Canoga Park Research Articles

Improvement in HLA-typing by new sequence-specific oligonucleotides kits for HLA-A, -B, and -DRB1 loci.

Polymerase chain reaction sequence-specific oligonucleotide is commonly used for HLA-typing. We replaced our LabType SSO HD (HD) kits with LabType SSO XR (XR) kits (One Lambda, Inc., Canoga Park, California) for HLA-A, -B, and -DRB1 following acquisition of a LABScan3D analyzer. The XR kits have more bead regions than the HD kits, allowing for an extended number of probes and exon coverage. They are claimed to improve typing resolution and to diminish the number of allele ambiguities, including common and well-documented (CWD) and null alleles to be resolved. We retrospectively selected patients who had their first HLA-typing performed with the HD kits and their second determination with the XR kits between 2015 and 2016. Forty-two patients were selected for HLA-A typing comparison, and 48 for HLA-B and 41 for HLA-DRB1. XR kits significantly decreased the number of allele ambiguities for HLA-A and -B. On the other hand, the improvement was limited for the HLA-DRB1 locus. The XR kits did not resolve all the CWD HLA allele ambiguities, which may be important for organ and/or hematopoietic stem cell transplantations. The XR kits eliminated 88%, 62%, and 27% of null allele ambiguities for HLA-A, -B, and -DRB1 loci, respectively. In conclusion, the XR kits allow for a significant improvement of HLA-typing resolution for HLA-A and -B loci in comparison with HD kits. In contrast, the number of oligonucleotides in the XR HLA-DRB1 kit should be extended to include exon 3 at the very least. It could also be interesting to include oligonucleotides allowing HLA-DRB3, 4, and 5 typing.

P116 Complement component 1q assay lacks sensitivity and trends with de novo donor-specific antibody titers

Aim Despite major advancements in immunosuppression, long-term allograft survival rates from deceased and living donors remain poor following post-kidney transplantation. The presence of de novo donor-specific antibodies (dnDSA) and complement component 1q (C1q) following transplantation contributes to repetitive microvascular injury that correlates with worsening graft function and graft failure. Immune monitoring post-transplantation of dnDSA and C1q is commonly employed to assess patient graft failure risk and can guide clinical decision making regarding the patient’s baseline immunosuppression. In this investigation, we sought to determine if the C1q assay provided added clinically relevant data as C1q positivity was often associated with dnDSA titer and the use of pooled patient serum for quality control varied greatly among different runs of the same lot number. Methods Kidney transplant patients were monitored for the presence of human leukocyte antigen (HLA) class I and class II antibodies using the Labscreen Single Antigen Antibody Detection Tests and the C1qScreen (One Lambda, Inc., Canoga Park, CA). C1q risk scores were delineated as N = C1q negative, 2 = C1q weak positive, 3 = C1q moderate positive, 4 = C1q strong positive, 5–6 = very strong positive based on C1q mean fluorescent intensity (MFI) cutoff ranges. Results DSA titration (1:16, MFI = 3136) trended as N = C1q negative. DSA titration (1:256, MFI = 13136) trended as 5–6 = C1q very strong positive. Poor delineation of C1q risk at intermediate titrations may be due to confounding quality control variability in the daily pooled plasma serum measurements, which can alter baseline risk scoring of C1q MFI values. Conclusions C1q and dnDSA are often tracked to make risk assessments regarding kidney allograft health post-transplantation; however, C1q MFI measurements may not be additionally informative for clinicians as it often tracked with dnDSA antibody titer and the C1q assay lacks sensitivity at lower concentrations and potentially makes C1q risk scores ambiguous or misleading.

P140Proper setting of cut-off value for the lymphocyte crossmatch by flow cytometry in kidney transplantation

Aim The flow cytometric lymphocyte crossmatch (FCXM) has become a routine test performed prior to kidney transplantation. Many studies have reported that a proportion of transplant recipients experienced early graft loss despite a negative complement-dependent cytotoxicity (CDC) crossmatch test. The FCXM is able to detect Human Leukocyte Antigen (HLA) antibodies of low titer or of a non-complement binding nature that may be responsible for graft loss. It is important to report positive and negative results using appropriate cut-off values. We reviewed the results of FCXM with CDC and panel reactive antibody (PRA) and attempted to set clinically useful cut-off values for FCXM. Methods From January 2015 to October 2017, 518 kidney recipients’ results of FCXM, CDC, and PRA (Screening test, Panel kit, and single antigen bead assay, LABScreen Luminex kits, One Lambda, Canoga Park, CA, USA) were analyzed. The PRA test was performed simultaneously from 403 patients, and 159 patients who were negative for HLA antibodies in the all PRA tests. Fifty-seven patients were positive for CDC test results and 37 patients were both positive in T cells and B cells. In 20 patients, only B cells CDC results were positive, and 13 patients were negative for FCXM test results. Results The final interpretation of FCXM result was decided through CDC, PRA result and existence of donor specific antibody (DSA). T cell FCXM result was interpreted as negative in 451 patients and positive in 67 patients according to DSA. The appropriate cut-off value of the Mean fluorescent intensity (MFI) ratio using the ROC curve for 518 patients was 1.86 (sensitivity 100%, specificity 98.2%, Youden’s index 0.982). However, patients who underwent desensitization treatment due to DSA or ABO blood type incompatibility were included. The cut-off value obtained using the ROC curve for 446 patients who have not experienced desensitization treatment was 2.45 (sensitivity 100%, specificity 99.3%, Youden’s index 0.993). Generally used, Median + 2 SD value of MFI ratio of patients judged as negative FCXM was 1.60. Conclusions Based on the statistical method used, various cut-off values could be obtained. It is important to determine the clinically significant cut-off value based on the data accumulated in each laboratory, not the simple median value from negative patients.

P060 Characterization of 108 genomic DNA reference materials for 11 HLA LOCI: A GeT-RM collaborative project

Improve availability of publicly available, renewable, and well-characterized genomic DNA reference materials to support molecular HLA typing assay development, validation and verification, quality control, and proficiency testing. The Centers for Disease Control and Prevention’s (CDC) Genetic Testing Reference Materials Coordination Program (GeT-RM) together with three clinical laboratories and the Coriell Cell Repositories characterized genomic DNA from 108 cell lines for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPA1, and DPB1. DNA was isolated by the Coriell Cell Repositories and distributed to the 3 laboratories. Methodologies used were: PCR-SSO-LABType® SSO (One Lambda, Canoga Park, CA), SSP – Olerup SSP® (Stockholm, Sweden), SBT-AlleleSEQR® HLA-SBT (Abbott Molecular, Des Plaines, IL) and NGS - HoloType HLA™ kits (Omixon Inc., Budapest, Hungary). Each of the 11 genes was tested by 2 or 3 methods. Consensus genotype was the NGS result, which provided the highest level of resolution, and was consistent with the results obtained by the parallel testing using other methods. HLA typing results using SBT, SSP, SSO and NGS were 99.95 % concordant. Of the 1905 alleles typed, only one discrepancy was observed. The discrepant result was found to be due to an error in SBT/SSO typing. Five unique novel alleles with differences in exonic regions were identified and three were submitted to the WHO Nomenclature Committee. Ambiguities persisted in 50/1905 allele calls, most due to alternative cis/trans combinations of exons 2 and 3 of DPB1 (n = 21 pairs: 42 alleles) and 8 caused by polymorphisms in a non-covered region (exon1) of the DRB1and DPB1 genes. Thirty HLA-A, 54-B, 30-C, 36-DRB1, 4-DRB3, 4-DRB4, 3-DRB5, 19-DQA1, 17-DQB1, 10-DPA1 and 29-DPB1 alleles are represented in the panel. These alleles cover a high percentage of HLA specificities present in five ancestry groups in the USA population. These genomic DNA samples are publicly available from the National Institutes of General Medical Science (NIGMS) Repository at the Coriell Cell Repositories. M. Bettinotti: 3. Speaker’s Bureau; Company/Organization; One Lambda Thermo Fisher. D. Ferriola: 7. Other (Identify); Company/Organization; Licensing proceeds of the HLA-NGS protocol by CHOP to Omixon. J.L. Duke: 7. Other (Identify); Company/Organization; receiving proceeds from the licensing of the HLA-NGS protocol by CHOP to Omixon. D. Monos: 4. Scientific/Medical Advisor; Company/Organization; Omixon. 7. Other (Identify); Company/Organization; receiving proceeds from the licensing of the HLA-NGS protocol by CHOP to Omixon.

P138 Clinical characteristics of patients who manifest unexplained crossmatch results for solid organ transplantation

Aim The flow cytometric crossmatch (FCXM) and the virtual crossmatch (VCXM) assess compatibility between solid organ donors and recipients. Theoretically, both methods should give the same results. Realistically, however, they can be discrepant due to limitations of assays used to detect alloantibodies, non-HLA alloantibodies, autoantibodies, and non-specific reactivity. We sought to identify clinical features of patients who manifest such discrepancies, hypothesizing an association with female gender, autoimmune disease, and potential heart recipients. Methods We retrospectively evaluated all living and deceased donor FCXMs (performed using BD FacsCanto flow cytometer, BD Biosciences, San Jose, CA) for solid organ transplant offers for patients at University of California San Diego from 2015 to 2017. Based on previous validation studies, a positive FCXM was coded as ”expected” when a single donor-specific HLA antibody (detected using LABScreen single antigen assay, One Lambda, Canoga Park, CA) demonstrated a mean fluorescent intensity of 3,000 or higher, or when the sum of multiple antibodies reached that threshold. Deceased donor typing was performed using LinkSeq real-time polymerase chain reaction kit (Linkage Biosciences, South San Francisco, CA). Living donor HLA genotyping was performed by SSO or NGS-based methods. FCXMs with unexpected positive results were investigated. Results 1594 FCXMs (1369 for kidney, 139 for heart, 47 for lung, and 39 for multiple organs) for 967 potential recipients (621 males, 346 females) were analyzed. Unexpected positive results occurred in 109 T cell FCXMs in 81 patients (8%) and 40 B cell FCXMs in 23 patients (2%). Analysis by both crossmatch and patient demonstrated correlations with female gender (T cell p Conclusions Unexpected positive FCXMs correlate with female gender, but not with transplant organ. In 5 cases with persistent unexpected positive FCXMs, we have successfully transplanted patients using the VCXM.

OR52 Ides desensitization therapy: Breaking down antibody barriers to transplantation

Despite increased transplant rates for highly sensitized candidates, inequities for those with CPRA >99.7% remain. This case highlights the efficacy of an endopeptidase (IdeS) to cleave IgG HLA-specific antibodies and complications for donor-specific HLA antibody (DSA) monitoring in the presence of therapeutic agents. DSA was evaluated using flow cytometric crossmatch tests (Flow-XM) and Luminex immunoassays (Lifecodes classes I and II single antigen; Immucor-Lifecodes, Stamford, CT and Single Antigen Beads; One Lambda, Canoga Park, CA). Sera were pretreated using Melon Columns™. A 43 year old male with a CPRA of 99.99% was entered into a clinical trial investigating the efficacy of IdeS to cleave IgG molecules and render them ineffective in eliciting complement activation or antibody-dependent cellular cytotoxicity. A deceased donor was identified to whom the patient had HLA-DP DSA yielding a positive prospective Flow-XM. Within 2 h of IdeS administration, immunoassays and a Flow-XM revealed no detectable DSA. Due to concern for DSA rebound, alternate day DSA monitoring and intense post-transplant immunosuppression were employed. Two single antigen panels confirmed DSA reappearance on POD7 prior to high dose immunoglobulin (IVIG) dosing. Administration of IVIG produced an increase in the negative and positive control beads and reactivity with self HLA-antigens (3400 mean fluorescent intensity, MFI). Utilization of multiple bead assays, Flow-XM tests, and HLA pattern analysis rather than MFI was needed to monitor further increases in DSA that would prompt therapeutic plasmapheresis (TPE). Cumulative DSA test analysis revealed a stabilization of DSA accompanied by declining serum creatinine values with no initiation of TPE. New therapeutics hold promise for removing DSA barriers; however, post transplant monitoring can be challenging due to test interference that accompany these biologics. This case illustrates the use of multiple assays and HLA pattern analysis to guide post transplant care.Download : Download high-res image (270KB)Download : Download full-size image A.M. Jackson: 3. Speaker’s Bureau; Company/Organization; Thermo Fisher Scientific.

Class II Human Leukocyte Antigen Epitope Mismatch Predicts De Novo Donor-Specific Antibody Formation After Liver Transplantation.

Formation of de novo donor-specific antibodies (dn-DSAs) has been associated with longterm immunologic complications after liver transplantation (LT). We hypothesized that human leukocyte antigen (HLA) epitope/eplet mismatch (MM) is a marker of immunogenicity and a risk factor for dn-DSA formation. Sera from 80 LT recipients were prospectively screened for dn-DSA by a Luminex single-antigen test (One Lambda, Inc., Canoga Park, CA) at 1, 2, 3, 6, 12, 18, 24, and 36 months after LT. HLA typing of the recipients and donors was performed using polymerase chain reaction (PCR)-SSP and PCR-SSOP Luminex low-resolution methods (One Lambda, Inc.). The HLAMatchmaker computer algorithm was used for identification of MM eplets at HLA-DRB1 and -DQA1/B1 loci. Luminex single-antigen bead solid phase assay was used for antibody analysis. Standard immunosuppression included thymoglobulin-rituximab induction and tacrolimus maintenance. There were 27 (34%) patients who developed dn-DSA. There were no episodes of antibody-mediated rejection, and 9 (11%) developed acute cellular rejection (ACR). A positive crossmatch status and a higher number of HLA-A, -B, -DR, and -ABDR MMs were not associated with dn-DSA formation. Patients developing dn-DSA had a significantly higher number of total (38 ± 2.7 versus 28 ± 2.3; P = 0.01) and antibody-verified (AbVer; 14 ± 1.1 versus 10 ± 1; P = 0.015) class II MM eplets. By a multivariate regression analysis, the number of class II MM eplets was strongly associated with risk of class II dn-DSA formation (odds ratio [OR], 1.2; P < 0.01). Patients with ACR had a significantly higher number of total (20.2 ± 1.3 versus 13.9 ± 0.9; P < 0.01) as well as AbVer (10.7 ± 1.1 versus 7.5 ± 0.6; P = 0.03) class I MM eplets. In conclusion, donor-recipient HLA epitope MM is associated with a risk of dn-DSA formation and rejection after LT. However, further studies are required to evaluate the clinical utility of epitope matching in LT.

Incidence of De-Novo Donor Specific Anti-Human Leucocyte Antigen (HLA) Antibodies (DSA) in Simultaneous Pancreas Kidney (SPK) Transplant Patients

Introduction De-novo Donor Specific Human Leucocyte Antigen (HLA) Antibodies (DSA) are detrimental to organ transplants. Few studies have explored the incidence of DSA’s and subsequent outcomes after SPK transplant, and none have compared different immunosuppression regimes. The aim of this study was to compare two different immunosuppression regimes (Alemtuzumab versus Basiliximab) with regard to the development of DSA and their long-term outcomes. Materials and Methods We introduced Alemtuzumab for all our SPK recipients from March 2008 onwards along with a Tacrolimus and MMF (from day 7) for maintenance. Prior to this, we used Basiliximab along with our standard immunosuppression regime of a CNI, MMF and steroids. We performed a retrospective analysis of all our SPK transplant patients between 2003 – June 2016. DSA were measured as early (within 2 years) and late (>2 years post-transplantation). HLA antibody testing was performed as per clinical need using a Luminex 200 flow cytometer (Luminex, Inc., Austin, TX). Samples were initially screened for the presence or absence of HLA antibodies using Labscreen mixed HLA antibody screening kits (One Lambda Inc., Canoga Park, CA, USA). For all positive and reactive results, HLA antibody specificities were determined using a combination of Lifecodes ID (Immucor USA) and Labscreen single antigen kits (One Lambda Inc.). Data was analysed using Microsoft Excel 2011 and SPSS 23. Chi-square test was used to compare the groups. Results and Discussion A total of 83 SPK transplants were performed Alemtuzumab (n=53) and Basiliximab (n=30). For early DSA, 20 patients were tested in the Basiliximab group; none developed DSA. In contrast, 34% patients (14 out of 41 tested) in the Alemtuzumab group developed early DSA (p=0.009). Of those 14 patients, 3 (21%) lost their kidney grafts, 6 (43%) lost their pancreas and 3 (21%) eventually died. For late DSA, 18 patients were tested in the Basiliximab group and 3 (17%) were found to be positive. 2/3 patients suffered pancreas graft loss, 1 lost the kidney graft and 1 died as a complication of post-transplant lymphoproliferative disorder. In the Alemtuzumab group, 12 (41%) out of 29 tested developed DSA (p=0.077). Out of those 12 patients, 4 (33%) kidneys and 5 (42%) pancreas were lost and 3 (25%) deaths were recorded. 11 patients (92%) out of the 12 who were positive for late DSA, had early DSA as well. Conclusion Patients on an Alemtuzumab based regime had a significantly higher incidence of early DSA in comparison to a Basiliximab based regime in those patients that were tested. A higher proportion continued to develop late DSA in the Alemtuzumab group. The presence of DSA was associated with very high rates of both pancreas and kidney graft loss.

369 Therapeutic Plasma Exchange in Small-Bowel Transplant Recipients With Acute Allograft Rejection

Therapeutic plasma exchange (TPE) is used in solid-organ transplant patients to treat antibody-mediated rejection (AMR) by removing donor specific antibodies (DSAs). While ASFA guidelines exist for treating AMR in other solid-organ transplants, no such guidelines exist for small bowel-allograft patients; the benefit of TPE in AMR of small bowel transplant patients has not been studied. Five patients were followed with serial surveillance ileoscopies and allograft biopsies. Tissues were processed using standard protocols (UNMC) for permanent histologic examination. Two H&E stained slides from each biopsy were examined microscopically by gastrointestinal/transplant pathologists for evidence of ACR. To assess for AMR, serial serum DSA levels were obtained via by Luminex LabScreen single antigen bead assay (One Lambda, Canoga Park, CA). Patients were treated with at least five TPE procedures. To determine whether TPE provided benefit, we examined three parameters in the post-apheresis period: DSA presence and strength, histopathologic rejection, and clinical response. All patients developed class I and/or class II DSAs following transplant. No clear change in DSA levels following TPE was seen. Sixty percent of patients (3/5) had histopathologic evidence of acute cellular rejection (ACR) prior to initiation of TPE. Following TPE, biopsies showed no evidence of ACR or did not meet the criteria necessary for evaluation. Management of rejection in small bowel transplant patients, caused by both antibody-mediated and T-cell driven processes, is extremely complex. We examined the impact of performing TPE in patients with histologic and/or clinical evidence of acute rejection. A clear trend in the burden or activity of DSAs, histopathologic improvement of allograft biopsies, or clinical improvement following administration of TPE, was not seen. Thus, in our study, TPE does not appear to have a clear benefit in the treatment of AMR and/or ACR in small bowel rejection. Additional studies will be needed to corroborate our results.

P076 Alloantibody desensitization outcomes: PRA levels, CPRA or antibody titers; how to use them best

Aim Desensitization protocols that include IVIG, plasmapheresis have been utilized by multiple groups with different levels of success. More recently, Rituximab and Bortezomib have been studied as part of desensitization therapies, but the data to support the utility and safety of these agents to reduce PRA is still debatable. This retrospective IRB approved study, looked at patients listed at Tampa General Hospital from 10/2011 to 10/2014. These patients were categorized into 3 different groups: non sensitized, sensitized non treated and sensitized treated. Sensitization was defined as a Percent Reactive Antibody (PRA) > 10%. The aim of this study was to evaluate the effects of PRA and HLA antibody specificity and titers, to best evaluate the efficacy of treatment. Methods % Class I and II PRA was measured by Flow PRA beads and antibody specificity/titers, using Luminex SAB (One Lambda, Canoga Park, CA). Results From 10/2011 to 10/2014, 164 heart transplant patients were listed for transplantation. Fifteen of the 58 sensitized patients (25.9%) underwent desensitization therapy. Patients on desensitization, all received plasmapheresis and IVIG, with a median of 3 monthly cycles. Eleven of the 15 patients (73.3%) also received Rituximab therapy. Two of the 15 (13.3%) received Bortexomib therapy after at least 2 cycles of plasmapheresis, IVIG and Rituximab with no significant decrease in HLA antibody by any of the methods tested. Twelve out of the 15 patients (80%) were transplanted. Eight out of 15 (53%) of patients showed no significant change in antibody reduction if %PRA or cPRA were utilized as end points. However, 12 out of 15 (80%) patients that proceeded to transplant after desensitization had a significant decrease in antibody titers. Conclusions When reporting the effects of desensitization therapy, the use of %PRA and/or cPRA can be deceiving and can possibly result in the misinterpretation of the efficacy of protocols among different transplant centers. The utilization of titers was the most reliable way to proceed to transplant. Patients that were transplanted after the desensitization protocol (80%) have shown no significant difference from non- sensitized patients in antibody mediated rejection (p 0.05) and have not developed significant DSA after transplant.

P224 Diversity of alleles and haplotypes of HLA class I and class II in Saudi population

Aim Human Leukocyte Antigens (HLA) play an important role in host immune responses to infectious pathogens, bone marrow and organ transplantation, cancer and autoimmune diseases. HLA association studies have shown that homozygosity was associated with worse clinical outcomes of transfusion associated-graft versus host disease (TA-GvHD). Here we conducted a retrospective study of HLA class I and class II genes homozygosity at allelic and haplotype levels in patients and healthy donors genotyped from 2012 to 2016 in a tertiary hospital at the capital of Saudi Arabia. Methods About 2773 cases were retrospectively analyzed for HLA-A, -B, -C -DRB1 and DQB1 homozygosity at allelic and haplotype levels. HLA molecular typing was performed using a commercial Sequence-Specific oligonucleotides (SSO) kit and following the manufacturer instructions (rSSO; One Lambda, Canoga Park, CA). Statistical analysis was done with Fisher’s exact test (two-tailed). The analysis was performed on Stata package. Results In contrast to other populations, we were able to identify only 46 HLA class I and 18 class II alleles demonstrating a very low genetic diversity in Saudi population. A total of 15 HLA-A, 20 HLA-B, 11 HLA-C, 13 HLA-DRB1 and 5 HLA-DQB1 alleles have been identified. In class I, the highest homozygosity is found in locus C followed by A and B (21.3% > 17.1% > 15.5%; p 20.3%; p Conclusions We found low genetic diversity of both class I and II alleles and haplotype in Saudi population which would have a significant impact in shaping the transplantation practices, transfusion associated graft versus host disease practices and host-pathogen interactions in the population.

P275 Using positive control bead MFI in HLA antibody identification as a quality measure

Aim This study is focused to determine the practicality of using the internal positive control bead as a qualitative measure in One Lambda’s Single Antigen Bead assay. Methods In the present study, forty samples (n = 40) were tested using One Lambda (Canoga Park, CA) Single Antigen Bead (SAB) Assay for HLA Class I and fifty-one samples (n = 51) were tested for HLA Class II antibody specificities using manufacturer’s suggested protocol. To treat serum with DTT, 5μL of 50 mM DTT was added to 195 μL of patient serum and incubated at 37 °C for 15 min. Results Forty samples were assayed to determine the identification of anti-HLA class I antibodies and 52 samples were tested to determine the identification of anti-HLA class II antibodies. For this study, 1000 mean fluorescence intensity (MFI) was considered positive. Data listed as in Table. Of the + to = beads, half were borderline, demonstrating low reactivity in an untreated sample but being 200–300 MFI from the positive threshold following treatment. A similar proportion followed the opposite path. Of the = to + group, half demonstrated borderline reactivity, just under the positive threshold. Following DTT treatment, the average MFI rose by only a few hundred (200–300) MFI. Conclusions Dithiothreitol treatment is a commonly used reducing reagent used to denature disulphide bonds in IgM molecules to remove background or prevent steric hindrance. This treatment, in regards to SAB testing, is performed to increase the positive control MFI. DTT is efficient at cleaving exposed disulfide bonds, thus eliminating IgM-based interference. DTT is also able to obviate C1 interference but does so poorly. EDTA prevents complement interference far more efficiently through Ca2+ chelation. Based on our results, we question the utility of using DTT to increase the positive control bead MFI. Due to lot-to-lot variability in the density of the IgG molecule on the surface of the positive control bead, One Lambda’s published positive control bead cutoff is sufficient to determine if the assay is valid. Download : Download high-res image (47KB) Download : Download full-size image

P151 Successful haploidentical hematopoietic stem cell transplantation of a patient with pre-existing donor specific anti-HLA antibodies

Aim Pre-existing donor specific anti-HLA antibodies (DSA) are implicated in graft rejection in HLA mismatched and haploidentical HSCT. We report a case of severe aplastic anemia whounderwent allogeneic HSCT from HLA matched sibling but developed primary graft failure. Due to the lack of matched unrelated donor (MUD), the patient was offered haploidentical HSCT from a sibling with the lowest number and strength of DSA. Methods Patient sera were tested using LABScreen PRA and LABScreen single antigen (SA) beads conventional IgG and C1q (One Lambda Thermo Fisher, Canoga Park CA). Flow cytometry crossmatch (FCXM) was performed with pronase treated T and B cell. FCXM was considered positive when the T cell and B cell were more than 38 and more than 50 MCS respectively. Desensitization was performed using 4 plasma exchange (1.5 volume) every other day followed by IVIG 1 g/kg on day −9 and Rituximab 375 mg/m 2 on day-8 followed by ATG/ fludarabine/TBI conditioning and post-transplant cyclophosphamide. Patient sera were tested again to determine the successfulness of the desensitization process. Results Initial anti-HLA antibody testing showed the patient was sensitized to HLA class I and II with three DSA. Anti-DRB1∗04:02 (8265 MFI) DSA, anti-DQB1∗03:02 (5419 MFI) DSA and anti-DR53 (566 MFI) DSA all of which were C1q negative. FCXM was positive for T- cell IgG (+75 MCS, most likely due to auto-antibodies) and positive B-cell IgG (+409 MCS). Post desensitization protocol, the DSA were as follows Anti-DRB1∗04:02 (3154 MFI) DSA, anti-DQB1∗03:02 (2141 MFI) DSA and anti-DR53 (1001 MFI) DSA. Patient had neutrophil and platelets engraftment on day 13 and 16 respectively. Currently, the patient is 60 days post transplant, alive with no graft versus host disease or other complications. Conclusions Despite the partial reduction of the DSA strength, the patient engrafted successfully, the question remains, was it successful because the antibodies were non complement fixing antibodies? The clinical significance of non complement DSA in HSCT waits for further studies.

Meaning of HLA Antibodies Frequently Detected by Single Antigen Bead Assay

Introduction: Single Antigen Bead (SAB) assay to detect HLA antibodies is the highly sensitive and specific technique. Nowadays, almost patients awaiting a transplantation performed SAB assay to determine unacceptable HLA antigens, and waiting time for transplantation from deceased donors was considered. Because of inherent limitation of SAB kits, which use recombinant technology, false reactivity to denatured antigens could happen. False reactivity affects donor selection and waiting transplantation time. We investigated SAB data to examine false reactive results. Methods: We tested 155 pre-transplanted and 45 post-transplanted sera for anti-HLA antibodies using panel kit and SAB kit (LABScreen Luminex kits, One Lambda, Canoga Park, CA, USA). Negative Control Beads (NC) value was below 500 mean fluorescent intensity (MFI) and Positive Control Beads (PC) value was over 5000 MFI in all results. Results: In panel kit analysis, 27% of patients were positive for HLA antibodies. Sixty-four percent of patients were positive at least one HLA antibodies on SAB assay even negative in panel kit analysis. MFI of detected HLA antibodies, which were from sera for negative in panel kit and positive in SAB kit, was under 3000 in 70.9% and between 3000 and 10000 in 25.6% in Class I SAB assay, and under 3000 in 80.7% and between 3000 and 10000 in 17.1% in Class II SAB assay. Comparison with allele frequencies from our laboratory HLA typing (n = 785), three-times high frequent HLA antibodies were HLA-A*11:02 (6%), A*80:01 (4.5%), B*15:12 (10.5%), C*17:01 (18.5%), C*18:02 (4.5%), DRB1*04:04 (6.5%), DRB1*16:01 (6.5%), DRB1*16:02 (5.0%), DQ2 (A1*05:01/B*02:01; 15.5%), DQ7 (A1*05:03/B1*03:01; 6.5%, A1*05:05/B1*03:01; 7.5%, A1*06:01/B1*03:01; 4.5%), DQ8 (A1*03:02/B1*03:02; 7.5%), DQ9 (A1*03:02/B1*03:03; 6.5%), DP1 (A1*01:03/B1*01:01; 8.5%, A1*02:01/B1*01:01; 5.0%), and DP5 (A1*02:02/B1*05:01; 7.5%). Conclusion: HLA antibody with higher detection frequency than allele frequency of population was considered false reactive antibody, because a possibility of exposure would be very low. Most of false reactive HLA antibodies showed under 3000 MFI. Erroneous interpretation about HLA antibodies for patients awaiting transplantation impact on donor selection and waiting time calculation. Studying for frequently seen false reactive HLA antibody could help relevant SAB results.

P156 An unexpected discovery complicates an otherwise uneventful HLA identical hemapoetic stem cell transplant

Oncology patients frequently require platelet transfusions during chemotherapy regimens. This case involves a 46 year old woman diagnosed with myelodysplastic syndrome, who developed HLA antibodies following platelet transfusions. The patient was then evaluated for a Hematopoietic Stem Cell Transplant (HSCT) with her sister. HLA typing was performed using rSSO, (One Lambda, Inc., Canoga Park, CA) and SBT with BigDye V1.1 Terminator reagents on a 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA). HLA-specific antibody was assessed using phenotypes, LMID, (Lifecodes®, Immucor, Norcross, GA) and single antigen panels (One Lambda Inc., Canoga Park, CA). Flow cytometric crossmatch tests were performed on a BD FACSCanto II. Intermediate typing failed to identify the patient’s A-Locus antigen using peripheral blood and buccal samples. SBT typing revealed a novel HLA-A∗02 allele that differed from the HLA-A∗02:01 present in her otherwise HLA identical sibling. Sequence alignments showed an apparent HLA-A∗02:01/A∗03:XX gene conversion event involving exon 3. HLA antibody assessments of the patient’s serum showed low level sensitization toward HLA-A2, A9, A28, A25, A26 and A66. A negative flow cytometric crossmatch confirmed the low strength of the HLA-A2 antibody. The urgency for transplant and low likelihood of finding a donor for which the patient was not sensitized (CPRA = 77) precipitated her placement into our desensitization HSCT protocol. A non-myeloablative, HLA-A mismatched HSCT with her sibling occurred following desensitization using plasmapheresis and low dose IvIg. Identification of HLA antibodies is crucial for HSCT programs that support HLA mismatched transplantation. New or rare alleles can pose additional challenges in HSCT with regards to matching with potential donors and avoiding sensitization to common HLA antigens found in blood products.

P018 Evaluation of labtype CWD and XR assays utilizing labscan3d for high throughput/resolution HLA typing

Aim High resolution HLA typing with clinical applications is preferred in some applications of transfusion and transplantation medicine. Concordance of results was determined between new reverse sequence specific oligonucleotide assays (rSSO; LABType CWD and LABType XR, One Lambda, Canoga Park, CA) and our current assays, rSSO (LABType SSO) and sequence-based typing (SBT; SeCore). Methods Forty-eight patient samples previously tested by our current rSSO and SBT assays were selected randomly. The 2 new assays and a new multiplex flow analyzer (LABScan3D TM ) were utilized to detect HLA-A common and well-defined alleles (LABType CWD) and HLA-B and DRB1 alleles at high resolution (LABType XR). DNA was amplified with group specific primers targeting exons 2, 3, 4, and 5 for HLA-A and HLA-B, and exon 2 for HLA-DRB1. Results We determined the concordance between the 2 new and our current assays (Table 1). The LABType CWD identified 6 out of 96 HLA-A alleles (6.25%) at the allele level (4-digit resolution) and was also able to resolve 1 ambiguous allele combination, exceeding the resolution obtained by our current LABType SSO. The LABType XR assay provided results with shorter allele strings in 59 out of 96 HLA-B alleles (61.5%) and 22 out of 96 HLA-DRB1 alleles (22.9%), respectively, exceeding the resolution obtained by our current SeCore SBT without reflux testing. Discordances were observed resulting from false bead reactions or ambiguous allele combinations. Conclusions The new LABType CWD and XR assays are suitable platforms for high throughput testing. Utilization of the LABType XR kits potentially could yield better results than SBT thus making them a consideration for clinical applications requiring quicker turn around time. However, even with LABType XR, additional testing may still be necessary to resolve any remaining discrepancies and ambiguities. Download high-res image (157KB) Download full-size image

P074 Donor specific anti-HLA antibodies are associated with subclinical allograft injury in heart transplant recipients

Aim Anti-HLA antibodies detected after heart transplantation have been associated with graft rejection and worse survival. However, the presence of donor specific antibodies (DSA) alone is not diagnostic of antibody mediated rejection (AMR). To better define the significance of circulating DSA, we investigated the relationship between DSA and relevant histo- and immunopathologic features of endomyocardial biopsies. Methods Sixty-three patients received heart transplantation between Jan 1st 2011 and Dec 31st 2013 and were followed at our center. Serum anti-HLA antibodies were monitored using the Single Antigen Bead assay (One Lambda, Canoga Park, CA). Endomyocardial biopsies were performed serially during the first year post-transplant and yearly thereafter, according to the existing clinical protocol. Results At a median followup time of 33 months, graft/patient survival was 98%. Sixteen (25%) out of 63 patients had at least 2 DSA positive tests after transplantation. DSA were predominantly directed to HLA class II antigens, with 13 out of 16 patients displaying anti-HLA-DQ DSA. A total of 906 endomyocardial biopsies were performed after transplantation. Out of 906, 305 biopsies had concomitant DSA results and were included in the analysis. Fifty-seven (19%) biopsies had positive DSA associated results. ACR was diagnosed in 60 (20%) out of 305 biopsies. There was no significant correlation between ACR and serum DSA. AMR was diagnosed in one (0.3%) biopsy, which had a concomitant positive DSA. The following histo- and immunopathologic parameters were analyzed in relation to DSA: perivascular inflammation, interstitial inflammation, myocyte injury, lymphocytic infiltration, interstitial edema, interstitial hemorrhage, fibrosis, microvascular changes and C4d staining. Microvascular changes included prominent endothelial cells and intravascular mononuclear cells and/or neutrophils. DSA were associated with microvascular changes (N = 9; p Conclusion Our results provide evidence that circulating DSA are associated with subclinical graft injury. Post-transplant DSA monitoring may serve as a useful, non-invasive tool to identify patients at risk for early graft loss.

P118 Association between the frequency of human Leukocyte antigen class I and class II genes in Saudi population and relative risk of developing nasopharyngeal carcinoma

Aim Genetic susceptibility and EBV infection have been reported as co-factors responsible for the risk of developing NPC. The incidence of NPC has a marked geographic variation, though rare in the west; it is high in countries such as Taiwan and China. While Human Leukocyte Antigen (HLA) have been associated with NPC predisposition in Asians, yet there have been no studies on HLA association to NPC in Saudis. Methods HLA-A, -B, -C and -DRB1 were examined for their association to NPC in case-control study of Saudi patients compared to healthy Saudi attending a tertiary hospital (n = 45 cases and n = 107 controls). HLA typing was molecularly performed using a commercial Sequence-Specific oligonucleotides (SSO) kit and following the manufacturer instructions (rSSO; One Lambda, Canoga Park, CA). Statistical analysis was done with Fisher’s exact test (two-tailed). The analysis was performed on Stata package. The genetic model was allele positivity corresponding to the dominant model. Results The results clearly demonstrated that while NPC protection was associated with HLA-DRB1∗11 allele (OR = 0.1412; p = 0.043), most NPC susceptibility was strictly associated with HLA-B∗08 (OR = 3.629; p = 0.017) and HLA-DRB1∗07(OR = 2.296; p = 0.041) alleles. Surprisingly and in contrast to the Taiwanese and Chinese studies, we could not find any significant association with any of the HLA-A or HLA-C alleles. Conclusions Our study show for the first time, that HLA-DRB1∗11 allele have protective association with NPC and HLA-B∗08 and HLA-DRB1∗07 have susceptibility association with NPC.

P006 Assessing the functional significance of HLA antibodies in renal allograft

Objective To assess potential function of the anti-HLA antibodies in terms of complement binding and non-complement binding in highly sensitized patients and to correlate the functional properties with in-vitro cytotoxicity assays and biopsy. Methods The Class I and II HLA antibody profiles of 78 patients were analyzed to determine antibody strength and specificity and to determine if any of the identified antibodies are complement binding. In vivo correlation was done in 37 patients who had a biopsy and donor-specific antibody (DSA) assessment. The HLA class I and II antibody specificities were determined by single antigen bead assay (SAB) and the C1q binding assay (C1q-B) using Luminex platform (One Lambda, Canoga Park CA). In vitro cytotoxicity assays were done by T-AHG and Amos CDC. Results Of the 2941 antibodies identified by SAB in 78 patients, 1940 (66%) were Class I and 1001 (34%) were Class II specific. In all, 758 (39%) of the Class I antibodies with ⩾ 5000 MFI and 484 (48%) of the Class II antibodies were with an MFI of ⩾ 5000. Of the total 2941 HLA antibodies, 238 (8%) were C1q-B. Of the C1q-B, 77 (32%) were directed against Class I HLA and 161 (68%) were directed against Class II. Importantly, 92% of all C1q Class I antibodies identified were defined as strong among the total 2941 HLA specific antibodies. Biopsy results were available from 37 patients and 15 of them had no C1q Class I or Class II DSAs and no AMR while 6 (16%) patients were C1q Class I and Class II positive DSA and AMR. Fourteen percent (n = 5) of the total patients were C1q Class I negative and C1q Class II positive and per biopsy were classified as C4d negative AMR. Of the 49 patients with CDC and C1q data available 19 (39%) nine were C1q Class I and Class II DSA negative and CDC B-cell and T-cell negative while 5 (10%) were C1q Class I and Class II DSA positive and CDC B-cell positive T-cell negative. In all, 4 were C1q Class I negative and Class II positive with CDC T-cell negative and CDC B-cell positive. Conclusions This limited retrospective analysis confers that assaying for C1q binding antibodies is very likely to have clinical relevance in terms of the functional effect of the multiple HLA antibodies on the allograft. The class II antibodies and antibodies with higher MFIs are are more likely to be C1q-B.

Failure of Calcineurin Inhibitor (Tacrolimus) Weaning Randomized Trial in Long-Term Stable Kidney Transplant Recipients.

Long-term renal transplant outcome is limited by side effects of immunosuppressive drugs, particularly calcineurin inhibitor (CNI). We assumed that some patients selected for a "low immunological risk of rejection" could be eligible and benefit from a CNI weaning strategy. We designed a prospective, randomized, multicenter, double-blind placebo-controlled clinical study (Eudract: 2010-019574-33) to analyze the benefit-risk ratio of tacrolimus weaning on highly selected patients (≥4 years of transplantation, normal histology, stable graft function, no anti-HLA immunization). The primary endpoint was improvement of renal function. Fifty-two patients were scheduled in each treatment arm, placebo compared to the CNI maintenance arm. Only 10 patients were eligible and randomized. Five patients were assigned to the placebo arm and five were assigned to the tacrolimus maintenance arm. In the tacrolimus maintenance arm, all patients maintained stable graft function and no immunological events occurred. Contrastingly, in the placebo arm, all five patients had to reintroduce a full dose of tacrolimus since three of them presented an acute rejection episode (one humoral, one mixed, and one borderline) and two displayed anti-HLA antibodies without histological lesion (one donor-specific antibodies [DSA] and one non-DSA). Clearly, tacrolimus withdrawal must be avoided even in long-term highly selective stable kidney recipients.