Complement-dependent Lymphocytotoxicity Research Articles

Characterization of novel HLA-A*24:608N allele discovered in Koreans.

A novel null HLA-A*24 allele, HLA-A*24:608N, was identified in five Korean subjects including three from a family and two separate individuals. This study was performed to discern its immunological function in transplantation settings. Because this null variant had deletions of approximately 12 k base pairs from intron 3 to 3' end of the HLA-A gene, low resolution HLA typing and amplicon-based next generation sequencing (NGS) typing methods had failed to assign it. Hybrid capture-based NGS method confirmed that this novel variant had a large deletion. T-lymphocyte crossmatching by complement-dependent lymphocytotoxicity and flow cytometry with a serum consisting anti-HLA-A24 antibody revealed negative results, implying that an individual with this allele would not carry a functioning A24 antigen. These findings highlight the importance of identifying a null HLA allele by employing appropriate molecular method and providing expected crossmatching outcomes in a real-world transplantation setting.

Evaluation of a new complement-dependent lymphocytotoxicity cross match method using an automated cell counter, the NucleoCounter® NC-3000™.

Complement-dependent lymphocytotoxicity cross match (CDC-XM) is the ultimate test of donor/recipient compatibility prior to organ transplantation. This test is based on cell viability, evaluated under fluorescence microscopy by an operator after proper staining. The determination of the positivity threshold may vary depending on the operator. We developed a new method in which the final step of determining cell viability is automated using the NC-3000™ (Chemometec®), an image cytometer able to precisely determine the percentage of dead/live cells in a suspension. After T and B donor cells isolation by negative selection, complement-dependent lysis was performed in macrovolumes in a PCR plate. Then, cell viability was measured by the NC-3000™. The sensitivity and routine CDC-XM results of this new method were compared to those of CDC-XM reference method using Terasaki plates. The sensitivity of CDC-XM expressed in the ASHI scoring system of this method was similar to the reference method results for a dilution range of the positive controls. Similarly, the results of the new method were comparable in a clinical situation to those obtained with the reference method after a study of 10 cross-matches, of which 5 cross-matches with DSA were positive and five cross-matches without DSA were negative. Moreover, ASHI scores were similar to those obtained using the reference method, and the mortality percentage was reproducible (CV < 15%). The assessment of cell viability by the NC-3000™ is easy to perform and highly reproducible but requires CDC-XM to be performed by the macrovolume method. The determination of a precise percentage of viability/mortality by the automation excludes operator variability and allows a better understanding of results close to the decision threshold.

Pretransplant donor-specific anti-human leukocyte antigen antibodies despite a negative complement-dependent lymphocytotoxicity crossmatch: Is it wise to desensitize before kidney transplant?

The significance of pretransplant donor-specific antibodies (DSAs) despite negative complement-dependent lymphocytotoxicity crossmatch (CDC-XM) would be useful for clinical decision-making. Hence, we aimed to determine the impact of pretransplant DSA despite negative crossmatch on the outcome of kidney transplantation. One hundred and eleven kidney recipients were prospectively enrolled in this study after being transplanted at Hamed Al-Essa Organ Transplant Center of Kuwait between January 2011 and December 2013. Of them, 50 recipients with positive DSA at the time of transplant were subjected to desensitization (Group 1). Three local protocols were utilized; first included plasma exchange, high-dose intravenous immunoglobulin (IVIG), and rituximab; second included immunoadsorption plus RTX, and the third included high-dose IVIG and rituximab. The second group included 61 recipients with negative DSA. All recipients had negative CDC-XM and flow cytometry crossmatch at the time of transplant. Panel-reactive antibody (±DSA) levels with mean fluorescence intensity and graft function were monitored along the first 24 months for all patients. There were no statistically significant differences between the two groups regarding early posttransplant graft function, patient and graft survivals. Pretransplant DSA with negative CXM carries a minimal clinical risk with optimized immunosuppression.

Comparative analysis of complement-dependent lymphocytotoxicity crossmatch and flow cytometry crossmatch results versus Luminex single-antigen bead-based donor-specific IgG class I antibody MFI values in live related renal transplant cases; a retrospective observation in 102 cases

ABSTRACT The aim of this study was to compare the results of solid phase assay and cell-based assay, and explore the near-accurate DSA-MFI-cutoff value detected on solid phase assay above which the cell-based assay would show a positive result. In this retrospective study, 102 prospective renal transplant recipients were tested for the presence of donor-specific antibodies (DSAs) by cell-based assay (T-cell-CDC-AHG-XM and T-cell-IgG-FC-XM) and solid phase assay (class-I-IgG-L-SAB) with their corresponding donor. Among the 40 patients in the group first (L-SAB-DSA-MFI<1000), one case was positive in IgG-T-cell-FC-XM while T-cell-CDC-AHG-XM was negative in all the cases. In the second group having L-SAB-DSA-MFI values between 1000 and 3000, 19 cases were positive and the remaining 11 cases were negative in IgG-T-cell-FC-XM. T-cell-CDC-AHG-XM showed a negative reaction in all 30 cases. In the third group having L-SAB-DSA-MFI values between 3000 and 5000, IgG-T-cell-FC-XM was positive in 18 cases while, two were negative. T-cell-CDC-AHG-XM demonstrated a negative result in 14 cases while reaming six cases demonstrated a positive result. In the fourth group having L-SAB-DSA-MFI values >5000, all 12 cases showed a positive result in both IgG-T-cell FC-XM and T-cell-CDC-AHG-XM. Our results indicated that the L-SAB-DSA-MFI values >2215 were significantly (P < .001) correlated with positive IgG-T-cell-FC-XM while L-SAB-DSA-MFI values >4689 were significantly (P < .001) correlated with positive CDC-XM.

Solid phase-based cross-matching for solid organ transplantation: Currently out-of-stock but urgently required for improved allograft outcome.

Transplant recipients who have undergone sensitizing events, such as pregnancy, blood transfusion or previous transplants, frequently develop antibodies directed against the highly polymorphous human leukocyte antigen (HLA)-molecules. These pre-formed, donor-specific antibodies (DSA) present a high risk of causing organ failure or even complete loss of the grafted organ as a consequence of antibody-mediated, hyper-acute or acute allograft rejection. In order to detect DSA, the so-called functional complement-dependent lymphocytotoxicity assay (CDC-XM) was established about 50 years ago. Although effective in improving the outcome of solid organ allo-grafting, for the last ten years this assay has been controversially discussed due to its low sensitivity and especially because of its high susceptibility to various artificial factors, which generally do not yield reliable results. As a consequence, novel immunochemical test systems have been developed using ELISA- or bead-based solid phase assays as replacements for the traditional CDC-based assays. Because these assays are independent of single or vital cells, which are frequently not available, they have provided an additional and alternative diagnostic approach compared with the traditional CDC-based and flow-cytometric analyses. Unfortunately, however, the AMS-ELISA (Antibody Monitoring System), which was the first system to become commercially available, was recently discontinued by the manufacturer after seven years of successful use. Alternative procedures, such as the AbCross-ELISA, had to be either considerably modified, or did not yield reliable results, as in the case of the Luminex-based assay termed DSA. We draw the conclusion that due to the unique features and fields of application reviewed here, the implementation of solid phase cross-matching still represents an urgent requirement for any HLA-laboratory's routine tasks.

Comparison of Cytotoxic Flow Cytometric Cross Match With Complement Dependent Lymphocytotoxicity and Flow Cytometric Cross Match in Renal Transplant Patients

Cytotoxic flow cytometric crossmatch (cFCXM), identified by detecting complement-mediated cytotoxic cell death in addition to the capability of showing the alloantibodies binding onto lymphocytes at the same time, can reduce the necessary time and workload in evaluating alloantibodies. More data from clinical samples are needed for cFCXM to be accepted by tissue typing laboratories. In this study, we compared cFCXM with complement-dependent lymphocytotoxicity and standard flow cytometric crossmatch in 41 renal pretransplant patients. A comparison of the obtained data was performed using Spearman's correlation test. We found that cFCXM showed no statistically significant differences with complement-dependent lymphocytotoxicity and flow cytometric crossmatch. We believe that cFCXM can be used in clinical laboratories in the near future following intra-laboratory validation.

Pretransplant Single Antigen Bead–Detected HLA Antibodies in Kidney Transplant Long-term Outcome: A Single-Center Cohort Experience

Single-antigen bead (SAB) platform permits the identification of antibodies not detectable by complement-dependent lymphocytotoxicity test, but their clinical significance is not completely understood. The aim of this study was to evaluate whether the presence of pretransplant SAB-detected antibodies is associated with the development of allograft failure.This is a single-center cohort study with 10-year follow-up in which 573 kidney recipients with negative pretransplant complement-dependent lymphocytotoxicity crossmatch who received transplants at the Kidney Transplant Center of Policlinico, Milan, from deceased donors between 1996 and 2005 were evaluated. Pretransplant plasma samples were retrospectively analyzed by SAB assay. Survival analyses were performed to assess the risk of allograft failures by SAB-detected antibodies.Pretransplant antibodies were found in 160 (28.0%) recipients, of whom 42 subsequently developed an allograft failure for a survival rate of 70.9% (95% confidence interval [CI), 63.5–78.4). Among those without antibodies, 58 (14.0%) returned to dialysis with a survival rate of 84.7% (95% CI, 81.0–88.4). In Cox regression analyses, patients with SAB-positivity had 2-fold higher risk of allograft failure than those who were SAB-negative (hazard ratio, 2.07; 95% CI, 1.39–2.79). Results did not change after adjustment for putative confounders.In conclusion, in this single-center cohort, 10-year allograft survival rate was significantly influenced by the presence of SAB-detected antibodies.

Donor-specific antibodies in kidney deceased donor transplantation

Aim Pre-sensitization against a broad array of HLA is associated with prolonged waiting times and inferior kidney allograft survival. Although the use of solid phase assay (SPA) for the detection and characterization of anti-HLA antibodies provides greater sensitivity than complement-dependent lymphocytotoxicity (CDC) assay, it often detects donor specific antibodies (DSA) which turn out to be clinically irrelevant. In this study, our aim is to determine whether kidney deceased donor recipients with preformed DSA detectable in serum obtained prior to transplant are at an increased risk of graft failure. Methods We studied 640 patients who received a kidney transplant from a deceased donor between January 1, 2009 and December 31, 2014 at Columbia University Medical Center. Sera collected from the recipients prior to transplantation were tested for DSA by CDC and SPA with single antigen coated beads. All patients were transplanted with negative CDC crossmatch. Results We analyzed the actuarial graft survival in patients with a primary allograft ( n = 525) and in patients with a secondary allograft ( n = 115). Kidney allograft survival was significantly higher in primary allograft recipients (85%) than in secondary allograft recipients (71%) at 6 years. ( P = 0.02) Forty-six out of 525 (8.7%) primary kidney allograft recipients and sixty-two out of 115 (53.9%) secondary kidney allograft recipients had SPA-detectable DSA in sera collected prior to transplantation. Six years following transplantation of primary kidney allografts the actuarial graft survival was 83% and 85%, respectively, regardless of whether DSA were detectable in recipients’ pre-transplantation sera. ( P = 0.39) In recipients of secondary kidney allografts with and without SPA-detectable DSA, the actuarial graft survival was 67% and 77% at 6 years, respectively. ( P = 0.12) Conclusions The presence of SPA-detectable DSA in recipients of kidney allografts from deceased donors is associated with lower long term (6 year) actuarial graft survival although the difference does not reach statistical significance.

Effects of Preoperative Positive Cross-Match and HLA Mismatching on Early Acute Cellular Rejection and Graft Survival in Living Donor Liver Transplantation

BACKGROUND We evaluated the effects of preoperative positive cross-match and HLA mismatching on early acute cellular rejection and graft survival in living donor liver transplantation (LDLT). MATERIAL AND METHODS We retrospectively reviewed data of 286 patients who underwent LDLT from 2008 to 2013. Cross-matching tests were performed by complement-dependent lymphocytotoxicity (CDC) and flow cytometry (FCX) methods. The CDC cross-matching test was performed using the National Institutes of Health (NIH) standard cross-match and antiglobulin (AHG) cross-match methods. RESULTS NIH, AHG, and FCX were positive in T-lymphocytes from 18 (6.3%), 21(7.3%), and 23 (8.0%) patients, respectively. T-CDC (T-NIH or T-AHG) results were positive in 23 (8.0%) patients. CDC and FCX results were positive in B-lymphocytes from 18 (6.3%) and 35 (12.2%) patients. All positive cross-match results were significantly associated with acute cellular rejection. Only a positive T-CDC cross-match was significantly associated with decreased graft survival (P=0.035). In a multivariate analysis, a positive T-CDC cross-match was the only independent risk factor with a decreased graft survival rate (P=0.041). An HLA mismatch was not associated with acute rejection (p=0.468 for HLA-A, p=0.644 for HLA-B, and p=0.811 for HLA-DR), graft survival (p=0.895 for HLA-A, p=0.580 for HLA-B, and p=0.969 for HLA-DR), and overall survival (p=0.862 for HLA-A, p=0.634 for HLA-B, and p=0.917 for DLA-DR). CONCLUSIONS Although a further prospective study with a larger cohort is required, it is not wise nor safe to perform LDLT in the setting of a positive T-CDC cross-match result.

Immunisation anti-érythrocytaire et anti-HLA au cours des hémoglobinopathies

AimEvaluate the anti-erythrocyte and anti-HLA immunization rates in hemoglobinopathies. Patients and methodsCross-sectional study (October 2009–March 2010) on 83 patients followed for hemoglobinopathies. The irregular antibodies research is realized by two techniques: indirect Coombs and enzymatic technique on gel cards. The search for anti-HLA class I antibodies is done by complement dependent lymphocytotoxicity. ResultsThe mean age was 30 years (14–64 years), the sex ratio M/F is 0.84. Our series included 42 cases of sickle cell disease (29 homozygous sickle cell anemia and 13 sickle-thalassemia) and 41 cases of thalassemia syndromes (26 major and 15 intermediate). The anti-erythrocyte alloimmunization rate is 10.84% without difference between thalassemia syndromes and sickle cell disease. The autoimmunization rate (22.89%) is higher in thalassemia syndromes (41.46%) than in the sickle cell disease (7.14%) (P<0.001). The anti-HLA immunization rate is 31.6% without difference between thalassemia syndromes and sickle cell disease. The young age, transfusion at a young age and the total number of transfusions are the factors that increase the risk of anti-erythrocyte autoimmunization. No clinicobiological parameter does influence the anti-erythrocyte and anti-HLA alloimmunization. There is no significant association between anti-erythrocyte and anti-HLA immunization. ConclusionThe erythrocyte and anti-HLA anti-immunization rates are high in our series. Preventive strategy is needed to ensure optimal blood safety.

P015 : EVALUATION OF THE RELATIONSHIP BETWEEN PRE-TRANSPLANT EVALUATION OF DSA BY VARIOUS METHODS AND INCIDENCE OF AMR IN KIDNEY ALLOGRAFT RECIPIENTS

Aim The aim of this study was to evaluate the prognostic value of the C1q-SPA assay in recipients of kidney from deceased donors. Methods The study population consisted of 123 recipients transplanted at Columbia University Medical Center between January 1 and December 31, 2012. None of the patients showed DSA that could be detected by complement dependent lymphocytotoxicity. Sera obtained from each patient prior to transplantation were evaluated for donor-specific anti-HLA IgG antibody (DSA) by Luminex single-antigen bead (SAB) testing (SAB-IgG). SAB-IgG positive sera were further tested by Luminex SAB-based assay detecting C1q-binding HLA antibodies (SAB-C1q). Results The one year kidney allograft survival was 96.6% in this patient population. Four recipients had an episode of AMR although 27 showed DSA detectable by SAB-IgG. C1q testing shows DSA in 7 sera among the SAB-IgG positive results (7/27, 26%), 3 of which were in patients with AMR and 4 in patients without AMR. Conclusions There was a positive correlation between the presence of C1q detectable DSA in pre-transplantation sera and development of AMR. However, the correlation between SAB-IgG, SAB-C1q and rejection was weaker, suggesting the need of further evaluation of methods that can predict the risk of AMR, without affecting the patients’ chances of receiving a transplant.

Su1394 Prediction of Sustained Remission After Discontinuation of Infliximab in Patients With Crohn's Disease

Background & Aim: Patients with Crohn's disease (CD) or ulcerative colitis (UC) who fail to respond to induction therapy with infliximab (IFX) are considered as primary non responders (PNR). We aimed at investigating the prevalence, prediction and management of PNR to IFX in patients with CD or UC. Patients & Methods: Retrospective analysis of prospectively acquired data at a single-center which included PNRs to induction therapy with 5 mg/kg IFX at weeks 0, 2 and 6. PNR was defined as no improvement or worsening of clinical symptoms through week 14 after starting IFX as judged by the CDAI and physician global assessment. Using RFLP or allele-specific PCR, single nucleotide polymorphisms in the promoter region of the TNF gene (-238 G/A, -308 G/A, and -857 C/T) and the -158 V/F polymorphism of the FcγRΙΙΙa gene were determined in 79 patients. In 48 patients, the HLA class II DRB1 and DQB1 alleles were also determined using the complement-dependent lymphocytotoxicity (CDC) and where available the sequence-specificoligonucleotide (SSO) and primer (SSP) HLA typing methods. Antinuclear antibodies and perinuclear anti-neutrophil cytoplasmic antibodies at baseline were also evaluated in 104 and 87 patients, respectively. Results: Between 1/7/2007 and 31/8/2012, of 160 IFX-treated patients [107 CD, 53 with UC, 94 males, median age at diagnosis 23 years (IQR 17-32)], 26 (16.3%) were PNRs [8/107 (7.5%) NC vs 18/53 (34%) UC, p<0.001]. Differences between CD and UC in PNR rates may be due to inclusion of patients with severe UC treated with IFX rescue therapy. CD/PNRs to IFX received adalimumab (ADA, n=4), azathioprine (AZA, n=1), methotrexate (MTX, n=1) or surgical treatment (n=2); 2 of these 4 ADA-treated CD patients developed also PNR to ADA but the other two achieved sustained clinical response (n=1) or remission (n=1) after escalation of ADA dose to 40mg weekly. UC/PNRs to IFX were mainly treated surgically (n=12); one of whom received ADA after surgery for refractory pouchitis, and 6 patients received AZA. Univariate and multivariate analysis (including variables with p<0.2) did not reveal any clinical, serological, demographical or genetic factors as predictors of PNR to IFX. PNR to IFX was the only predictor of PNR to ADA [ΟR=8.889 (95% CI: 2.30234.316), p=0,032] as at the end of the study, only one more patient [with secondary loss of response (SLR) to IFX] of the total 43 who were switched to ADA (of the initial pool of the 160 patients), due to PNR (n=5), SLR (n=21), intolerance (n=9), or adverse events (n= 8) to IFX, developed also PNR to ADA. Conclusion: This study which mirrors real life experience indicates that PNR to IFX in UC is more frequent than in CD; PNR to IFX predisposes to PNR to ADA. Larger prospective studies are needed to identify any predictive factors for PNR to anti-TNF therapy and the ideal management of this phenomenon.

Su1393 Primary Non Response to Infliximab in Patients With Inflammatory Bowel Disease

Background & Aim: Patients with Crohn's disease (CD) or ulcerative colitis (UC) who fail to respond to induction therapy with infliximab (IFX) are considered as primary non responders (PNR). We aimed at investigating the prevalence, prediction and management of PNR to IFX in patients with CD or UC. Patients & Methods: Retrospective analysis of prospectively acquired data at a single-center which included PNRs to induction therapy with 5 mg/kg IFX at weeks 0, 2 and 6. PNR was defined as no improvement or worsening of clinical symptoms through week 14 after starting IFX as judged by the CDAI and physician global assessment. Using RFLP or allele-specific PCR, single nucleotide polymorphisms in the promoter region of the TNF gene (-238 G/A, -308 G/A, and -857 C/T) and the -158 V/F polymorphism of the FcγRΙΙΙa gene were determined in 79 patients. In 48 patients, the HLA class II DRB1 and DQB1 alleles were also determined using the complement-dependent lymphocytotoxicity (CDC) and where available the sequence-specificoligonucleotide (SSO) and primer (SSP) HLA typing methods. Antinuclear antibodies and perinuclear anti-neutrophil cytoplasmic antibodies at baseline were also evaluated in 104 and 87 patients, respectively. Results: Between 1/7/2007 and 31/8/2012, of 160 IFX-treated patients [107 CD, 53 with UC, 94 males, median age at diagnosis 23 years (IQR 17-32)], 26 (16.3%) were PNRs [8/107 (7.5%) NC vs 18/53 (34%) UC, p<0.001]. Differences between CD and UC in PNR rates may be due to inclusion of patients with severe UC treated with IFX rescue therapy. CD/PNRs to IFX received adalimumab (ADA, n=4), azathioprine (AZA, n=1), methotrexate (MTX, n=1) or surgical treatment (n=2); 2 of these 4 ADA-treated CD patients developed also PNR to ADA but the other two achieved sustained clinical response (n=1) or remission (n=1) after escalation of ADA dose to 40mg weekly. UC/PNRs to IFX were mainly treated surgically (n=12); one of whom received ADA after surgery for refractory pouchitis, and 6 patients received AZA. Univariate and multivariate analysis (including variables with p<0.2) did not reveal any clinical, serological, demographical or genetic factors as predictors of PNR to IFX. PNR to IFX was the only predictor of PNR to ADA [ΟR=8.889 (95% CI: 2.30234.316), p=0,032] as at the end of the study, only one more patient [with secondary loss of response (SLR) to IFX] of the total 43 who were switched to ADA (of the initial pool of the 160 patients), due to PNR (n=5), SLR (n=21), intolerance (n=9), or adverse events (n= 8) to IFX, developed also PNR to ADA. Conclusion: This study which mirrors real life experience indicates that PNR to IFX in UC is more frequent than in CD; PNR to IFX predisposes to PNR to ADA. Larger prospective studies are needed to identify any predictive factors for PNR to anti-TNF therapy and the ideal management of this phenomenon.

Detection of donor-specific-antibodies by solid phase assay and its relevance to complement-dependent-lymphocytotoxicity cross-matching in kidney transplantation

Presensitization against a broad array of HLA is associated with prolonged waiting times and inferior kidney allogaft survival. Although the use of solid phase assay (SPA) for the detection and characterization of anti-HLA antibodies provides greater sensitivity than complement-dependent lymphocytotoxicity (CDC) assay, it often detects donor specific antibodies (DSA) which turn out to be clinically irrelevant. Our data reinforce the concept that these two types of assays should be used in parallel for pre-and post-transplantation monitoring of anti-HLA antibodies in recipients of solid organ allografts.

Sensitive Solid-Phase Detection of Donor-Specific Antibodies as an Aid Highly Relevant to Improving Allograft Outcomes

Transplant recipients who have had sensitizing events such as pregnancies, blood transfusions and previous transplants often develop antibodies directed against human leukocyte antigen (HLA)-molecules of the donor tissue. These pre-formed donor-specific antibodies (DSA) represent a high risk of organ failure as a consequence of antibody-mediated hyper-acute or acute allograft rejection. As a first assay to detect DSA, the complement-dependent lymphocytotoxicity assay (CDC) was established more than 40years ago. However, this assay is characterized by several drawbacks such as a low sensitivity and a high susceptibility to various artificial factors generally not leading to valid and reliable outcomes under several circumstances that are reviewed in this article. Furthermore, only those antibodies that exert complement-fixing activity are detected. As a consequence, novel procedures that act independently of the complement system and that do not represent functional assays were generated in the format of solid phase assays (SPAs) (bead- or ELISA-based). In this article, we review the pros and cons of these sensitive SPA in comparison with the detection of DSA through the use of the traditional methods such as CDC and flow cytometric analyses. Potential drawbacks of the alternative methodological approaches comprising high background reactivity, susceptibility to environmental factors and the possible influence of subjective operators' errors concerning the interpretation of the results are summarized and critically discussed for each method. We provide a forecast on the future role of SPAs reliably excluding highly deleterious DSA, thus leading to an improved graft survival.

Clinical impact of the baseline donor-specific anti-human leukocyte antigen antibody measured by Luminex single antigen assay in living donor kidney transplant recipients after desensitization therapy

The aim of this study is to investigate the clinical impact of donor-specific anti-HLA-antibody (HLA-DSA) baseline levels, measured using the Luminex single antigen assay (LSA), in living donor kidney transplantation (LDKT). Total 129 cases of LDKT were divided into four groups according to baseline mean fluorescence intensity (MFI) HLA-DSA values: Strong (n = 6), >10,000; Moderate (n = 8), 5,000-10,000; Weak (n = 11), 1,000-5,000, Negative (n = 104), <1,000. Pretransplant desensitization (DSZ) was performed to decrease the MFI to weak or negative values before KT. Clinical outcomes in the four groups were compared. After DSZ, HLA-DSA decreased to weak or negative levels in all patients; Acute rejections developed more frequently in strong group [5/6 (83.3%)] compared with other three groups (P < 0.05), and especially acute antibody-mediated rejection (AAMR) developed almost exclusively in strong group [4/6 (66.7%)]. Strong HLA-DSA levels at baseline were more predictive of AAMR than either type of XM (complement-dependent lymphocytotoxicity or flow cytometry) in ROC analysis. Allograft function in this group showed significant deterioration during follow-up compared with the other groups. In conclusion, strong HLA-DSA levels at baseline are associated with worse allograft outcome even after successful desensitization; therefore, strict monitoring and strong maintenance immunosuppression may be required in such patients.

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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

Structural aspects of HLA class I epitopes reacting with human monoclonal antibodies in Ig-binding, C1q-binding and lymphocytotoxicity assays

This study addresses the reactivity patterns of human cytotoxic HLA class I epitope-specific monoclonal antibodies in Ig-binding and complement component C1q-binding Luminex assays in comparison with complement-dependent lymphocytotoxicity data reported at the 13th International HLA Workshop. Some monoclonal antibodies reacted similarly with epitope-carrying alleles in all three assays but others showed different reactivity patterns. These reactivity differences were analyzed with HLAMatchmaker and we incorporated the concept that eplets are essential parts of structural epitopes which can contact the six Complementarity Determining Regions (CDRs) of antibody. The data show that technique-dependent reactivity patterns are associated with distinct differences between polymorphic amino acid configurations on eplet-defined structural epitopes. The findings have been viewed in context of antigen-antibody complex formation that results in the release of free energy necessary to stabilize binding and to induce conformational changes in the antibody molecule to expose the C1q binding site, the first step of complement activation. Moreover the amount of free energy should be sufficient to induce a conformational change of C1q thereby initiating the first stages of the classical complement cascade leading to lymphocytotoxicity. The complement-fixing properties of HLA antibodies require not only specific recognition of eplets but also depend on interactions of other CDRs with critical amino acid configurations within the structural epitope. Eplet-carrying alleles that lack such configurations may only bind with antibody. This concept is important to our understanding whether or not complement-fixing donor-specific HLA antibodies can initiate antibody-mediated rejection.

Superiority of AbCross Enzyme-Linked Immunosorbent Assay Cross-Match Over the B-Cell Complement-Dependent Lymphocytotoxicity Cross-Match

BackgroundThe AbCross enzyme-linked immunosorbent assay (ELISA) cross-match is a recently introduced solid phase cross-match technique with several technical advantages over the currently available Antibody Monitoring System ELISA cross-match. MethodsIn the present study, we investigated the potential superiority of AbCross over the traditional complement-dependent lymphocytotoxicity (CDC) B-cell cross-match (BXM). Pretransplant sera of 271 kidney transplant recipients who were transplanted at our center between 1998 and 2010 were tested in ELISA screening for the presence of human leukocyte antigen (HLA) antibodies and in AbCross and CDC for antibody reactivity against solubilized donor HLA class I and II antigens and donor B cells, respectively. ResultsPatients positive for HLA class I or II antibodies on ELISA screening had a significantly poorer graft outcome 2 years after transplantation than recipients who were negative for HLA antibodies (21% vs 6% graft loss; P = .002). Corresponding with this finding, 37 recipients positive for HLA antibodies in AbCross against donor HLA class I or II antigens had a 2-year post-transplant graft loss rate of 19%, which is significantly higher than the 8% rate in 186 recipients who were negative for both antibody classes in AbCross (P = .043). The 2-year graft loss rate in 34 AbCross positive but BXM negative patients was 21%, compared with 7% in 172 AbCross and BXM negative patients (P = .012) and 9% in 11 AbCross negative but BXM positive patients (P = .39). ConclusionsOur data indicate that the AbCross ELISA cross-match is superior to the CDC BXM, most likely because it detects antibodies against donor HLA antigens at a higher sensitivity.

Systematic Comparison of Four Cell- and Luminex-Based Methods for Assessment of Complement-Activating HLA Antibodies

Efforts to increase the specificity and sensitivity of human leukocyte antigen (HLA) antibody detection assays recently led to the establishment of two novel Luminex bead-based assays to detect complement-activating antibodies by the assessment of complement products C1q or C4d. Here, we present a systematic comparison of the four methods, complement-dependent lymphocytotoxicity (CDC) and C1q-, C4d-, and IgG-Luminex, to assess or predict the complement-binding capability of HLA IgG antibodies. Forty-five sera of highly immunized patients have been assessed by in-house modified C1q- and C4d-Luminex assays and compared with standard CDC and IgG-Luminex. Antibody specificities assigned by the C1q- and C4d-Luminex assay revealed an excellent concordance of 94% and 97% for HLA class I and II, respectively. Complement-fixing HLA class II antibodies were found less frequently among IgG antibodies compared with class I. Both C1q- and C4d-Luminex detected, on average, three times more specificities than CDC. Although we found a high correlation of mean fluorescence intensity values between C1q- and C4d-Luminex assays, IgG mean fluorescence intensity was not a suitable surrogate marker for the prediction of complement binding. C1q- and C4d-Luminex assays are characterized by an increased sensitivity and specificity compared with CDC, the current standard in detecting complement-fixing HLA antibodies. Pretransplantation risk assessment for transplantation but also posttransplantation monitoring are important applications for both assays to improve overall allograft survival.