Single Antigen Class Research Articles

Pathogenetic analysis of transfusion-related acute lung injury caused by human leukocytes antigen antibody against human leukocyte antigen

From September 2019 to October 2020, pathogenetic analysis of three patients clinically diagnosed as transfusion-related acute lung injury (TRALI) caused by human leukocyte antibodies was conducted by Guangzhou Blood Centre, including 2 males and 1 female, aged 56, 50 and 20 years old, respectively. Solid phase agglutination, anti-human globulin test and flow cytometry method were used to detect the presence of antibodies against patients. Sequencing-based human leukocyte antigen (HLA-SBT) typing technique was used to detect the human leukocyte antigen (HLA) genotypes of patients. Lifecodes single antigen class Ⅰ/Ⅱ kit (LSA-Ⅰ/Ⅱ) were used to detect the specificity of HLA-class Ⅰ and class Ⅱ antibodies in donor blood by Luminex 200 liquid suspension chip system. The HLA specific antibodies and corresponding epitopes in donors were also analyzed. The results showed that HLA class Ⅰ or class Ⅱ specific antibodies against TRALI patients were detected in the blood donors. The plasma of donor 3 received by patient 1 contained antibodies against the patient's HLA-DRB1*09∶01 antigen, and the epitopes mediating the antibody reaction of the donor and recipient were 70R, 31I, 70QA. There were antibodies against the HLA-A*11∶02, HLA-A*11∶01, DRB1*12∶02, and DRB1*09∶01 antigens of patient 2 in the plasma of donor 4, and the associated antigenic epitopes were 151AHA, 57V, and 16Y. Antibodies against the HLA-DRB1*14∶04, DRB1*11∶01, and DPB1*05∶01 antigens of patient 3 were present in the plasma of donor 6 and donor 7, and the associated epitopes were 96HK, 140TV, 13SE, and 111K. Three cases of TRALI were confirmed to be caused by HLA antibodies through laboratory analysis, and human leukocyte antibody detection should be paid attention in clinically suspected cases of TRALI, and targeted diagnosis and treatment should be given.

Predicting flow cytometry crossmatch results from single-antigen bead testing.

The aim of this study was to devise an algorithm that would predict flow cytometry crossmatch (FCXM) results using single-antigen bead (SAB) mean fluorescent intensity (MFI) levels using samples received through the National External Quality Assurance Scheme (NEQAS) 2B external proficiency testing scheme between 2019 and 2023. A total of 159 serum samples were retrospectively screened using LABScreen Single Antigen Class I and II (SAB), and 40 peripheral blood samples were human leucocyte antigen (HLA) typed with LABType SSO. Donor-specific antibodies were identified for each cell-serum combination tested, and cumulative MFI values were calculated for each test before correlating the screening result with the consensus crossmatch results for this scheme. HLA Class I MFIs were combined to predict the T cell crossmatch. For the B cell crossmatch prediction, two options were considered: (i) HLA Class II MFI values alone and (ii) HLA Class I+Class II MFIs. Receiver operating characteristic analysis was carried out to identify the combined MFI threshold that predicted NEQAS consensus results with the greatest sensitivity and specificity. HLA Class I combined MFI >5000 predicted T cell crossmatch results with 96% sensitivity, 100% specificity, 100% positive predictive value (PPV) and 92% negative predictive value (NPV). For B cell results, HLA Class I+Class II combined MFIs >11,000 gave the best model, showing 97% sensitivity, 82% specificity, 96% PPV and 85% NPV. However, for samples with only HLA Class II sensitization, combined MFIs >13,000 improved the B cell crossmatch predictions: 92% sensitivity, 95% specificity, 96% PPV and 91% NPV. Using this model, combined MFI can be used to predict the immunological risk posed by donor-specific antibodies when it is not possible to carry out an FCXM.

Comparison of HLA antibody identification methods for the selection of platelet products for HLA-mediated platelet refractory patients.

In an ineffective transfusion context, solid-phase immunoassays using the Luminex platform for the detection and characterization of HLA antibodies are currently used to select HLA-compatible platelet products. A new HLA antibody identification method, the HISTO SPOT® HLA AB test (BAG Health care GmbH, Lich, Germany), based on the detection of antibodies directed against a recombinant single antigen (SA) by colored spots detected by HISTO MATCH HLA AB module software, runs fully automated on the MR.SPOT®. The aim of this study was to compare the ability of the HISTO SPOT HLA AB and C1qScreen™ (C1q SAB) assays with that of the Labscreen single antigen class I (OL SAB) assay to detect anti-HLA class I antibodies in 56 serum samples from 54 platelet refractory acute myeloid leukemia patients who received HLA mismatch platelet concentrates at a single oncohematology center. In total, 1414 class I specificities, 433 HLA-A and 981 HLA-B, were detected by the OL SAB test. The mean fluorescence intensity (MFI) was >5000 for 874 antigens and <5000 for 655 antigens. The HISTO SPOT® HLA AB and C1q SAB tests identified 85% and 79% of OL SA-detected antigens with an MFI >5000, respectively, but did not identify 34% and 44% of OL SAB-detected antigens, highlighting the lower sensitivity of these techniques. Interestingly, the donor-specific antibodies (DSAs) identified by the HISTO SPOT® HLA AB and C1q SAB assays reacted against HLA mismatch platelet concentrates with the same specificity (86%) and positive predictive (77%) value as in the OL SAB test when the MFI threshold was >2000 for DSA detection. Although the HISTO SPOT® HLA AB test is less sensitive than the OL SAB test, this test could be used for the selection of HLA-compatible platelet products.

Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data.

Advances in next-generation sequencing (NGS) have improved the resolution of T-cell receptor (TCR) repertoire analysis, and recent single-cell sequencing has made it possible to obtain information about TCR pairs. In our previous study, cytomegalovirus (CMV) pp65-specific T-cell response restricted by a single human leukocyte antigen (HLA) class I allotype was observed in an individual. Therefore, to effectively clone an antigen-specific TCR from these T cells, we developed a TCR cloning system that does not require a single cell level. First, we established the improved Jurkat reporter cell line, which was TCRαβ double knock-out and expressed CD8αβ molecules. Furthermore, functional TCRs were directly obtained by reverse TCR cloning using unique CDR3-specific PCR primers after bulk TCR sequencing of activation marker-positive CD8 T cells by NGS. A total of 15 TCRα and 14 TCRβ strands were successfully amplified by PCR from cDNA of 4-1BB-positive CD8 T cells restricted by HLA-A*02:01, HLA-A*02:06, HLA-B*07:02, and HLA-B*40:06. The panels with combinations of TCRα and TCRβ genes were investigated using Jurkat reporter cell line and artificial antigen-presenting cells (APCs). In two TCR pairs restricted by HLA-A*02:01, one TCR pair by HLA-A*02:06, four TCR pairs by HLA-B*07:02, and one TCR pair by HLA-B*40:06, their specificity and affinity were confirmed. The TCR pair of A*02:01/1-1 showed alloreactivity to HLA-A*02:06. The one TCR pair showed a higher response to the naturally processed antigen than that of the peptide pool. This reverse TCR cloning system will not only provide functional information to TCR repertoire analysis by NGS but also help in the development of TCR-T therapy.

Successful Desensitization of Severe Alloimmune Platelet Refractoriness and Provision of Permissive Platelet Transfusions with a Novel IgG-Targeted Enzyme Therapeutic

AK and JCS contributed equallyBACKGROUND Alloimmune platelet refractoriness (APR), inadequate response to platelet transfusion secondary to alloantibodies targeting Human Leukocyte Antigens (HLA), affects ~25% of acute myeloid leukemia (AML) patients. For profoundly sensitized thrombocytopenic patients, HLA- or crossmatch-compatible platelet units may be unavailable, increasing the risk of thrombocytopenic hemorrhage. Imlifidase cleaves systemic IgG precluding complement fixation/phagocytosis and has been used to desensitize renal allograft recipients.1 Here we show that desensitization with imlifidase can significantly improve compatible platelet availability in APR and post-hematopoietic stem cell transplant platelet nadir. PATIENT CASE A 43-year-old woman with AML treated with cytarabine and daunorubicin was referred for matched-unrelated donor allogeneic hematopoietic progenitor cell transplantation. Her induction, at an outside institution, was complicated by bilateral retinal and mucosal hemorrhage secondary to APR and profound thrombocytopenia (platelet count to 0/µL). On presentation, her panel reactive antibody (PRA) showed exquisite sensitization (99.9%, i.e. ~99.9% of all donor platelet units predicted incompatible), precluding identification of HLA-compatible platelet units to support the patient through her anticipated platelet nadir. Desensitization included rituximab 375 mg/m2 (transplant D-13 and -6), therapeutic plasma exchange (TPE, D-2 and -1) and FDA-emergency-use-authorized imlifidase 0.25 mg/kg IV (D-1 and D+1). Following imlifidase administration, the number of unacceptable antigens and PRA decreased significantly (Figure) allowing sufficient compatible platelets to be identified and procured. During admission, the patient received 7 total platelet transfusions and experienced no hemorrhage (Table). She was discharged stable, post-platelet engraftment on D+19. STUDY DESIGN With IRB approval, the number of predicted clinically significant alloantibodies (antibodies with ≥ 3000 mean fluorescence intensity, MFI) and PRA (LABScreen single antigen and PRA Class I, One Lambda/ThermoFisher, West Hills, California) were compared at baseline, after rituximab/TPE, and after imlifidase administration. Corrected count increment (CCI) to platelet transfusions administered following imlifidase desensitization was calculated. CCI > 5000 was judged a permissive transfusion. Categorical variables were compared with Chi-squared followed by Fisher's Exact. P < .05 was significant. RESULTS AND CONCLUSION Compared to the baseline, unacceptable antigens (antigens targeted by antibodies at clinically significant levels) decreased significantly after rituximab/TPE (N=53/55 to 44/55 antigens, P = .015) and imlifidase (N = 44/55 to 16/55, P < .0001). PRA did not decrease significantly with rituximab/TPE (99.9 to 99.5%, P = .22) but did following imlifidase (99.5 to 87.2%, P < .0001). Of 7 transfusions given post imlifidase, the median CCI was 6000 (interquartile range: 500-7500), N=5/7 were judged permissive. In summary, the imlifidase-mediated decrease in PRA significantly aided the identification of numerous compatible platelet units for this patient and prevented further thrombocytopenic hemorrhage. Our limited data suggest a potentially novel application of imlifidase for desensitization of highly platelet allo-refractory patients requiring multiple platelet transfusions over a short period. We will perform western blot analysis of antibodies from in vivo samples collected over the treatment course to compare antibody fragmentation over time, as partially cleaved immunoglobulin detected by our microbead-based assays may not be pathologic.2 Additionally, as the patient's initial antibody levels exceeded the range of linearity of the bead-based assay, serial dilution studies of patient serum over the treatment course will more precisely semi-quantitate the true peak antibody levels. REFERENCES 1. Jordan SC, Legendre C, Desai NM, et al. Imlifidase desensitization in crossmatch-positive, highly sensitized kidney transplant recipients: results of an international phase 2 trial (Highdes). Transplantation. 2021;105(8):1808-1817. 2. Bockermann R, Järnum S, Runström A, et al. Imlifidase-generated single-cleaved IgG: implications for transplantation. Transplantation. 2022;106(7):1485-1496. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

HLA graph, a free and ready-to-use bioinformatics tool to explore anti-HLA eplets reactivity pattern.

HLA antigens are highly polymorphic, and their immunogenicity is dependent on the configurations of polymorphic amino acids. Monitoring anti-HLA immunization is essential in organ transplantation, as antibodies directed against HLA molecules are a major cause of rejection. Anti-HLA antibodies are not specific for HLA antigens, but recognize B-cell epitopes present on HLA molecules. To better understand antibody reactivity patterns, we calculated the Spearman correlation of the mean fluorescence intensity (MFI) of anti-HLA antibodies identified by a single-antigen assay performed using a Luminex® immunobeads assay on a large number of samples. Then, we built a computer tool analyzing antibody reactivity patterns with an accessibility by a web browser linked to the International Epitope Registry. We also extended our model to Onelambda® and Lifecodes® single-antigen class I and class II assays. The resulting MFI correlations reflect HLA antibody cross-reactivity and eplets similarity. We built HLA Graph, a computer tool that analyzes the eplets involved in antibody reactivity profiles. HLA Graph is usable with Onelambda® and Lifecodes® single-antigen class I and class II assays. The interpretation of reactivity against alleles not tested by the antibody assays and against the alpha and beta chains of HLA-DQ and HLA-DP loci were also developed. HLA Graph is a free and ready-to-use bioinformatics tool that can be used by all laboratories performing anti-HLA antibody identification by immunobead assay.

Donor Specific Allo-Antibody is Significantly Associated with Variability in Donor-Derived Cell-Free DNA Scores in Adult Heart Transplant Recipients

<h3>Purpose</h3> In this single-center retrospective pilot study heart transplant (HTx) patients were non-invasively monitored by donor-derived cell-free DNA (dd-cfDNA). We hypothesized that longitudinal dd-cfDNA variability scores correlate with preformed and de novo (dn) donor specific HLA allo-antibody (DSA). <h3>Methods</h3> 72 adult HTx patients with at least 3 dd-cfDNA results were included (AlloSure; CareDx Inc., Brisbane, CA). We determined dd-cfDNA variability (ASV) by standard deviation (SD) of longitudinal dd-cfDNA results, scaled x10 (ASV*10), and DSA by single antigen class I/II and MFI >1000 for HLA-A, B, DR, DQB/A and >2000 for HLA-C and DPB/A. <h3>Results</h3> Age at HTx was 49.1 years (SD 14.3). Study enrollment occurred at 270 days +/-SD 420.2 post-HTx. Biopsy-proven rejection (ACR ≥ 2R and/or pAMR ≥ 1) was detected in 16 (22 %), preformed DSA in 9 (12.5%), dnDSA in 13 (18%) and preformed + dn in 2 (2.7%) patients while 48 (66.7%) were DSA negative. Median time from HTx to dnDSA was 329d (range: 39-827). Including all values, mean ASV*10 was highest in patients with dnDSA (3.03 ± 4.74) compared to those with no DSA (0.65 ± 2.04) or preformed DSA (1.36 ± 3.40, p=0.001; <b>Table 1</b>). Patients who developed dnDSA showed a significant increase of ASV*10 after dnDSA detection (1.50 ± 2.06, p = 0.014), but not before dnDSA detection (1.24 ± 1.49, p=0.380) in comparison to patients with no DSA (0.65 ± 2.04, <b>Table 1</b>). <h3>Conclusion</h3> These findings highlight the value of longitudinal assessment of dd-cfDNA and DSA in HTx recipients. Our results show that increased dd-cfDNA variability is associated with the presence of preformed and dnDSA. Further, ASV*10 is significantly increased after identification of dnDSA, further supporting the mechanistic role of dnDSA in mediating poor outcomes after HTx. Confirmation in larger data sets may further advance the utility of longitudinal non-invasive surveillance using dd-cfDNA and dnDSA in transplant recipients.

Establishment of a novel cell-based assay using HLA-transfected cells to detect HLA antibodies

The detection of HLA antibodies is important in clinical practice, such as platelet transfusion refractoriness and transfusion-related lung injury. However, difficulties are associated with the preparation of panel cells for conventional HLA detection systems using intact cells, such as the immunocomplex capture fluorescence analysis (ICFA).Based on an ICFA analysis, HEK293 cells stably transfected with the HLA-A locus were used instead of peripheral blood mononuclear cells (PBMC). The reactivity, sensitivity, and stability of transfectants were examined.All 20 antisera to HLA-A identified by LABScreen® Single Antigen class I (LS-SA1) were reactive to our modified-ICFA (m-ICFA) and showed the same specificities as those in LS-SA1, indicating the cell surface expression and correct antigenicity of the HLA-A locus in transfectants. The expression of HLA class I antigens was similar between transfectants frozen for 6 years and those prior to freezing. In the reaction of the anti-A24 or anti-A33 antibody vs each transfectant, the index of m-ICFA was higher than that of WAKFlow® ICFA. Our m-ICFA also showed that false negative reactions sometimes observed in capture assays may be avoided.By using HLA-A transfectants as ICFA targets, we herein developed m-ICFA. Our m-ICFA may avoid false negative reactions of capture assay like enzyme-linked immunosorbent assay and can also be carried out in almost any laboratory without cell culture facilities.

Significance of Luminex-based single antigen class II bead assay and its mean fluorescence intensity in renal transplant cases; a retrospective observation in 97 cases

ABSTRACTThe objective of this study was to determine the mean fluorescence intensity (MFI) values of class II Luminex single antigen bead (L-SAB) assay and compare these MFI values with cell-based complement-dependent cytotoxicity crossmatch with anti-human globulin (CDC-AHG-XM) and IgG-B-cell flow cytometry crossmatch (FC-XM) results and explore the near-accurate MFI-cutoff values of positive cell-based crossmatch results. This retrospective study was an analysis in 97 renal transplant recipients, who were tested for the presence of DSA by CDC-AHG-XM and IgG-B-cell-FC-XM methods with their corresponding donor as well as for anti-human leukocyte antigen (HLA) antibody detection using a sensitive L-SAB assay. In the group having DSA MFI values <1000, none of the patients showed positivity for FC-XM and CDC-AHG-XM; in the group having MFI values between 1000 and 3000, 35.48% showed positivity for FC-XM but none by the CDC-AHG-XM method. However, in the group having MFI values >3000, 83.33% of cases were positive for FC-XM. Further, in those groups with MFI values between 3000 and 6000, 38.09% were positive for CDC-AHG-XM, while 86.66% showed positivity in the group with MFI >6000. Our results indicated that Luminex-DSA MFI value >1995 (P < .0001) significantly correlated with IgG-B-cell-FC-XM positivity while Luminex-DSA MFI value of >4247 (P < .0006) was significantly correlated with positive CDC-AHG-XM. MFI cutoff of 1995 exhibited a diagnostic sensitivity of 97.56% and specificity of 89.29% for predicting positive IgG B-cell FC-XM and MFI cutoff of 4247 exhibited a diagnostic sensitivity of 90.48% and specificity of 97.37% for predicting positive CDC-AHG-XM. However, a cutoff MFI of >5000 and >7000 for SAB assay had a sensitivity and specificity of 100% in detecting a positive IgG B-cell FC-XM and CDC-AHG-XM, respectively.

P111 Analysis of hla antibodies after native nephrectomy in renal allograft recipients

Aim Many centers avoid nephrectomy of chronically rejected kidneys because of fear of sensitization to HLA antibodies. It is not clear if the rise in antibodies is from an overall immune sensitizing event or specific to the removal of the donor organ. We analyzed anti-HLA antibodies following native nephrectomy as a control event to the allograft nephrectomy. Methods Twelve kidney allograft recipients who underwent native nephrectomy for cause had serum samples obtained before the nephrectomy and 1–32 weeks after nephrectomy. All patients remained on clinically indicated immunosuppression. The study was approved as an exempt study by the IRB. The serum samples were tested on a Luminex platform with Single Antigen Class I and Class II beads specific for HLA antibodies. Changes in calculated panel reactive antibody (cPRA) and peak mean fluorescence intensity (MFI) were measured by paired t-test. Results Three of twelve patients (25%) showed an increase in HLA antibodies. One of the sensitized and one of the non-sensitized patients required a blood transfusion during the procedure. Nine patients had no class l or class ll antibody before or after native nephrectomy. The 3 patients who had sensitization to HLA prior to the native nephrectomy all had an increase in antibody post-nephrectomy: pre cPRA = 65 and post cPRA 74, p = 0.02. Two of the 3 sensitized patients showed an increase of MFI in their highest antibody specificity class I MFI (7933 pre and 13733 post) and class II MFI (6200 pre and 8067 post), but this was not significant in this small group. One subject showed increased donor specific antibody, with no significant class I before native nephrectomy, but MFI of A3 of 14800 and B44 of 17600 after native nephrectomy; and class II DR1 MFI of 3300 before (no DR4) and DR1 MFI of 18,200 and DR4 MFI of 15500 after native nephrectomy. There was no incidence of allograft loss. Conclusions HLA antibodies may rise following a native nephrectomy in sensitized patients. Monitoring of HLA antibodies after native nephrectomy may be prudent.

P136 Successful adeptness of donor exchange program in renal transplantation at King Faisal Specialist Hospital Jeddah

Aim Different methodologies were established to overcome HLA immunological barriers to match the highly increasing number of patients with end-stage renal disease in Saudi Arabia, which is currently ranged between 120 and 150 patients per million per annum. At our hospital, King Faisal Specialist Hospital Jeddah, we therefore successfully established the desensitization and ABOi renal transplantation. Recently we have started to use a donor exchange program. We hereby report about our experience to overcome the immunological incompatibilities by using this program. Methods We used an IT program with high grade on flexibilities by performing different options including simple two-pair exchanges, more complicated domino exchanges and chain donations. All recipients with willing donors and with high titer HLA antibodies and/or strong positive cross match were included in the program. HLA typing was performed by using One Lambda SSOr and SSP. HLA Antibody Identification, Single Antigen Class I/II and C1q test, was performed by One Lambda. T and B cell IgG XM is performed by FACSCanto II flow cytometer, where the cut-off for positive XM is determined based on normal human studies. Results 20 patients were transplanted successfully by using the donor exchange program within a period of 12 months. Considering a total number of 220 transplanted kidneys during this time, which makes our hospital as the largest center for renal transplantation in Saudi Arabia, we were able to increase the number of transplanted organs significantly by 9% and we continue to extend and optimize our histocompatibility services at the hospital. Conclusions Our experience demonstrates the high efficiency of the donor exchange program in overcoming immunological barriers in renal transplantation. This program provides in some cases also an alternative to the invasive and costly desensitization protocols. Further efforts and thoughts are going on to include deseased donors as well in this successful donor exchange program.

P274 Discrepancy between DSA and HLA flow cross match associated with high frequency of prozone phenomenon in solid phase assay in organ transplantation

Aim Human Leukocyte Antigen (HLA) antibody identification solid phase assays play a key role in defining Donor Specific Antibody (DSA) which can determine the compatibility between the recipient and donor in cross match results. Luminex bead array test becomes high acceptance and wide reach in HLA antibody detection. However, HLA antibodies with high titer lead to activation of complement and might remove C1 complex on the bead. Subsequently, Interference of HLA antibodies and the secondary IgG antibodies might be occurred. This may lead to false-negative or weak antibody results, which is known as prozone phenomena. Technical methods, like treatment with EDTA, dithiothreitol (DTT), heat inactivation and dilution with normal saline can detect this phenomenon. Methods HLA Typing was performed for all recipients and donors before renal transplantation using One Lambda Sequence specific oligonucleotide revers and sequence specific primers (SSOr/SSP). HLA Antibody Identification, Single Antigen Class I/II and C1q test, was performed by One Lambda. T and B cell IgG XM is performed by FACSCanto II flow cytometer, where the cut-off for positive XM is determined based on normal human studies. Native serum samples of 11 recipients were diluted 1:10 with normal saline when clear discrepancy was found between DSA and HLA Flow XM (DSA 300 MCS). Results All cases (N = 11) showed strong elevating of DSA after 1:10 dilution, often >15000 MFI, which was correlated with strong positive HLA flow cross match results. Conclusions: Our results demonstrate the large incidence of prozone effect in solid phase assay. It is therefore strong recommended to treat the serum -in our cases dilution- for prozone effect always and whenever there is no correlation between strong positive FXM and weak or negative DSA.

P195 Single antigen dilution reveals interference and falsely low MFI for donor specific antibodies

A 48 year old man presented for second kidney transplant with a potential living donor, his brother. His first transplant was in 2000. At evaluation, the patient had HLA single antigen class I and II testing on neat serum treated with DTT, flow crossmatch (FCXM, ±Pronase) and cytotoxic crossmatch (CDC, ±DTT). Neat serum showed antibodies to HLA-A29 and B51, both repeat mismatches from his prior transplant, and an odd pattern of DQA1 antibodies (Table 1). The initial FCXM with his brother was positive on T (187MCS) and B cells (408MCS). The B CDC was also positive. The patient displayed DSA to HLA-A29 (8277MFI), B51 (1588MFI), and DQA1∗02 (8467MFI) in the same serum. However, the crossmatch was unexpectedly strong given the MFI. Importantly, the patient’s first transplant was from another brother, who was HLA identical to this potential sibling donor. We suspected that the patient in fact had strong antibodies to HLA-DQ2 and DQ7, which were repeat mismatches. It was curious that the beads with DQA1∗05 had the lowest MFI as this combination was exactly that present in the first donor. In addition, there were apparently allele-specific DQA1∗03:01 antibodies, which is unusual. Thus, we performed dilution of the serum at 1:10 and 1:100 to determine whether there was interference with very strong antibodies. Dilution of the patient’s serum resulted in a significant increase in MFI for DQ beads. For example, DQA1∗03:02/DQB1∗03:02 went from 944MFI to 10,533 MFI at 1:10. Dilution also showed that the patient’s DSA was to HLA-DQ2 and DQ7 rather than to DQA1 alleles. The donor was declined and paired exchange was recommended. We also noted on the single antigen reports that the patient exhibits “prozone” and the antibodies to DQ7 and DQ2 are much stronger than represented by the MFI in neat serum. This case demonstrates the utility of serum dilution to identify strong antibodies with falsely low MFI, and illustrates a pattern of antibody reactivity that should appear “suspicious” for interference: for example, apparent DQA1 allele-specific antibodies.Download : Download high-res image (208KB)Download : Download full-size image

HLA-typing analysis following allogeneic bone grafting for sinus lifting.

According to the Brazilian Association of Organ Transplants, in 2015, 19,408 bone transplants were performed in Brazil, over 90% by Dental Surgeons. The surgical technique itself has a respectable number of reports regarding its clinical efficacy, as measured by long-term survival of dental implants in grafted areas. Uncertainty remains, however, as to whether fresh frozen grafts from human bone donors remain immunologically innocuous in the body of the host. Six male with no previous medical history of note, including systemic diseases, surgery or blood transfusion were selected. These patients underwent reconstructive procedures (sinus lifting) using fresh frozen human bone from a tissue bank. All patients had venous blood samples collected prior to surgery and 6months after the procedure. Anti-HLA analysis for the detection of HLA (human leukocyte antigen) antibodies was performed using methods such as the LABScreen PRA Class I and Class II, LABScreen Single Antigen Class I and Class II, Luminex Platform. Reactive individuals to the screening tests (LABScreen PRA) were further investigated to determine the specificity of the antibodies detected (LABScreen Single Antigen) with a cutoff value of median fluorescence intensity ≥500. As a result, it was observed that two patients (33%) were positive in screening tests, one presenting with anti-HLA Class I and II sensitization and the other with anti-HLA class II. The specificity analysis showed that the patients sensitized to HLA class II presented 4 specificities, 3 of which immunologically relevant. In the second individual, 23 specificities were identified, 6 of which immunologically important for HLA class I and 4 specificities for HLA class II, 3 of these were immunologically important. All specificities detected had average fluorescence. These findings are suggestive that sinus-lifting procedures with allogeneic bone can induce immunological sensitization.

P009 Implementation of robotic methodology and instrumentation for the detection of HLA antibodies in single antigen beads assays

Aim The development of solid-phase assays allows the measurement of anti-HLA antibodies with high sensitivity. Our goal was to address MFI inconsistencies between Luminex instruments as well as tech-to-tech variation when performing Single Antigen Class I & II assays (SAB). Methods The SAB assay from One Lambda was used for this study. Run-to-run variability was assessed by testing a pool of serums from sensitized patients (PC) on every tray. SAB was performed manually or using the LABXpress Pipettor (LXP). Trays were then run using the same Luminex or in 1 randomly selected instrument out of 4 available. MFI values from informative beads with different MFI intensities were evaluated. We performed Instrument correlation studies by testing the same patient sera on the LXP and reading the same tray on two separate instruments at a time. MFI values for each specificity were compared. Results Comparison of PC individual beads MFI values between the manual and LXP methods read on the same Luminex showed an overall intra-run coefficient of variation (CV) of 17% and 10% respectively. An initial assessment of MFI values for PC using 4 different Luminex machines (CV13%) was higher than results obtained when the same Luminex was used (CV 10%). The run-to-run variability was greater for MFI ranges 9–15 × 103 (CV13%). Subsequent service (alignment & standardization) of all Luminex instruments did not show any improvement on MFI variance (CV 13%). MFI correlation studies revealed a 99% correlation coefficient for all instruments. Conclusion Our study revealed that implementation of the LABXpress significantly reduced the MFI variability and validates using this robotic approach for the SAB assay. Even though there was an excellent correlation between Luminex instruments and all QC parameters passed, Luminex controls do not assure instrument MFI comparability. Further efforts must be made to minimize MFI variations allowing the lab to compare MFI values from multiple instruments.

OR21 Clinical significance of C3D binding donor specific anti-HLA antibodies detected by single antigen beads after kidney transplantation

Aim Recent interest in characterizing the complement-fixing ability of post-transplant donor specific alloantibodies (DSA) resulted in the enhancement of Luminex solid phase alloantibody assays (SAB) to determine DSA complement-fixing capacity. Previous studies have reported a significantly worse clinical outcome after kidney transplantation for SAB C1q-binding versus non-C1q-binding DSA. A newly available SAB method detects C3d-binding rather than C1q. The purpose of this study was to compare the clinical outcomes in kidney transplant recipients that developed de novo DSA post-transplant stratified by C3d binding by SAB assay (Lifecodes, Immucor). Methods A cohort of 222 recipients transplanted between 2002 and 2013, with previously identified post-transplant de novo DSA, were retested by Luminex SAB IgG and C3d assay using the Immucor LSA Single Antigen Class I and Class II beads. This 222 patient cohort was 45.2 ± 12.1 (19–74) years old, 63% male ( n = 139), 35% African-American ( n = 77), 33% sensitized pre-transplant ( n = 73), and 11% retransplant recipients ( n = 25). The median time to first rejection episode experienced by 112/222 (50.4%) recipients was 18.8 months (range 8 days to 6.6 years). The median time of sera testing was 35.8 months post-transplant (range 8 days to 10 years). Results In 149/222 (67%) recipients, the SAB C3d assay was positive. Analysis of anti-HLA class specificity revealed 72 (32.4%) of recipients were SAB C3d negative, 16 (7%) were anti-HLA Class I only positive, 111 (50%) were anti-HLA Class II only positive, and 23 (10%) were positive for anti-HLA Class I and Class II. Death censored graft survival after sera acquisition for testing was significantly worse for all SAB C3d positive versus C3d negative recipients. The difference in graft survival was most pronounced when anti-HLA Class I SAB C3d binding was detected. Conclusion We conclude that C3d binding in this SAB assay identifies de novo DSA kidney recipients at highest risk for graft loss. I. Balazs: Employee; Company/Organization; Immucor, Inc.

Liver Allograft Provides Immunoprotection for the Cardiac Allograft in Combined Heart-Liver Transplantation.

When transplanted simultaneously, the liver allograft has been thought to have an immunoprotective role on other organs; however, detailed analyses in simultaneous heart-liver transplantation (SHLT) have not been done to date. We analyzed patient outcomes and incidence of immune-mediated injury in 22 consecutive SHLT versus 223 isolated heart transplantation (IHT) recipients between January 2004 and December 2013, by reviewing 3912 protocol- and indication-specific cardiac allograft biopsy specimens. Overall survival was similar (86.4%, 86.4%, and 69.1% for SHLT and 93.3%, 84.7%, and 70.0% for IHT at 1, 5, and 10 years; p = 0.83). Despite similar immunosuppression, the incidence of T cell-mediated rejection (TCMR) was lower in SHLT (31.8%) than in IHT (84.8%) (p < 0.0001). Although more SHLT patients had preexisting donor-specific HLA antibody (22.7% versus 8.1%; p = 0.04), the incidence of antibody-mediated rejection was not different in SHLT compared with IHT (4.5% versus 14.8%, p = 0.33). While the left ventricular ejection fraction was comparable in both groups at 5 years, the incidence and severity of cardiac allograft vasculopathy were reduced in the SHLT recipients (42.9% versus 66.8%, p = 0.03). Simultaneously transplanted liver allograft was associated with reduced risk of TCMR (odds ratio [OR] 0.003, 95% confidence interval [CI] 0-0.02; p < 0.0001), antibody-mediated rejection (OR 0.04, 95% CI 0-0.46; p = 0.004), and cardiac allograft vasculopathy (OR 0.26, 95% CI 0.07-0.84; p = 0.02), after adjusting for other risk factors. These data suggest that the incidence of alloimmune injury in the heart allograft is reduced in SHLT recipients.

Donor-specific HLA Antibodies Are Associated With Late Allograft Dysfunction After Pediatric Liver Transplantation.

The role of donor-specific HLA antibodies (DSA) after pediatric liver transplantation (LTx) is not clearly established. We completed a cross-sectional study to characterize DSA in long-term survivors of pediatric LTx and assess the impact of C1q-binding DSA on allograft outcomes. Serum samples were collected at routine clinic visits from 50 pediatric LTx recipients classified into 3 clinical phenotypes: nontolerant (n = 18) with de novo autoimmune hepatitis (DAIH) and/or late acute cellular rejection (ACR); stable (n = 25) on maintenance tacrolimus; operationally tolerant (n = 7). Samples were blinded, and antibody detection was performed using Luminex single antigen class I and II beads. Patients with positive DSA were tested for C1q-binding DSA. DSA were detected in 54% (n = 27) of the patients, with the majority directed at HLA class II antigens (DR, 41%; DQ, 53%). Patients with DSA were younger at the time of LTx (P = 0.016) and time of study (P = 0.024). Mean aspartate aminotransferase, alanine aminotransferase, total bilirubin, and gamma glutamyl transferase were higher in DSA-positive patients, though did not reach statistical significance. Nontolerant patients were significantly more likely to have DQ DSA (61%) compared to stable (20%) and tolerant (29%) patients (P = 0.021). The nontolerant phenotype was associated with DSA and C1q-binding DSA, with odds ratios of 13 (P = 0.015) and 8.6 (P = 0.006), respectively. The presence of DQ DSA was associated with DAIH and late ACR, with odds ratios of 12.5 (P = 0.004) and 10.8 (P = 0.006), respectively. Allograft dysfunction is not always evident in patients with DSA, but DQ DSA are strongly associated with DAIH, late ACR, and chronic rejection.

C1q-Binding HLA Antibodies Do Not Predict Platelet Transfusion Failure in TRAP Study Participant

Introduction: In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of the 530 subjects became clinically refractory (CR) to platelet transfusions in the absence of detectable antibodies against HLA as measured by the lymphocytotoxicity assay (LCA). Using more sensitive bead-based detection methods we have previously demonstrated that while many of these LCA- patients do have anti-HLA antibodies, that these low to moderate level antibodies do not predict refractoriness. In addition to being less sensitive then bead based methods, the LCA screen only detects complement-binding antibodies. As these antibodies could be important for platelet rejection, we assessed if previously undetected complement-binding antibodies could account for some of the refractoriness seen in LCA- patients.Methods: 169 LCA- (69 CR, 100 non-CR) and 20 LCA+ (10 CR, 10 non-CR) subjects were selected from the TRAP study. Anti-class I HLA IgG and C1q binding antibodies were measured in serum or plasma using two different multi-analyte, semi-quantitative, bead-based fluorescent antibody detection assays: the LabScreen mixed Luminex assay, and the LabScreen single antigen class I assay with or without added EDTA (One Lambda). Groups were compared using an unpaired t-test, a=0.05, and correlation between variables was also assessed.Results: New measurements of anti-class I HLA IgG antibodies reliably reproduced earlier data with a strong correlation between the old and new measurements (R2=0.9736, p<0.0001). While some of the LCA- subjects did have detectable C1q-binding anti-class I HLA antibodies, and some LCA+ subjects did not, levels of these antibodies were significantly higher among LCA+ subjects (p<0.0001). Levels of C1q-binding anti-class I HLA antibodies were not significantly different between CR and non-CR among either the LCA- or LCA+ subjects.Conclusions: While complement-binding anti-class I HLA antibody levels were higher in the LCA+ subjects, higher levels of these antibodies were not seen in CR LCA+ patients as compared with non-CR LCA+ patients. Complement-binding anti-class I HLA antibodies do not account for refractoriness seen among the LCA- TRAP subjects. This work confirms that low to mid level anti-class I antibodies do not drive refractoriness to platelets, and suggests that antibody-independent mechanisms cause refractoriness in patients lacking higher levels of anti-HLA antibodies. DisclosuresNo relevant conflicts of interest to declare.

P010 : COMPARISON OF TREATMENTS OF THE SERUM FOR ELIMINATING INHIBITORY FACTORS IN THE LUMINEX SINGLE ANTIGEN ASSAY

False-low and false-negative reactions may occur in the Luminex single-antigen bead assay, particularly when the tested serum is fresh. The purpose of this study was to compare our current method for eliminating inhibitory factors, i.e., heat 56 °C, 1 min (H-1 min), with the methods that most of the laboratories worldwide appear to use, i.e., treatment of the serum with DTT or EDTA. Two fresh sera with PRA I >80% (serum #1 and #2) and two fresh sera with PRA II >80% (serum #3 and #4), both with at least one reaction with >15,000 MFI, were tested with LABScreen® Single Antigen Class I and Class II (One Lambda), respectively. Serum treatments: H-1 min: the sera, already in Luminex plate, were heated, in a thermocycler, at 56 °C for 1 min; DTT: final concentration 5 mM, 37 °C, 30 min; EDTA: 10 mM. Differences ⩾50% in MFIs between treated and native sera were considered relevant. The different treatments did not significantly affect the reactions on negative nor positive control beads. Serum #1: 11 beads had higher MFIs in sera treated with H-1 min, DTT or EDTA and all these reactions could be explained by one antibody against an epitope shared by HLA-A2, -68, -69, -24, B-57 and B-58 molecules. Serum #2: no effect of any treatment was observed. Serum #3: six beads (five of them with DQB1 ∗ 03) had higher MFIs in H-1 min and EDTA treated sera (MFIs in native, H-1 min, DTT and EDTA treated sera: 13,854, 22,116, 16,494 and 23,744 respectively). Serum # 4: 18 beads had higher MFIs in sera treated with H-1 min, DTT or EDTA and, surprisingly, six reactions (all with beads with DQA1 ∗ 01/DQB1 ∗ 06) were practically abolished in EDTA-treated serum, being restored by the addition of calcium to the EDTA-treaded serum. Heat-1 min, DTT and EDTA treatments were in general very similar regarding their ability to eliminate the inhibitory factors of the serum. We see the following advantages of H-1 min over DTT or EDTA treatments: (a) the same treatment can be used if we want to test for IgM antibodies, which would not be possible with the DTT treatment; and (b) there is no interference with calcium-dependent epitopes, which can happen with EDTA. To the best of our knowledge, this is the first report of a calcium-dependent anti-HLA antibody or, better yet, of an antibody directed against a Ca[2+]-dependent conformational epitope on an HLA molecule.