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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.
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Page 7 and 8: Results: PHA-induced mRNA expressio
Page 9 and 10: conjugated IgM (BD Biosciences), Ig
Page 11 and 12: Methods: Peripheral blood and endom
Page 13 and 14: Aim: NK cells have an established r
Page 15 and 16: Methods: Sera (n=22) were tested fo
Page 17 and 18: expression on lymphocytes, we hypot
Page 19 and 20: Conclusions: Clean SA beads can eli
Page 21 and 22: goal is to re-examine our previous
Page 23 and 24: Aim: There is increasing evidence t
Page 25: esult in poor graft survival. The g
Page 29 and 30: 34-PCLINICAL AND PATHOLOGICAL SIGNI
Page 31 and 32: Nabil Mohsin, Isabelle G. Wood, Ind
Page 33 and 34: donor/recipient pairs for transplan
Page 35 and 36: and tested in the DynaChip assay fo
Page 37 and 38: average difference between Ave and
Page 39 and 40: Conclusions: The XM-One assay can b
Page 41 and 42: 59-PCYTOTOXIC AND NON-CYTOTOXIC ANT
Page 43 and 44: data suggests that DTT pretreatment
Page 45 and 46: Bhavna Lavingia 1 , Chantale Lacell
Page 47 and 48: ATG INTERFERNCE IN SINGLE ANTIGEN L
Page 49 and 50: 75-PPREFORMED LOW REACTIVE ANTI-HLA
Page 51 and 52: Sabarinathan Ramachandran 1 , Naohi
Page 53 and 54: 83-PALLELIC DIVERSITY OF KIR2DL4 AN
Page 55 and 56: São Paulo, Brazil; 3 Commissariat
Page 57 and 58: 91-PCHARACTERIZATION OF COMMON AND
Page 59 and 60: Conclusions: The extensive diversit
Page 61 and 62: 99-PPOTENTIAL COMMON NOVEL ALLELES
Page 63 and 64: 103-PTOWARDS A FUNCTIONAL HLA-DPB1
Page 65 and 66: 107-PWHOLE GENOME AMPLIFICATION (WG
Page 67 and 68: 111-PLOSS OF MISMATCHED HLA IN FAMI
Page 69 and 70: 115-PDONOR EPITHELIAL CELL CHIMERIS
Page 71 and 72: Caucasian patients. MRs vary signif
Page 73 and 74: Results: We obtained the total No.
Page 75 and 76: cord blood and platelet transfusion
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131-PASSOCIATION OF IL-2-330(G/T) W
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Institute, Children’s Hospital Oa
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Methods: To prove the robustness of
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populations.We studied the involvem
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amplicons generated from different
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Results: The sequence of the unexpe
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concentration of the Taq Polymerase
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160-PASSOCIATION OF HAPLOTYPES WITH
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164-PDETECTION OF A DE NOVO DONOR S
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168-PA NOVEL HLA-DQB1 ALLELE CONFIR
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172-PSCREENING FOR HLA-SPECIFIC ALL
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176-PIMPLEMENTATION OF THE WORLD HE
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epertoire of displayed ligands and
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mucosa. No other samples were avail
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Conclusions: In previously reported
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Conclusions: Our findings can demon
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Conclusions: In analyzing this data
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examined on microarrays from a huma
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Methods: Serial analysis of sera ob
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Conclusions: These new technologies
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allograft injury. Four of these 18
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A NOVEL HLA CLASS I SINGLE ANTIGEN
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Results: Approximately twice as man
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Methods: Molecular HLA typing was p
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