Higher MFI Values Research Articles

The challenge of HLA donor specific antibodies in the management of pancreatic islet transplantation: an illustrative case-series

Islet transplantation is a unique paradigm in organ transplantation, since multiple donors are required to achieve complete insulin-independence. Preformed or de novo Donor Specific Antibodies (DSA) may target one or several donor islets, which adds complexity to the analysis of their impact. Adult patients with type 1 diabetes transplanted with pancreatic islets between 2005 and 2018 were included in a single-center observational study. Thirty-two recipients with available sera tested by solid-phase assays for anti-HLA antibodies during their whole follow-up were analyzed. Twenty-five recipients were islet-transplantation-alone recipients, and 7 islet-after-kidney recipients. Seven recipients presented with DSA at any time during follow-up (two with preformed DSA only, one with preformed and de novo DSA, 4 with de novo DSA only). Only islet-transplantation-alone recipients presented with de novo DSA. Three clinical trajectories were identified according to: 1/the presence of preformed DSA, 2/early de novo DSA or 3/late de novo DSA. Only late de novo DSA were associated with unfavorable outcomes, depicted by a decrease of the β-score. Islet transplantation with preformed DSA, even with high MFI values, is associated with favorable outcomes in our experience. On the contrary, de novo DSA, and especially late de novo DSA, may be associated with allograft loss.

207.5: Impact of HLA Donor Specific Antibodies in the Era of Cell Therapy: Clinical Trajectories in Islet Allotransplantation Illustrate a Challenging Model

Introduction: Islet transplantation is a unique paradigm in the case of organ transplantation, since multiple injections and donors are required to achieve complete insulin-independence. Preformed or de novo DSAs may target one or several donor islets, which complexifies the analysis of their impact. Our objective is to discriminate illustrative clinical trajectories which can occur after transplantation. Methods: Adult type 1 diabetic pancreatic islet recipients transplanted between January 2005 and December 2018, were included in a monocentric observational study. Only patients with available sera tested by Luminex for Class I and II anti-HLA antibodies during their whole follow-up were analyzed. Results: 32 patients had a complete HLA antibodies screening during follow-up. Among them, 7 patients presented with DSA. Five clinical trajectories were identified according to the MFI-value, and the presence of preformed DSA, de novo DSA, or both. To simplify, we provide specific clinical-based trajectories considering an islet transplantation with 1- high MFI preformed DSA, 2- high MFI de novo DSA, 3- low-MFI preformed DSA, 4-low de novo DSA, and 5- a de novo DSA which becomes a preformed DSA because of multiple islet infusions. We analyzed also the specific case of islet-after-kidney transplantation. Recipients transplanted with preformed DSA had all favorable islet transplantation outcomes. The prognosis of de novo DSA was associated with unfavorable outcomes when following markers of unsteady glycemic control. Conclusion: Clinical-based trajectories and evidence from the literature reveal that islet transplantation with preformed DSA, even with high MFI values, can be associated with favorable outcomes. The fate of recipient presenting with de novo DSA is more questionable as both clinical data and preclinical studies show separate outcomes. Clinical phenotype and unsteady glycemic control before the emergence of DSA may lead the clinician to detect rejection.I-SITE ULNE.

P129 Flexmap 3d® generates a comparable result for lifecodes® LSA single antigen products with respect to luminex® 200 platform

Aim The objective was to demonstrate equivalency of qualitative results among the LIFECODES LSA Single Antigen products performed on two different Luminex Instrument Platforms: The Luminex 200 and the FLEXMAP 3D. Methods For each LIFECODES LSA Single Antigen Assay (LSA 1 and LSA 2), a panel of 32 samples was selected. Samples were chosen based on previously characterized reactivity to either HLA Class I or HLA Class II antigens. For the first incubation of the assay, replicate samples were pooled together (sera + beads) in sufficient volume to be subsequently divided and distributed to the designated wells to be run on the different instrument platforms. This was done to minimize any variation between the replicates that may occur during the initial incubation. For each LSA assay, samples were tested side by side on both the Luminex 200 Instrument and on the FLEXMAP 3D Instrument using the “Standard” reporter gain setting. Because the FLEXMAP 3D produces higher MFI values, an MFI divider was applied to obtain MFI equivalent to the Luminex 200. To identify the appropriate divider, a data simulation was performed by applying a variety of MFI Dividers with values in the range of 1.00 to 2.00. For each MFI Divider simulated, the sum of the MFI values for each sample was compared to the sum of the MFIs for the same sample run on the Luminex 200. A two-tailed T-test was then used to determine which simulated MFI divisor provided the most equivalent data points between the FLEXMAP 3D and LMX200 platforms. In a follow-up experiment, an assay was performed on FLEXMAP 3D by incorporating the most equivalent divider value. Results Over the evaluated MFI divider range of 1.0 to 2.00, a value of 1.67 gave the maximum p-value when compared to data generated by the Luminex 200 Instrument. A 2 × 2 analysis of antigen assignments for LSA1 & LSA2 show an overall concordance of >98% between FLEXMAP 3D (with a divider value of 1.67) and Luminex 200. Conclusions This study with 32 samples suggests that comparable results can be obtained between the Luminex 200 and FLEXMAP 3D instrument platforms when a divider value of 1.67 is applied to the MFIs generated by the FLEXMAP 3D. R. Kumar: 5. Employee; Company/Organization; Immucor. R. Clouse: 5. Employee; Company/Organization; Immucor. B.L. Ray: 5. Employee; Company/Organization; Immucor.

P131 Description of a new antibody-defined epitope on denatured HLA class I molecules

The purpose of this report is to describe a new epitope present on denatured HLA class I molecules. Serum from a kidney transplant candidate, male, and with only one previous blood transfusion was tested with the IgG-binding assay with single-antigen beads on a Luminex platform (LSA) (One Lambda - OL). The following positive reactions were observed: HLA-B- 13, 18, 37, 41, 44, 45, 47, 48, 49, 50, 52, 60, 61, 62, 72, 75, 76, 77. This reaction pattern could be explained by the HLA-Matchmaker eplet 66IS (65Q66I67S69T) which is considered an exposed eplet ( http://www.epregistry.com.br ). The serum was tested with flow-cytometry using three different cells carrying one of these specificities: HLA-B37 (Ac with MFI of 5250), B60 (Ac with MFI of 3661) or B50 (Ac com MFI of 4752) and the results were negative. In addition, no positive reactions were observed when the serum was tested with a LSA kit from another vendor. In order to investigate whether the OL Luminex reactions were false-positive, the serum was tested with OL beads bearing HLA molecules denatured by acid treatment and the same reactions were observed, although with higher MFI values. The reaction pattern observed on the beads with denatured HLA could not be explained by any cryptic epitope described by El-Awar, 2009. In order to search for the targeted epitope on the denatured HLA molecules, we analyzed the amino acid sequences and we found that the position 67S, cryptic in the HLA molecule, was present only in the molecules with which the serum reacted. In fact, this position is included in the polymorphic residues of HLAMatchMaker eplet 66IS. This finding underlines that not all reactions that can be explained by the HLA-Matchmaker are true-positive reactions.

P184 Next generation sequencing of small bowel biopsy reveals unresolved donor alleles and eliminates further augmented immunosuppression for antibody mediated rejection

Aim A young pediatric small bowel recipient was transplanted with a donor who was typed only at low resolution by an outside center. For 6 years, the patient received multiple blood transfusions and was monitored by IgG and C1q using LabScreen SAB (One Lambda). High MFI DSA to DQ2 was treated by IVIG desensitization; however, based on the low resolution typing, DQ2 DSA remained. To determine whether this was real DSA or whether the donor had a different DQ2 heterodimer, a fresh frozen biopsy sample was obtained to try to identify the donor HLA alleles by Next Generation Sequencing (NGS). Methods Full length genomic DNA from the patient and biopsy was sequenced for 11 loci (A,B,C,DRB1/3/4/5,DQA1/B1,DPA1/B1) using TruSight HLA v2 Sequencing Panel (Illumina), and analyzed by Assign v.2.1 (Illumina). Two to 3 field resolution was obtained. Results At each locus on the biopsy DNA, the sequence showed discontinuous alignments and much higher numbers of mismatched nucleotide positions than usual due to the mixture of both patient and donor. Individual sequenced reads containing mismatched nucleotides compared with the two predominant alleles were applied to BLAST using both the NCBI and IMGT websites. Once multiple alleles of the first field were obtained, polymorphic positions among exons were compared with other mismatched positions of the sequenced reads to determine the second and third allelic fields. Among all loci, more than two alleles were observed. By eliminating the recipient HLA alleles from the output, the donor’s HLA typing became clear. Typing of the donor by NGS from a biopsy sample revealed that the donor did not have the target heterodimer that had been giving high MFI values by both IgG and C1q. The DQ2 that had been followed was, in fact, not a DSA. Since a prior weak DR53 DSA had already resolved, continued treatment for AMR (Antibody-Medicated Rejection) was not indicated and augmented immunosuppression was discontinued. Conclusions This case decisively demonstrates the power of NGS technology to identify 2–3 field allele level HLA typing from a chimeric specimen using a frozen biopsy specimen. It also highlights the power of the technology to inform clinical decision making.

P212 EDTA fully overcomes the prozone effect in luminex-based HLA antibody assays regardless of technical modifications

Aim Our laboratory has recently reported that serum treatment with EDTA is sufficient to overcome the prozone effect. However, it has been reported that in rare cases, specimens are partially resistant to EDTA treatment, and serum dilutions are still necessary. Although we have not encountered such case in our laboratory, we sought to determine if differences in the technical aspects of the assay may contribute to EDTA’s effectiveness. Methods We screened samples sent to our laboratory for HLA antibody testing for suspected prozone effect, based on MFI values of >15,000. Selected samples were tested in three protocols. Protocol #1: to determine the presence of prozone effect, serum samples were frozen, thawed, centrifuged (14,000 rpm for 10 min), and tested at neat, 1:4, 1:16, 1:64, and 1:264 by single antigen (SA) bead assays (One Lambda). Protocol #2: to test the clinically-validated method used by our lab, samples were frozen, thawed, centrifuged, treated with EDTA (4 mM), and tested at neat only; incubation and washing was performed in a 1 μM filter plate on a vacuum plate-washing system (Biotek ELx50). Protocol #3: to test the effect of EDTA in fresh samples with the manual washing method, fresh serum samples were centrifuged, treated with EDTA, and then tested at neat only; incubation and washing was performed in a traditional round-bottom plate. Data were acquired on a Luminex-200 and analyzed with HLAFusion software (One Lambda). Results We identified prozone effects in 18 patients’ samples: 8 patients’ class I antibodies, 8 patients’ class II antibodies, and 2 patients’ class I and II antibodies. Following EDTA treatment, fresh and frozen samples showed similar MFI values for the corresponding assignments, including 138 class I and 103 class II specificities. In addition, all determined specificities showed higher MFI values in EDTA-treated samples than the peak MFI value in sample with dilution only, regardless of which technical protocol was used. Conclusions Samples with prozone effect resistant to EDTA treatment were not detected in our study, independent of technical variability for sample processing. EDTA treatment fully overcomes prozone effect in our testing system. Factors causing low efficacy of EDTA in rare cases remain unknown.

P096 Evaluation of immucor lifecodes® C3D detection assay in pre-transplant patients

Aim DSA contribute to hyperacute, acute or chronic transplant rejection. Therefore it is of major interest to define their ‘pathogenicity’, which could help to guide therapeutic interventions. As complement fixation is a primary mechanism for the induction of inflammation and organ damage during AMR, the CDC test has been used to detect DSA but its sensitivity has often been questioned. Most laboratories are now using more sensitive IgG-Luminex assays. They can’t however not distinguish complement-fixing antibodies. C3d-Luminex detects complement bound to antibody/antigen complex and the aim of our study is to evaluate this assay. Methods 63 sera were tested with CDC and C3d-Luminex. The specificities of two EPT CDC samples have also been compared as the CDC results can vary significantly between laboratories. Sera from 39 HLA-immunized patients waiting for a renal transplant were tested with IgG-Luminex with and without EDTA treatment (EDTA) and compared with C3d-Luminex. Results The % co-positivity between C3d-Luminex class I and CDC is 100% and % co-negativity is 90%. The amount of specificities of CDC was lower than those identified by C3d-Luminex. With regard to the EPT samples, one sample was not C3d positive for a consensus CDC specificity and a second sample was also not C3d positive for a CDC specificity detected by 29 of 39 participating labs. IgG-Luminex showed higher MFI values with EDTA than without. The prozone effect also disappeared after EDTA treatment and with C3d-Luminex. C3d positivity of one of the immunized patients was variable in time, DSA initially only positive with IgG-Luminex, 3 months later also positive with C3d and weakly positive with CDC and finally all the 3 assays were positive. Our pre-transplant data suggest that the C3d-binding capacity of antibodies is correlated with a higher MFI. C3d-Luminex was always negative/positive under/above a certain IgG-Luminex cutoff. Conclusions Antibodies directed against the graft can cause injury via different mechanisms, also in the absence of complement (ADCC and/or direct activation of endothelial cells). Technique-dependent differences between antibody reactivity could be associated with antibody titer (prozone), other DSA characteristics (avidity, IgM, IgG subclass) and the expression level of HLA (CDC).

De novo DQ donor-specific antibodies are associated with worse outcomes compared to non-DQ de novo donor-specific antibodies following heart transplantation.

Antibody-mediated rejection (AMR) resulting from de novo donor-specific antibodies (dnDSA) leads to adverse outcomes following heart transplantation (HTx). It remains unclear what role dnDSA to specific HLA antigens play in adverse outcomes. This study compares outcomes in patients developing dnDSA to DQ antigens with those developing non-DQ dnDSA and those free from dnDSA. The present study was a single-center, retrospective analysis of 122 consecutive HTx recipients. The primary outcome was a composite of death or graft dysfunction. After 3.3years of follow-up, 31 (28%) patients developed dnDSA. Mean time to dnDSA was 539days. Of 31 patients, 19 developed DQ antibodies and 12 developed non-DQ antibodies. Compared to non-DQ dnDSA, DQ antibodies presented with higher MFI values (P=.001) were more likely persistent (P=.001) and appeared later post-HTx (654 vs 359days, P=.035). In a multivariable analysis, DQ dnDSA was associated with increased risk of the primary endpoint (HR 6.15, 95% CI 2.57-14.75, P=.001), whereas no increased risk was seen with non-DQ dnDSA (P=.749). dnDSA to DQ antigens following HTx are associated with increased risk of death and graft dysfunction.

Case Report: Anti-HLA-DQα Antibodies Have Clinical Impacts in Highly Immunized Kidney Patients

Context and purpose: Organ exchange programmes are important in order to ensure organs to highly immunized kidney patients. In this case report, we describe a highly immunized patient in an acceptable mismatch program who had an unexpected positive Complement-Dependent Cytotoxicity (CDC) crossmatch with a deceased donor exchange.Main findings: Subsequent evaluation revealed donor specific antibodies characterized by serological epitopes, which included the HLA-DQα chain, were masked in the standard IgG Luminex assay unless the serum was diluted; however, the antibodies demonstrated high MFI values when the serum was pretreated with EDTA or analyzed with the C1q assay. These findings led us to evaluate the extent of masked anti-HLA-DQ antibodies that included HLADQα/ DQβ pairs rather than just the HLA-DQβ chains in 16 highly immunized patients on the kidney transplantation waiting list at our transplantation center. We determined that 25% of the patients had antibodies directed towards serological epitopes that involved the anti-HLA-DQα chain but were masked in the standard IgG assay.Principal conclusions: We conclude that masked anti-HLA-DQ antibodies directed towards serological epitopes that also involves the HLA-DQα chain are common and should be considered as they may cause a positive CDC crossmatch. Thus, donor specific HLA-DQ antibodies must be evaluated in relation to full HLA-DQα; DQβ pairs in the serological molecules of the donor.

OR5 Can you predict accurate crossmatch results with HLA-DP DSA?

Aim The new kidney allocation system has increased transplant rates for highly sensitized candidates in which HLA-DP antibodies are more commonly identified. Accurate crossmatch correlations with HLA-DP donor specific antibodies (DSA) can be difficult given the low and variable expression of HLA-DP antigens and the presence of additional DSAs. Our aim was to optimize a strategy for predicting positive flow cytometric crossmatches (FCXM) in the presence of HLA-DP DSA. Methods Hypotonic dialyzed sera from 22 patients containing antibodies to HLA-DP (MFI range 1217–22,270) in the presence or absence of additional DSA were analyzed using One Lambda Single Antigen Beads. FCXMs were performed on a BD FACS Canto and a ratio >2.5 (test serum to negative control serum) was used as a positive FCXM threshold. Surface HLA expression was measured using class I and II specific monoclonal antibodies. Correlation coefficients were calculated. Results All T-FCXMs were negative. Thirteen of 23 B-FCXM were positive and 7 of 13 positive B-FCXM had HLA-DP DSA MFI >10,000 (++7; +−6; −+3; −−7; r = 0.24). Six of 13 positive B-FCXM had HLA-DP DSA MFI values 1 HLA-A; B; DR or >3 total # DSA) were detected reconciling the positive B-FCXM results. Three B-FCXM were negative despite high HLA-DP DSA levels (>10,000MFI); however, HLA class II expression on donor cells was low in 2 of 3 FCXMs. Seven B-FCXMs were negative with MFI Conclusions The breadth of HLA sensitization in patients that develop antibody to DP antigens complicates accurate crossmatch correlations when HLA-DP DSA is present(r = 0.24). High MFI values in bead based assays serve as a tool in crossmatch prediction, but the added complexity of multiple DSAs and variable expression of antigen on cells impact virtual crossmatch accuracy. There is potential for improved correlations (r = 0.60) using this multifactorial approach, but these data must be further validated using a larger dataset.

Can solid phase assays be better utilized to measure efficacy of antibody removal therapies?

Antibody removal therapies are used for patients with antibody-mediated-rejection or those requiring desensitization to become transplantable. Accurate measurement of antibody levels prior to, and during treatment, are required to choose the best therapeutic approach, and to provide measure of treatment efficacy. Currently, the FDA does not regard solid-phase assays for HLA-antibody identification as a reliable surrogate-marker for treatment efficacy.Serum samples from 40 patients (58 assays; >2200 positive data points) undergoing antibody-removal-therapies were tested as sample-pairs, pre- and post-treatment. MFI values of IgG and C1q single-antigen-bead assays were compared with antibody titer values (serial dilutions). Antibody reduction was tracked and the differences in pre-to-post-treatment values were calculated as delta-reduction of antibody levels. Dynamic patterns of titration studies reduced effects of serum-inherent inhibitory factors (prozone-like); eliminated over-saturation limitations, and provided better estimation of antibody-binding strength compared with the other methods. Moreover, delta-reduction of antibody values using titration studies was significantly more uniform compared with either IgG or C1q tests. Analyzing antibody results using only C1q positive or only higher MFI values did not change the overall magnitude of results. Overall, titration studies provided better estimate of responsiveness to treatment and thus can serve as companion to monitoring efficacy of antibody-removal therapies.

Mechanical, flow and electrical properties of thermoplastic polyurethane/fullerene composites: Effect of surface modification of fullerene

Thermoplastic polyurethane (TPU) composites with fullerene loadings varying from 0.5 to 2 weight% were prepared by melt-mixing method. Nitric acid oxidation and silanization were applied to fullerene surface to improve interfacial interactions with TPU matrix. The influence of surface modifications of fullerene on mechanical, melt flow and electrical properties of TPU based composites were investigated. Incorporation of fullerene leads to nearly twofold increase in tensile strength and Young's modulus of the composites in addition to enhancing the flexibility. The best results are obtained in nitric acid and silane modified fullerene containing composites at the lowest concentration (0.5%). Higher MFI values were observed for composites loaded with surface treated fullerenes compared to pristine fullerene because of their better dispersion in TPU. Electrical properties of TPU also improved by the addition of surface modified fullerene particles. Surface oxidation and silanization gave rise to dispersion homogeneity which may be the reason of both tensile strength and strain improvements at the same time.

Complement Binding (C1q) and MFI of DSA at Time of Initial C4d+ Only Biopsies Are Not Associated With Progression of Subclinical Antibody Mediated Rejection in Heart Transplant.

Considering complement-binding (C1q+) donor-specific HLA antibodies (DSA) was reported to improve stratification of patients at risk for graft lose in kidney transplantation. In addition MFI values generated by Luminex-based solid phase assays of HLA antibody detection are commonly used semi-quantitatively as a surrogate marker of HLA antibody strength with high MFI values associated with increased risk of rejection. In this study we investigate HLA antibody characteristics at time of initial C4d+ (Subclinical AMR) in association with conversion to concomitant C4d+/C3d+ in heart biopsies that has been correlated with circulating DSA and graft dysfunction. Of 1589 transplants, 610 were screened for AMR in the last 6 years. We included patients with stored serum specimens collected ± one day of the biopsy date. Three groups were investigated including asymptomatic patients with C4d+ biopsies till last follow up (n=29), symptomatic patients with C4d+C3d+ biopsy (n=28) and patients who started as C4d+ then converted to C4d+C3d+ (n=11). We investigated HLA antibody characteristics including Class I vs. II, breadth (cPRA), presence of DSA, MFI of the highest reacting DSA, and C1q results. We also compared pre-transplant DSA, cPRA and crossmatch results. No significant differences were noted among groups in pre-transplant mean cPRA, incidence of DSA, or positive crossmatch. In contrast, at time of initial biopsy, significant differences in incidence of DSA and mean cPRA were noted (table 1).Table: No Caption available.C4d+ converters had significantly higher incidence of DSA (more dominantly class II) and higher mean cPRA compared to patients who remained C4d+ (P 0.2 for both). Having DSA at time of initial biopsy was associated with C4d+ conversion with high negative predictive value (1.0) but low positive predictive value (0.38). Interestingly, C4d converters had lower mean MFI (p NS) compared to those who remained C4d+ only and none of the converters was associated with C1q+ DSA. Some HLA antibody characteristics such as absence of DSA and lower cPRA correlate with C4d+/C3d+ patterns and may be relevant in determining which subclinical C4d+ patients may not progress to clinical AMR however C1q and MFI were not predictive.

62-OR

Aim The presence of C1q antibodies may be important for assessing the risk of antibody mediated rejection (AMR) in solid organ transplants. We studied the correlation between the C1q-single antigen bead assay (C1q-SAB) with the long-incubation complement-dependent lymphocytotoxicity assay (CDC) and the IgG-single antigen bead assay (IgG-SAB). Methods 60 reference sera (38 Class I and 22 Class II) from the UCLA serum exchange program were characterized using the CDC, Luminex PRA (Gen-probe Inc.) and IgG-SAB (One Lambda Inc.) assays. We also tested pre- and post-transplant sera from 16 renal allograft recipients. All patients had persistent posttransplant DSA as demonstrated by the IgG-SAB assay. Results The C1q-SAB assay showed 85% concordance with the CDC assay as 15% of the CDC positive antibodies were negative in the C1q test. Correlation between the SAB MFI and C1q positivity showed that 90% of sera with the MFI of >18,000 in the IgG-SAB assay were C1q binding. A retrospective study of 16 renal transplant recipients developing post-transplant donor specific antibodies (DSA) showed 5 of 9 patients diagnosed as C4d+ AMR displayed C1q positive DSA (P = 0.145). One out of 7 patients without AMR displayed C1q binding DSA. Conclusions Our data indicate that antibody strength correlates with the capacity to bind C1q (Fig. 1). However we identified a subset of antibodies that displayed high MFI values and CDC positivity, that failed to bind C1q. The development of C1q positive DSA was not significantly associated with positive C4d staining on the graft. Additional studies are required using a larger cohort of patients to verify these findings [Fig. 1]. Download : Download full-size image

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Aim It is widely believed that complement fixing donor specific antibody is more clinically relevant than non-complement fixing antibody. However, the C1q assay, which is used to determine if there is complement fixing Donor Specific Antibody (DSA) is time consuming and expensive. We wanted to study if the Luminex Single Antigen (SA) MFI level is related to the presence of C1q fixing antibody. Methods Twelve kidney recipient sera, demonstrating a total of 29 anti-HLA DSAs by the SA assay (One Lambda, Canoga Park, CA) were tested by the C1q assay (One Lambda, Canoga Park, CA). Results Five of these sera demonstrated C1q fixing DSAs. The average MFI of the SA for these positive sera was 17,920. The average MFI of the SA for the sera not having C1q antibody specificities was 4776. There were three sera that had relatively high MFI values that were not positive by the C1q assay (MFI average 12,269) [Fig. 1]. Download full-size image Conclusions The strength of the MFI in classical Luminex SA testing has a high predictive value for positivity in the C1q assay. We found that sera with greater than 13,000 MFI by Luminex SA had a high probability of being positive by the C1q assay. Therefore, the use of the classical Luminex SA assay to determine the strength of the MFI is beneficial to determine if reflex C1q testing is warranted.

28-OR

Aim A 53 year- old female with therapy-related myelodysplasia was a candidate for unrelated double-cord transplant. She had already received an autologous transplant, followed by a haploidentical transplant from her son. Multiple transfusions were required. STR analysis indicated that her cells were now 90-100% of patient origin. Anti-HLA antibodies were characterized to avoid cord-specific antigens. Methods Solid phase HLA antibody detection technology (One Lambda) was applied to screen and identify Class I and II anti-HLA. Results The patient was highly sensitized as shown by hemolysis after the haploidentical transplant. Further testing also detected anti Class I and II HLA antibodies, with very high MFI values starting at 14000. Specific antibodies to self antigens (A3, B51, 52) were not detected, except for an anti-HLA-A∗68. Raw data from single antigen beads (One Lambda), clearly showed the presence of the A68 antibody, however, the corresponding molecular specificity was not the patient’s A∗68:01, but A∗68:02, with high MFI values of 8500 and 3800, detected on 2 different samples 5 months apart. In both cases, anti A∗68:01 was negative. Conclusions There are 5 amino acid differences between A∗68:01 and A∗68:02, affecting the CDEF pockets, which may result in distinctly different peptide binding specificities. While the A∗68:01 allele is present everywhere, the A∗68:02 allele occurs mostly in Africa. A disease association with one, but not the other allele of the A68 group has been reported, and could result in functional differences. The previous transplant donor, the haploidentical son, was negative for A∗68:02 and therefore the presence of the A68 antibody would not have influenced the transplant outcome. Our institution uses HLA-A, B antigen and DRB1 allele matching for cords. In this particular case, our recommendation was to only mismatch antigens if the patient has no cord specific anti-HLA antibodies and to avoid mismatches for HLA-A∗68 alleles.

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Aim A 66 year old female with an end-stage NYHA class IV inotrope-dependent, ischemic cardiomyopathy received a heart transplant on 9-12 from a donor having antigens B13 and DR7. Pre-transplant antibody testing indicated only weakly positive, non-complement-fixing donor-specific antibodies. The patient suffered a cardiac arrest on POD#6 and was resuscitated. She was in cardiogenic shock from allograft failure and subsequently expired on POD#9 of multiorgan failure due to “acute humoral rejection”. These antibodies were investigated further. Methods Antibodies were evaluated using both Labscreen PRA (One Lambda) and Labscreen Single Antigen (One Lambda) beads, and crossmatching was performed using standard T and B cell CDC and flow cytometry methods. Results Pre-transplant antibody studies (Labscreen PRA) had shown the presence of clearly defined antibodies to B27, DR4 and a possible weak anti-DR7. Retrospective Labscreen Single Antigen(SA) (One Lambda) testing revealed antibodies to B13 and DR7, both donor-specific antibodies. MFI values were 2000 for B13 and 5000 for DR7. The pre-transplant crossmatches (CDC and flow cytometry) were negative except for a positive B cell CDC. The patient received blood products on POD#3. Crossmatching on POD# 3 showed only a weakly positive T flow crossmatch and negative DSA. However, SA antibody studies on POD#6 showed high MFI values of 19,000 (B13) and 23,000 (DR7), and strongly positive T and B CDC and flow crossmatches. C1q studies of sera from POD#0 and #3 showed the DSA were non-complement fixing. However, by POD#6, the DS antibodies were now clearly complement-fixing. The patient expired on POD#9. Myocardium tissue from the left ventricle taken at autopsy showed positive staining for C4d. Conclusions DSA with relatively weak MFI values can be a significant risk to both organ and patient survival. Non-complement fixing DSAs also pose a risk and can become expressed as complement-fixing antibodies in an acute immune response to mismatched donor antigens.

39-P

Aim Low level antibodies may not always produce a positive flow crossmatch and increase the risk for transplant. The aim of this study was to investigate the impact of increasing the MFI cutoff for listing the unacceptable antigens (UA) in UNET. UA are use in virtual crossmatches and exlude recipients without performing crossmatches. Methods Antibody detection was performed by One Lambda LABScreen Single Antigen HLA Class I and II beads. The normalized MFI was used to calculate the cutoff. The MFI cutoff for a positive result was increased from 1,500 to 3,000. T and B cell crossmatches were performed by flow cytometry with pronase treated cells and reported as MCS. The T cell cutoff for positive was 61 and B cell was 93 which is 3 standard deviations above our negative control serum. The ten highest ranking recipients on the kidney runs from the first 8 deceased donor workups following the change were studied. Results Fifty six of the 126 actively listed patients from CTHH had UA entered in UNET. Low level UA were removed from 50/56 recipients. After the modification, eight recipients fell below and 21 stayed above 80% CPRA. The remainder had a CPRA range from 5-73%. 48 of the top 80 recipients on recent kidney match runs were from CTHH and further studied. 14/48 (29%) had low level antibodies present. 3/14 (21%) were flow T and B cell negative and the DSA was to DQ7 in all three crossmatches. Eleven were T and/or B cell positive. Four of the positive crossmatches were due to Cw, DQA, or DP antibodies which are not reported as UA. Three were due to low levels of multiple antibodies producing a cumulative effect. One was due to Bw6 antibody that reacted with all Bw6 beads on the Luminex assay but reacted below 3,000 MFI. The remaining four positive crossmtaches were due to historically higher MFI values. Conclusions Low level antibodies should not be added as unacceptable antigens and automatically rule out a recipient for kidney transplantation. Some crossmatches will be negative.