assay may provide new insights regardingtransplantation in TCs presenting with negative B-cell CDC and positive FC CM.33-PA STATISTICAL TEST FOR SIGNIFICANCE OF CHANGE OF NORMALIZED MFI IN THELUMINEX SINGLE ANTIGEN TEST.Alan M. Luger 1 , Melissa Steele 1 , Bin Ge 2 . 1 Histocompatibility Laboratory, Jewish Hospital, Louisville, KY,USA; 2 Biostatistics, University of Missouri, Columbia, MO, USA.Aim: We present a statistical test of significance of changes in normalized MFI between runs in order toprovide clinicians with valid information about the effectiveness of desensitization of candidates fortransplantation.Methods: An aliquot of a serum from a sensitized patient was included in 23 consecutive runs of the assay.We rejected runs where positive and/or negative controls were unacceptable. In consultation withbiostatisticians we developed rules for decisions:• Outliers were removed using Grubb’s test. We calculated Spearman correlation coefficient between themeans of each test and corresponding standard deviation of each test to examine the relation between meanand SD. Results showed that the mean is positively correlated with SD (For Class 1: r=0.9255, p

34-PCLINICAL AND PATHOLOGICAL SIGNIFICANCE OF ANTI-DONOR ANTIBODIES INLIVER ALLOGRAFT RECIPIENTS.John G. Lunz 1 , Mariana Cajaiba 1 , Kristine Ruppert 2 , Adriana Zeevi 1 , Anthony J. Demetris 1 . 1 Pathology,University of Pittsburgh, Pittsburgh, PA, USA; 2 University of Pittsburgh Diabetes Institute, University ofPittsburgh, Pittsburgh, PA, USA.Aim: Anti-HLA antibodies (Ab) have been shown to have a detrimental effect on outcome for most solidorgan allografts. However, in liver transplantation (Tx), the clinical and pathological significance of antidonorAb is less certain. In this retrospective study, we sought to examine the relationship of crossmatch(XM) status and anti-HLA Ab with pathological and clinical parameters in liver allograft recipients.Methods: Analysis was performed on liver allograft recipients (N=809) from 10/03 through 6/09. Allpatients (pts) had at least a T cell (AHG) CDC-XM at the time of transplant and 1 post-Tx biopsy. HLA Abwas determined by CDC-PRA, ELISA, or Luminex single antigen. C4d was assessed byimmunohistochemistry in biopsies.Results: 100 of 809 pts were XM+. PRA was significantly higher in XM+ (62%) compared to XM- pts(3%) and was similar in both groups, irregardless of whether donor specific Ab (DSA) was detected. 59%of XM+ pts and 6% of XM- pts had DSA. However, sensitive solid phase HLA Ab testing was notroutinely employed. Both XM groups had similar graft and patient survival. And while both groupsreceived similar amounts of calcineurin inhibitors early after Tx, XM+ pts did require more post-Txsteroids. XM+ pts also had a more significant drop in platelets after Tx. Biopsies taken >1yr post-Tx fromXM+ pts displayed more injury and significantly higher rejection activity index (RAI) scores than XM- pts.More focal and diffuse endothelial cell C4d staining was seen in all liver vascular structures in XM+compared to XM- pts.Conclusions: While the presence of anti-donor Ab does not effect overall survival, evidence here suggeststhat they do contribute to liver allograft injury. More sensitive solid phase Ab testing and better markers ofcomplement activation and endothelial cell injury and reactivity may provide more insight to thepathological mechanisms of anti-donor Ab in liver allografts.35-PCELLS ARE NOT THE SAME AS ANTIGEN BEADS: CORRELATION BETWEEN MFI ANDMESF VALUES.Jennifer Mendiolina, Patricia Kimble, Marilyn Wetmore, Diane Hartzell, Michael Moritz, Robert Cirocco.HLA Lab, Lehigh Valley Hospital, Allentown, PA, USA.Aim: A study was initiated to identify a correlation between the Mean Fluorescence Intensity (MFI) valuefor a particular anti-HLA antibody in a sensitized patient’s serum, as determined by the Luminex ID andSingle Antigen bead array, and the MESF level from a positive flow crossmatch for the correspondingHLA antigen. If a correlation could be found, it would identify the level of reactivity on Luminex at whicha flow crossmatch would become positive. This data would help in determining the most useful cutoff forunacceptable antigens (UA). These UAs are exclusionary antigens that guide the determination of a suitabledonor for sensitized recipients.Methods: The patient sera chosen had specific antibodies that had been clearly defined over time on bothID and Single Antigen (Tepnel/Genprobe, Stamford,Conn.). Most had an MFI of 400 - 8000, with 3000used as the cutoff. These samples were then run on flow crossmatches with corresponding antigens onpronase treated lymphocytes from living and deceased donors. Each serum/cell combination was tested in aseparate assay to reduce interference from additive effects. The same specimen tested on Luminex wasused for crossmatching to reduce variability. Data from twenty crossmatches was entered into a spreadsheetand a scatter plot was generated.Results: There was a high degree of variability in reactivity among the different serum/cell combinations.There was no linear correlation (ID r=0.12, SA r=0.31).Conclusions: It was determined from the scatter plot that there was no linear correlation between the MFIand the MESF change. This may be due to differing antigen density from one cell to another, alteredantigen expression, or allele level antibodies. In addition, the beads may have a conformational change ofthe antigen as a result of the production process. Due to this lack of correlation, it can often be difficult topredict crossmatch outcomes based on MFI values alone.

36-PRE-THINKING THE ROP TRAYS IN REGION 1.Maureen M. Miller, Tod V. Alberghini, Michael Rewinski, Trish Michalski, Laurine Bow. TransplantImmunology Laboratory, Hartford Hospital, Hartford, CT, USA.Aim: To understand the usefulness and cost of creating and exchanging serum trays within Region 1. In2010 CTOP stopped exchanging sera trays monthly with MAOB and switched to exchanging sera quarterlyfrom actively listed patients with a CPRA of greater than 50%. Do we need to share sera this often or canwe stop sharing sera all together?Methods: To expedite kidney allocation, Region 1 has agreed that each OPO lab must generate a list of thetop15 patients with a negative crossmatch for each deceased donor. The frequency of using exhange traydata was assessed by reviewing deceased donor kidney matches. All of the deceased donor kidney matchesin 2009 from CTOP and donors from MAOB from 10/1/09 until 3/31/10 were reviewed in UNET.Results: In 2009 CTOP had 36 deceased donor kidney matches. MAOB crossmatch results were neededfor the kidney allocation in 26/36 (72%) kidney matches. MAOB had 104 deceased donors with kidneymatches in the six months reviewed and only once (1%, 91% CPRA, patient subsquently transplanted withnegative flow crossmatch) was the exchange tray data used in kidney allocation. This data mimicks MAOBdata from 2007-2009 presented regionally. The cost of the exhange trays for CTOP is approximately $2500every six months. This does not take into account the cost to run the trays for each donor which wouldinclude additional tech time and reagents.Conclusions: The low usage of the CTOP exchange tray data by MAOB suggests that they could beeliminated to cut costs. However, since data from the MAOB trays were used by CTOP 72% of the time,there usage should be continued. The difference in effectiveness of exchange tray use is most likely due tovarying numbers of patients on the list, antigen assignment vairation and local organ acceptance criteria forECD and DCD donors.37-PUNACCEPTBLE ANTIGENS: HOW ARE THEY WORKING?Maureen M. Miller, Tod V. Alberghini, Michael Rewinski, Trish Michalski, Laurine Bow. TransplantImmunology Laboratory, Hartford Hospital, Hartford, CT, USA.Aim: Determine if unacceptable antigens listed in UNET predict patients who should have a negative Tand B cell crossmatch against a particular donor. In 2009 the unacceptable antigens were reviewed when apatient was listed in UNET and then twice a year. An antibody must be detected in two different samplesby two different detection methods to be listed in UNET as unacceptable.Methods: Deceased donor kidney matches from 2009 were analyzed. Patients with a CPRA of ≥10% in thetop 15 crossmatch results on each kidney match were reviewed. Patients from CTOP had T and B cellcytotoxic crossmatches performed primarily and T and B cell flow crossmatches as needed, based on ourlab’s policy. Patients from MAOB had only T cell cytotoxic crossmatch performed.Results: 36 deceased donor kidney matches were reviewed. Of the 540 total crossmatches for the top 15patients on each list, 135 (25%) were patients with a CPRA of ≥10%. 76 of these were patients listed withinCTOP and were evalutated further. 24/76 (32%) of the patients with CPRA >10% had a T and/or B cellpositive crossmatch. The CPRA ranged from 12-99%. 52/76 (68%) of the patients had a negativecrossmatch. Their CPRAs ranged from 10-94%. 8/24 (33%) of the patients with an unexpected positivecrossmatch had donor specific antibodies identified by only one antibody detection method. Many of theantibodies were identified by Gen-Probe Luminex ID assays but were not confirmed by either Gen-Probeor One Lambda Single Antigen assays. Another 8 (33%) had donor specific antibodies but did not havetheir unacceptable antigens updated in UNET. The remaining 8 unexpected positive crossmathces weremostly likely due to C,DP or low levels of antibodies, possibly IgM.Conclusions: Unacceptable antigens do aid in the kidney allocation process. Since they play such animportant role more frequent review is necessary to accurately report them to UNET.38-PSEROLOGIC SUBTYPES OF DQ7 SUGGESTED BY PRINCIPAL COMPONENT ANALYSIS OFSINGLE ANTIGEN SCREENING DATA.

Nabil Mohsin, Isabelle G. Wood, Indira Guleria, Edgar Milford. Medicine, Brigham Hospital, Boston, MA,USA.Aim: Serum is often tested for antibodies using single HLA antigens as targets. WHO serologic entities areused to specify antibodies. Investigators have found sera which appear to react selectively with one or morealleles within a WHO specificity, but these have not been well defined. Methods for systematicallysearching for such new antibody specificities are needed.Methods: We analyzed 2750 sera from 1215 patients on Class II single antigen beads (One Lambda) usinga Luminex platform. We used Principal Component Analysis (PCA), a statistical method to find distinctpatterns of reactivity in multivariate space which account for the observed data.Results: For the five DQ7 beads, three principal components accounted for 99.3% of the MFI variance.Eigenvectors from PCA analysis showed the relative contributions of the beads to eachcomponent.[table1]The first component represented the 88.8% of sera which reacted equally with all of thefive alleles. The second component accounted for 9.2% of the data in which (DQA*03:01 DQB*03:01) hasa distinct pattern of reactivity from the other alleles. The third component, 1.3% of the data, were sera inwhich (DQA*03:03 DQB*03:01) had a unique pattern of reactivity.Conclusions: PCA is a powerful tool to identify candidate sera which can be used to detect new serologicalspecificities and might yield new WHO antigen definitions. The data suggests three serologic subtypeswithin DQ7.39-PANALYSIS OF A NEW CELL PRESERVATIVE FOR FLOW CYTOMETRIC CROSSMATCHES.Megan Moran, Patricia Brannon, Gebel Howard, Bray Robert. Pathology, Emory University, Atlanta, GA,USA.Aim: The importance of cell viability for crossmatching has increased, in part, due to the growth of paireddonor exchange programs. In this study we compared EDTA and ACD to the new Streck Cell Preservative(SCP). Samples were tested at day 1 and day 5 for antigen expression, viability and performance in astandard flow cytometric crossmatch.Methods: Peripheral blood from five healthy volunteers was collected into tubes containing ACD, EDTAor SCP and stored at room temperature. Mononuclear cells were isolated on day 1 and day 5 via ficollhypaqueseparation. Cell counts were performed and viability assessed. A flow crossmatch was performedusing two negative and one positive control sera.Results: At day 1 and 5, cell viabilities from all subjects were comparable (>98%) for all preservatives.SCP appeared to yield better separation and there was less red cell contamination compared to ACD orEDTA. In addition, light scatter was better preserved in the SCP tubes. However, cells from the SCPshowed significantly higher background fluorescence compared to ACD or EDTA. The high backgroundfluorescence did not affect the over all delta-MCF or MESF. In contrast, ACD and EDTA cells showed lowbackground fluorescence on day 1 and day 5 but, light scatter deteriorated, compromising the ability toidentify and gate lymphocytes. Nonetheless, there were no differences in crossmatch results for days 1 or 5.Conclusions: Peripheral blood collected into SCP, ACD, or EDTA yielded comparable results in the flowcrossmatch. Although the SCP tubes did appear better at preserving light scatter properties, the highbackground fluorescence florescence may be troublesome. While both ACD and EDTA showed loss oflight scatter integrity, they performed similarly in the crossmatch. In conclusion, ACD and EDTA areacceptable preservatives for shipping whole blood for up to 5 days. While SCP may be used, highbackgrounds will be a challenge for analysis.40-PDEFINING CUT-OFF FOR THE LUMINEX MFI THAT CAN PREDICT THE FLOWCYTOMETRIC CROSSMATCH (FCXM) RESULTS.Omar Moussa 1 , Howard M. Gebel 1,2 , Robert A. Bray 1,2 . 1 Pathology and Lab Medicine, Medical Universityof South Carolina; 2 Pathology, Emory University.Aim: At present, the definition of “clinically relevant” HLA antibody is the center of much debate. Formany centers clinical relevance is defined as producing a positive crossmatch while for other centersrelevance is defined by the presence/absence of donor-directed antibody and clinical outcomes. Solid phase

assays, such as Luminex single antigen beads, have greatly improved the ability to detect HLA antibodies,however, there is lack of agreement on how we define a cut-off threshold that correlates with the Flowcytometry crossmatch (FCXM). In this study we report our multi-center experience comparing theantibodies strength expressed in MFI and the FCXM.Methods: SA beads analysis was performed using the LABScreen® assay. Signal amplification wasperformed using biotinylated goat anti-human IgG secondary antibodies and StreptAvidind-PE. For eachpatient with donor specific antibody, SA data from 57 patients expressed in MFI were correlated to themedian channel shift (MCS) values.Results: With the exception of: (i) antibodies that are directed against public epitopes such as Bw4 andBw6, (ii) antibodies to shared epitopes, and (iii) antibodies to Cw antigens, FCXM positive was obtainedusing a cut-off threshold of ∼2000 MFI for class I antibodies and ∼3500 for class II antibodies. Thesevalues enabled the prediction of positive T and B cells crossmatch, respectively.Conclusions: In Luminex testing with using biotinylated goat anti-human secondary antibodies, the cut-offthreshold of 2000 MFI for class I antibodies and 3500 for class II antibodies enabled the prediction ofFCXM results. These data provides a significant attempt for using a biological approach to defineunacceptable antibodies. This approach may significantly aid the performance of the virtual CXM.41-PLIMITED EXPRESSION OF HLA CLASS I ANTIGENS ON ENDOTHELIAL CELLS.Arend Mulder 1 , Michael Eikmans 1 , Marry E.I. Franke 1 , Willem Verduijn 1 , Jacqueline D.H. Anholts 1 , DaveL. Roelen 1 , Sicco Scherjon 2 , Frans H.J. Claas 1 . 1 Immunohaematology and Bloodtransfusion, LeidenUniversity Medical Center, Leiden, Netherlands; 2 Obstetrics and Gynaecology, Leiden University MedicalCenter, Leiden, Netherlands.Aim: HLA alloantibody screening and crossmatching of patients’ sera are usually performed onlymphocytes or purified HLA molecules derived from lymphocytes or platelets. However, in HLAmismatched transplantation, the primary targets of the recipient’s antibodies (Abs) are the cells lining theendothelium. Therefore a more faithful representation would be to use endothelial cells (EC), or HLAmolecules originating from EC, as ligands for antibody detection.Methods: Here, we measured the expression of the individual HLA class I alleles on molecularly typed ECfrom umbilical cords (HUVEC) by flowcytometry (FCM) side-by-side with HLA allele specific mRNAlevels by quantitative RT-PCR. In FCM, individual alleles were addressed with human monoclonal Abs(developed in-house).Results: Total HLA class I protein expression was significantly lower on HUVEC compared to PBL.Theproducts of many (9/12) individual HLA-B alleles failed to be detected on HUVEC surfaces by FCM,whereas those of many (8/11) HLA-A alleles were readily detectable. Interferon-γ treatment restored cellsurface expression of previously undetectable HLA-B alleles. HLA allele specific mRNA was detectable in3/3 HUVECs, despite absence of FCM detectable expression of some HLA-B alleles, suggesting a lineagespecific mechanism that controls cell surface expression of HLA class I proteins.Conclusions: If extrapolatable to EC of Tx relevant organs, the upshot may be that when transplantingHLA alloimmunized patients, clinicians unnecessarily avoid organs that may harbour alleles in theirvasculature, that fail to be recognized by the recipients’ HLA Abs, provided that no local γ-interferon isproduced.42-PDEVELOPMENT OF A LARGE SINGLE CENTER KIDNEY PAIRED DONATION PROGRAM.Cathi L. Murphey 1 , Francis Wright 2 , Mathias Kapturczak 2 , Adam W. Bingaman 2 . 1 SouthwestImmunodiagnostics, Inc., San Antonio, TX, USA; 2 Methodist Specialty and Transplant Hospital, SanAntonio, TX, USA.Aim: Many patients are unable to take advantage of live donor (LD) kidney transplantation due tocompatibility constraints. A paired kidney donation program (KDP) was initiated in 12/07 to increaseaccess to LD transplantation.Methods: All consented patients with incompatible donors and non-HLA identical compatible pairs witholder donors were entered into the exchange database. The KPD database identified potential

donor/recipient pairs for transplant and flow crossmatching was performed to confirm immunologiccompatibility.Results: The KPD database contains 192 recipients and 290 donors. Of the incompatible recipients, 60%are crossmatch incompatible and 40% are blood type incompatible with their donors. A total of 63exchange transplants were performed including 18 double and 9 triple exchange transplants. Over the past12 months, KPD transplants accounted for 31% of our LD volume (51/162). The majority (37/63) of KPDrecipients were sensitized, of which 29/37 (78%) had calculated panel reactive antibody (cPRA) > 40%including 15/37 (35%) highly sensitized recipients with cPRA > 80%. Disadvantaged groups with regard tolevels of sensitization such as African Americans, females, and previous transplant recipients were alltransplanted at a statistically significantly higher rate in the KPD as compared with our compatible LDprogram. All recipients of KPD transplants had negative flow crossmatches at the time of transplant.Desensitization was required in 2 patients for low level incompatibility. There have been no episodes ofcellular or antibody mediated rejection and the median serum creatinine of recipients is 1.4mg/dl with amedian follow up of 6 months.Conclusions: In less than 2 years, we have established amongst the largest KPD programs in the US at asingle center. KPD has dramatically increased the volume of LD transplantation as well as access totransplantation for traditionally disadvantaged populations at our center.Murphey: One Lambda: Other: Instructor at Flow Workshop.43-PSOLID PHASE CROSSMATCH HELPS TO IDENTIFY FLOW CROSSMATCH POSITIVEPATIENTS AT MINIMAL RISK OF ANTIBODY MEDIATED GRAFT INJURY AFTERTRANSPLANTATION.Ronald Pelletier, Gregg Hadley, Annette Rearick, Patrick Adams. Tissue Typing Laboratory, Ohio StateUniversity Medical Center, Columbus, OH, USA.Aim: A positive B cell flow crossmatch (BFXM) with a negative T cell flow crossmatch (TFXM) issometimes due to clinically irrelevant non-HLA antibodies. In contrast, the DSA Luminex crossmatch(DSAXM) which uses donor-derived MHC proteins, detects only anti-donor HLA antibodies, and readilydistinguishes between antibodies directed to donor Class I and/or donor Class II antigen mismatches. In thisstudy, 11 live-donor renal allograft recipients who were BFXM positive were transplanted based on anegative Class II DSAXM. This study was undertaken to demonstrate the clinical efficacy of the Class IIDSAXM.Methods: We studied all living donor kidney transplants from October, 2007 to February, 2008 with anisolated positive BFXM. All recipients were TFXM and Class I DSAXM negative. 3-color FXM andDSAXM were performed immediately prior to transplant. The decision to transplant was based on anegative Class II DSAXM, so long as no evidence of anti-donor DQ DSA was indicated in Luminex singleantigen testing.Results: Mean follow-up time for this group is 2.3 years. All kidneys are functioning, with a mean serumcreatinine of 1.98. There have been no incidences of acute humoral rejection. One patient experiencedacute cellular rejection at 5 and 8 months post transplant, with suspected noncompliance. Post transplantmonitoring of this group has demonstrated complete lack of DSA elaborated during the follow-up period.Conclusions: The solid phase donor-derived DSAXM is clincally useful in identifying FBXM positivepatients at minimal risk for post transplant antibody-mediated complications.44-PEVALUATION OF LUMINEX SINGLE ANTIGEN BEAD ASSAY FOR USE AS A VIRTUALCROSSMATCH.Cecil L. Rhodes, Nancy Mata, Placida Abes, Joanna Szafran, Donna King, Julien Napoleon, Prakash Rao.NJ Sharing Network Transplant Lab, New Jersey Organ and Tissue Sharing Network, New Providence, NJ,USA.Aim: The use of Luminex Single Antigen Beads (LSAB) to predict Flow Cytometer Crossmatch (FLXM)and Complement Dependent Cytotoxicity Crossmatch (CDC) in solid organ transplantation remains achallenge. This study evaluates LSAB Mean Fluorescent Intensity (MFI) to FLXM and CDC in assessingthe effectiveness of predicting crossmatch results.

Methods: 272 T cell CDC and FLXM and 269 B cell CDC and FLXM’s were reviewed and the resultscompared to Mean Fluorescent Intensity (MFI) of LSAB Donor Specific Antibody (DSA).Results: DSA MFI’s were grouped into seven levels and are presented in the table below:[table1]Conclusions: From this data there was a very low frequency of positive crossmatches between 0 and 500MFI. At an MFI of 1000-2000, T cell FLXM and B cell FLXM were positive but CDC was still negative.FLXM did not achieve a 90% rate until the 5000-10000 MFI group. It is interesting that even at >15000MFI’s T and B cell CDC crossmatches were positive 62.5% and 77% respectively and B FLXM werepositive 92.3%. It is evident that LSAB can be used to predict FLXM and CDC within specific MFI rangesbut not with 100% accuracy. Comparing LSAB MFI’s to FLXM and CDC can aid in establishing effectivelimits for Unacceptable Antigens. LSAB MFI is useful as a guide for estimating the risk for a positiveFLXM and CDC.45-PDISCREPANT LUMINEX SINGLE ANTIGEN BEAD AND FLOW PRA RESULT IN RECIPIENTAWAITING HEART TRANSPLANT.Cecil L. Rhodes, Ijeoma Okere, Bryna L. Cook, Vijaya Hegde, Alpa Patel, Monica Ramirez, Prakash Rao.NJ Sharing Network Transplant Lab, New Jersey Organ and Tissue Sharing Network, New Providence, NJ,USA.Aim: To address unexpected results between Luminex Single Antigen Beads (LSAB) and Flow PRABeads (FLPRA).Methods: Patient serum with unexpected results between Luminex Single Antigen Beads (LSAB) andFLOW PRA (FLPRA) was tested by Flow Cytometer Crossmatch (FLXM) to confirm result.Results: Potential Heart Recipient MP was negative for both Class I and Class II antibodies by FLPRA butpositive for Class II LSAB. Subsequent testing by LSAB revealed many positive Class II beads with MFI’sranging from 2021 to 9041. Flow Single Antigen Class II Beads confirmed the LSAB result. Most LSABreactions appear as allele specificities with a lack of groups of positive alleles. Reactions that present asallele specificities are not confirmed by additional beads bearing the same antigen,i.e., theDQA1*0201/DQB1*0401 bead was strongly positive with an MFI of 9041 however the DQA1*0201 andDQB1*0401 are found independently on other beads and were both negative (0 MFI). Two flowcrossmatches (FLXM) were performed with donors matched for a positive allele specific DSA. One donorpositive for DRB1*0102 had a corresponding DSA of 4609 MFI. A second donor positive for DRB3*0202had a corresponding DSA of 6969 MFI. Expected crossmatch MCS for the DRB1*0102 and theDRB3*0202 were 100 and 150 MCS respectively, however, B cell FLXM’s were negative in both caseswith a MCS of 0.Conclusions: Reactions between DQA and DQB beads indicate that this antibody is not directed to HLAepitopes or depends on conformational characteristics of the DQ alpha and beta chains. Lack of reactivityby FLXM with normal lymphocytes may be due to cryptic epitopes that are only assessable in denaturedproteins on the beads.Important questions are raised by this data. 1. Are these antibodies relevant to graftsurvival. 2. What other method can confirm Flow Negative Luminex Positive specificities.46-PDETECTION OF DONOR SPECIFIC ANTIBODIES WITH DynaChip TECHNOLOGY: A PILOTSTUDY.Dave L. Roelen, Simone Brand-Schaaf, Marissa van der Linden, Marian Witvliet, Ilias I.N. Doxiadis, FransH.J. Claas. Dept. of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden,Netherlands.Aim: In the recent years many new technologies for HLA specific antibody screening have beendeveloped, among which the DynaChip assay (Invitrogen, UK). This Solid Phase Assay (SPA) makes useof a chip coated with purified HLA antigens of single donors. We have evaluated whether this assay ismore sensitive compared to the complement dependent cytotoxicity assay (CDC) and whether the definedantibodies are clinically relevant.Methods: In total 142 immunized kidney patients, transplanted between 1995 and 2004, with a negativeCDC cross-match (with current and historical sera) were tested. Sera before transplantation were selected

and tested in the DynaChip assay for Donor-Specific-HLA-Antibodies (DSA), which could not be detectedin the CDC.Results: In 51 patients the actual presence of DSA could not be determined because of their high PRA.These patients were excluded from the study. In 8 of the 91 informative patients DSA against HLA werefound. One patient lost the graft within 2 weeks with an anti HLA-A2. Three patients lost their graft within3 to 4 years, (two anti HLA-A9, one anti HLA-DR4). One patient, which died after 2 1/2 years with afunctioning graft, had DSA anti HLA-B18. In three cases DSA were found in patients with wellfunctioning organs (anti HLA-A2, HLA-DR11 and HLA-DR4).Conclusions: In conclusion, testing sera of sensitized patients on the kidney waiting list with the SPAbased DynaChip assay led to the identification of additional specificities not detected by CDC. The clinicalstudy confirms that the presence of DSA only detectable in the more sensitive SPA’s is rather a risk factorthan a contra-indication for transplantation. In some patients these antibodies may be associated withcomplications (one early rejection) whereas other patients have a functioning graft, one even more than 15years, in the presence of DSA. Future studies, including the titer and the antibody (sub)class of the DSA,should lead to a clearer identification of the patients at risk for early rejection.47-PTITER OF ANTIBODIES CAN BE ESTIMATED FROM MEDIAN CHANNEL SHIFTS OFPOSITIVE FLOW CROSSMATCHES.Jerry C. Rosenberg, Chak-Sum Ho, Mary Jackowski, Dorothy Levis, Christine Pieter, Kyle Putnam, A.Bradley Eisenbrey. Histocompatibilty Laboratory, Gift of Life Michigan, Ann Arbor, MI, USA.Aim: Transplant clinicians increasingly want to know the median channel shifts (MCS) when thecrossmatch is positive so as to gauge the strength of the antibody. Conventionally, the latter has beenassessed by the antibody’s titer. The following experiments were carried out to determine if the MCS canestimate the antibody’s titer and therefore the strength of the antibody or antibodies.Methods: Seven different serum samples that were highly positive on antibody testing were titered usingthe AHG cytotoxicity assay and then crossmatched to the same cell using flow cytometry. The resultingMCS and MESF were then correlated with the titer.Results:[figure1]A statistically significant correlation exists between the titer of an antibody and the MCSof a flow crossmatch. The correlation is best at the extremes of the titer, (when the titer is very high or verylow) and less reliable when the titer is between 1:4 and 1:8. The correlation with MESF was also significantat a p of 0.05. However, MESF is a linear function and MCS is a log function, resulting in a semi-log plotthat is not linear. Using the above formula for the linear relationship between titer and MCS, andcalculating the titer from the MCS, the average deviation of from the true titer was 17%. The differencewas greatest at a titer of 1:2 which was overestimated as a titer of 1:6.5. At titers of 1:128 and 1:1024 thedifferences varied between 0% and -7%.Conclusions: The titer of a highly sensitized serum sample against a lymphocyte can be reasonablyestimated in a linear fashion from the MCS obtained by flow cytometry.48-PCAN WE PREVENT SENSITIZATION AFTER GRAFT LOSS?Juan Scornik, Herwig-Ulf Meyer-Kriesche. Pathology and Medicine, University of Florida College ofMedicine, Gainesville, FL, USA.Aim: Transplant (Tx) loss, a major cause of sensitization, is generally considered a natural consequence offoreign antigen (Ag) exposure, but few if any studies have looked systematically to determine if that is thecase.Methods: In the present work HLA antibodies (Abs) were measured by Luminex single Ag and other solidphase tests in patients, originally unsensitized, who lost a kidney Tx (Tx duration: 1 day to several years).Results: Overall, 73/104 (70%) patients became sensitized (90% donor specific). Abs before Tx loss werepresent in 16/85 patients (19%), that is, sensitization is potentially preventable in 81% of the patients.Patients already sensitized were excluded from subsequent analysis. Tx nephrectomy was performed in 39patients and 31 (79%) became Ab positive, but 72% were also transfused. Conversely, 26/38 (68%)transfused patients made Abs, but 92% were also nephrectomized and most, in either group, were also offimmunosuppression (IS). To circumvent these confounding variables we identified 25 patients who had

only IS off, only nephrectomy, only transfusions, or none of the above, and 4 (16%) made Abs. I contrast,of 43 patients who had IS off plus nephrectomy and /or transfusions, 34 (79%) made Abs (p=10 -6 ). Thus, ISoff is a pre-requisite for sensitization, but it also requires nephectomy, transfusions, or both. 7/30 (23%)patients who did not undergo nephrectomy became sensitized, as did 12/31 (39%) patients who were nottransfused and 4/15 (27%) who were neither nephrectomized nor transfused. In contrast, none of 11patients who continued on IS with or without nephrectomy or transfusions became sensitized (p=0.025 vseither nephrectomy or transfusions with IS off).Conclusions: Thus, IS is a key factor in sensitization. This suggests the possibility of using IS, at Txmaintenance doses, in selected patients (such as before nephrectomy and/or transfusions) to avoid thegeneration of broad Ab reactivity.49-PCLINICAL RELEVANCE OF PRETRANSPLANT DONOR-SPECIFIC HLA ANTIBODIES INRENAL TRANSPLANTATION.Eun Young Song 1 , Yu-joo Lee 1 , Shin Young Joo 1 , Jung Won Hyun 1 , Yon Su Kim 2 , Jongwon Ha 3 , SangJoon Kim 3 , Myoung Hee Park 1 . 1 Laboratory Medicine, Seoul National University College of Medicine,Seoul, Korea; 2 Internal Medicine, Seoul National University College of Medicine, Seoul, Korea; 3 Surgery,Seoul National University College of Medicine, Seoul, Korea.Aim: This study was performed to evaluate the clinical relevance of pretransplant donor-specific HLAantibodies (DSA) and their acceptable levels, for which still controversies exist.Methods: A total of 28 patients showing positive flow crossmatch (T and/or B) results at the time oftransplantation or historical peak sera were selected and retrospectively tested for the presence of class Iand/or class II DSA at the time of transplantation using Luminex single antigen assay (LIFECODES LSA,Tepnel Lifecodes). Twelve patients underwent pretransplant desensitization therapy and all of the 28patients had negative T cell CDC crossmatch at the time of transplantation. DSA levels were determined bysum of MFI values to donor-specific HLA antigens: class I, class II, and total (class I + II). Correlation ofthe DSA levels with occurrence of biopsy confirmed acute rejection (AR) and graft survival was evaluated.Results: Sixteen of 28 patients (57.1%) had DSA. The incidence of AR was significantly higher in patientswith DSA than in patients without DSA (56.3% [9/16] vs. 8.3% [1/12]; P=0.02), but three-year allograftsurvival was not different between the two groups. The total DSA levels were significantly higher inpatients with antibody-mediated rejection (AMR) than in patients without AMR (17,667 vs 3,417,P

average difference between Ave and Allelic DSA was 65 +/- 2422 MFI (p=0.8, not significant). For multialleleantigens, reporting Max MFI overestimated MFI values for 65% of DSA, as compared to allelespecific MFI values. Ave DSA was inconsistent, under- and over-estimating MFI values 44% and 56% ofthe time, respectively. 43 Max DSA were re-classified as negative when reported as allele specific DSA.Variability was most notable for the DQ locus which comprised 30% of all DSA. For DQ DSA the averagedifference between Max and Allelic DSA was 3555 +/- 4627 MFI (p80%concordance.[table1]Table 2 lists ratio and % average consensus. Ratio ≥0.3 correlates with >80%consensus.[table2]Conclusions: We conclude that a MFI of ≥2000 results in >80% concordance and can be a standardizedMFI cutoff between labs. If one calculates ratio, cutoff based on this data and 80% concordance would be0.3. Antibodies identified with >80% consensus included A, B, and DR. Antibodies with

Conclusions: These results demonstrate that, despite markedly improved sensitivity, the use of solid phasemethods to identify HLA-specific antibodies may provide apparently aberrant results that require carefulclinical and laboratory investigation.53-PTHE IMPACT OF ALLOSENSITIZATION ON TIME TO TRANSPLANTATION ANDMORTALITY IN A COHORT OF PATIENTS AWAITING LUNG TRANSPLANTATION.Joseph T. Weber 1 , Bryan F. Meyers 2 , Alexander Patterson 2 , Elbert P. Trulock 1 , ThalachallourMohanakumar 3 , Ramsey Hachem 1 . 1 Division of Pulmonary & Critical Care Medicine, WashingtonUniversity School of Medicine, Saint Louis, MO, USA; 2 Division of Cardiothoracic Surgery, WashingtonUniversity School of Medicine, Saint Louis, MO, USA; 3 Department of Surgery, Washington UniversitySchool of Medicine, Saint Louis, MO, USA.Aim: To determine if allosensitization has an impact on the rate of transplantation, waiting time, or wait listmortality in those awaiting lung transplantation.Methods: We performed a retrospective cohort study of 64 consecutive candidates who were active on thewaiting list at our program between 01/2006 and 07/2007. Patients were prospectively screened forpreformed anti-HLA antibodies using the LABScreen Single Antigen assay at the time of listing. Acalculated Panel Reactive Antibody (cPRA) was obtained using the UNOS calculator for each candidate.We analyzed time to event using the Kaplan-Meier and Cox regression methods.Results: Of the 64 candidates, 22 (34%) were sensitized and 17 (26%) had a cPRA>50%. Sensitizedcandidates were significantly less likely to undergo transplantation using the virtual crossmatch approach(log rank p=0.0006).[figure1]In a multivariate Cox regression analysis, cPRA>50% was a significantnegative predictor of transplantation (OR=0.28; 95%CI: 0.1-0.6, p=0.001). Additionally, 4 of the 42 (10%)non-sensitized patients died while on the waiting list compared to 5 of the 22 (23%) sensitized patients (p =0.004). All 5 sensitized patients who died on the waiting list had a cPRA>50% while none of the sensitizedpatients with a cPRA

Conclusions: The XM-One assay can be used to detect donor specific anti-endothelial antibodies in asimple flow-cytometric cross match assay. The endothelial antigen system seems to be polymorphic. Theclinical significance of this assay is still under investigation.55-PDONOR SPECIFIC ANTIBODIES (DSA) – COMPLEMENT BINDING OR NOT? THE IMPACTON KIDNEY REGRAFT OUTCOMES.Isabelle G. Wood, Nabil Mohsin, Indira Guleria, Helen H. Mah, Edgar L. Milford. Tissue TypingLaboratory, Brigham and Women’s Hospital, Boston, MA, USA.Aim: Our goal was to evaluate the impact of DSA not detected by cytotoxic crossmatches (CDC) on graftoutcome in retransplant kidney recipients. Do flow cytometry crossmatches with T and B cells(TFXM,BFXM), solid phase Single Antigen (SA) screening (One Lambda), and SA modified to identifycomplement (C’) binding results predict graft outcome?Methods: We analyzed outcome in 103 kidney recipients from 14 centers in New England who had faileda prior transplant. At the time of transplant, the T cell cytotoxic screening and CDC T cell crossmatch withanti-globulin were the only immunologic exclusionary criteria. To determine the impact of DSA notdetected by CDC we tested pretransplant sera by TFXM, BFXM and SA for Class I and II anti-HLAantibodies. In addition, a modified SA technique (C’SA) was performed using a complement sourcefollowed by mouse anti-C4d and anti-mouse phycoerythrin.Results: The table shows % 1 year graft survival in regraft patients. PEL= those with early loss of a priorgraft. C’DSA data includes only patients with positive DSA.[table1]Conclusions: Previous early graft loss, T or BFXM+, DSA I+, DSA II+ and C’DSA I+ were risk factorsfor graft loss. PEL patients clearly benefit from SA and FXM testing. Complement binding antibodiesdetected with C’SA need further study in a larger population of primary and regraft patients to ascertaintheir significance.56-PFACTORS AFFECTING THE RELATIONSHIP BETWEEN SINGLE ANTIGEN ANTIBODYASSAYS AND CROSSMATCH RESULTS.Danny Youngs, Paul Warner, Karen Nelson. Immunogenetics Laboratory, Puget Sound Blood Center,Seattle, WA, USA.Aim: Prediction of crossmatch (XM) results from single antigen bead assays is imperfect. Two variablesthat might contribute to the problem were evaluated. Experiments were run to determine the effect bead-tobeadcompetition for antibodies had on single antigen MFIs, and the effect multiple target antigens on cellshad on XM strength.Methods: LABScreen Single Antigen Combi (LSAC) and LABScreen Single Antigen Singles (LSAS)(One Lambda, Inc.) were tested against mouse anti-HLA monoclonal antibodies (monoclonals werespecific for one, a few, many or all class I HLA beads). Monoclonal antibodies were used to ensure that theantibodies were single specificities, and diluted so that each gave an MFI of approximately 4000 against A2or A3 LSAS bead. Sera with known antibodies were flow crossmatched against cells bearing one, two,three or four antigens that were the targets of the antibodies.Results: For the LSAC assay, as the number of target beads for the monoclonals increased, the MFIsdecreased substantially, roughly 2-3% for each additional target bead (for Bw4, with 24 target beads, aroughly 50% decrease in MFI is predicted). In the XMs, as the number of antibody targets increased, theflow XM shifts increased.Conclusions: Competition for antibodies in the LSAC assay can cause significant reductions in MFIs whentarget epitopes are shared by antigens on many different beads. In contrast, multiple antigen targets on XMcells have the opposite affect: shifts increase with increasing numbers of targets. These differing dynamicscan significantly impact the relationship between an MFI level in an LSAC assay and the strength of a flowcytometry XM. The competitive effect in the LSAC assay and the additive effect in the MX make therelationship between the two tests highly variable, and adversely affect the ability to successfully predictflow XM results from single antigen test results.

57-PVIRTUAL CROSSMATCH SIGNIFICANTLY INCREASES ACCESS FOR SENSITIZEDPATIENTS TO HEART TRANSPLANTATION.Qiuheng Zhang 1,3 , Britt Jonathan 1,3 , Michael J. Cecka 1,3 , David W. Gjertson 1,3 , Jon Kobashigawa 2,3 , AbbasArdehali 2,3 , Elaine F. Reed 1,3 . 1 Department of Pathology and Laboratory Medicine, UCLA ImmunogeneticsCenter; 2 Division of Cardiology; 3 David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.Aim: Presensitization to HLA is a major barrier to heart transplantation. Recent advances in solid-phasetesting allow identification of anti-HLA antibodies with high sensitivity and specificity. This permitsaccurate crossmatch prediction and increases the transplantation of sensitized heart candidates and the useof hearts from distant regions.Methods: 238 patients transplanted from January 2006 to July 2009 were analyzed retrospectively;158heart recipients were transplanted based on flow cytometry crossmatches (FCXM) alone prior toimplementation of the virtual crossmatch (vXM) in June 2008, and since then 80 patients were transplantedaccording to vXM.Results: There were significantly more sensitized (PRA > 0%) patients transplanted by vXM (24/80, 30%)than during the previrtual period (28/158, 18%; p = 0.01). The previrtual transplant rate was 62% comparedto 75% with a vXM in the first year. In addition, 71/158 (45%) donors were imported from outside OPO inthe previtrual period compared to 40/80 (50%) in the vXM period. There was no significant difference inwaitlist mortality between the 2 periods. A comparison between actual FCXM with vXM showed that vXMaccurately predicted all crossmatch results. 20/82 vXM patients had a positive FCXM, 12 of whom had nodetectable HLA antibodies. The remaining 6 patients were presensitized to HLA but did not display anydonor-specific antibodies to HLA. 13/20 patients’ FCXM results were rendered negative with pronasetreatment. Although significantly reduced, 7 patients’ FCXM remained borderline positive after pronasetreatment.Conclusions: In conclusion, the comprehensive evaluation of HLA antibodies and the application of vXMincreased the transplantability of sensitized patients and the geographic size of the donor pool.[table1]58-POBSERVED INCREASE IN MFI OF DONOR SPECIFIC ANTIBODY UPONIMPLEMENTATION OF DTT SERUM TREATMENT.Carolyn L. Fisher, Heather A. Casey, Thomas L. Bement, Michele E. Lachance, Dennis F. Habig,Kimberly J. Goss, Justine L. Gaspari, Ronald E. Domen, Hiroko Shike. HLA Laboratory, Penn StateHershey Medical Center, Hershey, PA, USA.Aim: Naturally occurring serum substances can interfere with Luminex antibody assay, particularly singleantigen beads (SAB). Efficacy of hypotonic dialysis and DTT treatment were reported to inactivate suchsubstances (Zachary 2009; Kosmoliaptsis 2010). We made similar observation and implemented DTTtreatment for Luminex assay in March 2010. Observed increase in MFI of donor specific HLA antibody(DSA) upon DTT-implementation may be confused with true DSA increase, a risk indicator of antibodymediatedrejection. We evaluated the impact of DTT treatment in our post-transplant patient population.Methods: DTT treated serum (20µL 50 mM DTT + 180 µL serum) was tested by One Lambda LABScreenPRA Class I and II and LABScreen Single Antigen. Average DSA MFI from the last four consecutiveresults without DTT was compared with the first result after DTT implementation.Results: DSA has been detected by SAB for over a year in seven patients 2.3 to 15 years post kidneytransplant. One patient had Class I and II DSA and six patients had Class II DSA only. DSA-MFI (range:3,400 – 13,000) were stable before DTT implementation (CV of 1-28% in last four dates tested). AfterDTT implementation, DSA-MFI increased >1.5x by SAB but not by PRA beads in 5 patients. One patienthad increased DSA-MFI (2.2x) by PRA beads only. One patient had no changes by both SAB and PRAbeads. None presented antibody mediated rejection. Observed increase in DSA-MFI was attributed to DTTimplementation. Retrospectively, positive control bead MFI was not informative in predicting increasedDSA-MFI after DTT-treatment.Conclusions: DTT-treatment can increase MFI of Luminex-based HLA antibody detection including DSA-MFI. Observed increase in DSA-MFI coinciding with DTT-implementation should be communicated toclinicians to prevent a false alert. Once DTT is implemented, consistency in assay procedure is critical inpost-transplant monitoring.

59-PCYTOTOXIC AND NON-CYTOTOXIC ANTI-HLA ANTIBODIES IDENTIFIED IN LUNGTRANSPLANT RECIPIENTS.Geo Serban 1 , Eric Ho 1 , Hanna Dobrowolska 1 , Melissa Hallar 1 , Rodica Vasilescu 1 , Nicole Suciu-Foca 1 ,Selim Arcasoy 2 , Adriana I. Colovai 1 . 1 Pathology, Columbia University, New York, NY, USA; 2 Medicine,Columbia University, New York, NY, USA.Aim: The role of allospecific antibody production in lung transplantation is not clearly understood. Weevaluated the impact of anti-HLA IgG and IgM antibodies (Abs) on the survival of lung transplant patients.Methods: Sequential pre- and post-transplantation sera were obtained from 147 consecutive patientstransplanted at our institution from 2007 to 2009. Anti-HLA Abs were detected using complementdependentcytotoxicity (CDC) and solid-phase assay (Luminex platform). Mean follow-up posttransplantationwas 21 months.Results: All patients had a negative CDC crossmatch (XM) at the time of transplantation. The frequency ofpatients displaying HLA panel reactive IgG antibodies increased from 1.4% prior to transplantation to 6.8%post-transplantation (p

61-PANTI-HLA AND ANTI-MICA SPECIFIC ANTIBODIES DEVELOPED DURING THE FOLLOW-UP THREE YEARS CAN PREDICTIVE OF ACUTE REJECTION AND RENAL GRAFTFUNCTION.Jun He, Jian Quan Hou, Xiao Ni Yuan. Jiangsu Institute of Hematology, The First Affiliated Hospital ofSoochow University, Suzhou, Jiangsu, China.Aim: The purpose of this study was to detect dynamic for HLA and MICA could predict development ofacute rejection (AR) and kidney allograft function.Methods: 41 kidney transplant patients were tested for anti -HLA Abs and anti-MICA Abs. 37 patientswere screened using single-antigen beads to determine the HLA and MICA-specific antibody levels at 0,30, 90, 180, 360, 720 and 1080 days of posttransplantation. The patients and donors of HLA and MICAallele typing were determined by PCR-SSOP and DSA and NDSA.Results: 9 patients(21.95%) had pre-existing antibodies in 41 transplantation patients.among them 6patients had anti-MICA antibodies, 2 patients had anti-HLA antibodies, one patient had anti-MICAantibodies and anti-HLA antibodies.Patients with pre-existing anti-HLA Abs had higher acute rejection(66.7%) than patients with pre-existing anti-MICA Abs (33.3%)and patients without Abs (27.3%, (P

data suggests that DTT pretreatment of sera should be considered for all sera to minimize interference onSAB by Luminex.63-PPAINFUL FAMILY “MEMORIES”?Siva Kanangat 2 , Michele H. Prod 1 , Ina Kurbegovic 1 , Andre’s Jaramillo 1 . 1 Rush Medical Laboratories, RushUniversity Medical Center/HLA/Pathology, Chicago, IL, USA; 2 Pathology, St. Jude Children’s ResearchHospital, Memphis, TN, USA.Aim: Aim: To investigate two cases of acute renal transplant rejection despite total absence of HLAantibodies in recipients’ pretransplant sera and negative T/B cell cross match prior to transplantation.Methods: Patients/Donors: Two female living donor renal transplant recipients, a 62 year old female whoreceived kidney from her child, and a 58 year old female who received kidney from her husband, bothrejected the allograft shortly after transplantation. Alloimmune response analysis: Flow cytometry basedHLA antibody screening, Luminex-Microbead array based Labscreen PRA, Flow Cytometry based T and Bcell Cross match, and CDC-AHG Cross match were used for pretransplant HLA antibody detection. Posttransplant HLA antibodies were detected by single antigen microbead array (One Lambda).Results: Pretransplant sera were negative for both Class I and Class II antibodies and both Flow Cytometryand CDC-AHG cross matches using T and B cells prior to transplant were also negative in both patients.Post transplant serum from both patients had significant levels of multiple DSA as detected by Singleantigen bead assay.Conclusions: Both patients could have been exposed to their donor’s HLA prior to their transplants. Therecipient mother would have had exposure to the child’s paternally derived HLA during gestation.Similarly, the patient who received kidney from her husband would have had exposure to his HLA throughpregnancies. Although such exposure to foreign antigens during gestation could result in tolerization, itcould also result in alloimmunization. Both patients may have had memory cells to their donor’s HLA.Some long-lived plasma cells may not respond to certain immunosuppressants resulting in vigorousresponse upon contact with recall antigen after transplant. Therefore, a pretransplant negative cross do notcompletely rule out the possibilities of an undesired anamnestic alloimmune response.64-PA NEW RECIPE FOR RENAL ALLOGRAFT BIOPSY – A NOVEL TOOL FOR THEDIAGNOSIS OF ABMR.Janette Kwok 1 , Gavin S.W. Chan 2 , M.F. Lam 3 , Tony Yan 1 , Lydia Tang 1 , K.M. Kwong 1 , K.W. Chan 2 , T.M.Chan 3 . 1 Tranpslantation and Immunogenetics, Queen Mary Hospital, Hong Kong SAR, China; 2 Pathology,The University of Hong Kong, Hong Kong SAR, China; 3 Medicine, The University of Hong Kong, HongKong SAR, China.Aim: Renal allograft biopsy has always been used for morphological confirmation of acute or chronicrejection affecting the transplanted kidney. The donor Human Leucocyte Antigen (HLA) phenotypes areoften not available in patients who underwent transplantation outside of Hong Kong. Yet this information isessential for the determination of donor-specific antibodies (DSA) in the management of antibody mediatedrejection (ABMR), and in the selection of suitable donors for patients who require a re-transplant. Theobjective of this study is to determine mismatched donor HLA from allograft kidney biopsies in transplantrecipients with unknown donor HLA phenotype.Methods: Allograft kidney biopsies were obtained in 14 renal transplant recipients who presented withgraft dysfunction. Donor HLA phenotypes were known in 7 patients. DNA was extracted from freshallograft kidney specimens, and HLA typing was performed with PCR-SSP and PCR-SSO.Results: The deduced donor HLA phenotypes were confirmed in 7 allograft renal biopsies, i.e. HLA resultsbeing the composite of both the recipient and the donor HLA phenotypes. This methodology was validatedin 7 allograft biopsies with known donor HLA phenotypes.Conclusions: This novel methodology overcomes the challenge with unknown donor HLA in kidneytransplant recipients, and is thus a useful tool for the detection of DSA, the management of ABMR, and theselection of suitable donors for re-transplantation.

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.

Bhavna Lavingia 1 , Chantale Lacelle 1 , Christopher Lu 2 , Miguel Vazquez 2 , Juan Arenas 3 , Mouin Seikaly 4 ,Peter Stastny 1,5 . 1 Pathology, UT Southwestern Medical Center, Dallas, TX, USA; 2 Internal Medicine -Nephrology, UT Southwestern Medical Center, Dallas, TX, USA; 3 Surgery - Surgical Transplantation, UTSouthwestern Medical Center, Dallas, TX, USA; 4 Pediatrics, UT Southwestern Medical Center, Dallas, TX,USA; 5 Internal Medicine - Transplant Immunology, UT Southwestern Medical Center, Dallas, TX, USA.Aim: Kidney transplant recipients with acute dysfunction can be analyzed for events in tissue and blood.Tissue histology, complement deposition and donor-specific HLA antibodies (DSA) were studied tocorrelate between DSA and biopsy findings.Methods: Sera from 41 kidney transplant recipients with acute dysfunction, suspected to be rejecting, werestudied between 2004 and 2009 for de novo production of HLA antibodies, biopsy readings and C4ddeposition. All were transplanted with negative T cell AHG, and negative T cell and B cell flow cytometrycross-matches.Results: New antibodies against donor HLA developed in 24/41 (59%) recipients. Among the DSAnegativerecipients, 11 (27%) had no antibodies to HLA, 4 (10%) developed HLA antibodies not donorspecific and 2 (5%) had antibodies cross-reactive with donor HLA. In 6 of the 24 (25%), DSA were HLAclass I, in 9 (38%) HLA class II and in 9 (38%) HLA class I and class II. Interestingly, among the 18recipients with DSA HLA class II, 15 (83%) had only anti-HLA-DQ and not DR. Biopsies were availablein 37 recipients, 22 had DSA and 15 without DSA. Among the 22 with DSA, 12 (55%) recipients had C4ddeposition in peritubular capillaries. In the 15 without DSA, only one (7%) had deposition of C4d(p=0.002). All 13 recipients with C4d deposition had histological evidence of rejection. In the groupwithout C4d deposition, 5/25 (20%) recipients had histological rejection: 3 cellular and 2 humoral.Presence of DSA was significantly associated with rejection by biopsy (p

69-PSENSITIZATION BY HLA-C CAN RENDER HLA-B ANTIGENS AS UNACCEPTABLEMISMATCHES.Marilyn Marrari, Rene J. Duquesnoy. Transplant Pathology, University of Pittsburgh Medical Center,Pittsburgh, PA, USA.Aim: Recent reports have shown that anti-HLA-C antibodies (Abs) are associated with rejection and graftfailure. DNA-based methods can accurately determine HLA-C types, and sensitive screening assays such asLuminex can identify HLA-C Abs. HLA-C antigens (Ags) display considerable amino acid polymorphismthat can be translated into a structurally defined epitope repertoire. HLAMatchmaker can be used toidentify donor-specific Ab reactivity patterns against mismatched critical components of epitopes (eplets)found on HLA-C Ags.Methods: We analyzed Ab reactivity patterns in sera from patients mismatched for HLA-C with rejectedrenal transplants that were screened in Luminex assays using class I single allele beads. Specificabsorptions were performed using microbeads bearing single class I alleles and eluates were tested byLuminex.Results: We recently reported a case of sensitization of a B*4403 patient to the 156DA epitope on anHLA-Cw*0704 mismatch which led to Abs reacting with the 156DA epitope-carrying B*4402 allele andother HLA-B Ags, including B*0801, B*3701, B*4101, B*4201, B*4501 and B*8201 (Hum Immun 71:176-8, 2010). We report here four more cases whereby absorption/elution assays clearly demonstrate thatAbs induced by a HLA-C mismatch reacted with epitopes shared with HLA-B Ags:[table1]Conclusions: Our findings demonstrate the importance of HLA-C mismatching in humoralallosensitization. Some HLA-C Abs are specific for epitopes shared with HLA-B Ags which then becomeunacceptable mismatches although the patient may have never been exposed to them. HLAMatchmaker isuseful in understanding donor-specifc Ab reactivity patterns and the determination of HLA mismatchacceptability for transplantation.70-PAUTOANTIBODIES AGAINST NUCLEOLIN IN KIDNEY TRANSPLANT RECIPIENTS.Zhiqiang Qin 1 , Bhavna Lavingia 2 , Yizhou Zou 1 , Peter Stastny 1,2 . 1 Internal Medicine - TransplantImmunology, UT Southwestern Medical Center, Dallas, TX, USA; 2 Pathology, UT Southwestern MedicalCenter, Dallas, TX, USA.Aim: Nucleolin is expressed on the surface of endothelial cells in the course of angiogenesis.Autoantibodies against nucleolin have been previously described in patients with chronic graft-versus-hostdisease.We investigated antibodies from kidney transplant recipients which react with endothelial cells.Methods: Sera from 50 kidney transplant recipients were investigated for antibodies against humanumbilical vein endothelial cells (HUVEC). One serum was used for immunoprecipitation and proteinidentification. Presence of nucleolin was confirmed with a nucleolin-specific monoclonal antibody.Nucleolin isolated from endothelial cell lysates was used for analysis of patient sera by Western blotting.The effect of antibodies against nucleolin on HUVEC was studied by assessing cell proliferation, apoptosisand in vitro tube formation.Results: Antibodies against nucleolin identified a band of approximately 100 kDa by SDS-PAGE. Resultsof mass spectrometry showed that sequences of nucleolin were identified. We tested 50 sera obtained fromkidney transplant recipients after rejection and found that 7 (14%) had antibodies against nucleolinenrichedpreparations obtained from endothelial cell lysates by Western blot analysis. The frequency ofsuch antibodies should be higher if a more sensitive assay is used. After incubation with antibodies againstnucleolin the number of HUVEC was decreased, possibly by apoptosis. Tube formation by endothelial cellsin vitro was inhibited.Conclusions: Autoantibodies against nucleolin were identified in kidney transplant recipients who hadrecently undergone immunological rejection of their allografts. Such antibodies have previously beenreported in bone marrow transplant recipients with chronic graft-versus-host-disease. The potential role ofthese autoantibodies in organ allograft rejection is suggested by their effect on angiogenesis and inhibitionof endothelial cell proliferation.71-P

ATG INTERFERNCE IN SINGLE ANTIGEN LUMINEX TESTING: TO TREAT OR NOT TOTREAT.Marilyn Wetmore, Patricia Kimble, Michael Moritz, Robert Cirocco. HLA Laboratory, Lehigh ValleyHealth Network Hospital, Allentown, PA, USA.Aim: This study was conducted to determine if Anti Thymocyte Globulin (ATG) interferes with the meanintensity fluorescence (MFI) generated by the Single Antigen (SA) Luminex anti-HLA bead array. ATG isgiven as an induction agent to inhibit lymphocyte activation at the time of renal transplantation. It is presentin the sera for up to one month after administration. De novo antibody may not be observed in the SA if theinduction agent ATG masks low level production of anti-donor HLA antibodies. A similar study reportedno affects of ATG on the haplotype or ID kit sensitivity and specificity.Methods: Seven sera samples from post transplant patients treated with ATG were absorbed twice withSheep anti-Rabbit IgG Dynabeads® M-280 (Invitrogen, Deer Park, WI). The same sera samples were DTTtreated and run in parallel for the presence of both class I and class II antibodies by SA Luminex assay. Thepatients received ATG on day 0,3 and 5 post transplant. Each sample was tested before and after DTTtreatment and absorption with (Sheep anti-Rabbit IgG) Dynabeads® M-280.Results: The positive control value increased 1.5 times after treatment with DTT or M-280 over that of theuntreated sera. The negative control value is approximately the same after M-280 treatment and 1.5 timeshigher with DTT treatment Than that of the untreated sera. The PC/NC ratio is 1.2 times higher after DTTtreatment and 1.6 times higher after M-280 treatment. Most of the samples run had more antibodyspecificities identified after DTT treatment.Conclusions: After reviewing the data it is apparent that ATG does inhibit the MFI in SA assay. Aftertreatment with DTT and M-280 a modest increase in the average MFI is noted. In addition, DTT treatmentincreases the MFI of the positive controls and may increase the specifity of the assay. In conclusion, thetreatment of post transplant serum samples with DTT is effective and recommended.72-PEARLY DETECTION AND REDUCTION OF DONOR SPECIFIC ANTIBODY (DSA) IN POSTLUNG TRANSPLANT PATIENTS.Patricia Willey 1 , D. Phelan 1 , R. Hachem 2 , E. Trulock 2 , T. Mohanakumar 2 . 1 HLA Laboratory, Barnes-Jewish Hospital, St. Louis, MO, USA; 2 Dept. of Surgery, Washington University School of Medicine, St.Louis, MO, USA.Aim: Early detection of DSA (ratio ≥ 0.2) by Luminex Single Antigen (Lum SA) and subsequent treatmentof DSA lead to reduction and in some cases, total elimination of DSA.Methods: 556 post lung transplant (tx) patients; 490 living, 66 deceased were analyzed. Of 490 living, 164(34.1%) developed DSA. Of 66 deceased, 38 (57.62%) had DSA. Adult patients do not receive tx acrossknown DSA. In 2008, patients were protocol screened at 1, 3, 6 and 12 month intervals by Lum SA. Upondetection of DSA, treatment was initiated which included one dose Rituximab and six doses IvIg. 50% ofliving patients with DSA did well while the remaining had some degree of decreased lung function.Transplants ranged from 1992 to present, the majority since 2003.Results: Defined DSA is predominantly Class II, with the majority DQB*. With improved screeningmethods, DQB* identified in the past now includes DQA* antibodies. Of patients with DQB* antibodiesoriginally identified, n=60 (36.6%) have been redefined as DQA*. Class I antibodies are defined in anaverage of only 8.9% of recipients. Lum SA ratio were similar for all groups. Of living patients with DSAdetected in the first month, 48 of 71 or 67.6% totally eliminated their DSA post anti-rejection treatment.Subsequent screens showed the sustained absence of DSA. In contrast, only 10.5% of the patients who diedwere able to reduce or eliminate DSA. Delayed identification and/or treatment may very well be factors inour inability to eliminate DSA resulting in loss of graft.[table1]Conclusions: Improved detection methods and treatment at an early stage are beneficial in eliminatingDSA in post lung tx patients potentially decreasing the prevalence of chronic rejection.73-PPRELIMINARY DATA OF PCR-BASED CUSTOM ARRAY FOR ACUTE CELLULARREJECTION AFTER LIVER TRANSPLANTATION.

Tadafumi Asaoka 1 , Shigeru Marubashi 3 , Tomoaki Kato 4 , Seigo Nishida 2 , Panagiotis Tryphonopoulos 2 ,Akin Tekin 2 , Edie Island 2 , Jang Moon 2 , David Levi 2 , Gennaro Selvaggi 2 , Ichiro Takemasa 3 , HiroakiNagano 3 , Yuichiro Doki 3 , Masaki Mori 3 , Andreas Tzakis 2 , Phillip Ruiz 1 . 1 Transplant Laboratories,University of Miami Miller School of Medicine, Miami, FL, USA; 2 Surgery, University of Miami, Miami,FL, USA; 3 Surgery, Osaka University, Suita, Osaka, Japan; 4 Surgery, Columbia University MedicalCenter, New York, NY, USA.Aim: Despite recent advances in immunosuppressive therapy, acute cellular rejection (ACR) remains amajor challenge in liver transplantation. However, there are no specific biomarkers available to monitor thealloimmune response. We hypothesized that certain mRNAs provide critical regulation of an ensuingintragraft immune effector response. The aim of the present study was to identify molecular biomarkers forACR in liver allografts.Methods: We examined 21 liver biopsy samples consisted of Acute Cellular Rejection (AR: 10 cases, allgrade 2 or 3), Indeterminate ACR (IND: 7 cases, all grade 0) and Recurrent Hepatitis C (CHC: 4 cases,inflammatory grade 2 or 3) obtained from recipients after liver transplantation. RNA was isolated from theformalin-fixed paraffin-embedded (FFPE) biopsy samples and transcribed to cDNA. We utilized a PCRbased TaqMan Low Density Custom Array containing 96 assays for candidate transcriptomes selectedbased on our previous study (Differential transcriptome patterns for acute cellular rejection in recipientswith recurrent hepatitis C after liver transplantation. Liver Transpl 2009;15(12):1738-49). Relativequantification was done based on pooled normal liver using a comparative Ct method.Results: We successfully detected more than 80% of candidate transcriptomes using FFPE liver biopsysamples. We could simultaneously follow up selected candidate transcriptomes.Conclusions: It is sure that we need the large sample set to evaluate the utility of our custom plate anddetect the cut off values and predictive values of key molecules. But our method could potentially serve asnew appropriate diagnostic tool to monitor the rejection status.74-PCRITICAL ROLE FOR HOST T CELLS IN MIXED ANTIBODY MEDIATED REJECTION OFRENAL ALLOGRAFTS.Alice A. Bickerstaff Gaughan, Jiao-Jing Wang, Ronald P. Pelletier, Nicholas DiPaola, Patrick Adams,Sergey Brodsky, Tibor Nadasdy, Gregg A. Hadley. The Ohio State University.Aim: We have developed a mouse model of renal allograft rejection that mimics the common clinicalscenario of mixed cellular and antibody mediated rejection (mixed AMR) in sensitized recipients. The goalof the present study was to evaluate the role of T cells in promoting graft injury in this model.Methods: Mice were primed by donor skin rejection and then transplanted with renal allografts. To test therole of T cells in this model, primed recipients were treated with anti-CD8 and anti-CD4 mAbs on POD -3,-2, and -1. To test the role of donor-reactive antibodies and T cells, each were adoptively transferred intoimmunodeficient recipients of renal allografts.Results: Primed recipients acutely rejected renal allografts (mean survival = 8.6 ± 4.3 days) with allserologic and histologic features of mixed AMR. FACS analysis of graft infiltrating T cells revealed a vastpredominance of CD8 T cells. Surprisingly, CD8 T cell depletion did not prevent acute graft loss whereasCD4 T cell depletion prevented features of mixed AMR including elimination of donor-reactivealloantibodies from the serum long-term (>130 days). Analysis of long-term functioning grafts revealedgrafts void of PTC complement deposition but with evidence of chronic injury and an accumulation ofCD8+CD103+ cells. In transfer studies, anti-donor sera and MHC class I mAbs did not promote detectablerenal allograft injury in immunodeficient mice; in contrast, primed T cells were highly effective at elicitingfunctional graft destruction.Conclusions: These data are consistent with a critical role for host CD4 T cells as effectors that promoteacute destruction of renal allografts and thus challenge current dogma that anti-donor alloantibodies are thesole mechanism of graft injury during mixed AMR. These data further suggest a distinct mechanism forchronic rejection of renal allografts with CD8 T cells playing a key role.

75-PPREFORMED LOW REACTIVE ANTI-HLA ANTIBODIES ARE ASSOCIATED WITHMAJORITY OF EARLY ANTIBODY-MEDIATED REJECTION IN RENALTRANSPLANTATION.P. Brailey 1 , E.S. Woodle 2 , R.R. Alloway 2 , A.D. Tevar 2 , M. Cardi 2 , E. Portwood 1 , G. Mogilishetty 2 , A.Govil 2 , A.H. Rike 2 , R.C. Walsh 2 , G. Wall 2 , A.L. Girnita 1,2 . 1 Transplant Immunology Division, HoxworthBlood Center, University of Cincinnati, Cincinnati, OH, USA; 2 Division of Transplantation, Department ofSurgery.Aim: Early (

Leiden, Netherlands; 3 Biochemistry and Molecular Biology, Monash University, Melbourne, Australia;4 Microbiology and Immunology, University of Melbourne, Melbourne, Australia.Aim: The cross-reactivity of Epstein-Barr Virus (EBV EBNA3A) specific CD8 T-cells against allogeneicHLA-B*4402 has been shown to be dependent on presentation of self-peptide EEYLQAFTY by the targetantigen. Recently, we found that, allogeneic HLA-B*4402+ human umbilical vein endothelial cells(HUVECs) and proximal tubular epithelial cells (PTECs) are poor targets for EBV EBNA3A specific Tcells. We aimed to determine if this lack of recognition of HUVECs and PTECs (useful models of kidneytransplantation) was due to differential tissue presentation of the EEYLQAFTY self-peptide.Methods: The EEYLQAFTY peptide was exogenously loaded onto HLA-B*4402 and HLA-B*4403expressing HUVECs and PTECs. EEY peptide loaded, and unloaded, PTECs and HUVECs were thenincubated with serial dilutions of our EBV EBNA3A T-cell clone, in a four hour chromium release assay.Results: While HLA-B*4402 expressing PTECs were specifically lysed in proportion to the effector/targetratio by the EBNA3A T-cell clone, without peptide loading, lysis was greatly increased by exogenous EEYpeptide loading (15% vs 75%; p=0.0001). HLA-B*4402 expressing HUVECs were only lysed when loadedwith exogenous EEY peptide (0% vs 64%; p=0.0001). HLA-B*4403 expressing PTECs and HUVECs werenot lysed irrespective of peptide loading. The lack of recognition of endothelial and epithelial cells was notdue to the lack of ABCD3 gene expression. PTECs and HUVECs were also specifically targeted by anotheralloreactive T-cell clone (JS132) without exogenous peptide loading, suggesting that the lack of recognitionof HLA-B*4402+ endothelial and epithelial cells by the EBV EBNA3A T-cell clone was due to lack ofEEYLQAFTY peptide presentation.Conclusions: Organ specific (peptide dependent) alloreactivity may have important implications fortransplantation monitoring, rejection and tolerance induction.78-PCNI-FREE PROTOCOL IN RENAL TRANSPLANTATION: EVALUATION OF LONG TERMPOST-TRANSPLANT HUMORAL ALLOIMMUNE RESPONSE THROUGH PROTOCOLBIOPSIES WITH ANALYSES OF C4d DEPOSITION AND DETERMINATION OF HLAANTIBODIES BY LUMINEX ASSAY.K. Padros 1 , J. Petroni 2 , M. Carmen Rial 2 , M.B. Rodriguez 1 , O. Vanco 1 , O. Guardia 2 , D. Casadei 2 , E.Raimondi 1 . 1 PRICAI Fundacion Favaloro; 2 Instituto de Nefrología de Bs.As.Aim: CNI-associated nephrotoxicity and subclinical humoral rejection are important causes of long termallograft deterioration.We use a CNI-free immunosuppressive protocol based on Thymoglobulin induction,Sirolimus, MMF and steroids and evaluate the impact of post.transplant aloimmune response in a cohort oflong term (>3 years) CNI-free transplanted patients.Methods: 421 patients were treated with this immunosuppressive combination. 256 are on follow-up. Weselected a sample of 40 patients with 3 years of post-transplant evolution for Luminex testing of Class I andII HLA antibodies and for biopsies with determination of C4d. Biopsies were compared with implantbiopsy to evaluate the chronic evolution.Results: HLA antibodies: 11/40 were positive. 2 were donor specificity antibodies. No differences betweenAc + or – patients in terms of creatinine clearance (Ab - 54.44 ± 19.26 ml/min, vs Ab + 51.16 ± 21.18ml/min p=0.45) and proteinuria (Ab- 0.31± 0,92 mg/dl vs Ab + 0,17 ±0,27 mg/dl p= 0,65). Biopsies: Wereperformed 1703 ± 670 post transplant.We found CAN progression in 13 cases and no progression in 27cases. Histological progression was more frequent in HLA Ab + patients (5/11 vs 5/29, p= 0.07). C4ddeposition: 2 patients peritubular, with no antibodies ; 2 periglomerular, both had HLA Class II Ab and thebiopsy showed glomerulopathy.Conclusions: CNI-free immunosuppressive protocol is associated with: excellent results in terms of graftfunction and patient survival ; incidence of post transplant antibodies similar to that other IS combinations;low progression rate of histological lesions in the long term and better evolution in those patients Ab - byLuminex assay.The low positivity rate for C4d deposition may be due to the presence of no donor specificHLA antibodies.79-PIL-6 THROUGH INDUCTION OF AUTOIMMUNITY CONTRIBUTES TO DEVELOPMENT OFCHRONIC REJECTION.

Sabarinathan Ramachandran 1 , Naohiko Fukami 1 , T. Mohanakumar 1,2 . 1 Surgery, Washington UniversitySchool of Medicine, St. Louis, MO, USA; 2 Pathology & Immunology, Washington University School ofMedicine, St. Louis, MO, USA.Aim: In a murine model of chronic lung allograft rejection (Obliterative Airway Disease,OAD), wedemonstrated that administration of anti-MHC class I endobronchially results in activation of immuneresponses to self antigens K-a1 tubulin (Ka1T) and collagen V (ColV) resulting in epithelial metaplasia,cellular infiltration, fibrosis and occlusion of small airways. The objective of the study is to define earlyevents that contribute to induction of Th17 responses against self antigens which leads to development ofOAD.Methods: IL6 levels in serum and lungs of C57BL/6 mice treated with anti-MHC class I was measured byluminex and qRT-PCR. To define the role of IL6, IL6 KO mice were administered anti-H2kb or isotypeAbs. Lungs, spleen and serum were collected on day 30 and analyzed for development of immuneresponses to self antigens by ELISPOT, ELISA, neutrophil infiltration, H&E and trichrome staining.Results: Administration of anti-MHC class I resulted in a 4 and 5.3 fold increase in levels of IL-6 in serumand lungs. Analysis of frequency of Th17cells in IL-6 KO mice on stimulation with KA1T and COlVdemonstrated complete abrogation of induction Th17 T cells and antibodies to KA1T and COlV. IL6 KOmice demonstrated significant reduction in neutrophil infiltration in the lungs. In contrast, wild typeanimals demonstrated significant increase in levels of antibodies to KA1T,and COlV along with markedincrease in neutrophil infiltration. H&E staining of lungs from IL6KO mice demonstrated decreasedcellular infiltration, no detectable epithelial metaplasia or fibrosis.Conclusions: IL-6 through neutrophil infiltration increases the exposure of self antigens to the immunesystem and acts with TGF-b to induce Th17 responses that contribute to induction of autoimmunity anddevelopment of OAD. Blocking IL-6 signaling could prevent development of chronic rejection followinghuman lung transplantation.80-PLOW EFFICACY OF IVIG/RITUXIMAB IN DESENSITIZING EXTREMELY HLA-SENSITIZED PATIENTS.Paul Warner 1 , Karen Nelson 1 , Lisa Florence 2 . 1 Immunogenetics Laboratory, Puget Sound Blood Center,Seattle, WA, USA; 2 Dept. of Transplantation, Swedish Medical Center, Seattle, WA, USA.Aim: To provide an increased chance for deceased-donor renal transplantation, eight patients with anaverage cPRA of 99% (+/- 2%) were enrolled in a desensitization protocol, consisting of combinedIVIG/Rituximab therapy.Methods: High-dose IVIG (2 g/kg) was administered to each patient, followed by Rituximab (1 g) 14 dayslater, then a second high-dose of IVIG after an additional 14 days. The presence and strength of HLAantibodies before treatment, and monthly post-treatment, was determined with single antigen beads (SAB).Results: In the first month post-treatment, the mean cPRA demonstrated a slight decline (95% +/- 5%). Inthe second month, five patients demonstrated a mean cPRA of 93% +/- 8%. The greatest decline was seenin one patient, whose cPRA went from 95% to 81%. Two patients had no significant decline in cPRA at thetimepoints tested. Although all patients did demonstrate lower average MFI values at one- and two-monthspost-treatment, this decline was not enough to eliminate a significant number of unacceptable antigens(UA), as the majority of the antibodies were >10,000 MFI to begin with. By raising the cut-off forassigning UA from 2,500 to 4,000 MFI (and accepting the increased risk of a positive crossmatch), severalmore UA were eliminated from multiple patients. Two potential donors were identified for one patientwithin the same week. Unfortunately, each donor expressed two mismatched HLA that were the targets of2000-4000 MFI antibodies in this patient, and the crossmatches were strongly-positive, precludingtransplantation.Conclusions: In conclusion, this desensitization protocol appeared to confer no significant impact in thesepatients who had high cPRAs with high-titered antibodies. It is likely to be more useful in patients withmoderately-titered antibodies, or in identifying responders who may benefit from additional treatment.81-PIDENTIFICATION OF PROTEINS ASSOCIATED WITH INTEGRIN β4 IN THE HLA CLASS ISIGNALING PATHWAY IN ENDOTHELIAL CELLS.

Xiaohai Zhang, Elaine F. Reed. UCLA Immunogenetics Center, Department of Pathology, University ofCalifornia, Los Angeles, Los Angelse, CA, USA.Aim: Patients producing post-transplant anti-donor antibodies to MHC are at a higher risk of chronicrejection and transplant vasculopathy. Several studies suggest that the signaling events that occur inendothelial cells during interactions with MHC-I antibodies can contribute to the process of transplantvasculopathy. Previous studies have demonstrated that crosslinking of HLA class I molecules withantibodies activates intracellular signaling cascades and stimulates cell proliferation through the formationof a molecular complex with integrin β4. This finding prompted us to characterize the proteins associatedwith integrin β4 in the class I signaling pathway.Methods: Human aortic endothelial cells were stimulated with F(ab’)2 fragment of the class I antibodyW6/32 or control IgG. Cell lysates were immunoprecipitated with integrin β4 antibody or W6/32. Theimmuno-complex was directly digested by trypsin, or separated by SDS-PAGE and applied to In-geldigestion. Digested peptides were purified and analyzed by MALDI-TOF and tandem mass spectrometry toidentify new potential proteins in the complex.Results: MALDI-TOF analysis showed the immuno-complex of integrin β4 produced more than 20peptide peaks after trypsin digestion. We found 5 distinct peptide peaks only existed in the integrin β4complex in endothelial cells stimulated by class I antibodies, but not in cells treated with control IgG. Inaddition, In-gel digestion of HLA class I immuno-complex revealed a distinct peptide pattern.Conclusions: These peptide peaks may represent the novel proteins associated with integrin β4 after class Iligation. The proteomic approach described here will lead to a better understanding of class I signalingpathways which induce cell proliferation or survival in endothelial cells. This research will contribute to thedevelopment of new treatment options for prevention of transplant vasculopathy.82-PASSOCIATIONS BETWEEN HLA-C ALLELES AND MENIERE’S DISEASE.Mahsa M. Amoli 1 , Parvin Amiri 1 , Bagher Larijani 1 , Javad Tavakkoly-Bazzaz 2 , Nasrin Yazdani Yazdani 3 ,Hamed Emami 3 . 1 Otolaryngology and Head & Neck Surgery Research Centre, Tehran University ofMedical Sciences.Aim: Meniere’s disease (MD) is an autoimmune disorder characterized by fluctuating hearing loss andtinnitus, intermittent episodes of vertigo and aural pressure. Both genetics and environmental factors seemto play roles in the etiology of Meniere’s disease (MD). Several genes may be involved in susceptibility ofMD including Human Leukocyte Antigens (HLA). The associations between MD and HLA alleles havebeen previously studied in other populations and certain HLA alleles were shown to be predisposing. Theaim of this study was to determine the association between HLA-C allele frequencies and MD in an Iranianpopulation.Methods: Cases group consisted of (N=22) consecutive patients who were enrolled according to thediagnostic criteria of American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) for MD.Patients were recruited from the outpatient clinic in Amir-Aalam Hospital, Tehran, Iran. Controls werefrom the same area as cases. The study was approved by the Ethics committee of Tehran University.Informed consents were obtained from all of the patients attending the study.DNA from cases and controlswere extracted from anti-coagulated blood collected in EDTA using salting out method. HLA-Cw typingwas performed using Dynal AllSet+TM SSP kit.Results: The allele frequencies were significantly different between patients and controls. The allelefrequency of HLA-Cw*04 was 20% in the MD group, whereas it was only 1% in the control group [P =10-4, Pcorr = 0.0015, OR; 20, 95% CI (3.7-196.9)]. HLACw*16 allele frequency was also significantly higherin patients compared to the controls however the p value did not remain significant after Bonferronicorrection [P =0.01, Pcorr = 0.15, OR; 3.6, 95% CI (1.06-11.7).Conclusions: Our results revealed HLA-C is predisposing factor in definite MD in an Iranian population.

83-PALLELIC DIVERSITY OF KIR2DL4 AND KIR3DL2 IN THE POPULATION OF CURITIBA,BRAZIL.Danillo G. Augusto 1 , Marcia R. Pincerati 2 , Maureen P. Martin 3 , Mary Carrington 3 , Maria Luiza Petzl-Erler 1 . 1 Federal University of Paraná - UFPR, Curitiba, PR, Brazil; 2 University of São Paulo - USP, SaoPaulo, SP, Brazil; 3 National Cancer Institute/NIH, Frederick, MD, USA.Aim: Genetic polymorphism of killer cell immunoglobulin-like receptors (KIR) occurs at different levels.Most studied is the variation of gene presence/absence but there is little information about allelicpolymorphism. We aimed to describe the allelic diversity of KIR2DL4 and KIR3DL2 in a Brazilian urbanpopulation and to compare the frequencies to those of other populations.Methods: We analyzed 110 Euro-descendant individuals from Curitiba, capital city of Paraná State bysequence based-typing. For identification of 3DL2 alleles, we sequenced exons 3, 4, 5 and 7-9 and for2DL4 exons 3, 5 and 7-9. Allele frequencies were compared between populations by the exact test ofpopulation differentiation.Results: We have found 9 new 3DL2 alleles. The most frequent 3DL2 alleles were *002, *001, *007, *003and *005 (frequencies 0.268, 0.172, 0.141, 0.095 and 0.095 respectively). In addition, we observed thealleles *006, *008, *009, *010, *011 and *015. The allele frequency distribution did not differ significantlyfrom those of Northern Ireland and Euro-descendants from USA. It did differ from frequencies seen inJapan and China (P

85-PDIVERSITY OF KIR PROFILES IN COLOMBIAN POPULATIONS.Carolina Bonilla 1 , Emilio J. Yunis 3 , Juan J. Yunis 1,2,3 . 1 Instituto de Genética, Universidad Nacional deColombia, Bogotá, DC, Colombia; 2 Departamento de Patología, Facultad de Medicina, UniversidadNacional de Colombia, Bogotá, DC, Colombia; 3 Instituto de Genética, Servicios Médicos Yunis Turbay yCia, Bogotá, DC, Colombia.Aim: There are 16 Killer cell immunoglobin-like Receptors (KIRs) organized in haplotypes which arehighly variable in populations. This variability is define by differences in number and type of gene contentand by the allelic diversity of genes. Due to this variability, this gene family has been studied in severalethnic groups that is relevant to characterize human populations. Here we present the KIR haplotypes foundin a Caucasoid and an Afro-descendant populations from Colombia.Methods: KIR gene was estimated from 119 unrelated individuals randomly selected from Colombia: 70Afro-descendant (Pacific region) and 49 Caucasoid-mestizos (Andean region). The PCR amplification wasfollowed by liquid phase hybridization with Luminex technology (Lifecodes KIR-SSO typing kit). Thehaplotype frequencies and haplotype diversity were determined with Arlequin 3.11.Results: All 16 KIR genes were found in both Colombian populations, distributed in 59 different profiles.From framework genes, only KIR3DL3 gene was present in all haplotypes. Although haplogroup A isconsider the smallest haplotype, a haplotype formed by only 6 genes was found in the colombian Afrodescendantpopulation, a new haplotype not reported before for any other ethnic group. The caucasoidshowedmore haplotypes than the afro-descendant population (57% vs 29%, p

São Paulo, Brazil; 3 Commissariat à l’Energie Atomique, Service de Recherches en Hémato-Immunologie,Institut Universitaire d’Hématologie, Hôpital Saint-Louis, Paris, France.Aim: HLA-E, a non-classical major histocompatibility complex (MHC) class I molecule, presents fewpolymorphic sites in relation to other MHC-I molecules, exhibiting only nine alleles encoding threedifferent proteins described in worldwide human populations. Besides its known role on the innateimmunity, HLA-E has been reported as a ligand for T cells, potentially affecting allograft outcome. Due toits historical background, the Brazilian population is very suitable for polymorphic studies.Methods: Thirty-three samples randomly selected from a Brazilian normal blood marrow donor cohortwere evaluated in approximately 1200bp, encompassing the final portion of intron 2 and extending till exon4 of the HLA-E gene.Results: Four SNPs previously described were evaluated in the positions +756, +887, +906 and +1691.From these, only one (+756 G/A) was polymorphic in our sample, presenting a frequency of 0.6818 for theA allele, which in turn exclude the presence of the alleles E*01:03:03, *01:03:04 and *01:04 in the presentsample. More than 50% of the samples were homozygous for the E*0101 protein (A at the position +756).In addition, two new SNPs were detected, one at intron 2 and one at intron 3, which will be submitted torecognition by IMGT. Regarding linkage disequilibrium (LD) studies with neighbor genes (HLA-E, G andA), a high LD between A and G (coding and 3’UTR) was observed (p = 0.0000 for both); however, theSNP at HLA-E locus was not in LD with the HLA-A (p = 0.2031) or HLA-G (0.6294) loci, which mayreflect the presence of the high polymorphic HLA-A locus between HLA-G and HLA-E, or the longerphysical distance between HLA-E and HLA-A than between HLA-A and HLA-G.Conclusions: In conclusion, even in an admixed population such Brazilians, the HLA-E locus is veryconserved presenting only one polymorphic SNP in the coding region.88-PHLA-G 5’ REGULATORY REGION AND 3’ UNTRANSLATED REGION VARIABILITY IN THEBRAZILIAN POPULATION.Erick C. Castelli 1 , Celso T. Mendes-Junior 2 , Luciana C. Veiga-Castelli 1 , Philippe Moreau 3 , Eduardo A.Donadi 1 . 1 Division of Clinical Immunology, Department of Medicine, University of Sao Paulo, RibeiraoPreto, Sao Paulo, Brazil; 2 Departamento de Química, Faculdade de Filosofia, Ciencias e Letras deRibeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil; 3 Commissariat à l’EnergieAtomique/DSV/I2BM/Service de Recherches en Hémato-Immunologie, IUH, Hôpital Saint-Louis, Paris,France.Aim: The HLA-G gene is a non-classical class I HLA locus, predominantly expressed at the maternal–fetalinterface. It has been associated with maternal–fetal tolerance and in the inhibition of cytotoxic Tlymphocyte and Nk cytolytic functions. Several lines of evidence indicate that HLA-G polymorphisms atthe 5’ upstream regulatory region (URR) and 3’ untranslated region (3’UTR) may influence the HLA-Gexpression level. In contrast to the coding region, the 5’URR is highly polymorphic, with the presence ofseveral SNPs in transcription factors binding sites. Since the Brazilian population is a rich repository ofHLA variations, the variability of the HLA-G 5’URR and 3´UTR was characterized in 90 healthy donorsfrom Southeastern Brazil.Methods: The polymorphic sites were detected by direct sequencing and haplotypes were obtained byprobabilistic inferences.Results: Twenty-five variation sites were detected at the 5’URR in a region of 1350-bp before exon 1, allpresenting frequencies higher than 2% except for one SNP at the position -443, with a frequency of 0.5%.These SNPs were arranged in twelve haplotypes with frequencies varying between 0.5 and 32.7%. The3’UTR was also highly polymorphic, with 8 variation sites in a region of 350-bp. These 8 variation siteswere arranged in 8 specific 3’UTR haplotypes, with frequencies varying between 0.5 and 27.2%.Conclusions: In addition, high linkage disequilibrium among the variation sites of the 5’URR and 3’UTRwas detected, where each promoter haplotype was associated with a specific 3’UTR haplotype,demonstrating that the effect of each variation site in HLA-G expression may not be independent.FINANTIAL SUPPORT: CNPq/Brazil Grant 475670/2007-8 and CAPES/COFECUB Grant 653/09. E.C.Cis supported by FAPESP/Brazil, Grant 07/58420-4. L.C.V.C is supported by CNPQ/Brazil 150175/2009-4.

89-PHUMAN MONOCLONAL ANTIBODY REACTIVITY WITH HLA CLASS I EPITOPESDEFINED BY EPLET PAIRS.Rene J. Duquesnoy 1 , Marilyn Marrari 1 , Justin Mostecki 2 , Arend Mulder 3 , Ivan Balazs 2 . 1 TransplantPathology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 2 Gen-Probe TransplantDiagnostics Inc., Stamford, CT, USA; 3 Leiden University Medical Center, Leiden, Netherlands.Aim: It is now apparent that HLA antibodies (Abs) are specific for epitopes rather than antigens (Ags).Such epitopes can be structurally defined by HLAMatchmaker, which considers eplets as criticalcomponents of epitopes recognized by allo-Abs. We demonstrate here how eplets on immunizing Ags caninduce specific Abs.Methods: Fifty-four HLA class I human monoclonal antibodies (mAbs) derived from women sensitizedduring pregnancy were tested in Luminex assays using single allele beads. Epitope specificity wasdetermined from eplet differences between the immunizing Ag and HLA type of the Ab producer.Results: As expected, many mAbs (N=34) are specific for mismatched eplets predicted byHLAMatchmaker. In 20 cases the epitope corresponds to a mismatched eplet paired with a selfconfigurationpresent on an HLA Ag of the Ab producer. Examples of mAbs to eplets and epletpairs:[table1]In all pairs, the eplet and self-configuration are between 7 and 15 Ångstroms apart, sufficientdistance for contact by two separate complementarity-determining regions of Ab.Conclusions: The findings demonstrate that immunizing Ags have mismatched eplets that can form Abreactiveepitopes with self-configurations on the molecular surface. They suggest that HLA Abs are formedby autoreactive B-cells that have undergone receptor editing to accomodate recognition of non-self eplets,the driving force of a humoral alloresponse. This concept enhances our understanding of structural epitopeimmunogenicity and interpretation of Ab reactivity patterns.Mostecki: Gen-Probe Transplant Diagnostics Inc.: Employee. Balazs: Gen-Probe Transplant DiagnosticsInc.: Employee.90-PPOLYMORPHISM IN POSITION 156 INFLUENCES THE PEPTIDE REPERTOIRE OF HLA-B*4402 AND HLA-B*4403 MOLECULES.Hernando Escobar 1 , Eduardo Reyes-Vargas 2 , David K. Crockett 1 , Peter E. Jensen 1,2 , Julio C. Delgado 1,2 .1 R&D, ARUP Laboratories, Salt Lake City, UT, USA; 2 Pathology, University of Utah, Salt Lake City, UT,USA.Aim: Strong CTL-mediated allogeneic reactions have been reported between HLA-B*4402 and HLA–B*4403 alleles. These alleles differ only by a single residue located in position 156 of the alpha chain, withHLA- B*4402 expressing Asp and HLA-B*4403 expressing Leu. Position 156 influences the conformationof pocket D and E of the peptide binding groove of HLA class I molecules but has little influence on thespecificity of peptide anchor residues P2 and P9. This study was performed to investigate the influence ofposition 156 in the repertoire peptides presented by these two HLA-B44 subtypes.Methods: HLA-B44-peptide complexes were separated by immuno-affinity columns from whole celllysates of K562 cells expressing HLA-B*4402 and HLA-B*4403. Peptide pools were obtained by acidelution followed by HPLC fractionation and sequencing by collision-induced dissociation and time-offlightMS/MS.Results: We found that over 2/3 of peptides eluted from HLA- B*4402 differ from those eluted from HLA-B*4403. Close analysis of the peptide binding motif of non-over-lapping peptide sequences of these 2molecules revealed that in the non-anchor position P7, HLA-B*4402 prefers polar, uncharged and basicresidues such as Gly, Gln or His, whereas HLA*B4403 shows preference for non-polar residues Leu, Ile orVal. No differences were observed in the specificity of residues located in anchor positions P2 and P9.Conclusions: Position 156 has direct effect on the specificity of the residue located in position P7 of thepeptide binding motif of HLA-B*4402 and HLA-B*4403 alleles. Given that P7 position is important for T-cell receptor interaction and subsequent T-cell activation, we suggest that the polymorphism in position156between HLA-B*4402 and HLA-B*4403 alleles might play an important role in the alloreactivity betweenthese 2 alleles.

91-PCHARACTERIZATION OF COMMON AND WELL-DOCUMENTED HLA ALLELES INITALY: A MULTICENTER STUDY ON 24.000 UNRELATED INDIVIDUALS.Elisabetta Zino 1 , Annamaria Pasi 2 , Donata Mininni 3 , Elisabetta Sironi 1 , Marco Guarene 2 , GiovannaMongelli 3 , Benedetta Mazzi 1 , Cristina Capittini 2 , Roberta Morici 3 , Biagio Favoino 3 , Miryam Martinetti 2 ,Katharina Fleischhauer 1 . 1 Unit of Molecular and Functional Immunogenetics, Fondazione Centro SanRaffaele del Monte Tabor, Milan, Italy; 2 Immunogenetics Laboratory, Fondazione IRCCS Policlinico SanMatteo, Pavia, Italy; 3 Laboratory of Immunogenetics and Transplantation Biology, Azienda OspedalieraUniversitaria Policlinico, Bari, Italy.Aim: One of the major challenges in modern histocompatibility testing is the definition of common (genefrequency of >0.001) and well documented (observed in three or more unrelated individuals) HLA alleles(CWD) that need to be discriminated by high resolution typing because of their not negligible frequencyworld-wide. In 2007, the American Society of Histocomptibility and Immunogenetics (ASHI) reported atotal of 677 CWD HLA alleles for HLA-A,B,C,DRB1,DQB1,DPB1. The aim of the present study was todetermine CWD alleles in Northern and Southern Italy.Methods: A total of 24.000 unrelated individuals referring to three EFI accredited laboratories in Northern(Milano, n=7.000 and Pavia, n=14.000) and Southern Italy (Bari, n=3.000) were analyzed for CWD class Iand II alleles.Results: A total of 258 CWD alleles were found at the A (n=39/965; 4.0%), B (n=86/1543; 5.5%), C(32/626; 5.1%), DRB1 (55/762; 7.2%), DQB1 (19/107; 17.7%) and DPB1 (27/138; 19.5%) loci. Althoughthe percentage of CWD alleles in this cohort was lower (average 9.8%) as compared to the ASHI study(average 32.0%), 9 of the 258 Italian CWD alleles (3.4%) had not been reported as such before. These wereHLA-A*1112, B*1805, B*1807, DRB1*1124, DRB1*1321, DRB1*1325 (all with unique codingsequences for the antigen binding groove), and DPB1*0502, DPB1*0802, DPB1*0902. All these alleleswere described before 2006. Interestingly, the laboratory of Bari contributed 12.5% of total typings, but onethird (33.3%) of the new CWD alleles (B*1805; DRB1*1325; DPB1*0802) were found only by thislaboratory, in concordance with a high degree of unique polymorphisms in the gene pool from SouthernItaly.Conclusions: This study underscores the utility of integrated efforts to better define CWD alleles indifferent regions, in order to further improve the guidelines for resolution of ambiguities in 4-digit HLAtyping.92-PNON-CODING CLASS I SEQUENCES CONTAIN POLYMORPHISMS THAT ARE SPECIFICFOR CONSERVED EXTENDED/ANCESTRAL HAPLOTYPES.David C. Sayer 1 , Hayley S. Hogan 1 , Damian M. Goodridge 1 , Alison Castley 2 , Linda K. Smith 2 . 1 ConexioGenomics, Fremantle, Western Australia, Australia; 2 Clinical Immunology and Immunogenetics, RoyalPerth Hospital, Perth, Western Australia, Australia.Aim: The aim of the study was to determine the significance of novel HLA Class I non-codingpolymorphisms.Methods: Samples typed by SBT were retrospectively analysed using the non-coding analysis functionalityof Conexio Genomics’ Assign-SBT software.Results: Routine analysis of non coding HLA Class I sequences during SBT has revealed at least two novelalleles. One novel allele is identical to HLA-B*18:01:01 except for 3 differences located in intron 3, intron5 and the 3’ UTR. A retrospective analysis of intron 3 sequence data from 48 samples typed as B*18:01:01,or where B*18:01:01 is part of the ambiguity, revealed that 30/48 had the novel intron 3 polymorphism and18/48 were identical to the reference sequence. Additional analysis revealed that the novel intronpolymorphism was present on the A*25:01:01-B*18:01:01-DRB1*15:01:01 or 18.1 Ancestral Haplotypewhilst the alternative polymorphism was found on the A*30:02-B*18:01:01-DRB1*03:01:01 or 18.2Ancestral Haplotype. A second novel allele had a novel polymorphism in intron 1 of Cw*04:01:01. Aretrospective analysis of 62 samples typed as Cw*04:01:01 revealed that the novel intron 1 polymorphismwas seen in 10 of 34 samples that were Cw*04:01:01-B*35:01:01. The remaining 24/34 samples that wereCw*04:01:01-B*35:01:01 did not contain the novel polymorphism. Insufficient data was available to

determine if the novel polymorphism was Ancestral Haplotype specific. The novel polymorphism was notfound in any of the 28 samples where Cw*04:01:01 was present with alleles other than B*35:01:01.Conclusions: Non-coding analysis in SBT is important for unrelated BMT. Whilst some non-codingpolymorphisms may not be of functional significance, haplotype specific polymorphisms can be used indonor selection for unrelated bone marrow transplantation to improve matching.Sayer: Conexio Genomics: Employee; Stockholder. Hogan: Conexio Genomics: Employee. Goodridge:Conexio Genomics: Employee; Stockholder.93-PEXOGENOUS ANTIGEN CAN INDUCE CELL PROLIFERATION AND EXPRESSION OFMICA GENE IN ENDOTHELIAL CELL.Jian Quan Hou, Jun He, Yun Yan Wang. Urology, The First Affiliated Hospital of Soochow University,Suzhou, Jiangsu, China.Aim: To investigate the expression of major histocompatibility complex class I-related gene A (MICA) inendothelial cell stimulated with exogenous antigen.Methods: The endothelial cells were divided into control group and three experimental groups, which werestimulated with exogenous recombinant MICA antigen (group M) and heat shock protein-70 (HSP, groupH) at 5, 10 and 25ng/ml respectively for 48h. The expression of MICA mRNA was detected by real-timefluorescence quantitative PCR. The expression of MICA protein was measured by Western blot. Theexpression of MICA total protein on the cell surface was detected by using flow cytometry (FCM). Thelevels of soluble MICA (sMICA) in supernatant of all groups were detected by ELISA.Results: The expression of MICA mRNA and MICA protein were increased significantly after MICAstimulation (P < 0.05). The expression of MICA mRNA of group M 10 were remarkably higher than thegroup A 25 , but had no differences with group M 5 . The expression of MICA membrane protein of group A 10were higher than that of group M 25 (P < 0.05), but had no differences with group M 5 . The level of sMICAin three experimental groups were 35.8,27.4,21.8pg/ml respectively, which decreased obviously comparingwith that of control group (352.5pg/ml). These differences had statistical significances (P < 0.05). But therewas no significant difference among the experimental groups (P > 0.05). However, the expression of MICAgene and sMICA level didn’t change after HSP stimulation.Conclusions: The exogenous MICA antigen up-regulate the expression of MICA mRNA and protein,especially the membrane protein on the cell surface significant increase, but sMICA in supernatantdecreased.Hou: First Affiliated Hospital of Soochow University: Science Med Advisor. He: First Affiliated Hospital ofSoochow University: Science Med Advisor. Wang: First Affiliated Hospital of Soochow University: ScienceMed Advisor.94-PPHYSICAL LINKAGE OF GENES ADJACENT TO KIR2DL5 IDENTIFIES THIRTY ALLELE-LEVEL HAPLOTYPES IN AN AFRICAN AMERICAN POPULATION.Lihua Hou, Minghua Chen, Kanthi Kariyawasam, Jennifer Ng, Carolyn Katovich Hurley. GeorgetownUniversity, C.W. Bill Young Marrow Donor Recruitment and Research Program, Rockville, MD, USA.Aim: Natural killer cell immunoglobulin-like receptors (KIR) are a family of 15 receptors that detectmalignant or virally infected cells. Here, we determine the nature of KIR2DL5 gene polymorphism in 100random African Americans using DNA sequencing.More than half of the individuals (55%) carriedKIR2DL5 and the majority carried KIR2DL5B. Five KIR2DL5A alleles and 15 KIR2DL5B alleles wereidentified by DNA sequencing; 11 of these alleles were novel.Methods: The 20 KIR2DL5 alleles were physically linked to alleles at adjacent KIR loci to define thisregion of KIR haplotypes. Methods to obtain haplotype-specific long segments of DNA for sequencingincluded Haplotype Specific Extraction and long range PCR.Results: Based on linkage of KIR2DL5 with KIR2DS1, KIR2DS3, KIR2DS5, KIR2DL2, KIR2DL3, andKIR3DS1 alleles, 7 haplotypes of KIR2DL5A and 23 haplotypes of KIR2DL5B were observed. KIR2DL5alleles were linked to either KIR2DS3 (6 alleles) or KIR2DS5 (7 alleles). All of the KIR2DL5A alleles werelinked either to KIR3DS1*01301 or KIR3DS1*049N. The majority of the KIR2DL5B alleles were linked toseven KIR2DL2 alleles; two were linked to a novel allele of KIR2DL3.

Conclusions: The extensive diversity of KIR allele-level haplotypes present in this population isremarkable.95-PKIR3DL1/S1 ALLELES AND THEIR FREQUENCIES IN A SEX WORKER POPULATION INNAIROBI, KENYA.David La 1 , Rae-Anne Hardie 2 , Ma Luo 1,2 , Francis A. Plummer 1,2,3 . 1 HIV and Human Genetics, PublicHealth Agency of Canada, Winnipeg, MB, Canada; 2 Department of Medical Microbiology, University ofManitoba, Winnipeg, MB, Canada; 3 Department of Medical Microbiology, University of Nairobi, Nairobi,Kenya.Aim: NK cells are important in the innate defence against viruses, due to their ability to directly killinfected cells and rapid cytokine and chemokine secretion. Such functions enable NK cells to recruit andinduce stimulatory and regulatory effects on cells of the adaptive immune response. Killer cellimmunoglobulin-like receptors (KIR) are the most abundant surface receptors expressed on human naturalkiller cells and have specific HLA class I molecules as their ligands. This allows for NK cell recognitionand response towards cells that do not express self-HLA molecules, a common outcome of viral infections.Analysis of the KIR3DL1/S1 alleles and their frequencies in a Kenyan population will help to understandthe epidemiology of diseases in this region.Methods: Genomic DNA was isolated from whole blood samples. Exons 1-5, 7, and 9 of the KIR3DL1/S1loci were PCR amplified and sequenced using BigDye V1.1 Terminator Cycle Sequencing Kits (AppliedBiosystems), and genotyped using a Taxonomy-Based Sequence Analysis (TBSA) computer program.Frequencies were calculated using SPSS 13.0.Results: A total of 31 KIR3DL1 and 4 KIR3DS1 alleles were identified. The most common 3DL1 alleleswere 3DL1*01501 (15.44%), 3DL1*01502 (10.60%), 3DL1*01701 (9.22%), and 3DL1*00401 (8.41%).The combined frequency of 3DS1 alleles was 4.15% and the most common 3DS1 allele was3DS1*01301(3.57%). Our results show that the frequencies of activating KIR3DS1 alleles are much lowerin this population, whereas the frequency of 3DL1*01501 is much higher relative to other regions of theworld (4.92%).Conclusions: Our study shows that there is a diversity of KIR3DL1/S1 alleles in this region and confirmthe findings in other studies that KIR3DS1 are rare in the African population.96-PHAPLOTYPES CARRYING “UNCOMMON” ALLELES.Ana M. Lazaro, Yi Xiao, Carly Masaberg, Bin Tu, Jennifer Ng, Carolyn K. Hurley. CW Bill Young MarrowDonor Recruitment and Research Program, Georgetown University, Washington, DC, USA.Aim: We evaluated 88 “uncommon” alleles (25 HLA-A, 48 HLA–B; 15 HLA-C) with predictablehaplotypes and compared them with haplotypes carrying common alleles within the same allele family.Methods: Sequencing of cloned alleles or group-specific amplicons was performed to identify alluncommon alleles in this study. Haplotypes carrying uncommon alleles were predicted by shared HLAassignments from 3 or more unrelated individuals. Haplotypes carrying common alleles were obtained fromMaiers et al. 2007 (http://bioinformatics.nmdp.org).Results: Eighteen haplotypes carrying uncommon HLA-A alleles, were found to carry common HLA-B, -C,-DRB1 alleles. At the HLA-B locus, uncommon B*07 alleles [*07:36, *07:37 and *07:38 (all European)]showed the same associations as B*07:02--HLA-A*03:01, C*07:02, DRB1*15:01 (Eur freq:0.03563).B*27:20 (Asian /Pacific Islander) was found in the same haplotype as B*27:04--A*02:01g, C*12:02,DRB1*1202 (freq API :0.00025). B*27:36 (A) was found in the same haplotype as B*27:04--A*02:02g,C*03:04 (freq API :0.00028). B*35:11 (Hispanic) exhibited the same associations as B*35:01--A*24:04g,C*0304, DRB1*14:02 (freq Hisp:0.00025) and B*35:99 (American Indian) showed the same associationsas B*35:01--A*24:02g, C*03:05 (Hisp freq:0.00124). B*39:14 (Hisp) and B*39:01 shared the samehaplotype --A*24:02g, C*07:02 (Hisp freq:0.00051) and B*39:31(Eur) and B*39:01 shared A*02:01g,C*12:03 (Eur freq:0.00225). Uncommon HLA-C*02: 14 (AA) was associated with B*81:01/02 in contrastto the more common B*81:01, C*18:01 (freq AA:0.01101). C*05:14 (Eur) was observed with B*51:01+ ;the more common C*05:01 is also found with B*51:01g (Eur :0.00042, Hisp: 0.00075). The remainder ofthe uncommon HLA-C alleles were associated with common HLA-B alleles.

Conclusions: Uncommon alleles can share haplotypes with more common alleles. Patients without aperfect match will have a chance to find a common haplotype where the only mismatch is going to be theuncommon allele.97-PINTERLEUKINES GENES POLIMORPHISM IN PROSTATE CANCER PATIENTS.Alberto J. Leon 1 , Victoriano J. Leon 2 , Javier Garcia 3 , Manuel Urrutia 3 . 1 Division Immunology, InstituteInfection and Immunity, Shantou University, Shantou, Guangdong, China; 2 Servicio de Analisis Clinicos,Hospital Universitario de Salamanca, Salamanca, Spain; 3 Servicio de Urologia, Hospital Universitario deSalamanca, Salamanca, Spain.Aim: We have been studied 16 Prostate cancer patients, PCP, stage T1-T3, also 24 subjects control of ourcommunity for establized a genomic Interleukine polimorphism control group.Methods: The DNA was obtained from blood samples extracted with the kit DNA Direct II from Dynal,for the genomic studies were employed Cytokine genotiping Kit, Pel-freez.Results: The results are detailed. IL-1alpha/ - 889 (52.5 CC/40 CT/7.5 TT), IL-1beta/ -511 (35 CC/ 50CT/15 TT), IL-1beta/ 3962 (55 CC/35CT/10 TT),IL-1RA/mspa 11100 (0CC/50 CT/50 TT), IL1R/psti 1970(45 CC/52.5 CT/7.5 TT), IL-2/ - 330 (15 GG/ 50 GT/35 TT), IL-2/ 166 (55 GG/35 GT/10 TT), IL-4/ - 1098(10 GG/ 22 GT/ 70 TT), IL-4/ - 590 (75CC/25 CT/ 0 TT), IL-4/ - 33 (75 CC/20 CT/5 TT), IL-4Ralpha/1902 (75 AA/15 AG/10 GG), IL-6/ - 174 (15CC/ 45CG/40 GG), IL-6/ nt 565 (10 AA/50 AG/40 GG),IL-10/ - 592 (5 AA/45 AC/50 CC), IL-10/ - 819 (55 CC/35 CT/10 TT) IL-10/ - 1082 (20 AA/50AG/30GG), IL-12/ - 1188 (60 AA/35 AC/5 CC), IFN gamma/utr 5644 (35 AA/40AT/25 TT), TNF alpha/ - 238 (0AA/2O AG/80 GG), TNF alpha/ - 308 (5 AA/30 AG/65 GG), TGF beta1/codon 10 (25 CC/50 CT/25 TT),TGFbeta1/codon 25 (0 CC/10CG90 GG).Conclusions: The Chi-square test does not show differences P< 0. 01 inter PCP and control group.98-PA NOVEL HLA-A*66 VARIANT IDENTIFIED IN A BRAZILIAN VOLUNTEER BONEMARROW DONOR.Sibelle B. Mattar, Pryscilla F. Wowk, Maria da Graça Bicalho. Genetics, Universidade Federal do Paraná,Curitiba, Paraná, Brazil.Aim: Our HLA typing routine, accomplished by sequence specific oligonucleotide probe (SSOP), somegenotyping ambiguities are always observed. In order to resolve ambiguous assignments additional tests,such as, sequence-specific primer (SSP) and sequence-based typing (SBT) are also performed. The highresolutionallelic typing by Sequence-Based Typing (SBT) in association with other methods improves theaccuracy of results once the exact nucleotide sequence can be inferred and applied to the genotypeattribution. Here we describe a novel HLA-A allele identified in a Brazilian mestizo individual from theBrazilian Bone Marrow Donor Registry.Methods: Initial HLA-A* typing was performed using the LABType SSOP Kits (One Lambda, USA) anddonor genotype assignment was HLA-A*26:33*66 or *66*66, HLA-B*51*58:02, HLA-DRB1*11*15:03.Since HLA-A*26:33 was present in the Rare Allele Lists derived from IMGT HLA database with only onecase reported (API = Asian or Pacific Islander), we performed a SBT (AlleleSEQR Core Kit, AtriaGenetics, USA) in order to confirm allele typing.Results: The genotype A*66:01 A*66:01 was resolved. According to IMGT HLA database, the nucleotidesequence related to allele A*66:01 has a point mutation at codon 182 - exon 3 (G > G/A). Perhaps, this isindicative of there being only one heterozygous position for this special DNA sequence. Aiming to confirmthis result a new complete SBT was performed. The result revealed a variant of A*66:01, which differsfrom that by a single base substitution. This nucleotide change (ACG > CGC) locates at position 544(codon 182 Thr > Ala) and constitutes a conservative mutation (nonpolar > polar neutral).Conclusions: This new sequence might have arisen from HLA-A*66:01 through a point mutation. Thenucleotide sequence is available at the GenBank under accession number HM125713 and we are waitingfor the IMGT new report.

99-PPOTENTIAL COMMON NOVEL ALLELES FOUND IN BRAZILIAN POPULATION.Maria E. Moraes 1,2 , Ana M. Lazaro 3 , Yi Xiao 3 , Matilde Romero 1 , Margareth A. Torres 2 . 1 ImmunogeneticsLaboratory, JRM - Investigacoes Imunologicas, Rio de Janeiro, RJ, Brazil; 2 Immunogenetics Laboratory,LIG - Laboratorio de Imunogenetica, Sao Paulo, SP, Brazil; 3 CW Bill Young Marrow Donor Recruitmentand Research Program, Georgetown University, Washington, DC, USA.Aim: The aim of this study is to describe one HLA-A (A*68:02:01v) and three novel HLA-B alleles(B*18:10v, B*45:01v and B*52:01v) that were identified in Brazilians.Methods: PCR-SSO [One Lambda (OL) and InnoLiPA- Innogenenetics (ILP)] was initially performed.Futhermore, all alleles were isolated by cloning (HPT TOPO TA cloning kit, Invitrogen, Ca) or specifiprimers(in house). These novel alleles were sequenced using an ABI Prism Big Dye Terminator and a3730 xl sequencer (Applied Biosystems, Foster City, Ca).Results: HLA-A*68v was detected by a false positive reaction (fpr) with probes (p)26 and 33 (151R) fromOL and with p34 (149, 151) from ILP in a Brazilian Mestizo (BM). The SBT typing showed one nucleotidesubstitution at (cd 151.2 CAT>CGT), 151 H is changed to R. The new HLA-B*18:10v found also in BMexhibited fpr with p42 (171H) and 82 (163E, 171H) from OL and with p66 (171) from ILP. The SBTrevealed only one nucleotide substitution at (cd 171.1 TAC>CAC), 171Y is changed to H. The novelB*45:01v from a Brazilian Black individual displayed a fpr with p76 (109F) from OL. SBT detected thesame nucleotide at (cd 109.1 CTC>TTC), 109L is changed to F. The HLA-B*52:01v was detected in 10Mestizo individuals from SE and NE of Brazil, showing fpr with p72 and 96 (156W), and negative withp68 (156L) from OL and p59 (156W) from ILP showed a fpr. SBT detected 2 nucleotides substitutions at(cd 1561.2 GTG>TGG), 156L was changed to W. Interestingly, all individuals carrying B*52:01v,appeared to be associated with HLA-A*02, B*52:01v, DRB1*09:01 haplotype.Conclusions: Although the number of HLA new alleles is still growing, their frequencies are low. Further,high resolution typing could demonstrate that B*52:01v is very well represented in our population with itspredictable haplotype. This may have an impact on strategies search for a matching donor forHaematopoietic Stem Cell Transplantation.100-PASSOCIATION BETWEEN CLINICAL MANIFESTATIONS OF AUTOIMMUNE DISEASESWITH THE POLIMORFISMOS OF PTPN22, STAT4, CTLA-4 IN COLOMBIAN POPULATION.Martha Ramirez de Olano, Gerardo Quintana, Antonio Iglesias. Medicine, Universidad Nacional deColombia, Bogotà, Colombia.Aim: Autoimmune diseases (AD) are present in 5% of the human population and they are currently one ofthe most common and less understood health problems. We studied polymorphisms of three genes:PTPN22 C1858T, STAT4 rs 7574865 and CTLA4 CT60A/G and we evaluated associations between someAD such as Rheumatoid Arthritis (RA), Systemic Lupus Erithematosus (SLE) and Systemic Sclerosis(SSc)and their clinic manifestations and polymorphisms of genes before mentioned mediated a case-controlstudy.Methods: The patients and the controls in this study came from the Atlantic coast of Colombia(Barranquilla) and the central region of the country (Bogotá).Our study population consisted of 1023patients. We evaluated 394 RA patients, 94 SLE patients, 101 SSc patients and 434 healthy subjects. Allsubjects were of Colombian origin. Genotyping of the PTPN22 gene C1858T, STAT4 gene rs7574865,CTLA 4 gene CT60A/G polymorphisms was performed by real-time polymerase chain reactiontechnology, using the TaqMan 5_-allele discrimination assay.Results: After studying these genes, we found an association between the C1858T polymorphism of thePTPN22 gene with RA and SLE; however the polymorphism is not associated with SSc. This result isaccording with the studies previously. In addition, we found that the SNP rs7574865 from STAT4 isassociated to the RA and SLE in our population. Finally we did not find evidence of association betweenCT60A/G polymorphism of CTLA4 and AD.Conclusions: Our work contributed to establishment of PTPN22 and STAT4 genes as markers forsusceptibility for to develop AD. We suggest that CTLA4 CT60A/G polymorphism does not play a criticalrole in the genetic predisposition to develop RA, SLE or SSc in Colombian population.

101-PGETTING OUT OF THE GROOVE: PATTERNS OF SINGLE NUCLEOTIDEPOLYMORPHISMS WITHIN CLASS I EXON 4 SEQUENCES.Lois E. Regen 1 , Anajane G. Smith 1 , Shalini E. Pereira 1,2,3 . 1 Seattle Cancer Care Alliance, Seattle, WA,USA; 2 FHCRC, Seattle, WA, USA; 3 Lab Medicine, U of Washington, Seattle, WA, USA.Aim: In 2009, our laboratory submitted a new HLA sequence, A*02:97:02, which differed fromA*02:97:01 by a single nucleotide substitution (SNP) at codon 232 in exon 4. Garino et al (Tissue Antigens69(4):342) noted that this is a highly conserved residue, due to its proximity to CD8 contact residues 222-229. These residues encode the α3 domain region which binds to the CD8 coreceptor expressed bycytotoxic T cells. Given this, one would expect exon 4 to be highly conserved, but, until recently, exon 4sequence data has been sparse for many HLA alleles. We surveyed published sequences to determine thelevel of polymorphism in exon 4 of Class I alleles, especially near codons 222-229.Methods: The IMGT/HLA database was reviewed for Class I exon 4 sequences. We tabulated commonalleles, with emphasis on SNPs near codons 222-229.Results: Looking at exon 4 as a whole, both HLA-A and B are highly conserved from codons 208 to 227,while C is highly conserved from codons 229 to 247. These conserved regions appear to be “SNP deserts”.HLA-A has more polymorphism than B - rather surprising considering that there are so many more HLA-Balleles than A alleles. Both A and B have one synonymous SNP flanking the CD8 associated region - codon230 for A and codon 228 for B. HLA-C has five SNPs in this region at codons 219, 221, 224, 225 and 228.These SNPs were used to categorize Class I alleles into groups - two for A and two for B. For C, there werefive groups which corresponded to previously described clades.Conclusions: Exon 4 SNPs are consistently present near the CD8 associated residues. HLA-A, B, and Cdiffer in the patterns of these SNPs, which may indicate evolutionary lineages for some allele groups.Paradoxically, these “silent” SNPs occur within highly conserved exon 4 sequences and may point tounknown regulatory functions, while maintaining the amino acid sequences of the a3 domains.102-PTHE MOLECULAR BASIS OF AN HLA-A*24 INACTIVATION: A 4 kb DELETION TURNSHLA-A INTO A PSEUDOGENE.Christina E.M. Voorter, Nina Lauterbach, Marcel G.J. Tilanus. Transplantation Immunology, Tissue TypingLaboratory, Maastricht University Medical Centre, Maastricht, Netherlands.Aim: An unusual haplotype without a detectable HLA-A allele by serologic or molecular typing methodssegregates in a Caucasian family. Microsatellite analysis and fluorescence in situ hybridization implicatedthat the deletion encompasses a narrow region. In this study the molecular unraveling of the deletion isdescribed.Methods: To identify the deleted region a molecular strategy was followed in that amplification primerpairs were selected to surround highly polymorphic parts of the genomic region, both centromeric andtelomeric from HLA-A. Sequencing the generated PCR fragments revealed that all polymorphic sites werestill heterozygous in the individuals carrying the haplotype with the HLA-A deletion. Therefore, the 5’primer from the fragment closest to the centromeric side of HLA-A was combined with the 3’ primerclosest to the telomeric side encompassing an 11 kb region.Results: Sequencing revealed that a deletion of 4089 bp was present, located upstream of HLA-A,including exons 1 – 3 of the HLA gene up to exon 4. Sequence information of the 3’ part of HLA-A,downstream the deletion, identified that the deleted allele originates from an A*24 allele. This deletion wasdifferent from the previously detected HLA-A deletion found in 3 different Japanese families, in which thecomplete HLA-A gene was deleted accompanied by the insertion of a retrotransposon. Although differentrepeat sequences are present in the region both within and outside the newly found deletion, no evidencepoints to a retrotransposon mechanism.Conclusions: The here detected partial deletion of HLA-A turns this functional gene into a pseudogene,resembling HLA-T (HLA-16) in which also only exons 4 to 8 of HLA class I are present.

103-PTOWARDS A FUNCTIONAL HLA-DPB1 NOMENCLATURE BASED UPON FULL LENGTHPOLYMORPHISM AND EVOLUTIONARY RELATION OF THE HLA-DPB1 ALLELES. AJOINT WORKSHOP EFFORT PROPOSAL.Nina Lauterbach-Kunze 1 , Christina E.M. Voorter 1 , Pietro Crivello 2 , Lotte Wieten 1 , KatharinaFleischhauer 2 , Marcel G.J. Tilanus 1 . 1 Transplantation Immunology, Tissue Typing Laboratory, MaastrichtUniversity Medical Centre, Maastricht, Netherlands; 2 Department of Molecular and FunctionalImmunogenetics, Fondazione Centro San Raffaele del Monte Tabor, Milan, Italy.Aim: Functional polymorphism of HLA-DPB1 diversity in stem cell transplantation and antibodyproduction will be better defined by full length HLA-DPB1 polymorphism. Functional groups based onevolutionary relationship and correlation to cellular reactivity and antibody production will provide thebasis for a relevant functional HLA-DPB1 nomenclature.Methods: HLA-DPB1 alleles were studied in multiple B-cell lymphoblastoid cell lines and blood samples.Total RNA was isolated, cDNA prepared and amplified with locus specific primers from 5’UTR up to3’UTR. Full length sequencing enabled identification of polymorphism in the entire coding region of HLA-DPB1.Results: Analysis of 30 different alleles by this approach demonstrated that several alleles with identicalexon 2 sequences show polymorphism in the coding region outside exon 2. Preliminary data indicateadditional polymorphisms in different ethnic populations.Conclusions: Full-length sequences for as many DPB1 alleles as possible from different ethnic groups areneeded in order to clarify the evolutionary relationship of the alleles. Our results indicate thatpolymorphism in exons 3, 4 and 5, can group alleles based upon an evolutionary relationship of this allelepolymorphism. We propose a component of the 16th histocompatibility workshop, requesting participantsto collect as many different DPB1 alleles from different ethnic groups as possible, also those with identicalexon 2 DPB1 typing. Such a collaborative study would offer an excellent opportunity to expand the DPB1full length sequence information. Ultimately this coordinated action could lead to a functionally relevantDPB1 nomenclature.104-PESTIMATION OF HLA ALLELES AND HAPLOTYPES FREQUENCIES DISTRIBUTION INPOPULATION OF THE SAMARA REGION.Andrey Toropovskiy 1 , Olga Tyumina 1 , Larisa Trusova 1 , Stanislav Volchkov 1 , Loren Gragert 2 . 1 ClinicalCenter of cellular Technologies, Samara, Russian Federation; 2 National Marrow Donor Program.Aim: To calculate the HLA allele and haplotype frequency distribution for the Samara region of Russia.Methods: Cord blood is collected at all maternity hospitals of Samara after signing of informed consent.HLA typing was performed at medium-to-high resolution using SSO method with the Luminex systemfrom One Lambda. HLA typing was performed on the cord blood samples, with 3643 typings at HLA-A,3604 at HLA-B and 3715 at HLA-DRB1. Estimation of high-resolution A-B-DRB1 haplotype frequencieswas performed using the expectation-maximization (EM) algorithm.Results: The most frequent allele for the HLA-A locus were: A*02 (28.03%), A*03 (15.23%) and A*01(11.32%). For HLA-B: B*35 (11.78%), B*07 (11.61%) and B*44 - (9.76%). For HLA-DRB1: DRB1*13(13.7%), DRB1*07 (13.65%) and DRB1*01 (13.52%). The estimated frequencies of top 10 A-B-DRB1haplotypes are shown in Table 1.[table1]Conclusions: The HLA population genetics of the people of Samara shows their genetic similarity to otherSlavic peoples in Eastern Europe. This study is valuable for planning and development of stem cellregistries in Russia and for predicting high resolution typing in donor searches.105-PTHE MACAQUE MHC-DQB1 LOCUS IS A POLYMORPHIC SINGLE COPY GENE WITHALLELE GROUPS THAT ARE CLOSELY RELATED TO THEIR HLA-DQB1 ALLELE GROUPCOUNTERPARTS.Jin Wu, Sue Bassinger, Tenzin D. Tsewang, Michael A. Krencicki, Jr., Jennifer B. Woods, Thomas M.Williams. Pathology, University of New Mexico, Albuquerque, NM, USA.

Aim: Non-human primates are important as models for infectious disease, vaccine development, andtransplantation studies. The macaque MHC DQB1 locus is not very well understood. Our goal is to develophigh-throughput, high resolution, and robust genotyping molecular tools to characterize rhesus, pig-tail, andcynomolgus macaque DQB1 genes.Methods: Two sets of group specific PCR primers were designed and linked to M13 universal primers toamplify either Mamu-DQB1*06 or Mamu-DQB1*15/16/17/18/24 groups from rhesus genomic DNA. Exon2 was directly sequenced with M13 forward and reverse primers. These primers were also used to amplifyand sequence DQB1 alleles from pig-tail and cynomolgus genomic DNA. Sequences were analyzed withAssign software (Conexio, Perth, AU).Results: We used this method to genotype about 300 rhesus, pig-tail, and cynomolgus macaques. Fourteennovel and 32 known alleles were identified in rhesus, 31 novel alleles in pig-tail, and 26 novel and 22known alleles in cynomolgus macaques. Some allele sequences are identical across all 3 species. We wereable to define DQB1 allele frequencies for these animals. Non-human primate MHC studies have shownthat Class I A and B genes are duplicated loci with multiple A and B genes. We have not observed that theprimate DQB1 gene is duplicated. In addition, our phylogenetic tree analysis shows that the primate DQB1allele groups are closely related to their HLA DQB1 allele group counterparts.Conclusions: Our data show that the rhesus, pig-tail, and cynomolgus macaque DQB1 locus is highlypolymorphic and shares many similarities with HLA DQB1.106-PTHE ANALYSIS OF MICA ALLELE AND TRANSMEMBRANE REGION POLYMORPHISM INCHINESE HAN POPULATION.Xiaoni Yuan, Jun He, Xiaojing Bao, Chao Xu. Jiangsu Institute of Hematology, The First AffiliatedHospital of Soochow University, Suzhou, Jiangsu, China; Jiangsu Institute of Hematology, The FirstAffiliated Hospital of Soochow University, Suzhou, Jiangsu, China; Jiangsu Institute of Hematology, TheFirst Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China; Jiangsu Institute of Hematology,The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.Aim: The MICA gene polymorphism plays an important role in allogenic organ transplantation andhematopoietic stem cell transplantation. We analyzed to further assess of MICA polymorphism in the 137Chinese Han healthy people.Methods: All samples were analyzed using PCR-SSOP to measure the MICA gene exons 2, 3, 4, 5. Theambiguous alleles combination were distinguished by SBT. Meanwhile the MICA gene transmembraneregion microsatellite sequence were detected by the SBT specific primer.Results: From 137 Chinese Han healthy people, 13(19.40%) MICA alleles were detected in the 61 variousMICA alleles. MICA*008(23.36%) was the most common alleles, and then were MICA*010(21.53%),MICA*002 (19.34%), MICA*009(10.58%), MICA*027(7.30%), MICA*012(6.93%), MICA*016 (5.11%),MICA*007 (2.55%), MICA*045 (2.19%), MICA*004 (1.46%), MICA*017(1.46%), respectively. 42 allelecombination were detected, and most common alleles of MICA*008, MICA*010, MICA*002, MICA*009and MICA*027 respectively combination accounted for 63.51%. The common allele combination wereMICA*008, MICA*010 (10.95%); MICA*008,MICA*008(9.49%); MICA*008, MICA*027 (5.11%);MICA*010, MICA*010 (9.49%); MICA*002, MICA009(5.84%). In the MICA transmembrane regiontypes A5, A5.1, A9, A6, A4 were found, the frequency were A5(33.94%), A5.1(23.36%), A9(20.80%),A6(12.41%), A4(12.04%). The combination between A5, A5.1, A9 constituted 49.64% of transmembraneregion types. A5 associated with A5.1 or A9 were very common, account for 16.79%, 13.14% and 10.22%.Conclusions: The combination of PCR-SSOP and SBT can provide an accurate and reliable method todetect allele distribution of MICA polymorphism. The distribution of MICA polymorphism in Chinesepopulation are relatively focused. Our results provide frequency distribution data about MICA allelepolymorphism in Chinese Han population, The characteristic of MICA gene polymorphism is helpful to ourfuture research.Yuan: The First Affiliated Hospital of Soochow University: Science Med Advisor. He: The First AffiliatedHospital of Soochow University: Science Med Advisor. Bao: The First Affiliated Hospital of SoochowUniversity: Science Med Advisor. Xu: The First Affiliated Hospital of Soochow University: Science MedAdvisor.

107-PWHOLE GENOME AMPLIFICATION (WGA) AS A RENEWABLE SOURCE OF DNA ATNMDP’S RESEARCH REPOSITORY.Colleen Brady, Nadereh Jafari, Neng Yu, Stephen Spellman. Scientific Services, National Marrow DonorProgram, Minneapolis, MN, USA; Genomics Core Facility, Center for Genetic Medicine, NorthwesternUniversity, Chicago, IL, USA; HLA Services, American Red Cross, New England Region, Dedham, MA,USA; Scientific Services, National Marrow Donor Program, Minneapolis, MN, USA.Aim: The NMDP runs a dynamic, highly utilized Research Repository and strives to ensure thatirreplaceable samples are not fully depleted. WGA using commercially available validated kits provides analternative methodology to B-LCL transformation for generating a renewable source of DNA. This studyevaluated the effectiveness of WGA on sample types stored in the NMDP Research Repository.Methods: DNA extraction (Qiagen/Gentra Autopure LS) and WGA (GE’s Illustra GenomiPhi HY) wereperformed on 55 samples derived from: buccal swabs, DNA, frozen and dried blood. The WGA productswere evaluated by performing high resolution HLA- A, B, C, DRB1, DQB1, and DPB1 SBT. GenomicDNA (gDNA) was subsequently tested if typing issues arose using the WGA-amplified DNA.Results: WGA was performed on 10ng extracted DNA and yielded a median 48.2ug (range: 30-92ug) ofproduct with a median 260/280 ratio of 1.75 (range: 1.65-1.81). Of 540 alleles typed from WGA samples,99.8% were fully concordant with the HLA typing of record. WGA introduced a point mutation in onesample which was confirmed by gDNA assessment. The majority of the sample types produced problemfree testing results. However, degraded gDNA in buccal swabs and filter paper (peripheral blood, not cordblood) led to substantial allele dropouts at HLA Class I and amplification failures for HLA-DPB1 and wereexcluded from HLA typing accuracy assessement (18.2%).Conclusions: In contrast to B-LCL transformation, the WGA process is time and labor efficient, does notrequire viable cells, and has a high overall success rate. However, WGA use should be restricted to sampletypes that yield high quality gDNA to avoid allele dropout and amplification failure. While WGA doesproduce high fidelity replication of source gDNA, the rare potential to introduce point mutations must beconsidered. WGA provides a method to generate a renewable source of DNA for research applications.108-P8/8 HIGH-RESOLUTION (HR) HLA MATCH RATE: CAUCASIAN (CAU) AND AFRICANAMERICAN (AFA) PATIENTS.Jason Dehn 1 , Kelly Buck 1 , Soo Young Yang 2 , Alexander Schmidt 3 , Robert Hartzman 4 , Martin Maiers 1 ,Michelle Setterholm 1 , Dennis Confer 1 . 1 National Marrow Donor Program (NMDP), Minneapolis, MN,USA; 2 Histogenetics, Inc, Ossining, NY, USA; 3 DKMS, Tuebingen, Germany; 4 C.W. Bill Young MarrowDonor Recruitment and Research Program, Rockville, MD, USA.Aim: Estimation of the 8/8 (A, B, C, DRB1) HR match rate using real patient unrelated donor (URD)searches presents a biased sample for reasons including access to treatment, financial barriers andincomplete donor testing. A study was designed to estimate the true match rate for CAU and AFA groups,representing historical best and worst match rates respectively.Methods: 769 URD searches were performed for pseudopatients (PP) who were randomly selectedpreviously HR tested donors in the NMDP’s Be The Match Registry (BTMR). Searches were based on afixed BTMR file as of January 2009. Search results from CAU and AFA PP were classified as follows: 1)8/8 HR matched donor exists on BTMR 2) No potential 8/8 HR donors exist 3) Potential 8/8 HR donorsexist PP searches falling into category 3 (accrued until N= 200 per race) then had an HLA search strategyexpert rank potential donors within BTMR in order of their matching likelihood. Previously stored donorsamples were HR HLA tested in order of ranking and evaluated to determine match status. Consecutiverounds of donor sample testing were performed until either an 8/8 matched donor was identified or nopotential donors with stored samples remained.Results:[table1]Careful review of the cases “pending further testing” suggests that few additional caseswould yield 8/8 HR matches.Conclusions: This study provides a true 8/8 HR match rate estimate for CAU and AFA patients throughBTMR, which has not been accomplished previously. This study can be used to inform patients searchingBTMR and provides vital information for donor recruitment and availability efforts. Results provide abaseline match rate that can be further supplemented using the additional worldwide URD inventory.

109-PAMBIGUITY RATES IN HLA SEQUENCE-BASED TYPING.Angelica DeOliveira, Gansuvd Balgansuren, Bobbie Holeman, Dong-Feng Chen. Clinical TransplantationImmunology Laboratory, Duke University Health System, Durham, NC, USA.Aim: Group-specific sequence-based typing (SBT) offers us the best high resolution for HLA typingamong other DNA methods. However, the continuous addition of new alleles increases the ambiguity rates.The aim of this study was to evaluate the ambiguity rates of the SBT current in use.Methods: For each of HLA A, C, and DQB1 loci, we used 7 group-specific and 1 generic amplifications;and for each B and DRB1 we used 14 group-specific amplifications. Exons 2, 3, and 4 of A, B and C weresequenced using either reverse or forward primers. The exon 1 as the extension of exon 2 reversesequencing was utilized for allele assignments when needed. For DRB1 only exon 2 and for DQB1 bothexon 2 and 3 were sequenced. NMDP guideline was used to determine if ambiguities should be resolved.Results: SBT data obtained from 103 potential unrelated BMT recipients between Aug to Nov 2009 wereanalyzed. Of these typings, 5 ambiguities of HLA A were obtained. Three were caused by cis/trans linkagewith the same combinations of A*01 and A*11 alleles which were resolved by additional tests. The HLA Bambiguity rate was 24%. However, all of them did not need further tests except for 2 cis/trans ones. 22 oftotal 25 B ambiguities were B*07:02/61. Of the 69 HLA C ambiguities (69%), 21 were of C*04:01/09N/30,30 were of C*07:01/06/18, and 21 were of C*07:02/50. Among them only 5 needed to be resolved whichwere all C*04:01/09N/30 due to the linkage to B*44:03. None of 8 DRB1 ambiguities needed to beresolved. Four DQB1 ambiguities were all caused by the presence of two different DQB1*06 alleles, andonly one of them required a further typing.Conclusions: The SBT methods current in use generated very low ambiguity rates for HLA A, DRB1 andDQB1 typing. Although B and C typing showed higher ambiguity rates, majority of them was caused bypolymorphism out of exons 1-4 and did not need further typing. Our current SBT methods deliveredsatisfactory high resolution typing results.110-PDONOR-SPECIFIC (DSA) ANTI-DP ANTIBODIES ASSOCIATE WITH GRAFT FAILURE (PGF)IN UNRELATED DONOR HEMATOPOIETIC STEM CELL TRANSPLANTATION(MUDHSCT).Stefan O. Ciurea 1 , Pedro Cano 2 , Marcos de Lima 1 , Peter F. Thall 3 , Gabriela Rondon 1 , Partow Kebriaei 1 ,Edward Guerrero 2 , Fleur Aung 2 , Titus Barnes 2 , Dana Willis 2 , David Partlow 2 , Chitra Hosing 1 , Issa F.Khouri 1 , Richard Champlin 1 , Marcelo A. Fernandez-Vina 2 . 1 Stem Cell Transplantation, UT M. D.Anderson Cancer Center, Houston, TX, USA; 2 Laboratory Medicine, UT M. D. Anderson Cancer Center,Houston, TX, USA; 3 Biostatistics, UT M. D. Anderson Cancer Center, Houston, TX, USA.Aim: Our previous study showed that DSA associates with PGF in T-cell depleted haploidentical HSCT.3/4 patients with DSA had PGF compared with 1/20 patients without DSA. PGF was found in patients withHLA-A, B and DRB1 DSA; no PGF was found in 1 patient with anti-DP DSA. We propose that anti-DPDSA has a less deleterious effect.Methods: Here we describe 592 MUDHSCT cases performed since 2005. 88 % of the transplants matchedin 8/8 alleles of HLA-A,B,C,DRB1 in the HvG vector; approximately 76% of the transplants weremismatched in DPB1. DSA was determined by single antigen solid phase assays. Results were interpretedas fluorescence intensity (FI) against DSA mismatch.Results: 19 patients (3.2%) had PGF. The only DSA identified was directed against DP. Eight 8/8 patientshad DP-DSA; 38 DSA patients had PGF compared with 16/584 who did not have DSA (p=0.001,RR=21.9). The FI for DSA was similar in patients with and without engraftment (see table).[table1]Conclusions: These and previous findings, suggest that PGF occurs less often with anti-DP DSA (3/9)compared with DSA against HLA-A, B or DRB1. DP-DSA may confer a lower risk for PGF (p=0.05).DSA testing is warranted in HSCT when considering donors with HLA mismatches. Strategies for donorselection or DSA reduction may be needed to decrease the risk of PGF from partially HLA-matcheddonors.

111-PLOSS OF MISMATCHED HLA IN FAMILY HAPLOIDENTICAL AND UNRELATED HSCT.Luca Vago 1 , Cristina Toffalori 2 , Maria Teresa Lupo Stanghellini 1 , Daniela Clerici 1 , Alessandro Crotta 1 ,Benedetta Mazzi 2 , Magda Marcatti 1 , Consuelo Corti 1 , Massimo Bernardi 1 , Jacopo Peccatori 1 , ClaudioBordignon 3 , Chiara Bonini 4 , Fabio Ciceri 1 , Katharina Fleischhauer 2 . 1 Hematology and Bone MarrowTransplantation Unit, San Raffaele Scientific Institute, Milano, Italy; 2 Unit of Molecular and FunctionalImmunogenetics, San Raffaele Scientific Institute, Milano, Italy; 3 MolMed SpA, Milano, Italy;4 Experimental Hematology Unit, San Raffaele Scientific Institute, Milano, Italy.Aim: The antileukemic effect of Hematopoietic Stem Cell Transplantation (HSCT) from familyhaploidentical and unrelated donors (UD) heavily relies on donor T cell alloreactivity, targeting themismatched HLA molecules. Still, upon in vivo selective pressure by donor T cells, leukemia can undergogenomic loss of the patient-specific HLA, a mechanism which our group demonstrated to be frequentlyresponsible for leukemia relapse (Vago et al., NEJM, 2009).Methods: 119 patients underwent partially HLA-mismatched HSCT and donor T cell infusion for myeloidleukemia. For 81 patients the donor was family haploidentical, and for 38 was unrelated and mismatchedfor a median of 2/12 HLA alleles. Follow-up comprised monthly Short Tandem Repeat (STR) chimerismanalysis and HLA typing performed on bone marrow. In cases of relapse, STR chimerism and HLA typingwere performed also on purified leukemic blasts.Results: Disease relapse occurred in 36 and 11 patients after haploidentical and UD transplantation,respectively. After haploidentical transplantation, 13/36 relapses (36%) were due to leukemic blasts whichhad lost the patient-specific HLA haplotype. 1 of the 11 relapses after UD HSCT occurred through thesame mechanism, in a patient who had received two subsequent transplants from HLA-C-mismatcheddonors. Upon detection of the mutated leukemic blasts, 9 of these 14 patients were enrolled to receive asubsequent HSCT from a different donor, mismatched for the remaining HLA haplotype.Conclusions: Our data consolidate the clinical relevance of this escape mechanism from the antileukemiceffect of alloreactive donor T cells. Patients who relapse after HLA-mismatched HSCT should be screenedfor these mutations, to avoid inefficacious donor T cell add-backs, and quickly enroll patients to a salvagetransplant. Novel diagnostic and therapeutic tools are warranted, to provide earlier molecular detection andspecific targeting of these mutants.112-PSIMULATION OF CORD BLOOD UNIT (CBU) INVENTORIES SHOWS LOWER MATCHRATES THAN POPULATION-BASED MODELS BECAUSE OF DEPLETION OF HIGH TOTALNUCLEATED CELL COUNT (TNC) UNITS WITH COMMON HLA.Loren Gragert 1 , Martin Maiers 1 , Eric Williams 1 , Dennis Confer 1 , William Klitz 2 . 1 National Marrow DonorProgram; 2 University of California, Berkeley.Aim: Because few CBUs have sufficient TNC to provide a suitable dose for most adults, and larger celldoses lead to better outcomes, generally the largest (highest TNC) suitably matched CBU will be selectedfor any given patient. Consequently, the utilization and depletion of CBU inventories is skewed towards thelarger units, resulting in high contention for these desirable CBUs. Estimates of 4of6 A-B-DRB1 CBUmatch rates in the Be The Match Registry are over 90% using population-based models. In benchmarksearches of the actual CBU inventory, we observed much lower match rate estimates than in the models,which we hypothesized may result from the depletion of large CBUs.Methods: To more accurately model matching in CBUs, we simulated patients who select the largest bestmatchedCBU available and deplete a simulated inventory of CBUs. We recorded the match grade, size,and HLA-commonality of both the best available CBU and the best CBU from the original CBU inventory.Results: As patient searches depleted CBUs from inventory over time, we observed a steep increase in theproportion of subsequent patients losing all of their matches because of depletion. After depletion, the HLAcontent of the largest remaining CBUs also became highly enriched for more rare phenotypes. The HLA ofthese remaining large CBUs reflected neither population HLA frequencies nor the HLA frequencies of allCBUs recruited.Conclusions: We identify this HLA-biased depletion as the primary reason that current populationfrequency-based models overestimate match rates in real CBU registries. This larger CBUs with morecommon HLA phenotypes are likely selected and infused soon after they are listed. We conclude that cord

lood banks may want to focus more on adding CBUs with high TNC rather than aiming for large overallincreases in CBU inventory.113-PA NOVEL HLA-A*30 ALLELE IDENTIFIED BY SEQUENCE-BASED TYPING IN A GERMANSTEM CELL TRANSPLANTATION DONOR.Lawrence J. Jennings, James Hall, Edward Caparelli, Mercedes Silva, Min Yu. Pathology and LaboratoryMedicine, Children’s Memorial Hospital, Chicago, IL, USA.Aim: We present a novel HLA-A*30:36 allele from a stem cell donor from the Central German BoneMarrow Donor Registry (ZKRD).Methods: The initial sequence for exons 2, 3, and 4 of the HLA-A locus was obtained using anAlleleSEQR HLA-A kit obtained from ATRIA® Genetics (San Francisco, California, USA). Initialanalysis using ASSIGN® 3.5+ software (Conexio Genomics, Applecross, Western Australia, Australia)provided a type of HLA-A*02:71 which is rare and HLA=A* 30:01:01. To confirm the presence of the rareHLA-A*02:71, the HLA-A*02 and 30 alleles were amplified separately using allele-specific sense primers.The resulting PCR products were sequenced for exons 2 and 3 and the sequences obtained were analyzedusing ASSIGN® 3.5+ software.Results: This analysis revealed the presence of a HLA-A*02:01:01:01 allele which has adenine at position494 in exon 3 and a new allele of HLA-A*30 which has guanine at this position. This nucleotide differenceresults in a change in amino acid from glutamine to arginine in codon 141. The novel HLA-A*30 allele hasbeen officially named HLA-A*30:36 by the World Health Organization (WHO) nomenclature committee.This allele pair is ambiguous with the initially identified allele pair, HLA-A*02:71 which has a guanine atposition 494 and HLA-A*30:01:01 has adenine at this position.Conclusions: Of interest is the fact that due to the configuration of the nucleotides at position 494 in exon3, a 0 mismatch allele pair was identified with only 1 rare allele. It is the policy of our laboratory that allrare alleles must be confirmed using a different PCR methodology. In this case, failure to confirm thesingle rare allele would have led to incorrect typing of this donor.114-POUTCOME ANALYSIS IN PATIENTS RECEIVING PATERNAL VS. MATERNALHEMATOPOIETIC STEM CELL TRANSPLANTATION.Siva Kanangat, Victoria Turner, Terrance L. Geiger, Jie Yang, Chong Wong, Wing Leung.Pathology/HLA/Oncology/Statistics, St. Jude Children’s Research Hospital, Memphis, TN, USA.Aim: Cell trafficking in utero can induce tolerance to non-inherited maternal antigens (NIMA) in the fetusand may represent a benefit of using maternal donors instead of paternal donors in one-haplotypemismatched transplants. We compared outcome between patients who received a haplotransplant from theirbiological mother (NIMA mismatch) or other NIMA mismatched relatives to patients who received ahaplotransplant from their biological father (NIPA mismatched).Methods: The patients were those given a haplotype mismatched HSCT from 2005-2009 for leukemia orother hematopoietic disorders at St. Jude Children’s Research Hospital, Memphis, TN. This study wasapproved by the Institutional Review Board. Clinical data were collected from patients’ medical records.Statistical analysis was performed by SAS version 9.2 and StatXact,Windows version 8.Results: Patients who received a NIMA mismatched transplant (n=40), and patients who received a NIPAmismatched transplant (n=38) were similar in terms of disease status, recipient gender, product type, HLAmatch status, recipient age at transplant, and donor age at transplant. Patients receiving NIPA mismatchedtransplant received higher CD34+ cell numbers from their donors than those with NIMA mismatchedtransplants. NIMA-mismatched patients had a slightly better overall survival (OS), event free survival(EFS) and lower incidence of level 3 or higher aGVHD compared to NIPA-mismatched patients (notstatistically significant). The NIMA mismatched recipients with higher CD34+ counts had relatively lowerGVHD and a slightly better neutrophil/platelet engraftment compared to NIMA mismatched recipients withlesser CD34+ counts (not statistically significant).Conclusions: Outcomes for patients receiving NIMA mismatched transplants compared with thosereceiving NIPA mismatched transplants showed no statistical differences.

115-PDONOR EPITHELIAL CELL CHIMERISM IS A TRUE PHENOMENON: NUMBER OFDONOR-ORIGIN NASAL EPITHELIAL CELLS CORRELATES WITH TIMEPOSTTRANSPLANT BUT AT A FASTER RATE IN THE FIRST 3 MONTHS COMPARED TOLATER POSTTRANSPLANT.Faisal M. Khan 1,2 , Sarah Sy 2 , Polly Louie 2 , Megan Smith 2 , Judy Chernos 2 , Noureddine Berka 1,2 , Gary D.Sinclair 1,2 , Victor Lewis 2 , James Russell 2 , Jan Storek 2 . 1 Calgary Laboratory Services, Calgary, AB,Canada; 2 University of Calgary, Calgary, AB, Canada.Aim: Detection of donor-type epithelial cells (ECs) after allogeneic hematopoietic cell transplantation(HCT) (using XY-FISH and sex-mismatched donor-recipient pairs) is controversial given artifacts of XYbaseddifferentiation or the possibility of hematopoietic-nonhematopoietic cell fusion. As well, the kineticsof donor-type ECs (quantity at different time points after transplant) is unknown. Here, we documented theexistence of donor-type ECs using a method obviating the artifacts of XY-FISH, and studied their kinetics.Methods: We collected nasal scrapings and whole blood from 60 HCT survivors, either early (7-98 days,n=32) or late (6-22 years, n=28) posttransplant. DNA extracted from laser-captured nasal ECs(Cytokeratin + CD45 - cells) and blood leukocytes was PCR amplified for a panel of 16 short tandem repeat(STR) markers. FISH analysis for two autosomes was used in 10 patients to assess cell fusion.Results: Donor type ECs were identified in 88% of specimens. None of the nasal ECs in 10 HCT survivorsexhibited presence of cell fusion. Median percentage of donor-type ECs (among nasal ECs) were 0% onday 7, 0.6% on day 28-32, 2.0% on day 56-62, 2.8% on day 84-98 and 5.1% at 6-10 (median 9) years afterPBSCT, and 7.0% at 6-10 (median 9) years and 8.5% at 12-22 (median 17) years after BMT. A significantcorrelation was found between the time after transplantation and the number of donor-type nasal ECs.However, the rate of increase of donor-type ECs was faster during early (2.2% per 2 months) than late(0.19% per year) posttransplant period.Conclusions: Donor-type nasal ECs after HCT truly exists, their occurrence is significantly correlated withtime posttransplant and their percentage rises rapidly in the first 3 months posttransplant and slowlythereafter.116-PGENOMIC INSTABILITY AFTER ALLOGENEIC HEMATOPOIETIC CELLTRANSPLANTATION IS FREQUENT IN ORAL AND RARE IN NASAL MUCOSA: ISCHRONIC GRAFT-VS-HOST DISEASE RESPONSIBLE?Faisal M. Khan 1,2 , Sarah Sy 2 , Polly Louie 2 , Noureddine Berka 1,2 , Gary Sinclair 1,2 , Douglas A. Stewart 2 ,James A. Russell 2 , Jan Storek 2 . 1 Tissue Typing Laboratory, Calgary Laboratory Services, Calgary, AB,Canada; 2 University of Calgary, Calgary, AB, Canada.Aim: Genomic Instability (GI) is a precancerous/cancerous state but also occurs in non-neoplastic,chronically inflamed tissues. Hematopoietic cell transplant (HCT) recipients have a 2 to 4-fold higherlikelihood of developing a solid cancer compared to the general population. However, cancers of someorgans (eg, mouth) are more frequent than other organs (eg, nasal mucosa). Here we evaluated whether GIoccurs after allo-HCT, and if yes, does it occurs more frequently in oral epithellium than nasal epithelium.Methods: We examined GI in oral and nasal mucosal specimens from 105 subjects, including long-term(4-22 yrs, n=25) and short-term (7-98 days, n=32) allo-HCT survivors. Controls included autologous HCTsurvivors (n=11), patients treated with chemotherapy without HCT (n=9) and healthy individuals (n=27).DNA extracted from blood leukocytes and cells of nasal and oral mucosa was PCR amplified for a panel of15 microsatellite loci. Fragment size analysis was done to identify novel allele peaks (ones that were absentin the donor and recipient blood pre-transplant but were present in recipient mucosa post-transplant)indicative of GI.Results: GI was detected exclusively in the allo-HCT recipients. GI typically occurred late posttransplantand was frequently observed in the oral mucosal cells (60% long-term and 6% short-term allo-HCTsurvivors) and rarely in the nasal mucosal cells (4% long-term and 0% short-term allo-HCT survivors).None of the controls showed GI. Both presence of GI (p=0.04) and number of microsatellites showing GI(p=0.005) in oral specimens was significantly associated with history of oral chronic graft-vs-host disease(cGVHD).

Conclusions: GI after HCT is frequent in some (oral) but rare in other (nasal) epithelia. This may explainwhy some epithelia (especially those involved with cGVHD) are prone to develop cancer.117-PCOMPARISON OF HIGH-RESOLUTION HLA-A, B, Cw, DRB1, DQB1 ALLELEDISTRIBUTION OF CHINESE HAN WITH OTHER FIVE ETHIC GROUPS.Yang Li, Jun He, Xiao Jing Bao, Qiao Cheng Qiu, Xiao Ni Yuan. Jiangsu Institute of Hematology, TheFirst Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.Aim: High resolution donor-recipient HLA matching contributes to the success of stem cell transplantation.However, HLA genes are very polymorphic, so it is meaningful to study their distribution to assistant donorsearching program and donor registry plan.Methods: Data was obtained from 2540 unrelated Chinese Han individuals based on donor-recipient forCMDP. Alleles were determined by SBT, SSOP and SSP methods.Results: By large sample analysis, a total of 44 A, 83 B, 44 Cw, 61 DRB1 and 22 DQB1 HLA alleles wereobserved, covering 7%, 10%, 17%, 14% and 34% alleles defined in HLA dictionary. A*02, 11, 24, 33,B*40, 15, 46, 13, Cw*03, 07, 01, 08 and DRB1*09, 15, 12, 04 were the most common HLA allele inChinese Han. It is similar to Koreans and Japanese. But coverage of B*13, 46, Cw*01 and DRB1*09, 12 inCaucasians, African American and German was low even absent. Conversely, A*01, 03, B*07, 44 andDRB1*01, 13 covered a great part in Caucasians, African American and German, but little in Chinese Han.The most common HLA alleles in Chinese Han (frequency greater 0.05) : A*1101, 2402, 0201, 0207,3303, 0206, 3001; B*4001, 4601, 5801, 1302, 5101; Cw*0702, 0102, 0304, 0801, 0602, 0303, 0302, 0401;DRB1*0901, 1501, 1202, 0803, 0701, 0405, 0301, 1101; DQB1*0301, 0303, 0601, 0602, 0202, 0302,0401, 0502, 0201. Among these alleles, A*0206, 0207, B*4601 and DRB1*0803 were infrequent evenabsent in Caucasians, African American ; Frequencies of some alleles like A*2902, B*1402, Cw*0802 andDRB1*1303 were very low in Chinese Han but very common in Caucasians, African American.Conclusions: HLA distribution of Chinese Han is similar to Koreans and Japanese, but great differ fromCaucasians, African American and German. Chinese Han as a large ethic group in worldwide should bepaid much attention to. Our data will be valuable for stem cell donor searching program and donor registryplan.Li: The First Affiliated Hospital of Soochow University: Science Med Advisor. He: The First AffiliatedHospital of Soochow University: Science Med Advisor. Bao: The First Affiliated Hospital of SoochowUniversity: Science Med Advisor. Qiu: The First Affiliated Hospital of Soochow University: Science MedAdvisor. Yuan: The First Affiliated Hospital of Soochow University: Science Med Advisor.118-PMODELING EFFECTIVE PATIENT-DONOR MATCHING FOR HEMATOPOIETICTRANSPLANTATION IN UNITED STATES POPULATIONS.Martin Maiers 1 , Loren Gragert 1 , Eric Williams 1 , Dennis Confer 1 , William Klitz 2 . 1 National Marrow DonorProgram, Minneapolis, MN, USA; 2 University of California, Berkeley, CA, USA.Aim: In addition to the size and ethnic composition of the adult donor and cord blood unit (CBU)inventory, several factors affect the ability of stem cell registries to meet the needs of patients, includingavailability, CBU cell dose and HLA-matching stringency. We aim to determine the match rate (MR) forpatients now and under different future growth scenarios.Methods: We developed a model of the current NMDP-USA registry to determine HLA MR for patients in21 US population groups, as well as the “effective” (i.e., considering donor availability and CBU cell dose)MR under several growth scenarios. We utilized HLA haplotype frequencies derived from the registry todevelop MR projections using monte-carlo simulations that provide estimates of the MR across all possiblegenotypes under the assumption of Hardy-Weinberg Equilibrium.Results: Identification of at least 7/8 A,C,B,DRB1-allele-matched adult donor or 4/6 A,B-antigen- andDRB1-allele-matched CBU is possible for >90% of U.S. patients of European Caucasian ancestry, >70% ofthose of Asian or Hispanic ancestry, and >60% of those of African ancestry. Because of the very large adultdonor registry, the reduced matching requirements for CBUs, and the option for multiple CBU to addresscell dose requirements, an acceptable graft can be identified for most patients. However, matches at thehighest levels of stringency, known to provide superior transplant outcomes, are not available for most non-

Caucasian patients. MRs vary significantly by ethnic group, reflecting a combination of intrinsic diversitywithin populations and the ethnic composition of the registry. CBU MRs are improved by adding high celldose CBUs or decreasing CBU cell dose requirements.Conclusions: Improvements in donor availability and either increased CBU cell counts or decreased CBUcell dose requirements offer the potential for increases in effective match rates.119-PCHARACTERIZATION, ETHNICITY AND BANKING OF CORD BLOOD UNITS (CBU) OFTHE MEXICAN UNRELATED CORD BLOOD BANK-BACECU.Fernanda Pérez-V, Danaeé Rodríguez-D, Alejandra Vázquez-A, Gonzalo Lliguin, Carmen Aláez, HilarioFlores-A, Araceli Rodríguez, Andrea Munguía, David García, Elizabeth Solís, Clara Gorodezky. Dept. ofImmunology & Immunogenetics, InDRE, Secretary of Health, Mexico City, D.F., Mexico.Aim: Over 8000 CB transplants have been performed worldwide. BACECU belonging to the BMDW hascontributed with 50 of these transplants. Most of our CBU came from the NCBP and BACECU has given 4units. We have done 54 international searches. The aim was to analyze the characteristics and ethnicity ofour CBU. BACECU has collected 260 CBU. Written information on advantages/ disadvantages ofdonation is given to the mothers; they sign an consent, provide blood samples for HLA typing andinfectious disease markers and get the collection kit, instructions and the health questionnaire for thephysician.Methods: The collection bag is inspected when receiving it in the lab. We confirm that we have theminimum required vol., TNC and viability. The unit is processed and preserved at -196ºC. DNA, serum andcell aliquots for infectious disease control, determination of TNC, CD34 cells, viability and HLA typing arekept.Results: BACECU has cryopreserved 260 (97%); 27 have been discarded (10.1%)because of notcompliance. In the cryopreserved CBU, the viability is X=96.5%; TNCx10 7 =70.54; the final vol. X=56.31mL. The total CD34 cells is X= 2.4x10 6 ; with a conc. of X= 58.25 CD34/uL and X=91.9%. viability TheABO frequency is: O= 45.9%, A= 34.5%, B= 15.5% & AB=2.3%. 90.0% were collected at prívatehospitals. The prevalent HLA alleles are: A*02,24,68,01,11,30; B*35,39,51,65,62,08; Cw*07,03,06,08,04;DRB1*0701,1302,1104,0101,1501,1402/6;0407;DQB1*0301,0302,0202/0201,0402. Regarding ethnicity,87.7% are Mexican Mestizo, 6.4% Semitic, 3.6% Caucasian and 2.3% other Latin Americans.Conclusions: The HLA genetic pattern is compatible with a Meditterranean and Caucasian background.On the contrary, DONORMO, has more Mestizo and Amerindian genes. Our goal is to preserve CBU ofisolated populations of the country, since they will never become unrelated donors, because of their culturaland geographical isolation.120-PREAL-TIME PCR IN POST HEMATOPOETIC STEM CELL TRANSPLANT ENGRAFTMENTANALYSIS.Paula J. Peterson 1 , Charles M. Kellum 1 , Lois E. Regen 1 , Chris C. McFarland 1 , Anajane G. Smith 1 , ShaliniE. Pereira 1,2,3 . 1 Clinical Immunogenetics Laboratory, Seattle Cancer Care Alliance, Seattle, WA, USA;2 Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 3 Department of Laboratory Medicine,University of Washington, Seattle, WA, USA.Aim: RT-PCR methods of chimerism analysis in post-tx engraftment monitoring may confer a highersensitivity (0.05%) in detecting a minor component than STR (1-5%). We compared engraftmentmonitoring of BM samples by RT-PCR using the Celera AlleleSEQR® Assay with STR using the PromegaSytem. We also compared the number of informative indel markers in pt/donor pairs in the the RT-PCRmethod.Methods: DNA, from 86 mostly Caucasian HSCT pts and sib donors, was screened for informative indelsusing Celera Screening Plates. The number of informative markers observed per tx pair for RT-PCR andSTR were compared. Chimerism testing by RT- PCR was conducted on 21 samples from 16 pts where STRtesting had detected a range of 1-40% host. Additionally, BM samples from 5 post-tx pts who appeared tobe 100% donor but who subsequently revealed a recurrence of host, and 3 pts whose STR tests over timeshowed no host, were tested.

Results: Using the RT-PCR method, one tx pair had no informative markers, 19% had 1-2, 59% had 3-5,and 21% had 6-9. In the STR method, 2% had 1-3 informative loci, 44% had 4-6, and 54% had 7-11. Theaverage number of informative markers for RT-PCR was 4 and for STR, 7. Of 21 BMs tested by RT-PCR,those with the least minor component (1-5%) were most concordant with STR. At > concentrations (10-40%), RT-PCR results were sometimes discrepant. Host levels of < 0.75% were found in each of 5 BMsamples in which STR failed to find host until subsequent time points. Additionally, host levels of < 0.15%were found when STR could detect no host.Conclusions: The RT-PCR chimerism assay provides concordant results with STR at low concentrations ofminor component and detects minor component at levels below those detectable by STR. The number ofinformative markers per pt/sib pair for RT-PCR is slightly lower than that for the STR method. The utilityof RT-PCR may lie in predicting the onset of graft failure or relapse.121-PDynaChip HLA ANTIBODY ANALYSIS IN PATIENT’S SERUM UNDERWENT UNRELATEDONE ALLELE MISMATCHED BONE MARROW TRANSPLANT.Urszula Siekiera 1 , Miroslaw Markiewicz 2 , Tomasz Kruzel 2 , Monika Mietla 2 . 1 HLA & Immunogenetic Lab,Blood Center, Katowice, Poland; 2 Department of Bone Marrow Transplantation, Medical University ofSilesia, Katowice, Poland.Aim: We identified anti-HLA-A,B,C,DR,DQ,DP antibodies using sera collected from patients whounderwent bone marrow transplantation. The period of observation started in 2009 year. Each patientsreceived bone marrow from an unrelated donor. There were donor/recipient pairs mismatched. All thedonor/recipient pairs shared one different allele. Mostly there were alleles from HLA-C or HLA-DQ locus.Methods: To perform our research we used microchips spotted with purified HLA class I and HLA class IIantigens, pre-optimised reagents and DynaChip Processor (Dynal Invitrogen Corporation) for assayprocessing, data acquisition and analysis. Any HLA antibodies present in the test serum should bind to thesurface of the microchip. As yet we collected sera from 50 patients underwent bone marrowtransplantation. We started our research in 2009 year. Sera were collected and tested, beforetransplantation, three months, six months and one year after bone marrow transplantation.Results: We observed presence of the HLA antibodies class I in 40 % of patients whereas 10 % of patientsexpressed presence of antibodies against class I and class II antigens. In a group of 2 % of patients wedetected anti HLA antibodies before bone marrow transplantation. We were able to define specificities ofthe HLA- A, B, C, DR.DQ,DP antibodies on a low and high resolution level using DynaChip software.Wedefined specificity of antigens that masked the result of antibody identification. We did not define exactlywhether detected anti-HLA antibodies were donor specific.We used CREG’s analysis to compare antibodyreactivity and to make analysis easier. We did not detected anti HLA-DP antibodies in the group oftransplanted patients.Conclusions: There were a preliminary study results. We were not able to define donor specific antibodies.We intend continue the research using Luminex LabScreen method.122-PASSESSMENT OF CD34+ CELLS AND OTHER PARAMETERS IN HEMATOPOIETIC STEMCELLS (HSC) OBTAINED FROM DIFFERENT SOURCES.Alejandra Vázquez-A, Hilario Flores-A, Clara Gorodezky. Dept. of Immunology & Immunogenetics,InDRE, Secretary of Health, México, D.F., Mexico.Aim: CD34 used for HSC transplantation (Tx) are obtained from: Bone marrow (BM), MobilizedPeripheral Blood (PB) and Cord Blood (CB).The CD34 cells are relevant for engraftment and constitute 2-4% of the total nucleated cells (TNC) in the BM;

Results: We obtained the total No. (x10 6 ), % & CD34 + (cells/mL) conc., viability (%) & TNC. TrypanBlue staining (TB) was also used. We analyzed 171 CBU from BACECU, 12 CBU from the NY-NCBP,15 PBSC and 2 BM; 54 PBSC units & 2 BM were used for autologous Tx. The HSC Tx were done withunits from DONORMO or from international registries. Differences were found in CBU of BACECU Vs.NCBP in the total No. of CD34 cells (p=0.011, X=2.34; X=4.21) & in the TNC (p=0.002, X=633.12;X=1048.47); In the PB units (patients Vs. donors) the difference was shown in viability (TB) (p=0.006, X=86.2; X=98.14), in TNC (TB) (p=0.09, x=25,58; 67.91), TNC by FACS (p=0.02, X=577; X=945) and inthe vol. harvested (p=0.001, X=43; X=85.08).Conclusions: The results in the CBU were expected, since the units from the NCBP were selected with ahigh No. of TNC/CD34 + /Kg of weight of the patient, to assure engraftment. The difference found in PBmay be due to the fact that those used for autologous Tx are obtained from the patient in remission andthose used for allogeneic Tx are obtained from healthy donors.Our results are in agreement with theliterature and demonstrate that the QC used is optimal for transplantation.123-PILT3-Fc INDUCES ALLOSPECIFIC CD8+ T SUPRESSOR CELLS WHICH MEDIATE T CELLANERGY AND IMMUNOLOGICAL TOLERANCE.George Vlad, Chris Chang, Zhuoru Liu, Raffaello Cortesini, Nicole Suciu-Foca. Pathology, ColumbiaUniversity, New York, NY, USA.Aim: The aim of our study was to characterize the molecular changes which determine the conversion ofcytolytic CD8+ T cells into antigen specific CD8+ T supressor cells (Ts). The long term goal of ourinvestigation is to prevent the onset of allograft rejection and progression of autoimmune phenomena.Methods: Allospecific Ts were generated both in vitro and in vivo by exposure of primed T cells torecombinant ILT3-Fc molecule. ILT3-Fc-induced CD8+ T cells were tested for suppressor activity andcytokine production. Gene expression arrays were performed to identify changes which occur in ILT3-Fcinduced CD8+ Ts differentiating them from CTL. The in vivo effect of ILT3Fc was tested in hu-NOD/SCID mice tranplanted with human pancreatic islets. ILT3-Fc was administered before or after theonset of rejection.Results: ILT3-Fc induced Ts displayed significant upregulation of zinc finger transcritional repressors, andcell cycle regulators, while downregulating IL-2, IFN-g, IL-5, IL-10, granzyme B, and HLA class IImRNA. In vitro studies demonstrated that ILT3-Fc induced Ts inhibited CTL function of allospecific Tcells as well as the production of Th1, Th2 and Th17 cytokines in primary and secondary MLC. Ts areHLA class I allorestricted inducing the upregulation of inhibitory molecules and downregulation ofcostimulatory molecules in priming APC, but not in HLA-mismatched controls. ILT3-Fc treatment of hu-NOD/SCID mice transplanted with human islets, resulted in reversal of rejecton and induction of tolerancewhen administered within 2 weeks of humanization, and was mediated by CD8+ Ts.Conclusions: The efficiency of ILT3-Fc as an immunomodulatory agent which reverses allograft rejectionand induces transplant tolerance suggests the possibility of using this agent for treatment of autoimmunediseases and transplant rejection.124-PEXTENDED HLA HAPLOTYPES CONTAINING INFREQUENT DRB1 ALLELES.Georgena L. Wohlwend 1 , Lois E. Regen 1 , Maggie S. Sprague 1 , Anajane G. Smith 1 , Shalini E. Pereira 1,2,3 .1 Clinical Immunogenetics Laboratory, Seattle Cancer Care Alliance, Seattle, WA, USA; 2 FHCRC, Seattle,WA, USA; 3 Lab Medicine, U of Washington, Seattle, WA, USA.Aim: Our laboratory has identified several unusual DRB1 alleles in patients and potential donors typedprior to hematopoietic stem cell transplantation (HSCT) since 2003:DRB1*11:13,*11:29,*13:10,*13:12,*14:20,*14:21, and *14:48. We decided to type family members tolook for extended haplotypes in order to compare our data with published linkage studies.Methods: Archived samples of genomic DNA from patients and informative family members weresequenced using ABI-Big Dye® Terminator chemistry on a 3130XL capillary electrophoresis machine.Results were analyzed using Assign 3.5+ / Conexio software and linkages were inferred for five loci: HLA-A, B, C, DRB1 and DQB1.

Results: We characterized seven complete haplotypes (one for each allele) in seven independent families.(1) A*11:FXYE, C*12:03, B*18:RRG, DRB1*11:13, DQB1*05:03 (2) A*03:02, C*03:BPSK, B*40:01,DRB1*11:29, DQB1*03:01 (3) A*24:02, C*05:01, B*44:HTH, DRB1*13:10, DQB1*06:HPH (4)A*11:01, C*08:AEK, B*15:02, DRB1*13:12, DQB1*03:02 (5) A*03:01, C*16:02, B*51:08,DRB1*14:20,DQB1*03:01 (6) A*01:01, C*05:01, B*44:HTH, DRB1*14:21, DQB1*06:03 (7) A*24:02,C*01:02, B*15:30, DRB1*14:48, DQB1*03:01 Haplotype #4 appeared in a family self-described asChinese and haplotype #7 appeared in a family self-described as Hispanic. The other five familiesdescribed themselves as Caucasian.Conclusions: Knowledge of these rare haplotypes and alleles in different populations and ethnic groupsmay facilitate unrelated donor searches for patients lacking a family donor.125-PSTUTTER FOR SHORT TANDEM REPEAT TESTING IS A FUNCTION BOTH OF LOCUS ANDALLELE.Edgar Mlford, Rosalba Reyes-Gothers, Takako Watanabe, Doreen Sese. Tissue Typing Lab, BrighamHospital, Boston, MA, USA.Aim: Chimerism testing is done by genotyping individuals at nine to thirteen short tandem repeat geneticloci (STRs). Quantitation is done by capillary gel electrophoresis. Amplification produces a smaller artifactpeak at an amplitude from 2% to 12% of the allele from which it is derived. Stutter is subtracted fromoverlapping informative patient & donor peaks used in calculation of stutter.Methods: We calculated the percent stutter in patients and donors who were being genotyped in ourlaboratory and tabulated the results by gene and allele. 1736 stutter values were determined on 71 differentalleles at 9 STR loci.Results: Average stutter ranged from 4.5% for locus D7S820 to 7.6% for locus D18S51.[figure1]The rangeof stutter for alleles within a locus was even greater. Stutter in locus D18S51 ranged from 3.1% (allele 10)to 11.5% (allele 21). Within a locus there was a consistent increase in the stutter for alleles with highernumber of repeats.[figure2]Conclusions: Variance of stutter for a locus and allele between assays is substantial. When subtractingstutter from peaks attributable to a given allele, a more consistent approach would be to utilize the meanstutter for that locus and allele rather than a locus-specific stutter or stutter from prior genotyping of theparties in the mixed sample.126-PA SINGLE CENTER EXPERIENCE USING PRODIGY SSO PLATFORM.Nebila Abdulwahab, Scotty Lake, Nick DiPaola, Gregg Hadley. Clinical Histocompatibility Laboratory,The Ohio State University Medical Center, Columbus, OH, USA; Columbus, OH, USA; Columbus, OH,USA.Aim: We evaluated and compared the new SSO Prodigy platform with the current InnoLipa SSOtechnology for allele group assignment in terms of concordance, ambiguity, ease of the technology, techtime, and cost.Methods: Fifty patient, donor, and UCLA proficiency samples that were previously typed by InnoLipaSSO methodology were retested by Prodigy in batches of eight samples, due to the configuration of theProdigy chips. The analysis was performed using the Prodigy software and compared for allele resolutionand corresponding serology assignment of HLA-A, B, Cw, DRB1 and DQB1 loci. Additionally, bothplatforms were evaluated for hands- on tech time, efficiency of set-up and analysis, sample turn -aroundtime, and cost per sample including both reagents and tech time.Results: Concordance between the two methods was at 98%. Inno Lipa had 8% more ambiguitities thatneeded to be resolved further for HLA-A, B &34% for DRB1 loci. Prodigy had 20% more HLA-Cwserology ambiguities that needed resolution. All Prodigy tested samples that were homozygous forDRB1*07 required alternate testing. Total testing time for InnoLipa is about 8hrs, and Prodigy is 4.2hrs.Technologist hands- on time for InnoLipa was 89min(1.5hrs), and for Prodigy was 23min. The reagent costper sample for InnoLipa is $118/test, and for Prodigy is $109/test.Conclusions: From this initial experience we conclude that: 1-Prodigy has better resolution forintermediate level assignments. 2-Prodigy requires less DNA and has a shorter testing time. This allows for

cord blood and platelet transfusion samples with significantly low DNA yield to be typed efficiently. 3-Prodigy results in robust amplification and, less hands on manipulation. This translates to less chance forrepeats, errors, and less tech time. 4-Prodigy requires batch testing. In summary, the Prodigy assay platformoffers significant improvements for intermediate resolution HLA typing.127-PMBL2 HAPLOTYPES IN TRANSPLANT PATIENTS ARE SIMILAR TO OTHER POPULATIONFREQUENCIES AND CORRELATE WITH MBL PROTEIN LEVELS – IMPLICATIONS FOREVALUATION OF COMPLEMENT-MEDIATED EFFECTOR PATHWAY DEFICIENCY INTRANSPLANT RECIPIENTS.Jennifer McCue, Alexandra C. Amador, Folasade Amole, Gaetano Ciancio, Linda Chen, JunichiroSagashima, Giselle Guerra, David Roth, Warren Kupin, George W. Burke III, Andreas G. Tzakis, PhillipRuiz. Department of Surgery Tranplant Lab, University of Miami, Miami, FL, USA.Aim: To evaluate the distribution of MBL2 haplotypes and related MBL protein levels in our transplantpatient pool. Introduction: Several MBL2 gene polymorphisms appear to be related to complementdeficient defense mechanisms which may contribute to an increased incidence of infections in the posttransplantperiod as well as increased incidence of recurrent disease. Extended haplotype evaluation andMBL protein level testing for transplant patients may be a useful means of identifying high-risk patientsthat could be prophylactically treated to prevent infections.Methods: MBL2 genotyping for six SNPs using TaqMan® methodology was developed in our lab andDNA of 50 transplant patients and 25 controls was examined. MBL protein levels were determined usingELISA methodology. Haplotype data and protein levels were compared to published data.Results: Haplotype frequencies for our patient population closely mirrored those published for a Spanishpopulation. The prevalence of homozygous and heterozygous MBL-2 genotypes in the transplant patientswas comparable to our control population. MBL protein results correlated well with expected levels withsome variability, although serial samples were not determined in this study.Conclusions: Results for extended MBL2 haplotype frequencies and protein level analysis for ourtransplant patients mirrors published reports. Our data indicates that we can now monitor short- and longtermrelationships between the genotype for MBL-2 and the predicted vs. actual incidence of complicationsresulting from MBL protein deficiency.128-PLUMINEX BASED DETECTION OF IgG SUBCLASSES IN HLA ALLOANTISERA.Ozlem G. Ozturk 1 , Robert A. Bray 2 , Howard M. Gebel 2 . 1 Department of Clinical Biochemistry, MersinUniversity, Mersin, Turkey; 2 Pathology, Emory University, Atlanta, GA, USA.Aim: Recent studies suggest that not all donor directed IgG HLA alloantibodies (DSA) in renal allograftrecipients are pathogenic. One hypothesis is that pathogenicity is predicated on the ability of DSA tofix/activate complement, a process linked to IgG subclass specificity. This study was designed to develop asensitive, robust and reproducible Luminex-based phase assay to detect and quantify the IgG subclasscomposition of HLA alloantibodies in patients awaiting renal transplantation.Methods: Microparticles coated with either IgG1, IgG2, IgG3 or IgG4. Initially, flow cytometry was usedto document and validate the subclass specificity of commercially obtained biotinylated mouse monoclonalantibodies to IgG1, IgG2, IgG3 and IgG4. Conditions were then optimized for the Luminex platformwherein we analyzed sera from 10 highly sensitized renal transplant candidates (cPRA >80%). Sera werespecifically chosen such that at least two distinct antibody specificities were present.Results: In 5/10 sera, IgG1 was the only IgG subclass identified. For the remaining five sera, three wereadmixtures of IgG1 and IgG3, one was IgG1 and IgG2 and one was IgG1 and IgG4. Interestingly, 4/5 ofthese sera contained HLA specificities uniquely represented by subclasses other than IgG1.Conclusions: IgG2 and IgG4 isotypes are reported to poorly fix complement, while IgG3 has a short (7day) half life. It is tempting to speculate that these subclass attributes contribute to the stability and/orpathogencity of donor directed antibodies. For example, patients whose DSAs are mostly IgG2 or IgG4may be less likely to experience antibody mediated rejection, while fluctuations in HLA specificities maybe due to IgG3 antibodies. We believe this assay will be a useful tool to identify, classify and quantify IgGsubclass specificities of HLA antibodies in the sera of patients pre and post transplant.

129-PPOLYMORPHISM SER326CYS OF GEN hOGG1 IN RHEUMATOLOGICAL PATIENTS.Victoriano J. Leon 1 , Carlos Montilla 2 , Erica Polo-Hernandez 3 , Maria Luisa Hernandez Cerceño 1 . 1 Serviciode Analisis Clinicos, Hospital Universitario de Salamanca, Salamanca, Spain; 2 Servicio de Reumatologia,Hospital Universitario de Salamanca, Salamanca, Spain; 3 Instituto Neurociencias de Castilla y Leon,Universidad de Salamanca, Salamanca, Spain.Aim: The accumulation of damages in the DNA of the cerebral cells along the time, seems to play animportant paper in the pathogenesis of rheumatological diseases. hOGG1 is an enzyme responsible for therepair of DNA oxidative damage either in nuclei and mitochondria. The Ser326Cys polymorphism wasshown to diminish the hOGG1 activity in vitro, and it was suggested that the 326Cys allele might pose ahigher risk of 8-OHdG formation in DNA. Recently, some authors have demonstrated that the capacity torepair oxidative DNA damage was significantly decreased in healthy human subjects bearing the hOGG1Cys326Cys homozygous variant genotype, compared to wild type individuals.Methods: 88 patients with different rheumatological diseases (46 Rheumatoid Arthritis, RA;42, PsoriaticArthritis PA, and 94 controls were employed in this study. Genomic DNA was extracted from peripheralblood lymphocytes by means of the DNA direct II (Dynal) kit. Genotyping for the hOGG1 Ser326Cyspolymorphism was performed by PCR-RFLP technique, briefly, a 234 bp product was amplified using theforward: 5’-CCC AAC CCC AGT GGA TTC TCA TTG C-3’ and the reverse: 5’-GGT GCC CCA TCTAGC CTT GCG GCC CTT-3’ primers. PCR conditions were 4 min at 94 °C and followed by 30 cycles of30 s at 94 °C, 30 s at 60 °C, and 1 min and 30 s at 72 °C, and a final elongation step of 5 min at 72 °C.Overnight digestion at 37 °C with Fnu4HI resulted in 213- and 21-bp products for the wild type allele(Ser326), and in 164-, 49- and 21-bp products for the mutant allele (Cys326). Digestion products werevisualized after electrophoresis on a 2% agarose gel containing ethidium bromide.Results: The distribution of polymorphism in RA was: 64,3% SS, 32% SC 3,7 % CC, in PA 63,6% SS,32% SC 3,4 % CC the control group: 66,4 SS, 30,4 SC 3,2 CC. The Chi-square test, p< 0.01.Conclusions: There are not correlation in polymorphism in this study inter RA,PA and control group.130-PVALIDATION OF DNA DRY-STORAGE PRODUCT TO STORE SUFFICIENT AMOUNTS OFDNA FOR HLA PROFICIENCY TESTING.Arlene Locke, Cathy Huynh, Linda Dunn, Kathy Hatanaka, Kimiko Bibee, Maggie McGinley, RajaRajalingam, Elaine Reed, David Gjertson. UCLA Immunogenetics Center, David Geffen School ofMedicine at UCLA, Los Angeles, CA, USA.Aim: To assess a dry storage product’s ability to store 20-50 µg of DNA, comparing quantity/quality ofrehydrated product. To confirm that reconstituted DNA can be reliably typed for HLA-A,-B,-DRB1 bymultiple technologies.Methods: 10 DNA extracts were selected (Stds 1-5 B-cell lines, 6-10 spleen tissues). Aliquots wereproduced (100µL@200-500µg/mL) for inter/intra validations. Aliquots were placed in GenTegra matrix(GenVault Corp, Carlsbad CA), dried and stored at 22°C creating single-dry (SD) sets. After 6d, one setwas rehydrated with 100µL water, some DNA removed and remaining DNA was redried creating doubledry(DD) sets. DNA was reconstituted after 26d and typed via SSP, SSO and SBT.[table1]Results: Excluding a spill (Std 3), SD DNA amounts closely matched initial values (avg recovered = 97%).DD DNA quantities agreed with original amounts less DNA removed at 6d. Rehydrated DNA purityranged from 1.85-2.06 (260/280). Intra-assay variation was insignificant when comparing quantity andquality values from all aliquots within Stds 1 and 6. All HLA typing results from both SD and DD sampleswere consistent with known genotypes for these samples.Conclusions: GenTegra reliably stored 20-50 µg of DNA in dry state at 22°C over 4 weeks. Water elutionyielded high purity DNA samples with recovery rates exceeding 95%. Reconstituted DNA was correctlytyped for HLA-A, -B,-DRB1 using multiple technologies. The benefits include shipping or storing samplesat room temperature.

131-PASSOCIATION OF IL-2-330(G/T) WITH VOGT-KOYANAGI-HARADA´S DISEASE (VKH) INMEXICANS IS GENDER SPECIFIC. NO CORRELATION EXISTS WITH IFN-g 174-(A/T).Carmen Alaez 1 , Hilario Flores-A 1 , David García 1 , Luz Elena Concha 2 , Lourdes Arellanes 2 , ClaraGorodezky 1 . 1 Dept. of Immunogenetics, InDRE, Secretary of Health, Mexico City, D.F., Mexico;2 Inflammatory Eye Disease Clinics, Asociación Para Evitar la Ceguera en México, Mexico City, D.F.,Mexico.Aim: VKH is an autoimmune granulomatous panuveitis associated with systemic manifestations. A Thmediated autoimmune response Vs. melanocytes antigen(s) has been claimed. Proinflammatory cytokinesare increased in the patients’ sera and aqueous humor. SNPs present in the gene promoter region mayinfluence the amount of cytokines produced. The aim of this study was to assess the contribution of IL2-330(G/T) and IFN-g-174(A/T) SNPs to VKH susceptibility and to determine if a differential gendercontribution is present.Methods: Seventy five patients (59 females/16 males) and 202 controls (132 F/70 M) were included; all ofthem Mexican Mestizos. DNA extracted from peripheral blood was used for typing of the IL-2-330 (G/Trs2069762) and IFN-g-174-(A/T rs2430 561). Real time PCR with the 5´nuclease Taqman assay on a 7500platform was used. The analysis was done with SPSS17.Results: No significant differences were found in IL-2-330(G/T) alleles/genotypes distribution betweenpatients and controls. The prevalent allele was T (68.66% in VKH Vs. 60.89% in controls) The genotypedistribution GT(46.53%), TT(37.62%), GG(15.84%).was under HW expectation in controls. Genderstratification showed a significant increased frequency of TT in females (OR=1.98, p=0.04, pc=ns). A nonsignificant increase of GT genotype was found in males, possibly due to the small sample size of males(N=16). No difference was found for the IFN-g SNP.Conclusions: A gender association of IL-2 was shown for the first time in VKH etiology. Functional dataon this SNP are contradictory. TT and GT genotypes have been found in MS. The T allele and thecorresponding genotypes correlate with a higher IL-2 mRNA production in healthy individuals. Thisfinding supports a proinflammatory etiology for VKH and a gender differential genetic contribution toVKH as previously shown by us with HLA in VKH.132-PVERIFICATION OF B*08:06 DISPROVES EXISTENCE OF ALLELE.Elizabeth Beduhn 1 , Jane Kempenich 1 , Cynthia Vierra-Green 1 , Ana Lazaro 2 , Neng Yu 3 , Carly Masaberg 2 ,Lawrence Petz 5 , Alois Grathwohl 4 , Stephen Spellman 1 . 1 Scientific Services, National Marrow DonorProgram, Minneapolis, MN, USA; 2 Oncology, C.W. Bill Young/Dept. of Defense, Rockville, MD, USA;3 ARC-NE, Dedham, MA, USA; 4 HLA Service Team, DKMS German Marrow Donor Center, Tuebingen,Germany; 5 StemCyte Intl., Covina, CA, USA.Aim: B*08:06 is a rare allele, described on July 28, 1998 using a reference cell typed through the NMDPhigh resolution retrospective typing program. B*08:06 differs from B*08:20 by a single nucleotidesubstitution at position 302 (G to A), resulting in an amino acid change from serine to asparagine at codon77. B*08:06 and B*08:20 are further differentiated from B*08:01:01 at codons 152 and 156. The B*08:06reference cell line was selected for inclusion in an NMDP test panel, and subsequently typed by twoindependent labs, who confirmed the presence of B*08:20, not B*08:06, as previously reported. Since theIMGT reference cell sequence contained a likely error, the remainder of the donors on the Be The MatchRegistry containing B*08:06 were evaluated.Methods: Within the Be The Match Registry, eleven donors, typed between May 1999 and November2005, had B*08:06 assignments. Seven of the donors were re-typed by SBT and one donor was typed usingOne Lambda Luminex HD beads. No stored sample was available for three of the donors. The originalIMGT reference cell was also sent to a third laboratory, for cloning and sequencing of the putative B*08:06in isolation.Results: Five of the eleven donors and the IMGT reference cell retyped as B*08:20, two retyped asB*08:01, and one retyped as a novel B*08 allele (alanine to valine at codon 153). Two of the donors whocould not be tested were typed as B*07:04, B*08:06. It is likely these two donors actually carry B*07:02,B*08:01, because a cell carrying the same assignment was found to possess the more common alleles. It is

postulated the error may have been the result of a false negative probe. The remainder of the mistypingswere likely due to reagent limitations, as B*08:20 was not assigned until May 24, 2003.Conclusions: B*08:06 will be deleted from the IMGT database based on the resequencing results of thereference cell, which was found to contain an error at nucleotide position 302.133-PHLA-DQA1-DQB1 HAPLOTYPES IN THE US POPULATION.Eva Li, Stephanie M. Gibson, Elizabeth A. Mcconnell, Ainslie A. Fennell, Shanna M. Garrison, Trong-Thuy Tran, Maria P. Bettinotti. HLA and Immunogenetics, Quest Diagnostics Nichols Institute, Chantilly,VA, USA.Aim: The goal of the present study is to investigate the HLA-DQA1, DQB1 haplotype frequencies in theUS population. Our HLA laboratory receives a high volume of samples from individuals all over thecountry. The abundance of geographically diverse data allows us to obtain a collection of DQA1, DQB1haplotype frequencies reflective of the general US population.Methods: Genomic DNA of 5000 unrelated individuals was typed at the HLA-DQA1 and DQB1 loci byPCR-SSO. Haplotype frequencies were estimated using Arlequin 3.5 software.Results: Allele and haplotype frequencies including rare haplotypes will be presented.Conclusions: HLA-DQA1, DQB1 haplotype enumeration and estimated frequencies are helpful tools forHLA typing, for unrelated HSCT donor searches, for PRA studies as well as for estimating risk rates ofcertain diseases associated with DQA1, DQB1 loci.134-PWORLDWIDE RARE HLA ALLELES FREQUENTLY FOUND IN THE BRAZILIANPOPULATION.Pryscilla F. Wowk, Sibelle B. Mattar, Fabiana Poerner, Maria da Graça Bicalho. Genetics Department,Universidade Federal do Paraná, Curitiba, Paraná, Brazil.Aim: LIGH (Laboratory of Immunogenetics and Histocompatibility) from Universidade Federal do Paranáis part of a network of national laboratories that performs HLA typing to the Brazilian Bone Marrow DonorRegistry (REDOME). As a member of this network, sometimes we are faced with hard to resolve HLAgenotyping. This fact occurs mostly when rare alleles are involved. By analyzing our database (n= 82.000registries) we found that some HLA alleles, considered rare by the IMGT and the NMDP databases, seemto be frequent in the Brazilian population. In this abstract four of them are reported.Methods: Our HLA typing is performed with the SSOP Methodology (Sequence–Specific OligonucleotideProbe) supplied by LABType® SSOP Kits (One Lambda, USA) and genotyping is usually confirmed by asequence base typing (SBT - AlleleSEQR Core Kit, Atria Genetics, USA) when a rare allele is found.When the detection of a rare allele is confirmed this information is reported and submitted to thewww.allelefrequencies.net website. Up to March 2010, our laboratory reported to REDOME 82.000 HLAgenotyping of bone marrow volunteer donors.Results: The alleles reported in the present study do not represent allele frequencies, but the number oftimes a particular HLA allele appeared in LIGH database. Regarding HLA-A*0252, 43 individuals out of82000 were found to carry this allele, as to B*0720, 15 individuals out of 82 000 carry this allele. The samewas observed for HLA-DR*1356 (14/82000) and for HLA-DR*1413 (15/82 000).Conclusions: The Parana state region had a greater impact of the European immigration and has a largewhite majority population. LIGH data suggests that the some alleles considered rare around the world arerelatively frequent possibly due to the Brazilian ethnic history and Paraná state racial makeup.135-PTHE ENVIRONMENTAL DETERMINANTS OF DIABETES IN THE YOUNG (TEDDY):DENVER, CO (AN UPDATE).Alan Blair 1 , Jeff Post 1 , Lisa Ide 3 , Lavanya Nallamshetty 4 , Maria Alejandrino 2 , Misgana Bogale 2 , JudyBaxter 3 , Kathy Waugh 3 , Kathy Barriga 3 , Teodorica L. Bugawan 1 , Henry A. Erlich 1 , Marian J. Rewers 3 , TheTEDDY Study Group. 1 Human Genetics, Roche Molecular Systems, Inc., Pleasanton, CA, USA; 2 Research

Institute, Children’s Hospital Oakland, Oakland, CA, USA; 3 Health Sciences Center, University ofColorado, Denver, CO, USA; 4 Data Coordinating Center, University of South Florida, Tampa, FL, USA.Aim: TEDDY is an international program seeking to identify possible environmental triggers of T1D ingenetically susceptible newborns, using HLA genotyping of cord blood or blood spots. Individuals withhigh and moderate risk genotypes for DRB1, DQA1, and DQB1 are considered “Eligible” for follow-uptesting and monitored closely longitudinally for possible environmental exposures and the development ofautoantibodies against islet cell autoantigens (GAD-65, Insulin, and IA-2) and subsequent diabetes.Methods: We have developed a rapid DRB1, DQB1 genotyping assay to identify general population (GP)individuals in Denver, CO (one TEDDY center) with high and moderate risk genotypes using theimmobilized probe (Reverse LineBlot) strategy. In addition, first degree relatives (FDRs) of affectedprobands are genotyped using a DQA1-DQB1 assay. “Dual-probes” are used to set phase for 2 noncontiguoussequence motifs to distinguish the protective allele DRB1*0403 from the susceptible alleleDRB1*0407. StripScan software provides imaging of strips and automated allele assignment.Results: An “Eligible” GP subject has one of the following genotypes: DR03+04, DQB1*0201+0302;DR04+04, DQB1*0302; DR04+08, DQB1*0302+0402; or DR03+03, DQB1*0201. FDRs have anadditional 6 genotypes. From September, 2004 to April, 2010, 75,747 GP samples and 955 FDRs have beengenotyped with 4,170 “Eligible” GP individuals and 210 “Eligible” FDR individuals reported. Nineteen(19) subjects have developed T1D and thirty-two (32) subjects are autoantibody positive as of April, 2010.Of the 14 GP subjects who have developed T1D, 50% (7) have the high-risk DR03+04, DQB1*0201+0302genotype; 14% (2) have the moderate risk DR03+03, DQB1*0201 genotype; and 7% (1) have the moderaterisk DR04+08, DQB1*0302+0402 genotype.Conclusions: A method for determining high and moderate risk HLA genotypes is being combined withenvironmental data to assess risk for T1D.136-PSUBSTANTIAL INCREASE IN HLA-A,B,C AND DRB1 MATCHED UNRELATEDTRANSPLANTS FACILITATED BY THE NMDP FROM 2003-2009.Maria H. Brown 1 , Mike Haagenson 2 , Cynthia Vierra-Green 1 , Stephen Spellman 1 . 1 National Marrow DonorProgram, Minneapolis, MN, USA; 2 CIBMTR, Minneapolis, MN, USA.Aim: Current unrelated donor hematopoietic stem cell transplant matching guidelines recommend highresolution (HR) matching for HLA-A, B, C, and DRB1 8/8 based on publications by Flomenberg et al in2004 and Lee et al in 2007. The Lee study included transplants from 1988-2003 and approximately 50%were mismatched

5 Histogenetics, Inc, Ossining, NY, USA; 6 University of Maryland Medical Center, Baltimore, MD, USA;7 Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA; 8 ARC-Penn Jersey, Philadelphia, PA, USA.Aim: The NMDP maintains a publicly available list of rare alleles from HLA typings of patients, adultvolunteers, and cord blood units in the NMDP Registry. The typings of 104 of these rare alleles found in asmall number of donors were reexamined.Methods: Rare alleles were selected for study based on the following criteria: samples assignments showedprevious corrections, samples typed >4 years ago and allele not observed subsequently, probe data notconsistent with assignment, or sample carried two rare alleles. Laboratories were asked to review theirtyping; in many cases the analysis was repeated. Samples not resolved by this process were sent to alaboratory and typed by DNA sequencing.Results: 104 alleles were evaluated from 901 individuals. To date, the typings of 349 samples werechanged; the rare alleles in 95 samples were confirmed. The alleles impacted by this project are listedbelow.Conclusions: The results of this retyping project highlight the importance of continual data monitoring.Additional steps to confirm the presence of a rare allele prior to submission are crucial to ensure a correcttyping is reported.[table1]138-PCHARACTERIZATION OF A*24:23 AND A*30:10.Jane H. Kempenich, Jason Dehn. National Marrow Donor Program, Minneapolis, MN, USA.Aim: A*24:23 was described in a Native American (NAM) subject in Apr. 1999; A*30:10 was describedin Jan. 2001 per IMGT, www.ebi.ac.uk/imgt/hla/allele.html. Both alleles have a single nucleotidesubstitution, A*24:03/24:23 at codon 166 and A*30:02/30:10 at codon 99. Discrepancy review noted thatA*24:03 retyped as A*24:23 and A*30:02 retyped as A*30:10. A*30:10 was often seen withB*41/DRB1*04:05. A retyping project was done to evaluate if NAM samples with A*24:03 in fact carryA*24:23 and if A*30:10, not A*30:02, is in the haplotype with B*41/DRB1*04:05.Methods: 20 subjects listed as NAM with A*24:03 typed prior to 2001 and 117 subjects withA*30:02/B*41/DRB1*04:05 typed prior to 2002, with a stored sample, were retyped at HLA-A by SBT.Results: 70% of NAM samples with A*24:03 retyped as A*24:23; one sample carried A*24:33 which isidentical to A*24:03 within the antigen recognition site. 87% of the A*30:02 samples had A*30:10.A*30:10 was prevalent in subjects with Hispanic (HIS) ethnicity; there were no Asian (API) or NAMsamples in this dataset. The extent of A*30:10 linkage disequilibrium within the haplotypeA*30:10/B*41:01/DRB1*04:05 was evaluated. All subjects in the Be The Match Registry with A*30:10were examined. 80% of the A*30:10 subjects also carried B*41/DRB1*04:05. A*30:10 was reported withother B and DRB1 alleles, but the number for each was small and none indicated obvioushaplotypes.[table1][table2]Conclusions: A*24:23 was more common than A*24:03 in this NAM dataset. A*30:10 is the prevalentallele in the haplotype A*30 / B*41 / DRB1*04:05 in HIS and in African American (AFA) subjects; inCaucasians (CAU) A*30:02 is also common. This study highlights the importance of continual monitoringof HLA typing in order to keep current with new, often population specific alleles as they are discovered.139-PSEQUENCING OF HLA-DRB1 EXONS 2 AND 3 WITH INTRON BASED PRIMER DESIGNS.Tina Agostini, Dina Berchanskiy, Joel Shi, David Dinauer. Clinical Diagnostics, Life Technologies, BrownDeer, WI, USA.Aim: Many sequencing strategies for HLA-DRB1 utilize tailed amplification primers targeting bindingsites within the exon resulting loss of meaningful antigen binding site polymorphism. Tailed amplicon mayalso yield increased levels of baseline noise due to anomalous PCR fragment formation. We describe a newstrategy for HLA-DRB1 sequence based typing (SBT) that uses intronic primers for amplification andsequencing of complete exon 2 and exon 3 to increase resolution and ensure detection of novel alleles. Ourdesign produces uniform fragment lengths for each exon resulting in robust amplification and uses nestedsequencing primers to produce high quality, low background data.

Methods: To prove the robustness of the design, 174 proficiency samples were sequenced using thisstrategy followed by interpretation with uTYPE® sequencing analysis software.Results: 58% of samples had enhanced ambiguity resolution due to utilization of full exon2 and exon3sequence. All results were concordant with the consensus HLA types in the proficiency reports with theexception of 4 samples that exhibited novel alleles. Three samples showed novel exon3 sequence and oneshowed an unpublished polymorphism in intron 2.Conclusions: Complete exon 2 sequencing enables resolution of the following common alleles fromknown variants: DRB1*010101 from *0107/22; DRB1*030101 from *0342/50; DRB1* 040101 from*0464/66; DRB1*07010101 from *070102; DRB1*090102 from *0907; DRB1*110101 from *1130;DRB1*140101 from *1439; DRB1*150201 from *1519; DRB1*040501 from *040503; DRB1*110101from *110106; and DRB1*116501 from *116502. With the incorporation of exon 3, the strategy canfurther discriminate DRB1*1454 from 140101; and DRB1*120101 from *1206/17. The new HLA-DRB1SBT strategy described here produces reliable typing and novel allele discrimination for all exon2 andexon3 coding sequences as well as flanking intron sequences.Agostini: Life Technologies: Employee. Berchanskiy: Life Technologies: Employee. Shi: Life Technologies:Employee. Dinauer: Life Technologies: Employee.140-PANALYSIS OF CTLA-4 POLYMORPHISMS IN MEXICAN PATIENTS WITH VOGT-KOYANAGI-HARADA´S DISEASE (VKH).Hilario Flores-A 1 , Carmen Alaez 1 , David Garcia 1 , Luz E. Concha 2 , Lourdes Arellanes 2 , Clara Gorodezky 2 .1 Dept. of Immunology and Immunogenetics, InDRE, Secretary of Health, Mexico City, D.F., Mexico;2 Inflammatory Eye Disease Clinics, Asociacion para evitar la Ceguera en Mexico, Mexico City, D.F.,Mexico.Aim: VKH is an idiopathic multisystem inflammatory disease with bilateral uveitis. An autoimmunecellular immune response against melanocytes has been clearly shown. DRB1*0405 is strongly associatedbut does not fully explain the genetic risk. CTLA4 is a negative regulator of the T cell response whichinhibits CD28 T cell activation. Several autoimmune diseases have been associated to SNPs in the CTLA4gene. The aim of this study was to search for the role of four CTLA-4 polymorphisms in VKH.Methods: DNA from seventy five patients and 202 controls was analyzed. All of them were MexicanMestizos. For allele discrimination of CTLA4 -1661(A/G rs 4553808); -319(C/T rs5742909), +49(A/Grs231775) & +6230(A/G rs3087243) real time PCR with the 5´nuclease Taqman assay on a 7500 platformwas used. The analysis was done with SPSS17 and SNP Analyzer softwares.Results: No significant deviation in allele or genotypes distribution between patients and controls wasfound for any SNP or after gender stratification. The prevalent alleles in controls were -1661-A(87.12%), -319-C(93.3%); +49-A(55.54%); +6230-G(61.14%) All polymorphisms were under HW expectation incontrols. A total of nine haplotypes were found in the sample, but only four had a HF>5%. Haplotypedistribution was similar in patients and controls. The prevalent haplotypes were ACGG (45.20% vs45.25%) and ACAA (40.41% vs 40.75%). Strong linkage disequilibrium was present between the studiedSPNs. The SNP Analyzer software demonstarted that the ancestral haplotypes are ACGG & ACCA.Conclusions: CTLA-4 is not involved in susceptibility to VKH in Mexicans, contrasting with the findingsin Chinese where CTLA4-49-G and ACGG were increased. This haplotype correlates with susceptibility inother autoimmune diseases. The most frequent alleles for each SNP in Mexicans corresponds to theancestral one; their frequencies are similar to those reported for the Americas.141-PREGISTRY INTERPRETATION OF LABORATORY SUBMITTED PRIMARY HLASEQUENCE-BASED TYPING (SBT) DATA.John L. Freeman 1 , Jonathan Sorbie 2 , Gail Flickinger 3 , Loren Gragert 1 , Michelle Setterholm 3 , MartinMaiers 1 . 1 Bioinformatics Research, National Marrow Donor Program, Minneapolis, MN, USA;2 Bioinformatics Operations, National Marrow Donor Program, Minneapolis, MN, USA; 3 ScientificServices, National Marrow Donor Program, Minneapolis, MN, USA.Aim: The NMDP receives primary data from HLA sequence-based typing (SBT) in the form ofHistoimmunogenetics Markup (HML) files. Upon receipt, the NMDP performs interpretation against the

IMGT-HLA sequence database and validates the laboratory’s submitted interpretation. This primary HLAdata is useful for:• reinterpretation of primary data to the most current HLA allele database• creation of shorter genotype lists than allele code pairs• quality control on every sample.Methods: Using sample HML files from two different laboratories we developed an algorithm to interpretprimary SBT data. The reported interpretation was compared against ours. The HML files contained bothclass I and class II data, homozygous and heterozygous alleles, haploid and diploid interpretations, andsome diploid reads. Some alleles needed realignment due to insertion/deletion characters. Pairs of IMGTdefinedHLA alleles were matched against the SBT sequences to generate a genotype list. Comparison ofthe genotype lists from the submitted interpretation and NMDP interpretation ensured the algorithm wasworking correctly.Results: Of the 267 diploid samples, 255 cases (96%) were consistent, 6 failed interpretation, and 6 wereinconsistent. Of the 42 haploid samples, 18 were consistent, 2 failed interpretation, and 22 wereinconsistent because interpretation ruled out many invalid allele code pairs. Six samples had invalid data,which was communicated back to the laboratories.Conclusions: Significant progress has been made based on collaboration between laboratories and theNMDP. Successful SBT interpretation allows the NMDP to better refine matching predictions on patientsearch reports and reinterpret typing results to account for new allele discovery.142-PHLA CLASS I AND CLASS II ALLELES IN HELICOBACTER PYLORI POSITIVE PATIENTSWITH ACTIVE GASTRITIS AND DUODENAL ULCER.Sevim Gönen, Sinan Sari, Buket Dalgiç, Oguz Söylemezoglu. Pediatric Neprology, Gazi University,Ankara, Turkey; Pediatric Gastroentrology, Gazi University, Ankara, Turkey.Aim: Helicobacter pylori colonizes on the epithelial surface below the mucous membrane in the gastricantrum,causing chronic active gastritis and doudenal ulcer.HLA Class I and Class II antigens were studiedin H.pylori infected gastric cancer patients but we have no data in active gastritis and ulcer patients.Weinvestigated HLA Class I and Class II subtypes of H.pylori patiens.Methods: Forty H.pylori positive patients and 200 healthy controls were typed for Class I and Class IIantigens by PCR-SSO method using ‘Luminex’ technology. Chi-square test was used to comparephenotypic frequencies.Results: The genotypic frequencies in the patients were as follows: DRB1*04 (45 %),DRB1*13(32,5%),DRB1*11 (27,53 %),DQB1*03 (87,5 %),DQB1*05 (35 %),DQB1*02 (25 %), HLA-A*02(50%),A*24 (42,5%),B*51(40%),B*35(27,5 %),Cw*07(47,5 %),Cw12*(25%). Each genotype frequencywas compared to the control frequency of our group consisting of bone marrow transplantation donors. Thegenotyping frequencies of the control group were as follows: DRB1*11 (34 %),DRB1*04 (33,2%),DRB1*13 (18,4%),DQB1*03 (83,2 %),DQB1*05 (28,8 %),DQB1*02 (28,4 %). HLA-A*02(36%),A*24 (29,5%),B*51(16,5%),B*35(29 %),Cw*07(32 %),Cw12*(29%).Conclusions: Comparing the control group and patients DRB1*13 was found to be significantly morefruquent in patients.Although the patient number is limited we can suggest that carriying DRB1*13 antigenmay increase the suscepitiblity to H.pylori infections.143-PHLA AND CHRONIC IDIOPATHIC URTICARIA IN PATIENTS WITH POSITIVEAUTOLOGOUS SERUM SKIN TEST.Zamir Calamita 1 , Marcelo O. Ruiz 2 , Marcia Gamberini 2 , Dione G. Arevalo 2 , Wilson Baleotti, Jr. 3 , LarissaB. Lopes 3 , Antonio Fabron, Jr. 3 , Pelá B. Andrea 1 . 1 Faculdade Estadual de Medicina de Marília -FAMEMA, Marilia, SP, Brazil; 2 Laboratório de Imunologia de Marília - LIM, Marilia, SP, Brazil;3 Hemocentro de Marília, Marília, SP, Brazil.Aim: The major histocompatibility complex (MHC) encodes the human leukocyte antigens (HLA), whichacts as a marker for self during T-cell ontogeny and is associated with the pathogenesis of manyautoimmune diseases. The association between MHC class I and II alleles and chronic idiophatic urticaria(CIU) has been reported previously by Coban and col. (2008) and O’Donnell and col. (1999) in different

populations.We studied the involvement of HLA class I and class II (loci A, B and DRB1) in Brazilianpatients with CIU and positive autologous serum skin test (ASST).Methods: The DNA was extracted from blood of 37 patients with CIU (28 women and 9 men; mean + SDage: 43,2 + 14,8 years, range 19-70 years), and MHC class I and II type determined by a PCR and singlestrandoligonucleotide probe hybridization assay. The frequency of these alleles in CIU patients wascompared with donnor bone marrow from the center west of São Paulo State, Brazil. The diagnosis ofautoimmune chronic urticaria (ACU) was based on the patients’ history and routine laboratory tests furtherpositivity to ASST. The ASST was carried out as previously described by Greaves (2000). The allelicdistribution results between the patients and control groups were analyzed as odds rate (OR) withcalculation of 95% confidence intervals (CI).Results: We found a tendency (without statistical significance) toward association with ACU between theB*37 (OR 3.33; 95% CI: 0.91-12.17), B*45 (OR 2.91; 95% CI: 0.95-8.85), B*48 (OR 4.11; 95% CI: 0.81-20.7), B*50 (OR 2.17; 95% CI: 0.73-6.44) and DRB1*12 (OR 2,07; 95% CI: 0.59-7,22).Conclusions: Among the evaluated class I and II alleles, HLA-B seems to have the allele a more importantrole in chronic urticaria brazilian patients. This is a preliminary study and the sample number will beincreased.144-PHLA ASSOCIATIONS WITH BIRTH DATE AND AGE AND APPLICATIONS TO DISEASEASSOCIATION STUDIES.Loren Gragert 1 , Martin Maiers 1 , William Klitz 2 . 1 National Marrow Donor Program; 2 University ofCalifornia, Berkeley.Aim: Matching controls when conducting HLA disease association studies must be considered part of theresearch protocol to avoid erroneous conclusions. We have previously described significant HLAassociations within self-identified race/ethnic (SIRE) categories for geography, gender, and populationsubstructure. Our goal was to extend this to test for effects of birth date and age to see if these factorsneeded to be added to the matched controls protocol.Methods: In order to test for HLA disparities based on birth date and age, we split a control sample ofEuropean-American donors recruited by the NMDP between 1997 and 2002 into two parts and ran HLAassociation studies as a case-control study.Results: Comparing the HLA of donors under 50 with those over 50, we found several significant HLAassociations. The HLA haplotype most associated with older age was A*02-B*18-DR*04 with an oddsratio of 0.582 and a P-value of 0.0006 after Bonferroni correction. We also found a B-DR haplotypeassociated with younger age, B*35-DR*02 with an odds ratio of 1.25 and a P-value of 0.013. Whencomparing donors born before 1950 with those born after 1950, we also detected several significantassociations, some of which were distinct from the age association analysis.Conclusions: HLA differences based on age may come about from protective and/or predisposing effectsof HLA for disease, results in older subjects having a different HLA makeup than younger subjects. HLAdifferences based on birth date may reflect demographic changes in populations over time, a result of newimmigration or higher levels of admixture in younger subjects. Further investigation is needed to testhypotheses for the causes of these HLA disparities.145-PBOOTSTRAPPING THE EXPECTATION-MAXIMIZATION ALGORITHM TO CALCULATEHAPLOTYPE FREQUENCIES FROM HIGHLY AMBIGUOUS HLA TYPING DATA.Loren Gragert 1 , Martin Maiers 1 , William Klitz 2 . 1 National Marrow Donor Program; 2 University ofCalifornia, Berkeley.Aim: Calculating high-resolution HLA haplotype frequencies from mixed-resolution HLA typing data is asignificant computational challenge. Reducing the allele list to a minimal set required to explain all donorsin the cohort, the number of possible A-B-DRB1 haplotype pairs for a dataset of 679,521 DNA-typedEuropean-Caucasian donors is 131 trillion. We aim to reduce genotype lists inputted into the expectationmaximization(EM) algorithm to a tractable level. A very small fraction of donors have a disproportionatelyhigh number of haplotype pairs due to typing reported at the 2-digit DNA level and having noaccompanying genomic typing assay data.

Methods: Initially we calculated bootstrap haplotype frequencies using EM at the rollup race level on onlydonors with fewer than 500 haplotype pairs in their genotype list, excluding the other donors. The bootstrapfrequencies were then used to restrict the genotype lists when all donors were included in the second roundof EM at the detailed race and ethnicity level. For the larger cohorts, further constraints are placed on thevery largest genotype lists based on the bootstrap haplotype frequency. When this method restricts agenotype list such that it has no entries after this step, the EM defaults to using all possible haplotype pairsagain so that all donors are included in the estimation.Results: The bootstrapping technique reduced the number of possible haplotype pairs inputted to the EMalgorithm by up to eight orders of magnitude yet continued to achieve similar haplotype frequencyestimates.Conclusions: Bootstrapping helps to make calculation of high-resolution haplotype frequencies for entireregistries possible, even if all donors are not typed at high-resolution. These haplotype frequencies areuseful in improving prediction of high-resolution typing on search reports and in registry match ratemodeling.146-PRHEUMATOID ARTHRITIS (RA)-ASSOCIATED MHC GENES AND PRODUCTION OF AUTO-ANTIBODIES IN VITRO.Mei Han 1 , Maria Bellatin 1 , Donglan Xia 1 , Margarita Fallena 2 , Nancy Olsen 2 , David Karp 2 , Peter Stastny 1 .1 Internal Medicine - Transplant Immunology, UT Southwestern Medical Center, Dallas, TX, USA;2 Internal Medicine - Rheumatology, UT Southwestern Medical Center, Dallas, TX, USA.Aim: Autoantibodies against citrullinated proteins are associated with the development of RA. MHCrelatedgenetic factors and environmental stimuli, such as smoking, appear to play a role. We investigatedthe contribution of the RA-associated MHC alleles in the pathogenesis of RA by analyzing the productionof autoantibodies against citrullinated peptides in vitro.Methods: B-cells were isolated from 65 patients with RA and 60 healthy persons. Cultures of B cells withEL4B5 cells expressing CD40-ligand and a supply of T-cell factors were incubated for 14 days. Culturefluids were tested for antibodies against citrullinated peptides and non-citrullinated peptides by ELISA.RA-associated MHC genes were investigated by high resolution typing for HLA class II.Results: Antibodies against CCP were detected in 44/65 cultures from RA patients (67%). Production ofanti-CCP was also observed in 21/60 healthy subjects (35%). Patients with RA were more likely to have Bcells making anti-CCP if they were positive for an RA-associated MHC gene (Odds Ratio=5.8. p=0.005)and this was true also in the healthy controls (OR=4.6, p=0.01). Smoking was associated with a higherfrequency of anti-CCP production in RA patients (p=0.02). In vitro production of antibodies was specificfor the citrulline residue (CCP3) and did not react with peptides containing arginine instead (CAP3).Conclusions: Antibodies to citrullinated peptides were produced in vitro by B cells of RA patients andhealthy controls. RA-associated MHC alleles may facilitate this immune response. Study of the B cellrepertoire may offer a window to the events leading to the development of autoimmunity and disease inpersons with MHC-associated susceptibility for the development of RA.147-PHIGH RESOLUTION HLA GENOTYPING BY NEXT GENERATION 454 GS-FLXSEQUENCING EMPLOYING AUTOMATED AMPLICON HANDLING.Cherie L. Holcomb 1 , Suzanne Gelber 1 , Bryan Höglund 1 , Tom Chu 2 , Henry A. Erlich 1 . 1 Human Genetics,Roche Molecular Systems, Pleasanton, CA, USA; 2 Pharm Research and Early Development, Roche PaloAlto, Palo Alto, CA, USA.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. Achieving high throughput can be challenging due to the number of handling steps, includingamplicon cleanup, quality check, quantification, dilution and pooling, involved in amplicon preparationprior to sequencing.Methods: To achieve high throughput genotyping, we have used 14 PCR primer pairs (exon 2, 3, and 4 forclass I, exon 2 for DPB1, DQA1, and the DRB loci, and exons 2 and 3 for DQB1) with 11 multiplexidentifier (MID) tags to amplify HLA exons from individual samples. Use of MIDs allowed pooling of the

amplicons generated from different individuals prior to the emulsion PCR step. Using a liquid handler forpartially automated preparation of amplicon pools prior to sequencing, we have typed 64 DNA samplesderived from whole blood. To achieve a balance in read depth for the various amplicons, we employed avariation in construction of pools in which we added 2-4 times the molar concentrations of ampliconsgenerated by the primers for A (exon 4), B (exon 2), C (exon 3), and DRB (exon 2).Results: The clonal nature of this sequencing system reduces genotype ambiguities by allowing theassignment of “phase” to linked polymorphisms within the same amplicon. Typically, in a single run with adepth of 300 sequence reads per amplicon, we have run 22 samples in 5 days with resultant genotyping ofat least 98.3% of the 176 loci interrogated.Conclusions: The application of automation as well as construction of amplicon pools which producebalanced numbers of sequence reads can likely be applied generally to increase throughput of 454sequencing.148-PTHE IMPACT OF SPANISH COLONIZATION IN WORLD POPULATIONS.William Klitz 1 , Loren Gragert 2 , Maiers Martin 2 , Enric Carreras 3 . 1 School of Public Health, University ofCalifornia, Berkeley, CA, USA; 2 Bioinformatics, National Marrow Donor Program, Minneapolis, MN,USA; 3 Spanish Marrow Donor registry, Barcelona, Spain.Aim: Spain was one of the first European cultures to explore and occupy land from across the world’soceans, including several centuries of occupation in Mexico and the Philippines. One possible consequenceof that period is admixture with local populations. Here we assess the hypothesis of Spanish admixture inMexican and Filipino samples through the use of HLA-typed population stem cell donor registries, alsousing Japanese samples as an out group.Methods: Haplotype frequencies were estimated from the Spanish National bone marrow registry and forthe other populations from the National Marrow Donor Registry in the United States at two- digitresolution of HLA A, B and DRB1. The similarity index If was used to gauge overall haplotype identity.Results: Spanish A-B-DRB1 haplotypes have a similarity fraction of 48% with Mexico, 13% with thePhilippines and 11% with the Japanese. This fraction of European admixture in contemporary Mexicanshas been seen in studies using other genetic systems. The low and slight difference in If values between theFilipinos and Japanese suggest two possibilities. On the one hand little or no Spanish admixture may bepresent in the Philippines. The second possibility is that the availability of higher resolution haplotypesfrom each population might reveal a signature of Spanish admixture in the Filipinos largely invisible herebecause of the low resolution typing.Conclusions: We conclude that even at less than full allelic resolution HLA variation, the haplotype datacan provide an indication of historical admixture between Spain and its former colonies. Two avenues ofadditional research will be able to refine this initial effort: a) analysis of samples at four digit resolution andb) analysis of the Spanish sample by province, allowing a more precise consideration of populationdiversity and differential migration to the Spanish colonies.149-PTHE POWER OF HLA FOR THE DISCRIMINATION OF HUMAN POPULATIONS.William Klitz 1 , Loren Gragert 2 , Martin Maiers 2 . 1 School of Public Health, University of California,Berkeley, CA, USA; 2 Bioinformatics, National Marrow Donor Program, Minneapolis, MN, USA.Aim: While only a fraction of human genetic variation is distributed between populations, of that variationthe HLA complex appears to be the most powerful system in this regard for the discrimination of onehuman group from another. Here we utilized the high resolution HLA typing and exceptionally large andethnically specified population sizes of the National Marrow Donor Program adult donor database toportray the genetic relationships among a number of Asian population groups.Methods: We analyzed estimated A-B-DRB1 haplotype frequencies in eight Asian-American populations,including (abbreviation and samples size in thousands), South Asia (SA, 211), Filipino (F, 57), Hawaii (H,12), Vietnamese (V, 27), other SE Asia (SEA, 29), Chinese (C, 108), Korean (K, 97), and Japanese (J, 30).Results: The haplotype diversity in each population was very large: the ordered haplotype frequency listreached 50% of the sample ranging from 72 in the Filipino to 295 in the Other Southeast Asian group.Principal components analysis (PCA) and a clustering package (cluto) were used to reveal relationships

among the populations. PCA indicated that the South Asian and Other Southeast Asian groups fall at thecenter of the Asian populations. Three pairs of populations diverge in different directions, including aPacific island group (F and H), an east Asia group (C and V) and a derived East Asian group (K and J). Thecluto analyses revealed the HLA similarity of these same pairs, along with the specific most frequentlyassociated haplotypes for each population.Conclusions: These results mirror the conclusion of a recent genome wide analysis of these same groupspointing to a founding population source for East Asian peoples from Southeast Asia. More generally thisstudy points to the power of a single complex genetic system, the HLA complex, to define populationdifferentiation in our species.150-PUNEXPECTED ADMIXTURE LEVELS REVEALED BY HLA.William Klitz 1 , Loren Gragert 2 , Martin Maiers 2 , Janesz Walaszeski 3 , Marcelo Fernandez-Vina 4 , ChaimBrautbar 5 , Shoshana Israel 5 , Gil Benedek 5 . 1 School of Public Health, University of California, Berkeley,CA, USA; 2 Bioinformatics, National Marrow Donor Program, MInneapolis, MN, USA; 3 POL Transplant,Warsaw, Poland; 4 MD Anderson Center, University of Texas, Houston, TX, USA; 5 Hadassah MedicalSchool, Hebrew University, Jerusalem, Israel.Aim: We utilize HLA haplotype frequencies from Polish and Jewish populations to assess degrees ofadmixture and estimate average admixture rates necessary to achieve observed levels of haplotype sharing.Methods: Three-locus HLA A, B and DRB1 haplotypes were utilized from the POL Registry in Warsawand the Hadassah registry in Jerusalem. Samples consisted of 13,556 Poles and 1,676 Ashkenazim havingparents from Poland. We utilized a new approach for estimating admixture that averages the frequencydifferences between to populations between haplotypes assigned to a population of origin.Results: As anticipated, the polymorphism levels of the three locus haplotypes were very high in bothpopulations. Assuming all haplotype frequencies differences as due to admixture, the index of similarity, I f ,was used to assess the degree of haplotype sharing. The estimated frequency of Ashkenazi HLA haplotypesin Poles is 24%, while the frequency of Polish HLA in the Polish derived Ashkenazim is 31%. Theseestimates, along with number of generations at which admixture may have occurred, are sufficient todetermine the mean rate of admixture per generation necessary in each population. For example, assuming40 generations (or ∼1,000 years) of co-residence in Northern Europe, per generation admixture rates of0.92% of Ashkenazim into Poles and of 0.68% of Poles into Ashkenazim explain the contemporarypopulation compositions. The levels of admixture in the Polish and Ashkenazi populations observed heregain independent confirmation from recent genome wide and Y chromosome studies on Jewish populationsin Europe.Conclusions: Despite culturally restricted gene flow between the two groups, the HLA evidence shows thaton the order of just one admixed pregnancy per generation accumulated over time can have a sizeableimpact on population composition.151-PA CASE OF A*24 VARIANT SHOWING DISCREPANCY BETWEEN HIGH- AND LOW-RESOLUTION HLA TYPING DUE TO COMPLETE LOSS OF ALL INTRONS.Sun-Young Ko 1 , Sung-Eun Choi 1 , Heung-Bum Oh 1 , Yong-Hak Sohn 2 . 1 Department of LaboratoryMedicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea; 2 Departmentof Laboratory Medicine, Eulji University Hospital, Daejeon, Korea.Aim: We encountered a very rare case showing a discrepancy between sequence-based typing (SBT) andsequence-specific primer (SSP) HLA molecular typing. The results were A*0201/A*0201 and A*02/A*24,respectively. The HLA type of the case’s father was A*0201/A*2601 and A*02/A*26, but showed anadditional amplification with A*24 primers. Serological typing showed A2/blank for the case and A2/A26for the father. It was assumed that an A*24 variant of the case was inherited from the father.Methods: To characterize the A*24 variant, we extracted genomic DNA from the case’s peripheral blood,amplified it with A*24 specific primers, and sequenced the PCR products. The case showed an A*24-specific PCR product, but its size was smaller than expected. Accordingly, the case was considered to haveboth a normal HLA-A*02 allele and an abnormal A*24 variant allele. To identify the nature of thisabnormal A*24 allele, we cloned and sequenced these PCR products.

Results: The sequence of the unexpected PCR products revealed a complete loss of all introns of theA*2402 allele from exon 1 to exon 7. Thus, it could be reasoned that the A*2402 variant was not detectedby the PCR-SBT, which employs primers in the intron region, whereas it was detected by PCR-SSP, whichuses primers in the exon region. The A*2402 variant of our case is presumed to be a processed pseudogeneby reverse transcription of A*2402 mRNA.Conclusions: Though rare, this type of variant is likely one of the cause showing discrepancies in HLAtyping results among SSP, SBT and serology.152-PNEPHROGENIC SYSTEMIC FIBROSIS AFTER GADOLINIUM-BASED CONTRAST AGENTSIS NOT ASSOCITATED TO HLA-A,B OR DR TYPES.Pernille Koefoed-Nielsen 1 , Christian Erikstrup 1 , Tina R. Elmholdt 2,3,4 , Bjarne K. Møller 1 . 1 Department ofClinical Immunology, Aarhus University Hospital, Skejby, Aarhus N, Denmark; 2 Institute of ClinicalMedicine, Aarhus University Hospital, Skejby, Aarhus N, Denmark; 3 Department of Dermatology, AarhusUniversity Hospital, Aarhus N, Denmark; 4 MR Research Center, Aarhus University Hospital, Skejby,Aarhus N, Denmark.Aim: Nephrogenic systemic fibrosis (NSF) is an iatrogenic connective tissue disease. The condition ischaracterized by painful and debilitating, progressive fibrosis and thickening of the skin, with occasionalinvolvement of other organs and tissues such as the lungs, heart, liver, esophagus, testes, dura mater andmuscles. Until now it has only been described in people with acute or chronic kidney disease. The role ofgadolinium- based contrast agents in the development of NSF is now widely accepted. Gadolinium is usedin MRI to visualize structures in the body e.g. the vessels. Because of the nature of the disease, wespeculated whether there could be an association between the development of the disease and HLA types ofthe patients.Methods: 35 patients diagnosed with NSF and 189 control patients who were treated with gadoliniumbasedagents without developing NSF were included in the analysis. The NSF diagnosis was based onclinical presentation, skin biopsy, medical history and blood samples (e.g. auto antibodies to exclude otherconnective tissue diseases). Serological HLA tissue typing was performed using a complement-dependingcytotoxicity (CDC) technique.Results: We analysed all HLA head types for a possible association to NSF. For HLA-A types and DRtypes we found no statistically significant association. Regarding HLA-B types we found 13 of the 35patients with NSF to have the HLA-B12 antigen and 39 of the 189 control patients (p=0.048). Because ofmultiple comparisons, we corrected results with Bonferroni constant, leading to a non-significant result.Conclusions: Our results demonstrated no association between the development of NSF and HLA tissuetypes.153-PHLA ASSOCIATION WITH ALLOPURINOL-INDUCED SEVERE CUTANEOUS ADVERSEREACTIONS IN KOREANS.Kyung Wha Lee 1 , Hye-Ryun Kang 2 , Young Koo Jee 3 , Yon-Soo Kim 2 , Chang-Hwa Lee 3 , Heung-WooPark 2 , Yoon-Seok Chang 2 , Sang-Heon Cho 2 , Kyung-Up Min 2 , Sang-Heon Kim 4 . 1 Hallym Institute forGenome Application, Hallym University School of Medicine, Anyang-Si, Kyunggi-Do, Korea; 2 InternalMedicine, Seoul National University School of Medicine, Seoul, Korea; 3 Internal Medicine, DankookUniversity College of Medicine, Cheonan-Si, Choongchung-Do, Korea; 4 Internal Medicine, HanyangUniversity College of Medicine, Seoul, Korea.Aim: Allopurinol (AP), a drug used in the treatment of symptomatic hyperuricemia and its complications,can induce life-threatening severe cutaneous adverse reactions (SCAR). Recent investigations suggest thatHLA class I alleles including B*5801 are associated with allopurinol-induced SCAR (AP-SCAR).However, the strength of association seemed to be quite variable to disease phenotypes and ethnicbackgrounds. In this study, we analyzed HLA class I association with AP-SCAR in Koreans expressinghigh frequency of B*5801 (phenotype frequency = 13.0%).Methods: A total of 25 Korean patients with AP-SCAR (20 hypersensitivity syndrome (HSS), 5 Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN)) and 57 AP-tolerant controls, were enrolled andhigh-resolution HLA class I genotyping was performed by the direct DNA sequencing analysis.

Results: In the HSS group, the frequencies of three alleles comprising a closely linked haplotype inKoreans were significantly increased compared with tolerant controls: B*5801 (95.0% vs. 10.5%,OR=161.5, Pc

concentration of the Taq Polymerase to 0,1 U/tube, instead of the 100 ng of DNA and 0,4 U /tube of TaqPolymerase recommended by the kit instructions. The conditions of the PCR had been: 1) 94ºC, 2 min., 1cycle. 2) 94ºC 10 sec., 65ºC 60 sec. 10 cycles. 3) 94ºC 10 sec., 61ºC 50 sec., 72ºC 30 sec. 30 cycles. 4)72ºC 3 min.Results: The allelic distribution in our study are for HLA DQB1*02, *03, *04, *05,and *06 in controlgroup (n=126), 29,7; 27,3; 1,5; 19,4; 21,8. in AR group (n=42) 32,3; 26,2; 1,1; 18,3; 22,0.Conclusions: The Chi-square test show differences P< 0.001 inter AR, and control group.156-PREFERENCE HIGH-RESOLUTION HLA HAPLOTYPE FREQUENCIES FOR 21 USPOPULATIONS DERIVED FROM THE ENTIRE DNA-TYPED DONOR REGISTRY.Martin Maiers 1 , Loren Gragert 1 , William Klitz 2 . 1 Bioinformatics Research, National Marrow DonorProgram, Minneapolis, MN, USA; 2 University of California, Berkeley, CA, USA.Aim: We aim to calculate 5-locus high-resolution HLA A-C-B-DRB1-DQB1 haplotype frequencies for 21US populations using the entire DNA-typed Be The Match USA bone marrow donor registry. Previouslypublished results used a much smaller set of donors who were all typed at high-resolution.Methods: The new cohort consists of 3.89 million donors, 2.23 million of which have primary DNA typingdata. 10.1% of the donors were typed at the C locus, and 2.3% typed at the DQB1 locus. The size of thepopulations at the detailed race level range from 679,000 (EURCAU) to 1,100 (ALANAM). Wereinterpreted all HLA typings with primary data to the IMGT-HLA v2.24 allele list and used a modifiedexpectation-maximization (EM) algorithm to calculate high-resolution haplotype frequencies fromgenotype lists of varying levels of ambiguity.[figure1]Results: Genetic distance measurements show high levels of HLA genetic divergence between the 21detailed race categories, to the greatest degree among the 8 Asian-American populations.[figure2]Conclusions: These haplotype frequencies constitute a fundamental improvement for improving matchpredictions for donor selection algorithms and for improving the accuracy in modeling registry match rates.157-PHUMAN LEUCOCYTE ANTIGEN GENE (HLA-A, HLA-B, HLA-DRB1) FREQUENCIES IN ACOLOMBIAN POPULATION.Diana Carolina Martin-Gamez, Claudia Bibiana Mendez, Martha Ramirez de Olano. ImmunogeneticSection, Laboratorio Medico Echavarria, Bogotá, Colombia.Aim: To study HLA system has provided great information about genetic composition of the populationsand has been of great usefulness for to identify alleles associated with the predisposition to develop someautoimmune diseases and in the organ transplant for selection of recipients and donors. The objective ofthis work was to determinate the allelic, genotypic and haplotypic frequencies of HLA loci evaluatingHLA-A*, B* and DRB1* alleles in a Colombian population.Methods: 476 individuals that were included in this study were evaluated in a period of 4 years (2006-2009) in Laboratorio Medico Echavarria S.A. HLA were typified using sequence specific primerpolymerase(SSP PCR). Frequencies were estimated with Genetic Data Analysis Version 1.0 andARLEQUIN 3.0.Results: 20, 36 and 16 different alleles were identified for HLA-A, -B and –DRB1 loci respectively. Themost frequent were A*24 (28.7%), A*02 (25%), B*35 (19.3%) and DRB4* (29.3%). The most frequentgenotypes were A* 04,24 (14.4%), B* 35,40 (4.2%), B* 35, 39 (3.5%) and two of DRB1 loci: *04,07 and*04,11 (5.4%) with the same frequency. For the three loci, the most frequent haplotypes were A*24, B*35,DRB1*04 (7.7%) and A*24, B*40, DRB1*04 (4.4%).Conclusions: Population studied in this work is predominantly Caucasian. Frequencies calculated confirmthe population heterogeneity and are similar with previous studies in Colombia.

158-PHLA CLASS II AND TOTAL IMMUNOGLOBULIN E ASSOCIATION WITH PEDIATRICBRONCHIAL ASTHMA.Mahendra N. Mishra 1 , Rakesh K. Gupta 2 . 1 Dept. of Pathology, Command Hospital (SC), Pune,Maharashtra, India; 2 Dept. of Pediatrcs, Armed Forces Medical College, Pune, India.Aim: This study was carried out at our centre to examine the association of pediatric bronchial asthma withHLA Class II alleles and IgE in an Asian Indian population.Methods: One hundred and four cases of Bronchial asthma diagnosed on basis of clinical symptoms whichshowed reversibility with β 2 blockers and 144 adult controls with no history of asthma were included inthis study. DNA was extracted from whole blood using Qiagen, (Germany) kits and HLA Class II DRB1and DQB1 typing was performed by Sequence specific primers (SSP) using kits from Olerup (Austria) forall patients. DPB1 typing was done for 45 samples. High resolution DQB1 typing has been done for 40samples so far. IgE estimation was carried by ELISA in duplicate. The protocol mentioned in product insertwas followed for all three procedures.Results: Sixty-eight males and 36 females patients included in this study with aged three months - 13years. Serum IgE levels were normal in 28% and 67% of patients and controls respectively, with peakvalues of 3865 and 627 IU/ ml in asthma patients. A positive family history was present in 40% of patients.The absolute eosinophil count was elevated in 23% of patients. The commonest alleles in patients / controlsexpressed as percentage were DRB1*07 (35/24), 1*15 (35/50), 1*04 (25/10) and 1*11 (17/14) respectively.DPB1 typing and high resolution DQB1 typing are under way and the data shall be presented.Conclusions: Raised serum IgE levels were found to be associated with pediatric asthma and the peaklevels were much higher in patients. The alleles DRB1*04 and 1*07 are possible associated with anincreased risk of asthma while DRB1*15 was could have a protective role.159-PTO AUTOMATE OR NOT TO AUTOMATE PROCESSES IN THE LAB.Alessandro Pirri, Sonia M.C.M. Costa, Sibelle B. Mattar, Maria da Graça Bicalho. Genetics Department,Universidade Federal do Paraná, Curitiba, Paraná, Brazil.Aim: The LIGH-UFPR has been making genotyping analysis since the end of the 90’s reaching up to 2000exams/month for the Brazilian Bone Marrow Donor Registry totalling more than 80000 HLA genotypinganalyses. Given this workload, there is a great concern in establishing new high-throughput techniques inorder to guarantee celerity and efficiency in the triage of samples and workflow ensuing precision in therelease of HLA genotyping results.Methods: Twenty samples/h were processed with the use of manual commercial kits for the extraction ofDNA and later with the use of an automated DNA extractor. In the beginning of 2010 an automatedpipetting system (Liquid Handling Workstation) customized for the use of several techniques and manualextraction kits was acquired.Results: The workstation is capable of processing 64 samples/h. The apparatus is customized to allow theextraction with magnetic beads as well a vacuum system with built-in stirring and temperature controllers.In addition, the system counts with a bar code reading capability, which guarantees traceability to theanalyses workflow. This Liquid Handling Workstation allows the processing of pre-PCR stages through itscapability to perform both DNA extraction and amplification independently and with equal outputcapability of process around 750 samples daily.Conclusions: This equipment fulfils current laboratory demand a of an increasing number of samples and itis capable of tracking samples workflow and frees the operator to perform more complex routine analyses.The substitution of manual for automated methodologies requires previous analyses of several factors, suchas the cost-benefit ratio, versatility and interface with other equipment. In our case, these and other factorssignalled favourably towards routine automation, what we believe will bear positively in terms ofefficiency, lower error probability and a higher reliability of the HLA analyses obtained.

160-PASSOCIATION OF HAPLOTYPES WITH SUSCEPTIBILITY AND RESISTANCE TO LEPROSYIN BRAZILIAN PATIENTS.Matilde Romero 1,2 , Maria E. Moraes 3 , Cynthia C. Cardoso 1 , Milton O. Moraes 1 . 1 Leprosy Laboratory,Instituto Oswaldo Cruz - FIOCRUZ, Rio de Janeiro, RJ, Brazil; 2 Immunogenetics Laboratory, InstitutoNacional do Cancer - INCA, Rio de Janeiro, RJ, Brazil; 3 Immunogenetics Laboratory, JRM InvestigaçõesImunológicas, Rio de Janeiro, RJ, Brazil.Aim: Nearly 250,000 new cases of leprosy have been reported to the World Health Organization (WHO) inthe past years. Several genetic studies of different populations have been conducted to correlate HLA-DRB1 association with leprosy; whereas HLA class I studies are limited. In this study, a case control studytesting HLA class I association with leprosy in Brazilians was analyzed.Methods: The study included 424 unrelated patients with leprosy and 489 controls from de samegeographic area of Rio de Janeiro. HLA-A and HLA-B alleles were genotyped by PCR-SSOP(Innogenetics and One Lambda) low/intermediate resolution.Comparisons between the frequencies ofalleles/haplotypes in cases and controls were performed by using logistic regression models withadjustment for gender and ethnicity non-genetic covariates. Analyses were carried out with R for Windows2.9.2.Results: HLA-A*02 and HLA-B*35 were the most frequent alleles in patients and controls. Results havesuggested a borderline association of the HLA-A*11 and HLA-A*30 alleles and leprosy susceptibility (OR= 1.852; CI = 0.977 – 3.512; p = 0.059 and OR = 1.770; CI = 0.993 – 3.148; p = 0.052, respectively),whereas HLA-B*50 appeared strongly associated with resistance to Leprosy (OR = 0.184; CI = 0.046 –7.432; p = 0.017). Analyses of the haplotypes demonstrated significant association of HLA-A*30-B*15with susceptibility to leprosy (OR = 6.60; CI = 1.15 – 37.99; p = 0.03) and borderline association of HLA-A*02-B*50 with resistance (OR = 0.19; CI = 0.04 – 1.04; p = 0.05).Conclusions: Significant association of the haplotype HLA-A*30-B*15 with susceptibility to leprosysuggests that HLA-A*30 allele acts in combination with other alleles to confer susceptibility to leprosy.However, the borderline significant association haplotype HLA-A*02-B*50 with resistance to leprosysuggests that HLA-B*50 may be playing the major role in conferring resistance to leprosy.161-PHLA DIVERSITY OF THE ESTONIAN POPULATION SHOWS UNIQUE CHARACTERISTICS.Anu Tamm 1 , Christina E.M. Voorter 2 , Eva Mulkers 2 , Astra Västrik 1 , Kadri Raudsepp 1 , Ingrid Tagen 1 ,Marcel G.J. Tilanus 2 . 1 United Laboratories, Tartu University Hospital, Tartu, Estonia; 2 TransplantationImmunology, Maastricht University Medical Centre, Maastricht, Netherlands.Aim: Historically the population of Estonia was subjected to Finnish and Russian influences. SNP analysis,used to characterize population diversity, identified unique Estonian characteristics. In this study weexamined if HLA diversity is as unique as the SNP pattern of the Estonian population.Methods: HLA typing data from 350 individuals were analyzed for their allele frequency, B-C and DRB1-DQB1 associations.Results: Although the alleles found are similar to those identified in the West-European population, theirfrequencies differ. Unique HLA-B-C associations, infrequent or absent in the West European Caucasianpopulation, were encountered such as B*07-Cw*06; B*18-Cw*04, B*35-Cw*02, B*39-Cw*03 and B*44-Cw*08. Haplotype analysis of these 350 individuals reveals that 5 of the top 10 haplotypes encountered inthe Estonian population were present among the top 10 haplotypes of European Caucasian, Hispanic andAfrican-American populations. Five of the predicted top 10 Estonian haplotypes were unique in a sensethat they are not or not frequently represented in the three other populations mentioned. Predicted frequentEstonian haplotypes are currently confirmed in family analysis. Considering the data obtained it is worth toexplore the high resolution typing characteristics and to extend family analysis in the Estonian populationto gain insight in the population diversity.Conclusions: The BMDW database represents more than 13 million potential donors for stem celltransplantation. Despite these numbers no HLA matched donor with match grades 10/10 or 9/10 isavailable for about 40% of the patients requiring a stem cell transplantation due to the presence of rarealleles or rare associations. From the data obtained in this study it can be concluded that it is worthwhile toinclude potential stem cell donors from Estonian origin to increase the diversity in stem cell donor registry.

162-PHLA DIVERSITY IN THE GUADELOUPE POPULATION; THE RELEVANCE OF INCLUDINGTHE GUADELOUPE POPULATION IN A DONOR STEM CELL REGISTRY.Christina E.M. Voorter 1 , Frantz Agis 2 , Fausto Palusci 1 , Eva Mulkers 1 , Marcel G.J. Tilanus 1 .1 Transplantation Immunology, Tissue Typing Laboratory, Maastricht University Medical Centre,Maastricht, Netherlands; 2 Immunodiagnostics and Immunogenetics, University Hospital Pointe-a-Pitre,Morne a L’eau, Guadeloupe.Aim: Donor registries included in the BMDW represent more than 13 million potential donors for stem celltransplantation. The level of resolution obtained by different HLA typing approaches to characterize thedonors varies from antigen to allele assignments. When HLA-C and –DQ typing results are lacking, donorscan be selected based on HLA-B-C and DRB-DQB associations. Still, for about 40% of the patientsrequiring a stem cell transplantation an HLA matched donor is not available. The underlying reason is thatthose patients have either unique HLA-B-C or DR-DQ associations, unique HLA haplotypes or HLAalleles, that are considered rare in the available databases. To increase the diversity of a donor registry,exploration of other populations is required. The Guadeloupe population today is a mixture of Black andCaucasian ethnicity. We studied the HLA diversity of 250 individuals from the Guadeloupe population.Methods: The frequency of alleles was defined and HLA-B-C and DRB-DQB associations weredetermined.Results: Among the 51 HLA*B15 alleles 25% is B*1503, 34% is B*1510 and 22% B*1516; the remaining19% includes B*1501, 1517, 1518, 1520 and a new B*15 allele. Haplotype analysis of these 250individuals, reveals that of the top 10 of most frequent HLA-A/B/DR haplotypes of Guadeloupe only 3correspond with HLA-A/B/DR haplotypes defined in the top 10 of the European American, Hispanic andAfrican American population. Among the unique haplotypes of Guadeloupe, the A*74-B*15-DRB1*13,A*68-B*53-DRB1*07 and A*30-B*53-DRB1*15 haplotypes were frequently present. Family analyses andcomplete high resolution typing will refine allele assignments and confirm the predicted frequenthaplotypes.Conclusions: In the Guadeloupe population many different haplotypes are encountered at a commonfrequency which are rather rare in the European American, Hispanic and African American population.163-PINFLUENCE OF CLASS I AND II HLA ALLELES ON INHIBITOR DEVELOPMENT INHAEMOPHILIA A PATIENTS FROM THE SOUTH OF BRAZIL.Morgana F. Barros 1 , Juliana C.M. Herrero 1 , Ana M. Sell 2 , Fabiano C. de Melo 2 , Marco A. Braga 2 , CinthiaB. Pelissari 3 , Júlio Machado 3 , Sandra S. Schiller 4 , Loide S. Hirle 4 , Jeane E.L. Visentainer 2 . 1 Departamentode Análises Clínicas, Universidade Estadual de Maringá, Maringá, Paraná, Brazil; 2 Departamento deCiências Básicas da Saúde, Universidade Estadual de Maringá, Maringá, Paraná, Brazil; 3 Laboratório deHematologia, Centro de Hematologia e Hemoterapia do Paraná (HEMEPAR), Curitiba, Paraná, Brazil;4 Hemocentro Regional de Maringá, Universidade Estadual de Maringá, Maringá, Paraná, Brazil.Aim: To identify the Class I and II alleles that may be influencing the formation of inhibitors.Methods: Genotyping of the Class I (HLA-A, -B and -C) and Class II (HLA-DRB1, -DQA1 and -DQB1)alleles of 171 patients with haemophilia A, including 40 who had developed antibodies to factor VIII, wasperformed.Results: After the comparison of the group without inhibitors and the group with inhibitors, HLA-A*03(OR = 2.10; P = 0.0432), HLA-B*42 (OR = 6.84; P = 0.0285), HLA-C*16 (OR = 5.23; P = 0.0192) andHLA-DRB1*14 (OR = 3.49; P = 0.0245) were found to be positively associated with the formation of theinhibitors.Conclusions: These results confirm that HLA is involved in inhibitor production and could be used as atool for recognition of groups at high risk of possible inhibitor development in Southern Brazilianhaemophilic patients.

164-PDETECTION OF A DE NOVO DONOR SPECIFIC ANTI-DQA1 ANTIBODY IN A LUNGTRANSPLANT RECIPIENT.Gansuvd Balgansuren 1 , Adella Clark 2 , Laurie Snyder 3 , Barbara Burgess 2 , Dong-Feng Chen 1 . 1 Pathology,Duke University Medical Center, Durham, NC, USA; 2 Clinical Laboratories, Duke Health System,Durham, NC, USA; 3 Medicine, Duke Health System, Durham, NC, USA.Aim: Post transplant monitoring of donor specific antibody (DSA) is important to prevent rejection and/orcomplications. HLA class II molecules consist of α and β chains and both of them are immunogenic. Herewe report a de novo DSA against DQA1*05 in a lung transplant recipient who returned to the hospital withchronic rejection.Methods: PRA is examined by flow cytometry and identification of Ab specificity is performed byLuminex single antigen bead assay.Results: The patient received bilateral lung transplant from her parents in 2000. Anti-DR52 and DR17were detected in 2001 which were specific to her mother’s antigens (Ag). Over the years her PRA wasgradually diminished with some fluctuations and DSA was disappeared. However, an anti-DQ7 Ab bindingwas revealed in 2006 which appeared to be against her own Ag. Due to her transfer to another hospital, theAb monitoring was discontinued. When she returned to our hospital in 4 years, her class II PRA wasdramatically increased from 3% to 57% and the Ab specificity was solely against DQA1*04, *05 and *06.All 3 beads against DQA1*05 showed stronger (MFI>3000) reaction than others and it was donor (mother)specific. The serum sample of 2006 was re-examined by the current technology. All beads carryingDQA1*05 were strongly positive (MFI>9000) and the presence of anti-DQ7 (DQB1*0301) could beexcluded. These results imply that the Ab binding to DQ7 detected in 2006 was actually against DQA1.Conclusions: Anti-DQA1*05 DSA was already developed in 2006 which could not determined by the testperformed at that time. The new technology allows us to distinguish anti-DQA1 from anti-DQB1. Ourfindings suggest that monitoring DQA1 Ab might have clinical implication for acute/chronic rejection inLung transplant recipients.165-PDISCREPANCY IN DNA TYPING ANALYSIS DURING DECEASED DONOR WORKUP.Gansuvd Balgansuren 1 , Angelica DeOliveira 2 , Adella Clark 2 , Candace Young 2 , Bobbie Holeman 2 , Dong-Feng Chen 1 . 1 Pathology, Duke University Medical Center, Durham, NC, USA; 2 Clinical Laboratories,Duke Health System, Durham, NC, USA.Aim: The correct HLA typing information in UNOS is very important for identifying compatible donorsfor potential transplant recipients. Due to short time allowed, HLA typing on deceased donor is normallyperformed by low resolution SSP. Recently we observed a discrepancy between UNOS reported and ourHLA typing on an import deceased donor. Here, we aimed to discuss the origin of the discrepancy toprevent from the possible recurrences.Methods: HLA typing of import deceased donor was confirmed by serology. Upon transplant the importeddonor was further typed for HLA by Luminex-rSSOP. Any typing discrepancy was confirmed by SSPand/or SBT.Results: The HLA-A locus typing of the donor was reported in UNOS as HLA-A3 and A68. However, ourinternal serology typing detected HLA-A2 and A3 but not A68, and the Luminex-rSSOP resulted HLA-A*9222 (serological equivalent is A2) and A*03XX. To confirm the discrepancy we ran SBT and SSP. TheSBT results unambiguously showed HLA-A*0301 and A*9222. However, “A High Res SSP” presentedHLA-A*0301g and A*6815, the same antigen assignment as in UNOS. Further, we ran “A*02 SSP” thatresulted HLA-A*9222 and A*6801g. Since, the discrepancy was in “A High Res SSP” we carefullyscrutinized gel documentation and its worksheet. According to the worksheet, the lane 6 should have areaction in order to be A*9222, however it was clearly missing.Conclusions: Based on the SBT and Luminex-rSSOP typing results, we assigned A*9222 and A*0301.The descrepancy observed in SSP needs further investigation. Our findings suggest that relying only on onemethod or vendor for HLA typing in deceased donor workup is not sufficient to cover consistentlyincreasing HLA polymorphism to get accurate typing results.

166-PANALYSIS OF PLATELET ELUATE FOR THE ELUCIDATION OF SENSITIVITY IN AKIDNEY TRANSPLANT CANDIDATE: CASE REPORT.Hugo Mendonça Mundim 1 , Maria Aparecida Caixeta Lins 1 , Eridane Botelho Guzmán 1 , Heline Leal Titan 1 ,Sueli Donizete Borelli 2 . 1 Fundação Hemocentro de Brasília, Brasília, Distrito Federal, Brazil;2 Departamento de Ciências Básicas da Saúde, Universidade Estadual de Maringá, Maringá, Paraná,Brazil.Aim: Discrepancies in results from ELISA and CDC-AGH methods and the patient’s history ofsensitiveness triggered the hypothesis of a possible positive false result by ELISA. The patient’s serumadsorption method with platelets and study of eluate as a confirmatory test was suggested to confirm abovehypothesis.Methods: Suspension of platelets: platelets were obtained from a serum-type HLA-A2 donor. Adsorptionof Antibodies: suspension of platelets was incubated with 500 µL serum under analysis, during 60 min, at22ºC. After two washings and centrifuges at 3000 rpm, the final volume was adjusted to 300 µL with PBS.Elution of Antibodies: suspension of adsorbed platelets was acidified with HCl 10N up to pH 3.0, during10 minutes and then neutralized with NaOH 10 N. Supernatant with eluted antibodies was separated foranalysis after being centrifuged at 3000 rpm. Study on adsorbed serum with platelets and on the eluateof adsorbed platelets: adsorbed serum and eluate were submitted to research protocols for anti-HLAantibody by ELISA LAT1240-One Lambda INC, according to instructions by manufacturer.Results: ELISA method showed that adsorbed serum with platelets did not reveal antibodies for HLA-A2specificity. Eluate showed antibodies for HLA-A2 specificity since it was reactive up to 1/8 dilution. Noantibodies for HLA-A2 specificity in the eluate were detected by CDC-AGH method.Conclusions: The assay suggested is a contribution for sensitivity evaluation between CDC-AGH andELISA methods in characterizing antibody specificity in the patient’s serum. Further evaluations of theprotocol should be undertaken to evaluate their worth in discrepancies between different lab tests for thecharacterization of antibodies.167-PIMPROVEMENT IN PROTEINURIA AFTER BORTEZOMIB-BASED THERAPY IN APATIENT WITH TRANSPLANT GLOMERULOPATHY.D. Dadhania 1,2 , C. Hartono 2 , R. Friedlander 2 , J. Lee 2 , A. Menon 2 , V. Sharma 1,2 , M. Suthanthiran 1,2 .1 Department of Transplantation Medicine, NYPH-Weill Cornell Medical Center, New York, NY, USA;2 Immunogenetics and Transplantation Laboratory, The Rogosin Institute, New York, NY, USA.Aim: Chronic Ab-mediated injury manifesting as transplant glomerulopathy (TxG) is associated withincreased risk of graft loss. Optimal management for TxG are being investigated.Methods: We report the outcome of a sensitized patient who developed nephrotic range proteinuria andtransplant glomerulopathy at 21 months post renal transplant and treated with a combination therapy ofbortezomib, plasmapheresis, IVIG and steroid pulse. The patient received 2 doses of bortezomib 1.3mg/m 2(Rx#1 and #2).Biopsy#1 Grade III TxG, C4d+Biopsy#2 Grade I TxG, mild C4d+Figure 1 outlines the Therapy (Rx).[figure1]Results:[figure2]Post-therapy, his proteinuria improved, anti-A2 Ab decreased and remained low andcreatinine remained at baseline. There was a positive correlation between reduction in anti-A2 Ab andreduction in proteinuria (r s =0.86, P=0.02). Six months post therapy, his creatinine is 1.3mg/dL andproteinuria is

168-PA NOVEL HLA-DQB1 ALLELE CONFIRMED ON BLOOD AND BUCCAL SWAB DNA.Angelica DeOliveira, Bobbie Holeman, Alexandra Baumbach, Gansuvd Balgansuren, Dong-Feng Chen.Clinical Transplantation Immunology Laboratory, Duke University Health System, Durham, NC, USA.Aim: The polymorphism of HLA genes and the number of alleles is continuously increasing. Many ofHLA class II alleles in the IMGT database have only exon 2 sequence data available, which sometimesmakes the typing difficult. Recently, we observed a potential new DQB1 allele with a novel sequence inexon 3 in a potential BMT recipient. Here, we describe the discovery of this novel allele.Methods: HLA-DQB1 was sequenced by using 7 PCR reactions for exon 2 and one single PCR reactionfor exon 3 of all HLA-DQB1 alleles.Results: The HLA DQB1 SBT demonstrated a reverse sequence of exon 3 with one single base mismatchfrom all know HLA-DQB1 alleles. Data obtained for exon 3 combines the alleles present in a heterozygousformat. The analysis software was not able to identify a DQB1 allele having an “A” in position 192 ofHLA-DQB1 exon 3. The sequence allignment showed that codon 159 which encodes for Val (GTG)changed to Met (ATG). The sequence data obtained from exon 3 was in a heterozygous format containingboth alleles. Upon reviewing data, position 192 was called as an “R” which indicated the presence of an“A” in one of the two alleles. The analysis software was unable to identify any allele present when exon 3data was included. When exon 3 data was excluded from analysis, the software identified HLA-DQB1*02:01/04 and DQB1*04:02 as the alleles present based on the exon 2 sequencing data.Conclusions: We discovered a novel sequence in HLA-DQB1 exon 3 which caused a change of aminoacidfrom Val to Met at codon 159. This finding was confirmed in DNA from the patient blood and his buccalswab.169-PIMPORTANCE OF RENAL TRANSPLANT BASELINE SENSITIZATION IN INTERPRETINGPOST-TRANSPLANT HLA ANTIBODIES: A CASE STUDY WITH WEAK CREG ANTIBODY.Kim Larlee 1 , Luz Stamm 1 , Kevin McLaughlin 2 , Noureddine Berka 1 . 1 Tissue Typing Laboratory, CalgaryLaboratory Services, Calgary, AB, Canada; 2 Medicine, University of Calgary, Calgary, AB, Canada.Aim: Our Laboratory is conducting routine post-renal transplant HLA antibody monitoring concomitantwith any protocol or for cause biopsy. The appearance of de novo donor specific antibody is immediatelycommunicated to physician to be interpreted with graft function and history.Methods: We recently tested a 56 year old female with HLA phenotype A3,-: B7,38; DR11, 13; DR52;DQ7, 6 that was transplanted in 2003 with a deceased donor 6/6 HLA antigen mismatch kidney. The donorHLA phenotype was A26, 31; B37, 51; DR12, 14; DQ3,-. Frozen Cell Tray and ELISA method was usedpre-transplant for detecting antibodies and Luminex based screening and single antigen method posttransplant.Final crossmatch was done using AHG-CDC and later on a retrospective Flow crossmatch wasrun on both pre-transplant samples.Results: The screening of the pre-tranplant sera showed a week B12 CREG antibody and included B51antigen by frozen cell tray. The final AHG-CDC crossmatch was negative and the patient was successfullytransplanted. Recently we tested a post transplant sample during a routine screen and find out that the sameB12 CREG with DSA to B51 at MFI 5000. We then pulled a pre-transplant sera and tested it by Luminexand find out that it contained the same exact B12 CREG and B51 antibody at but at higher MFI 15000.Conclusions: This case study shows that Luminex antibodies should be interpreted in the light of the baseline pre-transplant serum. It also raise the question about the clinical significance of CREGs.170-PANGIOTENSIN II TYPE 1 RECEPTOR ANTIBODIES IN A CASE OF HYPERACUTE RENALALLOGRAFT REJECTION.John G. Lunz 1 , Ron Shapiro 1 , Parmjeet Randawa 1 , Harald Heidecke 2 , Duska Dragun 3 , Adriana Zeevi 1 .1 Departments of Pathology and Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA;2 CellTrend, Lukenwalde, Germany; 3 Nephrology and Intensive Care Medicine, CharitéUniversitaetsmedizin Berlin, Berlin, Germany.

Aim: Sensitive pre-transplant (Tx) anti-HLA antibody (Ab) testing has minimized the incidence ofhyperacute antibody mediated rejection (AMR) in renal Tx. However, pre-Tx non-HLA Ab screening lagsbehind HLA Ab testing, and the contribution of non-HLA Ab to hyperacute AMR has not been widelydemonstrated. Here we report a case of hyperacute AMR where angiotensin II type 1 receptor (AT1R) Abwas detected pre and post-Tx in the absence of anti-HLA or MICA Ab and suspected to contribute to thegraft failure.Methods: HLA typing was performed by SSOP (One Lambda) and SSP (Invitrogen). Anti-HLA andMICA Ab was detected by Luminex beads (One Lambda). Crossmatches (XM) were performed bycomplement dependent cytotoxicity ((CDC) AHG (T cell) and Amos Modified (B cell)) and flowcytometry. AT1R Ab was detected by ELISA (CellTrend, Lukenwalde, Germany).Results: A 34 yo male underwent a 5th renal Tx with a HLA-A, B, Cw, DR, DQB1 and DQA1 matchedorgan. The absence of HLA-DP Ab precluded HLA-DP typing. No HLA DSA or MICA Ab was presentprior to Tx. T and B cell CDC and flow XM were negative. Following reperfusion, gross evidence ofhyperacute AMR was observed and an intraoperative biopsy revealed significant acute tubular necrosis(ATN). No renal blood flow was detected and the organ was removed 1 day after Tx. Pathology on thefailed allograft again revealed ATN and patchy, focal C4d. Post-Tx Ab testing was again negative for HLADSA or MICA Ab and XM were again negative. Anti-AT1R Ab was found to be positive in both pre(32U/ml) and post (55U/ml) Tx samples. Ab over 19U/ml is considered predictive. The patient is currentlyawaiting a sixth allograft.Conclusions: This case is the first published report where AT1R Ab was implicated in the hyperacuteAMR of a renal allograft. While other non-HLA Ab cannot be ruled out, the presence of AT1R Ab wasassociated with clinical features of hyperacute AMR.171-PPLATELET REFRACTORY PATIENT WITH AN ALLELE SPECIFIC HLA ANTIBODY.Amber R. O’Shields 1 , Karen A. Cellars 1 , Dana A. Gates 1 , Carissa Hagy 1 , Elise M. McPherson 1 , Tomas G.Thompson 1 , Sheree H. Waslaski 1 , Omar Moussa 1 , Howard M. Gebel 1,2 , Robert A. Bray 1,2 . 1 MedicalUniversity of South Carolina; 2 Emory University.Aim: Serological HLA typing still widely used in Blood Banks to aid in the selection of HLA matchedplatelets for patients experiencing platelet refractoriness due to the presence of HLA antibodies. Howeverlow resolution typing may not be sufficient for in highly sensitized patients and patients with HLA allelespecific antibodies. This case report focuses on a a 62 year old Caucasian female with 99% PRA andplatelet refractoriness. The patient’s HLA type is A*30, A*--, B*65, B*72, Bw6, Bw6, Cw*02, Cw08.Methods: Intermediate resolution typing was performed by Labtype SSO. SA beads analysis wasperformed using the LABScreen assay.Results: All but one of the A locus beads were positive with the exception of the A*3002 bead, theA*3001 bead was positive at 15,213 MFI. The American Red Cross HLA laboratory currently typesplatelet pheresis donors by serologic methods and therefore does not have high resolution, allele leveltyping on donors. The HLA haplotype frequency did not provide addition helpful information sinceA*3001 and A*3002 are both common on many ethnic backgrounds. MUSC transfusion services requestedcrossmatched platelets from the American Red Cross for this patient. The American Red Cross performsplatelet crossmatching by a solid-phase red cell adherence method. This patient was crossmatched with 45donors, of which 2 were compatible. The HLA type for donor #1 was A2--; B45, B49. The HLA type fordonor #2 was A25, A26; B44, B53. Interestingly, this donor was compatible although the antibodies to A25and A26 were 15,000 and 13,000 MFIs respectively.Conclusions: For platelet refractory patients with allele specific antibodies, high-resolution typing onplatelet pheresis donors is needed. Further studies are needed to determine the correlation between thestrength of HLA antibody, the platelet crossmatch compatibility by the red cell adherence method and theincrease in platelet survival in vivo.

172-PSCREENING FOR HLA-SPECIFIC ALLOANTIBODY IN NEONATAL ALLOIMMUNETHROMBOCYTOPENIA. ARE WE SENSITIVE ENOUGH? CASE REPORT.E. Portwood, P. Brailey, A. Gaddini, A.L. Girnita. Transplant Immunology Divison, Hoxwort BloodCenter, University of Cincinnati, Cincinnati, OH, USA.Aim: Alloimmune thrombocytopenia is the most frequent cause of severe thrombocytopenia in thenewborn. It is mediated by maternal IgG antibodies directed towards fetal platelet-specific and/or HLAantigens.Methods: We report here on a case with alloimmune thrombocytopenia in a newborn (age = two days)from a 30 year-old mother. The screening for anti-HLA and platelet specific antibodies was performed byELISA (GTI), while antibody specificity was obtained by Luminex single-antigen beads (One Lambda).Serial determinations were available both in mother and in newborn.Results: The initial screening by ELISA has shown concordant results in mother and child. Anti-HumanPlatelet Antigen (HPA) 1a, 1b, 3a, 3b, 4a, 5a, 5b and GPIb/IX IgG antibodies were not detected. Thescreening for HLA-antibodies by ELISA provided positive results for newborn, but negative for mother, inthe first sample - two days after birth. Luminex was positive for mother and child, with identical anti-Bw4/Aw4 epitope pattern (mother typed as Bw6 only: HLA-A1,3; B8,55). Interestingly, early after birth,the antibody strength was higher in newborn (dominant MFI = 9000) than in mother (MFI = 5700), whichexplained discordant initial ELISA results. The following samples exhibited a decrease in antibody strengthin newborn (15oo MFI in four days), and an increase in maternal antibody strength, which was associatedwith positive-ELISA screening in maternal follow-up samples.Conclusions: While anti- HPA-1a is considered the most frequently involved platelet antigen in neonatalalloimmune thrombocytopenia, a sensitization towards HLA epitopes may also be detected. ELISAplatforms allow for a combined single-run screening for HLA and platelet-specific antibody. However,single-antigen bead arrays should be considered in combination with ELISA for an accurate determinationof alloantibody pattern in ambiguous cases.173-PCASE STUDY: DISCREPANCIES BETWEEN DSA SPECIFICITY ASSIGNMENTS ANDDONOR CROSSMATCHES.Paula Steller, Patrick Adams, Annette Rearick, Nicholas DiPaola, Gregg Hadley, Ronald Pelletier. TissueTyping, Ohio State University, Columbus, OH, USA.Aim: A heart transplant candidate (typing DR11,13) was tested by Luminex single antigen beads (SAB) on3 sera obtained over a 3 month period. All SABs for DRB1 were positive, except DR8. Bead ranking andMFI values for all three samples were comparable (3,000-5,000 MFI range). These sera were furtherinvestigated to determine their ability to cause a positive B cell flow crossmatch (BFXM). Additional seracontaining only Class II antibodies assigned by SAB were likewise studied for their ability to cause apositive BFXM.Methods: 3-color flow cytometry crossmatch testing was performed with seven consecutive donors. Alldonors were HLA-DR mismatched with the heart recipient by at least one DR antigen. The seven donorsrepresented all DR types with the exception of HLA-DR8 and DR9. Additionally, this patient’s serum wassent to another laboratory for SAB testing using an alternate vendor SAB. Additional Class IIspecificity/BFXM comparisons were performed. Sera were included if >50% of the SAB beads for a givenantigen were > 4,000 MFI. 15 sera were selected and crossmatched with 10 donors in a variety ofcombinations, resulting in 51 Class II specificity/BFXM comparisons.Results: Flow cytometry crossmatches with all seven donors were negative. SAB testing by an alternatevendor was in agreement with these crossmatch results. SAB testing with the alternate vendor demonstratedthe complete absence of anti-DR antibodies. Subsequent crossmatch comparisons for a variety of Class IIspecificities (assigned using both vendor products), demonstrated poor specificity (63%), using the BFXMas the gold standard.Conclusions: This case and the subsequent Class II specificity/BFXM comparisons demonstrated poorspecificity in making positive BFXM predictions. Therefore, especially in the case of heart transplantcandidates, crossmatch studies are warranted to confirm both sensitization and antibody specificityassignments.

174-PUSE OF STRs TO HELP RESOLVE ENIGMATIC SERUM ANTIBODY RESULTS.Brian Susskind 1 , Rana Saad 1 , Judith E. Baker 1 , Nicholas Onaca 2 , Lawrence Melton 2 , Goran Klintmalm 2 .1 Pathology, Baylor University Medical Center, Dallas, TX, USA; 2 Baylor Regional Transplant Institute,Baylor University Medical Center, Dallas, TX, USA.Aim: Patient X, offered a 0-Ag mismatch kidney, had a historical serum with donor specific antibody(A66), based on single antigen beads (SAB). Per protocol, flow crossmatch (FCXM) was performedbecause X was “sensitized,” but with current serum only (1 st transplant). FCXM was negative, but anti-A66enigma still required explanation.Methods: See below.Results: Retrospective historical serum FCXM was positive, but pre-transplant and POD3 sera werenegative for anti-A66 by SAB. Allele-specific antibody in historic serum was ruled-out by SAB (both anti-A6601 & -6602 present); SSO typing showed both X and donor as A*6602. Sample mix-up might be theexplanation. If serum switched, then typing specimen received at the time may also have been, and thus Xwould not be 0 antigen mismatched vs renal donor. Because of implications for immunosuppression,patient was re-typed; original results were verified. It was still important to determine whether historicalsample was from X because based on our algorithm, if X were re-listed in the future, the suspicious serum -which contains numerous anti-HLA specificities, would potentially be used in FCXM as X’s “peak PRA.”We compared STRs among samples of X’s DNA derived from whole blood, current serum (200ul extractedon Qiagen EZ-1, total recovery=1400ng) and the historic serum in question (2200ng recovered). Resultsobtained on a Beckman-Coulter Vidiera NsD system showed only the former two were STR-identical.Therefore the historic serum was not from patient X.[table1]Conclusions: HLA labs receive numerous serum samples daily, therefore large potential for sample mix-upexists. When there is reason to suspect that one has occurred, serum-derived DNA and STR analysis can beused to prove or disprove patient of origin.175-PCOMPLETE RESOLUTION OF FALSE “HIGH PRA” RESULTS.Shikha Arora 1 , Hae Pang 2 , Donna L. Phelan 2 , Thalachallour Mohanakumar 2 , James Meegan 1 , JefferyRossio 1 . 1 Clinical Diagnostics, Life Technologies Corp, Brown Deer, WI, USA; 2 HLA Laboratory, BarnesJewish Hospital, St. Louis, MO, USA.Aim: Accurate identification of antibodies to Human Leukocyte antigens (HLA) is just one of the factorsutilized within the organ transplantation and post-transplant monitoring regimen. DynaChip® HLAAntibody Analysis Products are intended for use in the detection and identification of HLA antibodies inserum samples using an ELISA-based Chip Technology. Variable percentages (5-20%) of patient samplesare known to contain interfering substances that may bind non-specifically to the proteins on the microarrayor interact with assay reagents, resulting in a false high PRA result. The goal of this study was to develop amethod to resolve these samples and allow determination of true specificities. DynaChip HLA AntibodyAnalysis is For In Vitro Diagnostic Use Only.Methods: We treated 11 patient samples, previously known to exhibit the high PRA phenomenon and 4normal patient samples, with a combination of blocking and precipitating agents and tested them pre- andpost-treatment in an optimized DynaChip assay. Three of the 11 samples tested also had high negativecontrol values.Results: Treatment of these samples with the combination of agents resulted in significantly reduced PRAvalues in all 11 cases. In addition, the treatment also resulted in lowering of the control values for the 3discrepant samples. The results were confirmed independently at 2 different sites.[table1]Conclusions: We have shown that pre-treatment of known high PRA serum samples with a combination ofblocking agents can improve the accuracy of the results in a DynaChip assay.

176-PIMPLEMENTATION OF THE WORLD HEALTH ORGANIZATION (WHO) HLANOMENCLATURE CHANGES WITHIN MULTIPLE INTERFACED SOFTWAREAPPLICATIONS.Natalie L. Chambers, Lisa M. Hallaway, Brittany A. Schneider, Manish J. Gandhi. Mayo Clinic, Rochester,MN, USA.Aim: When new HLA nomenclature was introduced, the laboratory had to plan on implementing changeswithin testing and reporting systems. Laboratory Information Systems (LIS) and vendor applicationsneeded to be upgraded, tested and validated. SOPs needed to be modified along with education and trainingof technologists and physicians. The lab needed to implement a HistoTrac/Fusion interface during thissame timeframe.Methods: Patient testing could not be interrupted. Implementation followed these steps: 1. InformationTechnology (IT) resources were assigned to software upgrades, interface development and software qualityassurance (SQA). Lab personnel were assigned to process validation, SOP writing and training. Supportfrom the Medical Director for physician training was needed as well as vendor support for softwareupgrade activities. 2. Using past software implementation and upgrade experience, the amount of timeneeded for each phase of the project was estimated. A detailed timeline was created with all major tasks tobe completed before go-live. (See Figure 1) 3. Weekly meetings kept everyone on task.[figure1]Results: With advance planning, assignment of responsibilities and clear expectations, a go-live date ofmid-August 2010 is anticipated. We expect to maintain a top level of service to our patients with no impactto daily workflow.Conclusions: A project of this scope requires advance planning and coordination for routine work tocontinue as normal. Support from individuals knowledgeable in IT, SQA, and laboratory processes isessential to success.177-PISOLATION OF HIGH CONCENTRATION (>100 ng/MICROLITER) ELUATES FROM WHOLEBLOOD ON THE Maxwell® 16 INSTRUMENT.Paraj V. Mandrekar 1 , Zheng Ma 1 , Steven T. Krueger 1 , Cristopher A. Cowan 1 , Erin P. McCombs 2 .1 Integrated Solutions and Engineering, Promega Corporation, Madison, WI, USA; 2 Integrated Solutions,Promega Corporation, Madison, WI, USA.Aim: Recently, Promega Corporation developed a DNA isolation kit that combines an automated methodand a new paramagnetic particle chemistry designed to isolate microgram amounts of genomic DNA fromwhole blood, and elute these volumes into 50 microliter volume eluates. This DNA isolation kit wasdesigned for use on the Maxwell® 16 instrument, and can obviate laborious DNA isolation from the buffycoat required to obtain eluates in excess of 100 nanograms per microliter.Methods: Whole blood stored fresh or frozen, or in EDTA, sodium heparin, or sodium citrate collectiontubes were purified with the Maxwell® 16 LEV Blood Kit. Eluates were assayed by Spectrophotometricmethods, electrophoretic methods, and PCR-amplification based methods to determine the quality of theDNA isolation method introduced.Results: For all storage conditions tested, the Maxwell® 16 LEV Blood Kit isolated DNA from wholeblood in excess of 100 ng/microliter. DNA eluates were high molecular weight, and were amenable toPCR-based amplification.Conclusions: The Maxwell® 16 LEV Blood Kit routinely isolates high concentration eluates from wholeblood under a variety of storage conditions.Mandrekar: Promega Corporation: Employee. Ma: Promega Corporation: Employee. Krueger: PromegaCorporation: Employee; Stockholder. Cowan: Promega Corporation: Employee. McCombs: PromegaCorporation: Employee.

178-PCOST EFFECTIVE STREAMLINED IMPLEMENTATION OF THE 2009 ASHI MANDATEDBONE MARROW AND STEM CELL TRANSPLANTATION CONFIRMATORY HLA TYPINGSTANDARD.Brittany A. Schneider, Lisa M. Hallaway, Natalie L. Chambers, Cindy M. Kroning, Manish J. Gandhi.Laboratory Medicine and Pathology, Tissue Typing, Mayo Clinic, Rochester, MN, USA.Aim: Implementing the NMDP/ASHI mandated bone marrow confirmatory standard was a significantundertaking involving additional costs and increased complexity for the recipients/donors. We present ourexperience of a cost effective streamlined implementation.Methods: Collaboration between the laboratory, clinical team, and administration allowed a focused planto be developed. This covered patient care, billing, laboratory process flow, laboratory information systems(LIS), and staff education.Results: Billing patients for the confirmation typing was not possible as this was a regulatory requirement.To conserve costs, only Class I typing was performed. Coordinating patient visits and clinical care concernsto collect an appropriate sample (adequate WBC count) was another priority. To avoid repeat visits forblood draws, the decision was made to implement a same day draw, with mail-out kits as an alternateoption. Upon approval from ASHI, our information technology department placed rules in our LIS toensure the following: the confirmatory typing was automatically ordered with the initial typing, the orderwas automatically separated into two draw appointments at least 60 minutes apart, and the possibility ofmanual manipulation by phlebotomists was eliminated. With education being critical to success, ourdirector gave presentations to the clinical staff which was incorporated into their ongoing educationcurriculum. A communiqué was distributed to the phlebotomy supervisors who educated over 200phlebotomists. Laboratory education included weekly updates and focused training on the modifiedprocedure.Conclusions: On December 22, 2009 we implemented this process. This has been a smooth transitionwithout affecting patient care. Although this will increase laboratory testing costs, we significantlydecreased expense by performing only the Class I typing.179-PSTRUCTURE OF A CLASSICAL MHC CLASS I MOLECULE THAT BINDS NON-CLASSICALLIGANDS.Chee Seng Hee 1 , Song Gao 2 , Marcia M. Miller 3 , Barbara Uchanska-Ziegler 1 , Oliver Daumke 2 , AndreasZiegler 1 . 1 Institut für Immungenetik, Charité-Universitätsmedizin Berlin, Berlin, Germany; 2 Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany; 3 Department of Molecular and Cellular Biology,Beckman Research Institute, Duarte, CA, USA.Aim: The MHC-B region of the chicken resembles the mammalian MHC, while little is known about theMHC-Y region. To obtain insights into the role of MHC-Y-encoded YF1 molecules, we chose a structuralapproach.Methods: The complex of YF1*7.1 heavy chain and beta2m was reconstituted either without adding aligand or in the presence of palmitoyloleoylphosphatidylcholine (POPC). The complexes were analysed byX-ray crystallography and calorimetry.Results: The YF1*7.1 complex exhibits the typical architecture of classical MHC class I molecules, but thebinding cleft is lined by many hydrophobic residues. Both an unidentified ligand as well as POPC could bemodelled in the YF1*7.1 groove in two independent complexes. In-depth comparative analysesdemonstrated that these complexes exhibit a low degree of stability, and that only a small portion of POPCmight be accessible to a receptor on an effector cell. Therefore, the binding of both ligands to YF1*7.1 isdistinct from that observed for comparable ligands to mammalian CD1b and CD1d molecules.Conclusions: Classical MHC class I antigens may thus bind also hydrophobic ligands. The YF1*7.1molecule appears as a hybrid, favoring the interaction with non-classical ligands through the combinationof a hydrophobic binding groove with a classical framework. As there is only a single fully functional,polymorphic classical MHC class I gene in most MHC-B haplotypes and just one CD1 molecule isavailable to display complex lipids, the antigen-presenting capabilities of the chicken might be limited.Together with the unusual, massive expansion of highly polymorphic CHIR loci within the leukocytereceptor complex, lipid-binding YF1 molecules might be part of a strategy to overcome inadequacies in the

epertoire of displayed ligands and thus improve the ability of this species to fight successfully againstinfections.Tuesday, September 28, 20102:00 PM - 3:30 PMWorkshop 4: Case Studies in Stem Cell Transplantation25-ORCOMPLETE GENOMIC SEQUENCING OF HLA-C*04:09N USING NEXT-GENERATIONSEQUENCING TECHNOLOGY.Curt Lind 1 , Deborah Ferriola 1 , Kate Mackiewicz 1 , David Sayer 3 , Damian Goodridge 3 , Dimitri Monos 1,2 .1 Department of Pathology & Laboratory Medicine, The Children’s Hospital of Philadelphia; 2 Departmentof Pathology & Laboratory Medicine, Univeristy of Pennsylvania; 3 Conexio Genomics, Fremantle,Australia.Aim: To determine the complete genomic sequence of HLA-C*04:09N from a single long-range PCRamplicon using next generation sequencing (NGS).Methods: Long-range PCR was performed (Qiagen) to amplify the entire HLA-C gene from 5’UTR to3’UTR (3,349 bp). The amplicons were prepared for shotgun sequencing using the GS FLX Standard DNALibrary Preparation protocol (Roche). Briefly, amplicons were fragmented, ligated with adaptors, andcaptured on beads for emulsion PCR. Following emPCR, clonally amplified DNA bound beads wererecovered, enriched, and sequenced on the GS FLX Genome Analyzer. Sequence Data were analyzed usingAssign-NG software (Conexio Genomics).Results: 8,974 individual sequence reads aligned to the HLA-C region. The average read length was 243bases producing >2Mb of sequence. 100% of the 3,349 bases of the HLA-C gene were covered with anaverage read depth per position of 613. Allele assignment for this sample was C*04:09N,15:02:01. Bothexonic and intronic sequenced were used in the analysis. While the complete genomic sequence ofC*15:02:01 is known, the intronic sequence of C*04:09N was previously unknown. Given the on-phasesequence generated by this technology the intronic sequence was easily retrieved from the sequence data.The intronic sequence of C*04:09N is nearly identical to C*04:01:01:01, however the C*04:09N possessesa single base substitution (T>C) 19 bases from the end of intron 6 at position 2659. This substitution is 66bases away from the deletion at position 2725 in exon 7 that results in the lack of expression of this allele.Conclusions: NGS technology offers the means to fully characterize HLA Class I genes from a singleamplicon using a shotgun sequencing approach.This approach may prove to be a meaningful tool tocomplete the sequences of all known HLA alleles and may further advance efforts to develop a morecomprehensive HLA typing strategy than current methods.26-ORLEUKEMIC CELLS CAN INFLUENCE QPCR RESULTS: A CASE STUDY.Laima Gaidulis, Ketevan Ghendzekhadze, David Senitzer. HLA Laboratory, City of Hope Medical Center,Duarte, CA, USA.Aim: We use Quantitative Real Time PCR(QPCR) to monitor chimerism and minimal residual disease(MRD) in patients post allogeneic stem cell transplant(SCT). QPCR is a rapid, highly reproducible methodthat can detect 0.01 % recipient cells. As a result, patients at higher risk for relapse can be identified earlier.Methods: The QPCR method (QPCR-Celera) utilizes 34 polymorphic indels for identifying informativemarkers between patient and donor.At least 2 markers are necessary to quantitate engraftment. The %recipient cells is calculated by the software AlleleSeq (Celera) and is reported as an average. If two markersdiffer by more than 15 %, the test is repeated with other markers or confirmed by PCR-STR (ABIIdentifiler).Results: Recently we encountered a patient with only 2 informative indels, on chromosome(Chr) 1 and 2.Complete or weak chimerism (less than 2%) was detected post-transplant on days 77, 83, 111 in peripheralblood (PB), bone marrow (BM) or CD 3 cell subsets. The difference between 2 markers was less than0.5%, But at day 169, one marker showed relapse and/or rejection (40-50 %R BM and BMCD3), but weak

mixed chimerism with the Chr 2 marker. Ten informative SNP were identified using PCR-STR, with nineindicating relapse (∼30%R), and one, D2S1338 on Chr2, 6-9% mixed chimerism. The Cytogenetic reportshowed 34.1 % host cells with numerous chromosomal abnormalities, including tetrasomy 1 and 2 copiesof chromosome 2,consistent with relapse of the patient’s ALL.Conclusions: With QPCR (only 2 informative patient markers) interpretation was between “Weak MixedChimerism” and “Relapse and/or Rejection”. For STR, with 10 informative patient markers, thechromosome 2 marker could be excluded without affecting the over-all interpretation of relapse.In cases ofsuch discrepant indel results, cytogenetic abnormalities should be considered in the final interpretation ofthe results.27-ORLEUKEMIA SPECIFIC LOSS OF HETEROZYGOSITY AT MHC LOCUS DETECTED ATCONFIRMATORY TYPING OF HSCT RECIPIENT WITH CLL.Myra Coppage 1 , Angela Busacco 1 , Nufatt Leong 1 , Paul Rothberg 1 , Gordon Phillips 2 , Michael Becker 2 .1 Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA;2 Medicine, University of Rochester Medical Center, Rochester, NY, USA.Aim: A 63 WM with refractory B-CLL presented for allogeneic HSCT evaluation; HLA typing wasperformed on PBL at time of WBC= 53K, ALC= 47K and revealed homozygosity at Class I locus andheterozygosity at Class II locus (Table 1). Two siblings were full mismatches with the recipient and anunrelated search initiated. Patient was treated with Campath complicated by aplastic anemia andbacteremia. Prior to transplant, confirmatory typing performed on PB revealed two full haplotypes at ClassI and II.Methods: Sample identification error and the presence of third party lymphocyte engraftment as a result ofprior red cell or granulocyte transfusion(s) were ruled out by STR analysis of 8 loci (D11S554, D12S391,D16S539, FGA, Penta E, SE33, THO, VWF-B) of all samples. T and B cells from cryopreserved PBcontaining tumor blasts were HLA typed by SSO and SSP independently.Results: T cell typing yielded both complete haplotypes (genotype verified by offspring HLA typing); Bcells typed for homozygous haplotype indicating loss of heterozygosity of MHC locus. Microarray basedcomparative genomic hybridization of tumor cells confirmed LOH at 6p.Conclusions: Confirmatory typing revealed presence of second haplotype masked by tumor LOH.[table1]28-ORUNUSUAL CASE OF CHIMERISM AFTER BONE MARROW TRANSPLANTATION.Juan J. Yunis 1,2 , Guiselle A. Cuervo 2 , Emilio J. Yunis 2 . 1 Patología, Facultad de Medicina e Instituto deGenética, Universidad Nacional de Colombia, Bogotá, DC, Colombia; 2 Instituto de Genética, ServiciosMédicos Yunis Turbay y Cia, Bogotá, DC, Colombia.Aim: Genetic chimerism is usually used after BMT in order to determine engraftment or graft failure.Usually, STR analysis is used in order to determine the degree of chimerism in a post-transplant samplewhen compared to the donor’s and recipient’s genetic profile. In the present case, an unusual geneticchimerism was found during a routine paternity testing case from a male individual that received a BMTtransplant from his sister eight years before.Methods: STR analysis was carried out with the Identifiler kit (Applied Biosystems) and analyzed in a3130XLS genetic analyzer.Results: In a paternity testing case, a paternal exclusion was found. The individual typed XX withAmelogenin. A new sample of the alleged father was requested. We were informed that he had received aBone Marrow transplantation (BMT) eight years before. His HLA identical sister was the donor. Wecontacted the alleged father to obtain a bucal swap. Once the sample was analyzed, a mixture XX/XYprofile was obtained. In the mixture, the obligated paternal alleles that did not exclude the paternity werefound. Due to the mixture, three new samples were obtained, one from the inner right chick, one from theinner left chick and one from the tongue. Two of the three samples showed an identical genetic XX profileas found in the first blood sample without evidence of the XY profile. In the third sample, an XY geneticprofile was found with minor peaks for some alleles found in the XX profile.Conclusions: Our results indicate that the post-trasplant chimerism was not limited to the bone marrowderived cells. The stem cells from the HLA identical donor had also replaced a good part of the mouth cells

mucosa. No other samples were available for testing. Recently, after a Liver transplantation case inAustralia, the recipient’s bone marrow was replaced with donor specific stem cells. These cases indicatethat graft derived Stem cells could replace different tissues of the body.29-ORIDENTIFICATION OF A RECOMBINATION SITE BETWEEN THE HLA LOCI DRB1 ANDDRB3.Pedro Cano, Edward Guerrero, Dana Willis, David Partlow, Marcelo A. Fernandez-Vina. LaboratoryMedicine, UT M. D. Anderson Cancer Center, Houston, TX, USA.Aim: The HLA system represents one of the most polymorphic and complex genetic systems in humans.The classical loci present extremely high genetic diversity and high rates of heterozygosis. Another featureof the system is the extent and strength of linkage disequilibrium (LD) between alleles of different lociresulting perhaps from selective pressures thatrelationships between particular alleles. LD is maintained inspite of an overall recombination fraction higher than 0.5 percent. No recombination hot spots have beenidentified in the DR-DQ region.Methods: We analyzed the segregation of alleles at seven loci in 2739 families and observed 49recombinations. Two families bore recombinant haplotypes that resulted from crossing-over eventsbetween DRB1 and DRB3 loci. In both of them, the telomeric block of the recombinant haplotypesincluded C*07:01-B*08:01-DRB3*01:01 while the telomeric fragments included DRB1*14:01-DQB1*05:03 or DRB1*14:54-DQB1*05:03. The haplotypes donating the class I and DRB3 allelesincluded DRB3*01:01-DRB1*03:01-DQB1*02:01 while the haplotypes donating the centromericfragments included the highly homologous DR-DQ blocks DRB3*02:01-DRB1*14:01-DQB1*05:03 andDRB3*02:02-DRB1*14:54-DQB1*05:03. The analysis of 120 non-recombinant haplotypes bearingDRB1*14:01/14:54 had either DRB3*02:01 or DRB3*02:02 indicating that the recombinant haplotypesidentified here are less common or rare.Results: Our results show that for this specific recombination site only specific alleles or blocks appear tobe involved suggesting that for recombination between haplotypes specific cis/trans pairing is required.Depending on specific population distributions some segments of the HLA region may behave as ‘frozenrecombination blocks’.Conclusions: Our findings also indicate that even though the alleles of DRB3 may be useful markers topredict the alleles of DRB1, some exceptions may occur as the result recombination.30-ORIDENTIFICATION OF 3 MICA ALLELES IN A HEMATOPOITIC PROGENITOR CELLTRANSPLANT CANDIDATE.Aiwen Zhang, Dawn Thomas, Raymond Jurcago, Paul Kawczak, Stacey Macecevic, Diane J. Pidwell,Medhat Askar. Allogen Laboratory, Cleveland Clinic Foundation, Cleveland, OH, USA.Aim: MICA sequence-based genotyping (SBT) was attempted on a peripheral blood sample collected froma candidate for HSC retransplant. An electropherogram pattern that suggested carriage of more than 2distinct MICA alleles was observed. We sought to identify the MICA alleles carried in this patient’sgenotype through TA cloning technology.Methods: SBT and Sequence-Specific Oligonucleotide Probes (rSSOP) were used for MICA moleculartyping. Short Tandem Repeat (STR) was performed to rule out mixed donor chimerism from the previousHSCT donor. Molecular TA cloning of MICA gene products and SBT analysis of MICA clones wereperformed to identify the MICA alleles carried in this patient’s genotype.Results: DNA sequencing results from peripheral blood and buccal swab DNA samples consistentlydemonstrated a triple-peak electropherogram pattern. STR testing excluded the possibility of DNA samplecontamination from other sources. Combining the typing results of SBT from exon 2, exon 3, exon 4 ofMICA gene and rSSOP, the ambiguous heterozygous MICA alleles of the subject were typed asMICA*008/027/048/019 and MICA*002/007/020/052/055. Molecular TA cloning of MICA gene productand SBT analysis of MICA clones assigned three MICA alleles as MICA*008:01/008:04/A5.1,MICA*007:01/A4, and MICA*00201/A9. One sibling of this patient carries homologousMICA*008:01/008:04/A5.1.

Conclusions: The molecular cloning and sequencing data obtained from peripheral blood and buccal swabDNA samples confirmed the existence of three MICA alleles, MICA*008:01/008:04/A5.1,MICA*007:01/A4, and MICA*00201/A9. The origin of this third extra MICA allele remains unknown andrequires further investigation.31-ORSINGLE NUCLEOTIDE DELETION ON EXON 2 CAUSES A NOVEL NULL DRB1*01 ALLELE.Andrea A. Zimmerman, Laura K. Spruit, Walter F. Herczyk, Janine M. Ternes, Jerome G. Weidner, SusanaR. Marino. Transplant Immunology & Immunogenetics Laboratory, University of Chicago Medical Center,Chicago, IL, USA.Aim: Sequenced based typing (SBT) was performed on a stem cell transplant (SCT) recipient (mother) anda potential related donor (son). Initial results indicated DRB1*01:01 with a possible deletion on exon 2which was seen in both the recipient and donor. To confirm and characterize the new allele, additionalsequence specific primers were designed and applied and phenotype testing was performed by cytotoxicityand flow cytometric methods.Methods: DNA samples from both Caucasian family members were amplified for DRB1 locus and exon 2was sequenced using group specific sequence primers. DRB1*01 allele was sequenced in isolation from thesecond allele by newly designed sequence specific primers. The sequence data was collected using the ABI3730 and analyzed with SBTengine software. Both samples were also typed by rSSOP and presence of DRmolecules on the cell surface was tested by complement-dependent cytotoxicity (CDC) and by flowcytometry testing.Results: Initial sequencing results indicated DRB1*01 variant allele and DRB1*03:01 for the recipient andDRB1*13:02 for the related donor. A single nucleotide deletion in exon 2 at nucleotide position 123 atDRB1*01:01 was identified. The deletion was confirmed for the DRB1*01:01 allele by SBT testing. Theresults obtained by rSSOP yielded a false negative bead whose binding site corresponds to the suspecteddeletion. Lack of expression of the DR1 molecules on the cells surface was confirmed by serological typingusing CDC method and by flow cytometry.Conclusions: A novel DRB1*01 null allele has been characterized. Lack of expression of this allele hasimportant implications for donor selection.32-ORDETECTING CHIMERISM FROM HLA CONFIRMATORY TYPING OF CORD BLOOD UNITSAMPLES.Wei Dong 1 , Lesa Foley 2 , Judy Davis 2 , Carly Carozza 1 , Michelle Setterholm 2 , Susan Hsu 1 . 1 American RedCross Penn-Jersey Blood Services Region, Philadelphia, PA, USA; 2 National Marrow Donor Program,Minneapolis, MN, USA.Aim: Data on the sensitivity of methods used to detect maternal chimerism (MC) and its frequency in cordblood unit samples (CBUS) are limited. NMDP samples and a simulated mixing study were performed todetermine frequency and sensitivity rates.Methods: A pilot study was designed to simulate CBUS by mixing blood samples from three sets ofmother and child pairs in 5 serial dilutions of 1-20%. DNA was extracted for HLA and short tandem repeat(STR) testing to determine the minimum detection level of maternal (M) sample present. HLA typing wasperformed at HR for HLA-A, B, C by SBT or IR for HLA-A, B, C by LABType SSO. HR DRB1 by SBTwas performed on all samples. STR was performed with ABI kits. The percent of M DNA present in CBUSwas then estimated.Results: Out of 15075 CBUS received for confirmatory typing, 3 filter paper samples had evidence of MC(rate of 0.02%). Failure to obtain clear HLA typing in the 3 samples due to extra probe reactions was theonly indication MC was present in the samples. The pilot study demonstrated that indications ofcontamination appeared upon SBT when maternal DNA exceeded 15%. For IR typing, a high backgroundfor 3-4 probes appeared for some but not all loci at 10% and MC and was evident in almost all loci when MDNA reached 20%. In contrast, STR results demonstrated unequivocally the presence of M DNA at 1%.Results of STR testing on the three un-manipulated CBUS with corresponding M DNA samples indicatedthat 41, 16 and 7%, respectively, of M DNA was present.

Conclusions: In previously reported studies, the frequency of maternal contamination (0-38%) detected inCBUS and blood/DNA mixing studies was obtained by identifying maternal HLA class II types or VNTRsnot shared by the fetus. Our 0.02% rate from HLA typing may be an underestimate for the true MC rate.Tuesday, September 28, 20102:00 PM - 3:30 PMAbstract Session 3: Antibodies and Solid Organ Transplantation33-ORDECREASED EARLY ACUTE HUMORAL REJECTION IN RENAL TRANSPLANT PATIENTSFOLLOWING TERMINAL COMPLEMENT INHIBITION.Manish J. Gandhi, Steven R. DeGoey, Suresh Raghavaiah, James M. Gloor, Mark D. Stegall, Lynn D.Cornell. Mayo Clinic, Rochester, MN, USA.Aim: Renal transplant (Tx) candidates with donor specific alloantibody (DSA) are at increased risk ofacute humoral rejection (AHR). Current protocols for performing crossmatch positive kidney Tx inalloimmunized patients have high rates of AHR which may contribute to shortened allograft survival. AHRappears to occur secondary to activation of complement cascade by high levels of DSA. We hypothesizedthat eculizumab; a humanized anti-C5 monoclonal antibody that blocks the activation of terminalcomplement might prevent AHR.Methods: Recipient donor pairs were evaluated by T-cell and B-cell flow cytometric crossmatch (FXM).Candidates selected had a B-cell FXM (BFXM) mean channel shift (MCS) between 200 and 450. Pre-Txplasma exchange (PE) was performed to achieve a BFXM

difference in the strength of class II was not found in the focal and negative C4d deposition categories. Incontrast, the strength difference of class I DSA was not significant in the diffuse, focal, and negative C4dpatients.Conclusions: In kidney transplant patients with AMR, HLA class II DSA and their strength correlate withthe degree of C4d deposition. These results underline the value of HLA antibody strength as an importantmarker in the diagnosis of AMR.35-ORANTIBODY RESPONSES TO HLA CORRELATE WITH THE TYPES OF HLA-DR INRESPONDERS.Qingyong Xu 2,1 , Hyun J. Lim 3 . 1 Pathology and Laboratory Medicine, University of Saskatchewan,Saskatoon, SK, Canada; 2 Laboratory Medicine, St. Paul’s Hospital, Saskatoon, SK, Canada; 3 CommunityHealth & Epidemiology, University of Saskatchewan, Saskatoon, SK, Canada.Aim: Antibodies specific to donor HLA are critical for rejecting organ transplant. Does the ability of anHLA to induce antibodies differ from one HLA to the other? We hypothesize that the types of HLA-DR ina responder determine whether antibody to a specific HLA class I or II can be easily generated or not.Methods: We studied DR-homozygous patients on kidney waiting list from 2000 to 2009 in UNOS(United Network for Organ Sharing) database. Patients (n=1637) with positive class I PRA less than 80%were selected for studying antibody responses to A, B and Bw. Patients (n=2626) with anti class IIantibodies were selected to study the antibody response to DR and DQ. Risk ratio and p-value (by two-sideFisher’s Exact Test) are calculated with SAS software.Results: We found the ability of a responder to produce antibody to a specific HLA correlates with theresponder’s DR type. The correlations could be either positive or negative. For example, patientsexpressing DR5 are more likely to produce antibodies to A2 and A28; patients expressing DR4 are easier toproduce antibodies to B8, B59, DR3 and DQ1. Anti-DR3 antibodies are less frequently produced inpatients bearing DR6. HLA-DR can be categorized into three groups: DR1-4-7-9, DR3-5-6-8 and DR1-2-10. Anti-DR antibodies are easier to be generated for sensitizations between the groups than within thegroup.Conclusions: The ability to generate antibodies to a particular HLA is dependent on the DR type in theresponder. This is the first time that such study is done with big and accurate data. These findings are veryuseful to 1) analyze the antibody screen data; 2) predict whether a donor/recipient pair is more likely tohave de novo donor-specific antibodies generated post-transplantation vs. other pairs. For high riskdonor/recipient pairs, transplantation can either be avoided or managed with stronger immunosuppressionand more careful post-transplantation monitoring.36-OREVALUATION OF HUMORAL IMMUNE RESPONSE TO DONOR HLA AFTERIMPLANTATION OF CRYOPRESERVED VERSUS DECELLULARIZED HUMAN HEARTVALVE ALLOGRAFTS.Carolina Kneib, Michelle F. Susin, Francisco da Costa, Cristina Q.C. von Glehn. ImmunogeneticsLaboratory, Pontificia Universidade Catolica Do Paraná, Curitiba, Parana, Brazil.Aim: We have evaluated the development of humoral antibodies in response to donor allograft valveimplant in patients who received cellularized and decellularized allografts and determined possibleimmunogenic epitopes considered responsible for antibodies reactivity.Methods: Sera samples from all recipients who received cellularized allografts (CRYO) or decellularizedSDS allografts (DECEL) were collected before valve replacement and at 5, 10, 30 and 90 days postoperatively and stored at -20C until required. Tests were performed using Luminex LABScreenSingleAntigen Class I and II. To determine possible immunogenic epitopes, the samples that presented donorspecific antibodies were analyzed using the software HLAMatchmaker.Results: We have observed that decellularized grafts elicited lower levels of class I and class II anti-HLAantibody formation after implantation than cellularized allografts. Furthermore, all patients from CRYOgroup presented DSA class I and II within 3 months of observation period. In HLAMatchmaker analysis,the CRYO group had significantly higher numbers of immunogenic epitopes than DECEL group for bothclass I and II (p: 0,002 – cl I / p: 0,009 – cl II / p: 0,004 – cl I and II).

Conclusions: Our findings can demonstrate that the possible immunogenic epitopes in the cellularizedgroup were against donor HLA molecules and that in the decellularized group the possible immunogenicepitopes were not against donor HLA molecules. These findings lead us to suggest that choosing SDSdecellularization process is the best alternative to decrease the immunogenicity of allograft valvetransplant.37-ORHLA CLASS II REACTIVE SERA FROM KIDNEY ALLOGRAFT RECIPIENTS: DETECTIONOF ANTIBODIES SPECIFIC FOR EPITOPES OF DQ AND DP ALPHA/BETA CHAINS.Antonina Piazza, Elvira Poggi, Giuseppina Ozzella, Valentina Imbroglini, Daniela Caputo, Rosa Cremona,Domenico Adorno. National Council of Researches, Institute of Organ Transplantation, RegionalTransplant Center - Lazio, Rome, Italy.Aim: All preformed HLA antibodies have a deleterious impact on kidney graft outcome. Alpha and betachains of DQ and DP molecules can elicit a humoral immune response.Methods: In 191 HLA class II-positive renal transplant recipients we investigated the incidence of DQ andDP alloantibodies by Single antigen beads coated with 29 DQA1/DQB1 and 24 DPA1/DPB1 heterodimers(One Lambda Inc). We also analyzed the putative epitopes for antibody formation by analysis of aminoacid sequences of molecules corresponding to detected specificities. All patients were DQA1/DQB1 and/orDPA1/DPB1 typed by SSP assays.Results: Anti-DQ antibodies were detected in 126 patients. Ninety-four (75%) had had a previoustransplant; 14 of these only developed anti-DQ antibodies. Seventy-one percent of patients had anti-DQB1antibodies, 2% had anti-DQA1 and the remaining 29% had both. As for the epitopes that might determineantibody-patterns development, 28 epitopes were identified (11 for DQA1 and 17 for DQB1); 7 epitopeshad not been reported yet (DQA1: 41K/130A=0103; 50L=02, 03; 52R=03, 04, 05, 06; 69T=04, 06; 75I=01,02, 03, 04, 06; 160D=0302, 0303. DQB1: 87Y=05, 0604/05/07/09). Anti-DP antibodies were detected in64 patients. Fifty-one (80%) had had a previous transplant; 6 of these only developed anti-DP antibodies.Eighty-one percent of patients had anti-DPB1 antibodies, 3% had anti-DPA1 and the remaining 16% hadboth. As for the putative epitopes, we identified 1 epitope characteristic of DPA1 and 10 epitopescharacteristic of DPB1 molecules. Nine DPB1 epitopes were located in the 6 hypervariable regions of exon2 of the DPB1 alleles, while one epitope (96R:*02, 04, 17, 23, 28) was outside of these regions.Conclusions: These data show that both alpha and beta chain epitopes of DQ and DP molecules areimmunogenic and indicate the importance of epitope-identification studies.38-ORTHE COMBINED OCCURRENCE OF IgG1+IgG3+IgG4 SUBCLASSES IN POST-TRANSPLANTSERA WAS FOUND TO BE MOST CORRELATED WITH FAILURE OF KIDNEYTRANSPLANTS.Michiko Taniguchi, Paul I. Terasaki, Naomi Andersen. Terasaki Foundation Laboratory, Los Angeles, CA,USA.Aim: A major question about HLA antibodies present after a kidney transplant is whether they are going tobe deleterious or not. We examined IgG subclasses to see whether they might give a clue as to whether theantibodies will be immediately deleterious or not.Methods: We selected a total of 35 renal transplant patients with HLA class II-donor specific antibodies(DSA) (failed group: n=20, functioning group: n=15). A total of 152 post-transplant sera was tested withLABScreen Single Antigen beads with detection by four PE-mouse anti-human IgGs specific to each IgGsubclass. C1q-fixing after SA-antibody binding was also tested.Results: As shown in the figure, IgG3 development showed a distinct subclass trend between failed andfunctioning patients (65% vs. 27%, p=0.041). IgG1 was present in both groups (95% and 100%,respectively). Of the other subclasses, IgG4 was also increased in failed patients (65%) compared withfunctioning patients (27%). As shown in the figure, the highest discrimination between failed andfunctioning patients was noted when the frequency of IgG1+3+4 combination in each DSA specificity wascompared (P=0.001). All DSAs in this combination (11 DSAs) were in vitro C1q-fixing positive(100%).[figure1]

Conclusions: In this longitudinal study of 35 renal transplants (total 152 sera) for IgG subclasses, IgG 1 byitself was not very discriminating, but IgG 3 was better correlated with failure. However, IgG 1, 3 and 4 incombination provided the highest degree of discrimination between failure and functioning grafts.Terasaki: One Lambda Inc.: Stockholder.39-ORANTIBODIES TO HLA PRECEDE ANTIBODIES TO MICA AND SELF-ANTIGENS – MYOSIN,VIMENTIN, COLLAGEN-V – IN HUMAN CARDIAC ALLOGRAFT RECIPIENTS WITHACUTE ANTIBODY MEDIATED REJECTION.D.S. Nath, V. Tiriveedhi, H. Ilias Basha, N. Angaswamy, S. Ramachandran, D. Phelan, G.A. Ewald, T.Mohanakumar. Washington University in Saint Louis.Aim: Antibodies (Abs) to HLA, MICA and cardiac self antigens have been implicated in the pathogenesisof acute allograft rejection. Our hypothesis was to test whether Abs to HLA precede the induction of Abs toMICA and cardiac self antigens following human adult cardiac transplantation (HTx) in acute antibodymediated rejection (AMR).Methods: Development of Abs against myosin, vimentin and collagen-V were determined using ELISA in65 pts during the early post-HTx (80%). 9.4% of the sensitized kidney recipients had antibodies to HLA-DPantigens. Considering just those patients suffering acute or chronic kidney allograft loss, 58% had apretransplant CPRA of 0%. Of the 42% of graft loss patients who were sensitized at time of transplant,33% had a donor-specific antibody (DSA) to an HLA-DQ antigen, and 22% had a Class I DSA in their pasthistory. The remaining 45% were sensitized pretransplant, but not to donor-specific HLA antigens. Nosensitized patient having pre-formed HLA-DP antibodies suffered a graft loss, regardless of otherconcurrent sensitization with HLA Class I or other Class II antibodies.

Conclusions: In analyzing this data, we hoped to measure the impact of HLA-DP antibody on kidneyallograft survival in our transplant program. Unexpectedly, we found not only no impact of pre-formedHLA-DP antibodies, but offer the following conjecture. Could having pre-formed HLA-DP antibodies beprotective in terms of kidney allograft survival? Further investigation is underway.Tuesday, September 28, 20102:00 PM - 3:30 PMAbstract Session 4:Markers and Mechanisms of Pathology41-ORPHOSPHORYLATED S6 KINASE IS ASSOCIATED WITH HLA ANTIBODY AND DIAGNOSISOF ANTIBODY-MEDIATED HEART ALLOGRAFT REJECTION.Fang Li, Qiuheng Zhang, David Gjertson, Michael C. Fishbein, Chi Lai, Jennifer Wei, Elaine F. Reed.Immnuogenetics Center, University of California, Los Angeles.Aim: Ligation of class I molecules on the surface of endothelial cells (EC) by anti-HLA antibody (Ab)activates FAK, p70S6 kinase (S6K), S6Ribosomal protein (S6RP), ERK and Akt. We hypothesized thatanti-HLA Ab are immune mediators of Ab-mediated rejection (AMR) by binding to the endothelium of thegraft and transducing signals leading to the activation of cell proliferation pathways.Methods: 107 heart allograft recipients were monitored for post-transplant anti-HLA Ab. The specificityand quantity of DSA (MFI) was determined by single antigen luminex beads. Immunohistochemicalstaining of endomyocardial biopsies was performed using Ab against phosphorylated S6RP, S6K and ERK.Cardiac allograft rejection was diagnosed using ISHLT criteria. AMR was characterized by the presence ofCD68+ macrophages and/or C4d+ staining. Statistical associations were tested utilizing Fisher’s exact testand logistic regression analysis.Results: 67 heart transplant recipients diagnosed with acute rejection (33 with AMR, 18 with acute cellularrejection (ACR), 16 with AMR+ACR) and 40 recipients with no rejection were studied for DSA andphosphoproteomic capillary endothelial staining. A significant association was observed between positiveS6K (p=0.001) and S6RP (p=0.008) staining in the biopsy and diagnosis of AMR. Diagnosis of AMR wasalso associated with development of DSA to HLA-A (p=0.008), DR (p

Results: This analysis revealed that 27 variants of Ii-derived peptides are associated with HLA-DQ2,whereas remarkably, only 2 Ii-derived variants are associated with HLA-DQ6 and none are associated withHLA-DQ1 and -DQ8. In HLA-DQ2 Ii-eluted peptides we identified 3 different Ii-derived peptide bindingregisters (Ii 91-99, Ii 96-104, Ii 99-107), whereas in HLA-DQ8 Ii-eluted peptides we identified only one Iiderivedpeptide binding register (Ii 96-104). HLA-DQ-Ii peptide binding and stability studies showedhigher binding affinity and stability of Ii-derived peptides with HLA-DQ2 compared to other HLA-DQmolecules.Conclusions: We conclude that celiac disease-associated HLA-DQ2 has increased binding affinity foralternative Ii-derived peptides in comparison to other HLA-DQ molecules. This property might beresponsible for an alteration of negative selection of auto-reactive T- cell clones involved in thepathogenesis of celiac disease.43-OREVIDENCE FOR NATURAL KILLER CELL INDUCED GRAFT LOSS IN HLA FULLCOMPATIBLE KIDNEY TRANSPLANTATION.Ilias I.N. Doxiadis 1 , Allan Thompson 2 , Geert W. Haasnot 1 , Joke I. Roodnat 3 , Johan W. de Fijter 4 , FransH.J. Claas 1 , Fritz Koning 1 , Jeroen van Bergen 1 . 1 Immunohaematology and Blood Transfusion, LeidenUniversity Medical Center, Leiden, Netherlands; 2 Internal Medicine, Division of Nephrology, ErsamusMedical Center, Rotterdam, Netherlands; 3 Nephrology, Leiden University Medical Center, Leiden,Netherlands.Aim: Natural Killer (NK) cells are cytotoxic lymphocytes of the innate immune system with the ability todetect HLA class I disparities via Killer Immunoglobulin-like Receptors (KIR). We questioned whetherNK cells contribute to the rejection of human kidney allografts.Methods: We did a retrospective cohort study of 397 HLA-DR compatible kidney transplantationsperformed between 1990 and 2004 and determined the KIR and HLA genotypes of recipients and the HLAgenotypes of donors. Univariate and multivariate statistical methods examined the effect of mismatchesbetween donor and recipient HLA that could be detected through recipient KIR (KIR-ligand mismatches)on death-censored graft survival.Results: In transplantations fully compatible for HLA-A, HLA-B and HLA-DR (n=137), in which a rolefor T cells and HLA antibodies in rejection was minimized, KIR-ligand mismatches were associated withan approximately 25 % reduction in 10-year graft survival (p=0.043). This NK cell effect was comparableto the effect of classical HLA-A and HLA-B incompatibility, and in HLA-A,-B incompatibletransplantations (n=260) no significant additional effect of KIR-ligand mismatches was observed.Multivariate Cox regression analysis considering risk factors that are known to influence transplantoutcome confirmed the effect of KIR-ligand mismatching as an independent risk factor in HLA-A,-B,-DRcompatible transplantations (hazard ratio 2.29, range 1.03-5.10, p=0.043).Conclusions: This finding constitutes the first evidence that alloreactive NK cells hamper successfulkidney transplantation, and suggests that suppression of NK cell activity improves the survival of HLAcompatible kidney grafts in which NK cell alloreactivity is predicted based on KIR and HLA typing.44-ORMICROARRAY ANALYSIS OF RENAL TRANSPLANT BIOPSIES IDENTIFY TRANSCRIPTSSELECTIVELY ASSOCIATED WITH DSA AND FIND NK CELLS IN PERITUBULARCAPILLARIES IN BIOPSIES DIAGNOSED AS ANTIBODY MEDIATED REJECTION.Luis G. Hidalgo 1 , Banu Sis 1 , Patricia M. Campbell 1 , Joana Sellares 2 , Philip F. Halloran 2 . 1 LaboratoryMedicine and Pathology, University of Alberta, Edmonton, AB, Canada; 2 Alberta Transplant AppliedGenomics Centre, University of Alberta, Edmonton, AB, Canada.Aim: The pathogenesis of ABMR is the result of damage incurred onto microvascular endothelium byDSA. The presence of mononuclear cells and neutrophils in the glomerular or peritubular capillariessuggests that cells could mediate the endothelial injury through Fc receptors and/or complement receptors.We used microarrays and the patient’s DSA status to define transcripts selectively associated with DSA togain insight into the mechanisms of ABMR pathogenesis.Methods: 145 clinically indicated biopsies from 145 renal transplant patients were studied. DSA wasidentified using single antigen beads by flow cytometry. Transcripts selective for DSA were defined and

examined on microarrays from a human cell panel. Immunohistochemistry (IHC) was performed onavailable tissue sections from biopsies diagnosed as ABMR (n=12) or T cell mediated rejection (n=6) forCD3, CD68, and CD56.Results: 23 DSA selective transcripts were identified reflecting endothelium and NK cells. IHC resultsshow CD56+ NK cells (and CD68+ macrophages) selectively increased in peritubular capillaries of ABMRbiopsies compared to TCMR.[figure1]Conclusions: Microarray analysis of clinically indicated biopsies show selective increases of NK cell andendothelial transcripts in ABMR biopsies. IHC confirms that NK cells (and macrophages) are bothincreased in peritubular capillaries in ABMR but not TCMR. This study suggests that NK cells andmacrophages are the main effector cells involved in antibody-mediated microcirculation injury.45-ORFINE-MAPPING OF MHC REGION VARIANTS IN JUVENILE IDIOPATHIC ARTHRITIS (JIA)REVEALS EVIDENCE OF ADDITIONAL PREDISPOSING SITES TELOMERIC TO CLASS I.Jill A. Hollenbach 1 , Jarek Meller 2 , Susan Thompson 2 , Carl Langefeld 3 , Teodorica Bugawan 4 , MarcSudman 2 , Glenys Thomson 5 , Mimi Ryan 2 , Henry Erlich 4 , David Glass 2 . 1 Center for Genetics, Children’sHospital Oakland Research Inst., Oakland, CA, USA; 2 Cincinnati Children’s Hospital Medical Center,Cincinnati, OH, USA; 3 Wake Forest University Health Sciences, Winston-Salem, NC, USA; 4 RocheMolecular Systems, Pleasanton, CA, USA; 5 University of California, Berkeley, Berkeley, CA, USA.Aim: We previously characterized HLA class I and class II haplotypes in a large cohort of JIA patients andcontrols and identified several haplotypes that were strongly associated with disease, with extensivevariation between disease subtypes. In the current study we identify additional, non-classical HLApredisposing variants within the MHC region.Methods: We have expanded our original sample to increase statistical power. Patients (n= 924) andcontrols (n=504) were genotyped for ∼2700 single nucleotide polymorphisms (SNPs) in the MHC regionusing the Affymetrix SNP6.0 Genechip; high resolution HLA typing was available for all individuals.Results: SNP association peaks were superimposed upon those obtained for patterns of correlation betweenthe tested SNPs and classical HLA loci in order to identify those associations that may be independent oflinkage disequilibrium (LD) with HLA. After eliminating SNPs in LD with the classical HLA, weidentified a cluster of SNPs strongly associated in JIA approximately 3 MB telomeric to the class I region.To further control for LD, individuals were stratified according to disease subtype and JIA associatedhaplotypes prior to further association testing of the SNPs within this cluster. Analysis of older-onsetpatients with polyarticular JIA, in whom HLA-mediated disease is limited, confirmed association of a SNPhaplotype with JIA (p= .0002); sliding window analysis localized the association to two SNPs in completeLD, rs2494705 and rs4591839 (p=0.008), with OR = 1.51 (CI 1.1-2.1) in individuals with no predisposingDRB1 alleles. While weaker, these associations were corroborated in other disease subtypes aftercontrolling for all classical HLA associations.Conclusions: These results for the first time show clear evidence in a large cohort of additional variantsmediating predisposition to JIA within the MHC.46-ORTHE ERYTHROID MEMBRANE-ASSOCIATED PROTEIN (ERMAP) THAT CARRIES THESCIANNA BLOOD GROUP ANTIGENS INDUCES A POTENT CYTOTOXIC T CELLRESPONSE.Constanca Figueiredo 1 , Yarua Jaimes 1 , Britta Eiz-Vesper 1 , Axel Seltsam 2 , Rainer Blasczyk 1 . 1 Institute forTransfusion Medicine, Hannover Medical School, Hannover, Lower Saxony, Germany; 2 German RedCross Blood Service NSTOB, Institute Springe, Springe, Germany.Aim: The erythroid membrane-associated protein (ERMAP) is expressed on erythrocytes where it carriesthe Scianna blood group antigen and on leukocytes. Until now there are no data on the function of thismolecule. While homologous to proteins located in the MHC locus on chromosome 6, ERMAP resides onchromosome 1. Here, we investigated the effect of ERMAP in the cytotoxic T cell activity.Methods: ERMAP was expressed in HEK cells and used to stimulate CD8T cells alone or in combinationwith IL2. The cytokine secretion profile and proliferation rate of CD8T cells was characterized in presence

of ERMAP. Cytotoxic assays were performed using allogeneic B-LCL cells as target cells and nonstimulatedor CD8T cells pre-stimulated with ERMAP as effector cells.Results: Stimulation of CD8 T cells with ERMAP plus IL2 caused a significant increase of IFN-gammasecretion (1098.4pg/mL) in comparison with IL2 stimulation alone (456.4pg/mL). Stimulation withERMAP alone or in conjugation with IL2 induced an increase of IL1β secretion to 38.7pg/mL and37.2pg/mL respectively, in comparison to IL2 alone (13.2pg/mL). IL5 secretion strongly increased to107.4pg/mL upon IL2 plus ERMAP stimulation in comparison to 7.5pg/mL produced by CD8+ T cellsstimulated with IL2 only. ERMAP stimulation also led to 5-fold higher secretion of IL6 (13.9pg/mL) inCD8T cells than stimulation with IL2 (2.4pg/mL). The proliferation rate of CD8T cells was increased by upto 30% upon ERMAP stimulation. In the presence of ERMAP the cytotoxic capacity of CD8T cells againstB-LCL cells increased by up to 50%. A 5-fold upregulation of Granzyme B mRNA levels was observed inthe CD8T cells after ERMAP stimulation.Conclusions: This study demonstrates for the first time that ERMAP induces a potent cytotoxic T cellresponse, identifying ERMAP as an important effector molecule in cell-based immunity.47-ORREPEAT MISMATCHING FOR HLA-DQ EPITOPES INCREASES REJECTION OF LUNGRETRANSPLANTS.Adriana Zeevi 1 , Marilyn Marrari 1 , Kathy J. Spichty 1 , Maria Crespo 2 , Joseph Pilewski 2 , Diana Zaldonis 3 ,Christian Bermudez 3 , Yoshiya Toyoda 3 , Rene J. Duquesnoy 1 . 1 Pathology, University of Pittsburgh,Pittsburgh, PA, USA; 2 Pulmonary Medicine, University of Pittsburgh Medical Center, Pittsburgh, PA,USA; 3 Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.Aim: Donor-specific HLA antibodies cause rejection and decrease lung transplant survival;retransplantation is often required. Since HLA antibodies react with epitopes, we undertook a study todetermine whether repeat class I and class II HLA epitope mismatching is associated with rejection of lungretransplants.Methods: Thirty-two patients were retransplanted between 01/2003 and 02/2009. All patients and donorswere typed for HLA-A, B, DR and DQ by molecular and/or serological methods. The eplet version ofHLAMatchmaker was used to determine the number of mismatched epitopes on each donor. An eplet wasconsidered a repeat mismatch (RMM) if it was shared between the 1 st and 2 nd donor. Retrospective CDC-AHG crossmatches were negative.Results: The cohort consisted of 18 surviving patients, with mean follow-up of 44±22 months, and 14deceased patients with mean follow-up of 27±16 months. Nineteen patients had at least one rejectionepisode and 13 patients did not show rejection. The table below shows the median numbers of RMM epletsin the rejection and no-rejection groups. There were no significant differences between the numbers ofRMM eplets for A,B and DRB, but the rejection group had a significantly higher number of DQB RMMeplets (p=0.027 by Mann-Whitney U test).[table1]Moreover, there was a higher incidence of rejection inthe group with >5 vs. ≤5 repeat DQB RMM eplets (13/19 vs 2/13, p=0.01, x 2 test). We found nodifferences in rejection between the numbers of non-repeat eplets for DQB, as well as for A,B and DRB, orbetween repeat serological antigen mismatches for all loci.Conclusions: These findings suggest that HLA-DQB epitopes are important in lung transplantation andthat repeat exposure may be considered a risk factor for rejection.48-ORCOLLAGEN V EPITOPE CONSTRAINT LEADING TO CYTOKINE SWITCH FOLLOWINGALLOIMMUNE RESPONSES TO MISMATCHED MHC CLASS I ANTIGENS WHICHINDUCES AUTOIMMUNITY AND CHRONIC REJECTION.Venkataswarup Tiriveedhi 1 , David Brand 2 , Ramsey Hachem 1 , Elbert P. Trulock 1 , G. Alexander Patterson 1 ,T. Mohanakumar 1,3 . 1 Surgery, Washington University School of Medicine; 2 University of Tennessee;3 Pathology & Immunology, Washington University School of Medicine.Aim: Alloimmunity induced autoimmune responses to self antigen collagen-V (ColV) play a critical role inpathogenesis of chronic rejection post human lung transplantation (Post-LTx). The study objective is todetermine the immunodominant portions of ColV and its role in Th-phenotype switch and cytokinesecretion leading to loss of peripheral tolerance.

Methods: Serial analysis of sera obtained from 12 BOS(+) and ColV Abs(+) LTx recipients were selectedin the study. Immunodominant epitopes were defined using western blot, cytokine secretion by ELISPOT,Abs to ColV by ELISA, and Treg cells by FoxP3 demethylation assay.Results: Western blot analysis revealed that patients prior to onset of BOS (-2 yrs) developed Abs to bothCol-α1(V) and α2(V) fragments. However, at the time of BOS and 6 months after BOS, sera had Abs onlyto α1(V). Based on bioinformatics approach 14 epitopes of ColV were chosen to determine theimmunodominance. ELISA based competitive inhibition assay revealed that before onset of BOS epitopes6,7,9, and 14 were immunodominant and stimulated IL-10. However, at the time of BOS, theimmunodominance switched to epitopes 1,4 and 5, and resulted in IFN-γ and IL-17 secretion with loss ofIL-10. FoxP3 assay defining Treg in the peripheral blood revealed marked reduction in the Treg frequencyfrom 7.8 ± 2.1% (-2 yrs) to 0.7 ± 0.3% (+6 mo) (P100 peptides per allele and found the B*44/156 variants to share a highpercentage of their peptide repertoire. In LCL 721.221 a strong surface expression of B*44/156 variantswas observed. In LCL 721.220 only B*4428 156Arg was detected on the cell surface reaching an expressionlevel of >40%, suggesting that B*4428 is less dependent on TPN.Conclusions: These functional differences in PLC dependence confered by sequence variations at AA 156is likely to cause different capabilities of combating viral infections and might need to be considered intreatment regimens of immunosuppressed patients and the donor selection process in stem celltransplantation.

50-ORHLA CLASS I INDUCED SIGNAL TRANSDUCTION AND CELL PROLIFERATION ISDEPENDENT UPON MOLECULAR ASSOCIATION AND PHOSPHORYLATION OFINTEGRIN β 4.Xiaohai Zhang, Elaine F. Reed. UCLA Immunogenetics Center, Department of Pathology and LaboratoryMedicine, University of California, Los Angeles, Los Angeles, CA, USA.Aim: Patients developing anti-donor HLA antibodies following transplantation are at increased risk oftransplant vasculopathy and graft loss. Several studies suggest that the signaling events that occur inendothelial cells during interactions with MHC-I antibodies can contribute to the process of transplantvasculopathy. Previous studies have demonstrated that crosslinking of HLA-I molecules with antibodiesactivates intracellular signaling cascades and stimulates cell proliferation through the formation of amolecular complex with integrin β4. This finding led us to investigate the mechanisms underlying howintegrin β4 transduces proliferation signals upon anti-HLA antibody ligation of HLA-I molecules.Methods: Primary cultures of human aortic endothelial cells were stimulated with F(ab’)2 fragments of theHLA-I monoclonal antibody W6/32 or control IgG. Cell lysates were immunoprecipitated with integrin β4antibody and the immunecomplexes were assessed for tyrosine phosphorylation of integrin β4 andactivation of SHC and ERK by Western blot.Results: Treatment of endothelial cells with HLA-I antibodies stimulated molecular association withintegrin β4 and cell proliferation. siRNA knockdown of integrin β4 blocked anti-HLA-I mediated cellproliferation. Ligation of HLA-I molecules with anti-HLA antibodies triggered phosphorylation of integrinβ4 on tyrosine residues and increased complex formation between integrin β4 and an adapter protein SHC.Conclusions: Crosslinking of HLA-I molecules stimulates signal transduction by associating with integrinβ4. HLA-I ligation stimulates phosphorylation of integrin β4 which in turn recruits the adaptor proteinSHC and activates ERK signaling. Elucidation of the HLA-I signaling pathway has the potential to identifynovel therapeutic strategies to prevent transplant vasculopathy.51-ORTARGETED ENRICHMENT FOR COMPLETE CHARACTERIZATION OF 1.4Mb OF THEMHC WITH NEXT GENERATION SEQUENCING.Deborah Ferriola 1,2 , Katarzyna Mackiewicz 1 , Curt Lind 1 , Xiaowu Gai 3 , Monica D’Arcy 3 , JohannesDapprich 2 , Dimitri Monos 1,4 . 1 Pathology and Laboratory Medicine, The Children’s Hospital ofPhiladelphia, Philadelphia, PA, USA; 2 Generation Biotech, Lawrenceville, NJ, USA; 3 Bioinformatics CoreFacility, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 4 Pathology and LaboratoryMedicine, University of Pennsylvania, Philadelphia, PA, USA.Aim: Comprehensive, detailed characterization of the MHC region, which harbors a large number ofimmunologically relevant genes, is needed because of its importance in transplantation and many diseases.Our objective was to evaluate the use of an enrichment technology and Next-Generation Sequencing (NGS)to fully sequence the MHC.Methods: We enriched and sequenced a contiguous 1.4Mb region including HLA-C through DQB1. Thehomozygous PGF cell line was used, which is the established reference sequence for the MHC of thehuman genome. Region Specific Extraction, an enrichment technology was used by first hybridizing oligosto the region of interest and then enzymatically extending the oligos with biotinylated nucleotides. TheDNA was then captured and sequenced on a single lane of an Illumina platform, producing 72 base pairedendreads. The reads were aligned with Burrows-Wheeler Aligner and differences between theexperimentally determined sequence and the reference were identified with Sequence Alignment/Maptools.Results: A total of 170,730,300 bases were mapped to 738,945 unique bases in the target region. 99.8% ofthe targeted region of the MHC was covered with a mean depth of 68X. 92 % of the target region had readdepth of at least 10. A comparison between the consensus sequence and the reference revealed 32discrepancies. Sanger sequencing revealed that 3 were in agreement with NGS, 23 were in agreement withthe reference and 6 were heterozygotes, differing from both reference and NGS. Overall, 99.996% of basesin the target region were correctly assigned with NGS.

Conclusions: These new technologies allow detailed characterization of the MHC with significantly lesscost and time, opening new opportunities for the comprehensive evaluation of this region. This in turn mayidentify the inter-relationship between genes that makes this genomic region so unique and important toimmune response.Dapprich: Generation Biotech: Employee.52-ORALLELE SPECIFIC ANTI-HLA CLASS I ANTIBODIES DIFFER IN THEIR CAPACITY TOTRANSDUCE CELL SURVIVAL AND PROLIFERATION SIGNAL TRANSDUCTION INENDOTHELIAL CELLS.Yi-Ping Jin 1 , Xiaohai Zhang 1 , Arend Mulder 2 , Frans Claas 2 , Jia-How Lee 3 , Elaine F. Reed 1 . 1 Pathology andLaboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA;2 Immunohaematology and Blood Transfusion, Leiden University Medical Center, Leiden, Netherlands;3 One Lambda, Thousand Oak, CA, USA.Aim: Antibody (Ab)-mediated rejection (AMR) is an important clinical problem after transplantation and isestimated to affect ∼15% allografts. Recipients producing posttransplant HLA Ab are at a higher risk foracute and chronic AMR. Ligation of HLA class I molecules on endothelial cells (EC) with anti-HLA Abactivates intracellular signaling cascades including Src, FAK, PI3K/Akt and mTOR/S6K leading toproliferation. In this study, we determined the capacity of allele-specific anti-HLA class I mAb to stimulatesignal transduction pathways leading proliferation and survival of human aortic EC.Methods: Protein phosphorylation was measured by Western blot, cell proliferation was measured byCSFE labeling and FGFR expression was tested by flow cytometry. The antigen (Ag) binding capacity(ABC) of the HLA mAb bound to the EC was measured by MESF (molecular equivalent solublefluorochrome) and simply cellular beads.Results: We found that HLA-A locus Ag are expressed at 7-fold higher than B locus Ag on EC (p

patients’ HLA type is A*02, A*3, B*35, B62*,--, Bw6, Bw--, DR4, DR1, and DQ2, DQ5. Single antigenbeads analysis was performed using the LABScreen® assay. Sequence analysis was performed usingClustalW2 multiple sequence alignment program for proteins (EMBL-EBI).Results: RESULTS: For case 1, the patient (89% PRA), while multiple beads reacted, they were at levelsbelow 1000 MFI. The majority of this antibodies were directed against HLA-Bw6 antigens. Additionally,two Cw* locus antibodies were detected (Cw*09 and Cw*10). Sequence alignment analysis revealed thatCw*09 and Cw*10 expressed the Bw6 epitope. FCXM with surrogate Bw6 donors were positive. In Case2, the patient had 67% PRA, all of antibodies were directed against HLA-Bw4 antigen. FCXM with hispotential donor and surrogate donors that are Bw4,-- were all positive. Surrogate FCXMs with Bw6, --donors were negative.Conclusions: Although the signal strength is considered a major determinant regarding the clinicalrelevance of antibodies identified by single antigen beads, the pattern of reactivity should not beoverlooked. Additionally, these data may provide useful information regarding shared epitopes acrossdifferent HLA loci.54-ORON THE MONITORING OF DONOR SPECIFIC ANTIBODIES USING SOLID PHASE ASSAYS:EXISTING CHALLENGES.Rex A. Friedlander 1 , Elder F. Diaz 1 , Vijay K. Sharma 1,2 , Arvind Menon 1 , Blanca M. Ponce 1 , DarshanaDadhania 1,2 , Manikkam Suthanthiran 1,2 . 1 Immunogenetics and Transplantation Laboratory, The RogosinInstitute, New York, NY, USA; 2 Department of Transplantation Medicine, NYPH-Weill Cornell MedicalCenter, New York, NY, USA.Aim: The Luminex platform is the protocol of choice for detecting and monitoring antibody. The levels arequantified by mean fluorescence intensities (MFIs). Because different lots of HLA-coated beads may beused clinically, we investigated whether there are significant differences in the MFIs across the assay lots.Methods: We used anti-HLA Class I monoclonal antibody, W6/32 and collected the trimmed MFIs for 97single antigen beads (SABs) from two lots of Class I SABs from a single vendor. The MFIs were comparedutilizing Student’s t-Test, and the ranking of the SABs (alleles) in the two lots was compared usingSpearman’s rank-order correlation.Results: We found significant differences in the MFIs of 88 of 97 Class I SABs. Table 1 lists the P-valuescalculated following comparison of the MFIs of identical alleles from the two lots; importantly, 20 of 97SABs had their MFI value increased by 50% or greater. The lack of perfect correlation was reflected bySpearman’s rank-order correlation of 0.538, P

allograft injury. Four of these 18 had B-cell flow crossmatches performed: 3 were positive. Six subjectsdeveloped DSA to both HLA-DR and HLA-DQ. Four of these 6 subjects had histological evidence ofantibody mediated rejection and were C4d positive.Conclusions: DSA restricted to HLA-DQ were not predictive of C4d deposition in the kidney allograft inthis cohort; the presence of antibodies to HLA-DR increased the incidence of C4d deposition. This mayreflect lower expression of HLA-DQ in stable allografts. However, due to the incidence of chronic allograftinjury in these subjects, further study of the correlation between DSA specificity and graft function iswarranted.56-ORCOMPARISON BETWEEN TWO DIFFERENT METHODS FOR THE ANALYSIS OF SINGLEANTIGEN DATA.Chantale Lacelle 1 , Ian-Michael Salvador 1 , Daniel Hines 1 , Lama Hussein 1 , Peter Stastny 1,2 . 1 Pathology, UTSouthwestern Medical Center, Dallas, TX, USA; 2 Internal Medicine - Transplant Immunology, UTSouthwestern Medical Center, Dallas, TX, USA.Aim: There is no standard method to analyze Luminex single antigen (SA) data raising some concern thatdifferences in analysis may affect results. We investigated the differences in HLA antibodies detected usingeither normalized ratios (NR) or a fixed normalized MFI (NMFI) value of 1000.Methods: Class I and II SA data for two consecutive sera from 30 patients were assessed using an in-houseanalysis which calculates NR and considers NR >2X as positive and the OneLambda software using a fixed1000 NMFI cutoff. NR were determined by multiplying the raw MFI by an antigen density correctionfactor and dividing by the mean +3 SD of NHS panel. NMFI values were raw data – NC, minus the serumnegative control (LSNC).Results: Antibody specificities identified by NR and not by NMFI were seen in 21/30 (70%) of the patientsfor class I and 14/30 (47%) of the patients for class II antigens. We found that 81% (17/21) of the patientsfor whom NR identified additional class I and 57% (8/14) class II specificities and not detected by NMFI,remained positive in subsequent serum samples; while 52% (11/21) of patients negative by NMFI for classI and 36% (5/14) for class II became positive in the later serum. Crossmatches in 2 patients with DSAidentified by NR but not NMFI were confirmed as positive in both cases by flow cytometry.Conclusions: Our study shows that the method used to analyze SA tests can impact the antibodyspecificities identified. NR identified more positive class I and II specificities and yielded morereproducible results when comparing sequential serum sample than NMFI. We also showed thatspecificities only identified as positive using NR and not by NMFI can cause a positive flow crossmatch.57-ORVIRTUAL CROSSMATCHING: UNACCEPTABLE ANTIGEN ANALYSIS VERSUS FLOWCROSSMATCH RESULTS DEMONSTRATE UNIQUE CHALLENGES IN SENSITIZEDPATIENTS.Robert Cirocco, Patricia Kimble, Marilyn Wetmore, Jen Mendolina, Michael Moritz. HLA Laboratory,LVHN, Allentown, PA, USA.Aim: The determination of the Unacceptable Antigen (UA,used in UNET) is the challenge of the modernHLA laboratory for sensitized individuals. A higher MFI cutoff will allow more crossmatches for thesepatients concomitantly there will be more positive flow crocssmatchs. Do lower cuttoffs result in more txs?Methods: Twenty-five positive flow crossmatches were analyzed from recipient pairs that were thought tobe compatible by virtual crossmatching. We empolied the MFI single antigen assay to determine theUnacceptable Antigens for the virtual crossmatch. we then compared the MESF values of these Pronaseflow B cell crossmatches. We determined a plausible reason for the positive crossmatch retrospectivlly.Results: Between 5-1-09 and 3-31-10, 114 sensitized recipients were flow cross-matched. Thirty two outof 51 (69.3%) were positive from 5-1-09 until 8-1-09 (no virtual crossmatch) (four were transplamted)From 9-1-09 until 12-1-09, we had seven out of 39 positive crossmatches (17.9%) resulting in eighttransplants. In the positive flow crossmatches eight were additive from two or more antigens below cuttoff.eight were from anti-HLA C or DQ antibodies. DP caused two (18%). The results demonstrate salientfactors that contribute to donor/recipient incompatibility.

Conclusions: In sensitized potential recipients demonstrating a positive crossmatch: 1) virtualcrossmatching results in less crossmatches and more sensitized recipients transplanted. 2) Anti-HLA C andDQ should be used as UA’s (new UNOS histo committee proposal). 3) There is a poor correlation betweenMFI and MESF. 4) Thirty-two % were additive UA antigens that could not been known prior to donortyping. 4) anti-DP was relavant. Other factors will be, unknown antibodies (Endothelial, anti-phospholipid,Anti-DNA, and/or MICA) and variable cell HLA Ag expression, resulting in a positive crossmatch.58-ORCOMPLEXITY OF ALLOANTIBODY REACTIVITY WITH HLA-DQA1/DQB1 HETERODIMERMOLECULES.Jane Kearns, Thanh-Mai Bui, Malek Kamoun. Pathology and Laboratory Medicine, University of PA,Philadelphia, PA, USA.Aim: The high degree of amino acid variability among the DQA1/ DQB1 allele heterodimers at surfaceexposed residues contributes to the complexity of anti-DQ antibody specificity. We compared reactivity ofanti-DQ antibodies using a Luminex Single Antigen bead (SAB) assay to reactivity with B cells.Methods: Reactivity of 6 anti-DQ specific sera were evaluated by SAB and flow cytometry using a cellpanel from 21 unrelated donors who were typed for HLA class I and class II alleles using SSO methods.All sera were non reactive with other HLA class I or class II antigens.Results: The correlation between the strength of antibody reactivity with DQ alleles assessed by Luminexbeads versus reactivity with B cells varied significantly with different DQA/DQB allele combinationssuggesting allele-specific variations in the level of surface expression of DQ molecules. Furthermore, tissuespecific structural variations of the heterodimer-peptide complex may influence the reactivity of anti-DQantibodies.[table1]Conclusions: Anti-DQ antibody reactivity level determined by SAB assays may not accurately reflect thelevel of reactivity with cell surface DQ molecules.59-ORUSE OF A CALCULATED RELATIVE RATIO TO NORMALIZE LUMINEX SINGLE ANTIGENBEAD RESULTS.Deborah O. Crowe, Sallyanne C. Fossey. Transplant Immunology, DCI Laboratory, Nashville, TN, USA.Aim: With the Luminex Single Antigen Bead assay, run-to-run variations limit the use of mean fluorescentintensity (MFI) as a true “quantitative” value. Likewise, the simple ratio of the test MFI/Neg varies greatlyamong different runs and samples. The intent of this study was to normalize the results in order tostandardize the data.Methods: The Pos/Neg ratio can be used as a gauge for variability between runs. If the Pos/Neg ratio is setat 100%, the test ratio can be normalized proportionally. Relative Ratio = Ratio x 100 divided by P/N IfRel Ratio for a Positive reaction is set at 10, then the cutoff Ratio on printout is: Cutoff = P/N divided by10 (all Ratios > than this number should be Positive).Results: The normalized or “Relative Ratio” was calculated for antibodies identified by Luminex singleantigen beads and correlated with Flow crossmatch results. There was good correlation between theRelative Ratio and the Flow crossmatch shift. Looking at 65 positive Flow crossmatches and the relativeratios of the antibodies to unacceptable antigens, we were able to set ranges for the strength of the antibodyresponse. These were: Relative response of 5-10 = Borderline; 10-30 = weakly positive; 30-50 =moderately positive; and >50 = strongly positive.Conclusions: Relative Ratio (RR) can be used to predict a positive crossmatch. It can also be used toestablish a cutoff ratio when analyzing HLA antibody results. RR was used in post-transplant monitoring toassess the effect of treatment. The advantage is that multiple samples do not need to be testedsimultaneously on the same run to prevent run-to- run variation. The power of the Relative Ratio to identifypotential candidates for desensitization protocols was also examined. While the acceptable risk may varyamong transplant programs, the strength of the antibody as determined by Relative Ratio can be used as amarker to predict success of the treatment.60-OR

A NOVEL HLA CLASS I SINGLE ANTIGEN BEAD PREPARATION ELIMINATES FALSEPOSITIVE REACTIONS ATTRIBUTED TO NATURAL ANTIBODIES – IN THE SERA OF POSTTRANSPLANT PATIENTS.Nadim R. El-Awar 1 , Paul I. Terasaki 2 , Cristina von Glehn 3 , Matthew Everly 1 , Judy Hopfield 1 , AnhNguyen 1 . 1 One Lambda Inc.; 2 Terasaki Foundation Laboratory; 3 Pontificia Universidade Catolica.Aim: Single antigen beads (SA) have been shown to produce “false” irrelevant reactions in the sera of nonimmunizednormal males. We tested a new bead preparation produced to eliminate these reactions in thesera of post transplant kidney patients.Methods: Fifty nine sera from post transplant patients were tested with SA beads, EB treated beads andClean SA Beads (patent pending) - free of denatured class I antigens (One Lambda Inc.). All values >1000MFI of the adjusted and normalized trimmed mean values were considered positive.Results: Examples of HLA class I specificities of post transplant sera are shown in the figure below.Reactions of the Clean Beads become negative as compared to the reactions with SA beads. Reactions withthe EB treated beads are positive indicating the antibodies target cryptic epitopes. Arrows in the figurepoint to clean beads MFI values that are too low to show.[figure1]Conclusions: A novel preparation of the SA Beads (Clean beads) can eliminate “extra” reactions attributedto natural anti-HLA antibodies in post transplant sera. Reactions that are positive with the SA beadsbecome negative with the clean beads. Clean beads have no effect on the reactivity of alloantibodies (datanot shown) and can potentially simplify sera analysis.El-Awar: One Lambda Inc: Employee; Stockholder. Terasaki: One Lambda Inc.: Stockholder. Everly: OneLambda Inc.: Employee. Hopfield: One Lambda Inc.: Employee. Nguyen: One Lambda Inc.: Employee.Wednesday, September 29, 20104:00 PM - 5:30 PMAbstract Session 6: Tolerance, Immunity and Graft Survival61-OREFFECTS OF PRETRANSPLANT MICROCHIMERISM ON ALLOGRAFT SURVIVAL IN HLA-HAPLOIDENTICAL FAMILY DONOR RENAL TRANSPLANTATION.Shin Young Joo 1 , Myoung Hee Park 2 , Eun Young Song 2 , Yunsu Shin 2 , Jongwon Ha 3 , Sang Joon Kim 3 .1 Department of Laboratory Medicine, Seoul Paik Hospital, Seoul, Republic of Korea; 2 Department ofLaboratory Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea;3 Department of Surgery, Seoul National University College of Medicine, Seoul, Republic of Korea.Aim: Microchimerism (MC) may develop after solid organ transplantation or transfusion and fetalmaternalMC may develop between mother and child and between siblings, which can persist lifelong. Theeffects of pretransplant MC on the outcomes of solid organ transplantation are not known and weinvestigated the effects of pretransplant fetal-maternal MC on allograft outcomes in HLA-haploidenticalfamily donor renal transplantation.Methods: A total of 106 cases of family donor renal transplantation performed between 1996 and 2004were studied. The study group included 63 cases of HLA-haploidentical and the control group included 43cases of HLA-identical transplantation. Pretransplant MC against mismatched donor HLA-DRB1 allelewas detected in the recipient’s peripheral blood using nested PCR-single strand conformationpolymorphism method. The allograft outcomes of HLA-haploidentical MC (+) and (-) groups werecompared with those of HLA-identical group.Results: Pretransplant MC in the recipients was detected in 22.2% (14/63). Compared with HLA-identicalgroup, MC (-) group (n=49) showed significantly inferior allograft outcomes: higher acute rejection rate(14.0% vs. 40.8%, p=0.005), higher serum creatinine level (5 yr-posttransplant, 1.1 vs. 1.4 mg/dL, p

Conclusions: In HLA-haploidentical family donor renal transplantation, pretransplant MC present in therecipient may have beneficial effects on rejection-free allograft survival.62-ORNOVEL REGULATORY MECHANISM OF THE HLA CLASS II ANTIGEN PROCESSINGPATHWAY.Yarua Jaimes, Britta Eiz-Vesper, Constanca Figueiredo, Rainer Blasczyk. Institute for TransfusionMedicine, Hannover Medical School, Hannover, Lower Saxony, Germany.Aim: The HLA class II processing pathway provides a mechanism to selectively present peptides generatedin the endosomal compartments of antigen presenting cells. Viral and parasite infected cells or tumour cellsescape immunosurveillance by preventing the cell surface expression of HLA class I and class II antigens.Here, the expression of the proteins involved in the antigen processing pathway was evaluated in theabsence of either HLA-DR or HLA-DM.Methods: Lentiviral vectors were designed to express short hairpin RNA sequences (shRNA) targetingHLA-DM or HLA-DR. Silencing of HLA-DM and HLA-DR was measured at mRNA level by Real TimePCR and at protein level by Flow Cytometry. Also, the cell surface expression of the Class II associatedinvariant peptide (CLIP) was assessed by flow cytometry.Results: Moreover, the Class II transactivator (CIITA) and HLA class I levels were measured by RealTime PCR. The expression of HLA-DR and –DM was knocked down up to 85% and 75% respectively.One week after knocking down the expression of HLA-DR, the levels of other proteins involved in theantigen processing pathway such as HLA-DM were not affected. However, 6 weeks after thedownregulation of HLA-DR expression, the levels of HLA-DM molecules were reduced by up to 50%.Also, in the absence of HLA-DM molecules the expression of HLA-DR decreased by up to 70%. Inaddition, the transcript levels of the CIITA molecules were downregulated by up to 35% in the absence ofboth HLA-DR and –DM proteins. In the absence of HLA-DR or HLA-DM the CLIP cell surfaceexpression decreased by up to 85%. The expression levels of β-actin and β2 microglobulin were notaffected by targeting either HLA-DM or HLA-DR.Conclusions: Here, we describe a novel outside-in regulatory mechanism of the HLA class II processingpathway. These findings may contribute to improve or develop therapeutic approaches against tumor cellsor infections.63-ORSIGNIFICANT CORRELATION BETWEEN DONOR-RECIPIENT HLA-DPB1 T CELLEPITOPE MATCHING AND SURVIVAL IN 4490 UNRELATED 10/10 MATCHEDHEMATOPOIETIC STEM CELL TRANSPLANTS ANALYZED WITHIN THE 15 THINTERNATIONAL HISTOCOMPATIBILITY WORKSHOP.Katharina Fleischhauer 1 , Bronwen Shaw 2 , Mari Malkki 3 , Ted Gooley 3 , Elisabetta Zino 1 , StephenSpellman 4 , Yasuo Morishima 5 , Andrea Velardi 6 , Peter Brady 7 , Jean-Denis Bignon 8 , Alejandro Madrigal 2 ,Effie Petersdorf 3 . 1 Unit of Molecular and Functional Immunogenetics, Fondazione Centro San Raffaele delMonte Tabor, Milan, Italy; 2 Anthony Nolan Research Institute, Lundon, United Kingdom; 3 FredHutchinson Cancer Center, Seattle, WA, USA; 4 National Marrow Donor Program (NMDP), Center forInternational Blood and Marrow Transplant Research (CIBMTR), Minneapolis, MN, USA; 5 Japan MarrowDonor Program (JMDP), Nagoya, Japan; 6 European Group for Blood & Marrow Transplantation(EBMT), Perugia, Italy; 7 Australian Bone Marrow Donor Registry (ABMDR), Adelaide, Australia; 8 SociétéFrance Greffe de Moelle (SFGM), Nantes, France.Aim: Donor-recipient HLA-DPB1 matching for functional T cell epitopes (TCE) involving 3 (TCE3) or 4(TCE4) groups of DPB1 alleles has been suggested to improve survival after 10/10 matched unrelatedHSCT. The aim of this study was to test this model in a large number of transplants facilitated through theworld-wide registries.Methods: In the 15 th International Histocompatibility Workshop, we have tested the TCE3 and TCE4algorithms in HLA-A,B,C,DRB,DQB1 allele-matched transplants with (n=4490; 10/10) or without(n=1348; 12/12) allelic DPB1 disparities. Regression models were adjusted for different parametersincluding disease severity, transplant conditioning, and number of mismatched DPB1 alleles.

Results: Approximately twice as many pairs were scored as permissive by TCE3 (60%) as compared toTCE4 (30%). Using TCE3 permissive pairs as reference, the hazards of overall mortality (OM) weresignificantly higher among transplants with non-permissive TCE3 both in the graft versus host (GvH; HR1.19; 95CI 1.07-1.31; p=0.0008) and in the host versus graft direction (HvG; HR 1.12; 95CI 1.01-1.25;p=0.02), and were comparable to 12/12 transplants (HR 0.97; 95CI 0.88-1.06; p=0.56). Similarly, thehazards of OM were significantly higher in TCE4 non-permissive as compared to TCE4 permissivetransplants, both in the GvH (HR 1.16; 95CI 1.05-1.28; p=0.002) and in the HvG vector (HR 1.10; 95CI1.00-1.21; p=0.04), and comparable to 12/12 transplants (HR 0.99; 95CI 0.89-1.10; p=0.89).Conclusions: Our data demonstrate in a large number of international transplants, that survival after 10/10allele-matched unrelated HSCT correlates with donor-recipient match status for functional TCE. Theseresults provide a rationale for prospective DPB1 allele assessment of potential 10/10 matched donors.64-ORDIFFERENTIAL EFFECT OF PRE-TRANSPLANT BLOOD TRANSFUSIONS ON IMMUNEEFFECTOR AND REGULATORY COMPARTMENTS IN HLA-SENSITIZED- AND NON-SENSITIZED RECIPIENTS.Michael Eikmans 1 , Marloes M. Waanders 1 , Dave L. Roelen 1 , Paula P.M.C. van Miert 1 , Jacqueline D.H.Anholts 1 , Johannes W. de Fijter 2 , Anneke Brand 3 , Frans H.J. Claas 1 . 1 Dept. of Immunohematology andBlood Transfusion, Leiden University Medical Center, Leiden, Netherlands; 2 Dept. of Nephrology, LUMC;3 Sanquin Blood Bank Southwest Region, Sanquin, Leiden.Aim: Blood transfusion (BT) may elicit both harmful and beneficial immune responses against subsequentorgan grafts. Our goal was to investigate the effect of BT on the cellular phenotype and function in bloodsamples of two recipient groups.Methods: Immune parameters of a single, non-leukocyte-depleted BT were investigated in: non-HLAsensitizedrecipients with a one-HLA-DR matched donor (PBT), and females with previous exposure toHLA alloantigens through pregnancy (donor specific transfusion, DST).Results: Thirty-five percent of the DST recipients and 9.5% of the PBT recipients developed HLAantibodies after BT. Analyses were performed in pre-BT-, 2 wk-post-BT-, and >10 wk-post-BT samples(PBT: n=10; DST: n=14). The number of BT donor-reactive IFN-γ-producing memory T cells increased 2wk post-BT, but only in the DST group increased frequencies persisted beyond 10 wks (P

this study FCXM analyses and survival data in 624 consecutive deceased donor or live donor renal allograftrecipients (1998 to 2008) were evaluated.Methods: Patients received induction with ALG and maintenance therapy of a calcineurin inhibitor,prednisone, and MMF. A mean channel shift (MCS) above a NEG serum > 49 was recorded as a POS T orB cell FCXM (+ pronase).Results: T - B - FCXM occurred in 468 recipients whereas 156 (25%) had a POS FCXM, 82 T + B + and 74 T -B + . 10 yr death censored actuarial graft survival of patients with a T - B - FCXM was 83% whereas graftsurvival was 67% for T + B + patients and 53% for T - B + recipients (Log Rank (Mantel-Cox, p

Methods: Molecular HLA typing was performed on the patient’s specimens of peripheral blood (PB),buccal epithelial cells (BE), and bone marrow aspirate (BM) collected at different times. The deceaseddonor’s HLA type was obtained from the organ procurement organization.Results: HLA class I SSP typing on PB (post-transplant day 71; WBC count of 200/µl) showed a pattern ofmixed chimerism: strong bands corresponding to the donor’s phenotype of HLA-A*01,*68; B*08,*35 anda second set of much weaker bands representing HLA-A*02,*31; B*57,*58. The latter phenotype was thepatient’s own as revealed by the BE (day 77) result. The patient and donor were identical for the class IIphenotype DRB1*03,*15; DQB1*02,*06. Additional HLA typing of PB and BM from days 91-97, whenPB WBC counts increased above 3,ooo/µl (86-92% of neutrophils) showed only the donor’s HLA type.Chimerism studies using an informative VNTR locus D1S80 consistently showed only the donor origin inspecimens of PB (day 99) and BM (days 97, 112, and 174; mostly myeloid cells).Conclusions: Cutaneous and gut GVHD developed following liver transplant from an HLA-DR and -DQidentical deceased donor. The class II-identity possibly permitted uninhibited proliferation of passengerlymphocyte population leading to development of GVHD. Partially recovered neutrophils were determinedto be of donor origin, suggesting that partial myelopoiesis was sustained by the hematopoietic progenitorcells passively transferred with the allograft.68-ORHUMAN CD4 + CD25 HIGH FOXP3 + CELLS REGULATE CTL REACTIVITY.Yuming Yu 1,2,3 , Joshua Miller 1,2 , Joseph R. Leventhal 1 , Anat R. Tambur 1 , James M. Mathew 1,2 .1 Comprehensive Transplant Center, Northwestern University, Chicago, IL, USA; 2 ComprehensiveTransplant Center, Jesse Brown VA Medical Center, Chicago, IL, USA; 3 Department of OrganTransplantation, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China.Aim: To determine if human CD4 + regulatory T cells (T4regs), generated in a Treg MLR assay previouslyreported, can also regulate cytotoxic T cells (CTL).Methods: T4regs were generated from bulk MLRs of healthy DR mismatched volunteers. These were thenadded as third components in donor-specific and non-specific MLRs and micro-CMLs, the latter- modified51 Cr release assays previously described. In micro-CML we first used PBMC as responder CTL. We thenselected CD8 + purified cells supplemented with IL-2 for this readout. Also, a CFSE-based CD8 +proliferation assay assessed inhibition of proliferation by T4regs.Results: Among the T4regs, >90% were CD4 + CD25 high , and of these >95% were CD4 + CD25 high FOXP3 + .When these T4regs were added to MLRs in Modulator:Responder ratios of 1:10, 1:50 and 1:250 donorspecific (original stimulator) vs. non-specific (HLA mismatched indifferent stimulator) inhibitionpercentages were respectively 84%±19% vs 68%±17%, 77%±18% vs. 50%±14%, and 54%±13% vs.20%±10% (n=4). When T4regs were added to PBMC responders in micro-CML assays in the same ratios,inhibition percentages of target cell lysis in donor specific vs. non-specific CTLs were 93%±31% vs.75%±11%, 52%±32% vs. 31%±33%, and 16%±19% vs. 7%±12% respectively (n=3). When PBMCresponders were replaced by CD8 + cells in the presence of 10U/ml of IL-2 inhibition percentages of targetcell lysis in donor specific vs non-specific assays at each ratio were 80% vs 93%, 35% vs 59% and 0% vs.0% respectively, i.e. regulation did not appear to be donor specific(n=1). Finally, T4regs did not suppressCD8 + proliferation, either with or without IL-2.Conclusions: T4regs (CD4 + CD25 high FOXP3 + ) generated in MLR can regulate both (CD4 + ) proliferationand CTL (CD8 + ) lysis. There is “partial” donor specificity needing clarification. CTL (purified CD8 + )regulation does not appear to involve impaired proliferation, in contrast to regulatory effects on CD4 + cells.