Development of ELISA-detected anti-HLA antibodies precedes the development of bronchiolitis obliterans syndrome and correlates with progressive decline in pulmonary function after lung transplantation

BACKGROUND: Development of anti-HLA antibodies after lung transplantation (LT) is thought to play an important role in the etiology of bronchiolitis obliterans syndrome (BOS). However, a cause-effect relationship between anti-HLA antibodies and BOS has not been established. This study was conducted to determine the temporal relationship between the development of anti-HLA antibodies and BOS after LT, and to determine the antigenic specificity of the antibodies developed in BOS patients. METHODS: Sera from 15 BOS+ LT patients and 12 BOS- LT patients were obtained before LT and collected again at 6, 12, 24, 36, and 48 months after LT. Anti-HLA antibodies were detected by the PRA-STAT ELISA system and by complement-dependent cytotoxicity assays. Anti-HLA reactivity was further characterized by flow cytometry and absorption/elution with human platelets. RESULTS: When analyzed by ELISA, 10 of 15 BOS+ patients developed anti-HLA antibodies, whereas 0 of 12 BOS- patients developed anti-HLA antibodies (P<0.001). When analyzed by complement-dependent cytotoxicity, only 2 of 15 BOS+ patients developed anti-HLA antibodies and 1 of 12 BOS- patients developed anti-HLA antibodies (P = 0.99). There was a significant difference of 20.1 months between the time of anti-HLA antibody detection and the time of BOS diagnosis (P = 0.005). A progressive decrease in pulmonary function correlated with a progressive increase in the anti-HLA reactivity 36 months after LT. The anti-HLA reactivity was directed to one of the donor HLA class I antigens and to other unrelated HLA class I antigens. No anti-HLA reactivity was found against HLA class II molecules. CONCLUSIONS: Our study indicates that anti-HLA class I antibodies play an important role in the pathogenesis of BOS and that monitoring of anti-HLA class I antibody development by a highly sensitive assay such as the PRA-STAT ELISA after LT can provide an early identification of an important subset of LT patients with an increased risk of developing BOS.     

Frequency of Human Leukocyte Antigens and Donor Specific Antibodies in Long-Term Living Donor Kidney Transplantation

PurposeHLA antibodies have been shown to be associated with late graft loss. In this study, we defined the incidence and profiles of anti-HLA antibodies and their impact on graft outcome in long-term kidney recipients.MethodsThe sera of 118 kidney transplant recipients were screened for anti-HLA antibody presence. The antigen specificity of the detected HLA class I and class II antibodies was identified using a Luminex assay (Luminex Corp, Austin, TX, United States). Presence of donor specific antibodies (DSA) was examined in individuals with anti-HLA antibodies using the Luminex method.ResultsAnti-HLA class I and/or class II antibodies were detected in serum of 16.1% of the kidney transplant patients. The antibodies were directed against HLA class I antigens in 4 patients (21.1%), HLA class II antigens in 9 patients (47.4%), and both class I and class II antigens in 6 patients (31.6%). The overall prevalence of DSA was 10.2%. Anti-HLA antibodies were significantly associated with higher rate of cyclosporine use. Presence of DSA was associated with a lower rate of tacrolimus use, a higher rate of cyclosporine use, and lower donor age. Presence of anti-HLA antibodies was associated with higher acute cellular rejection and higher chronic active humoral rejection rates. Presence of DSA was associated with chronic active humoral rejection.ConclusionThe presence of either HLA antibodies or DSA significantly correlated with lower graft survival, poor transplant function, and proteinuria.     

The effect of antithymocyte globulin on anti-human leukocyte antigen antibody detection assays

BACKGROUND: We evaluated the effect of antithymocyte globulin (ATG) on anti-human leukocyte antigen (HLA) antibody assays. METHODS: We tested sera from six in vivo ATG-treated kidney transplant patients after measuring serum concentrations, as well as six nonsensitized sera with ATG added in vitro. T- and B-cell complement-dependent cytotoxicity (CDC), flow cytometric (FXM), and solid-phase HLA class I and II assays based on antigen-coated microspheres and enzyme-linked immunosorbent assay (ELISA) were studied. Sera were then retested after treatment to remove ATG. RESULTS: We found that ATG affects test results differently depending on whether sera is obtained from in vivo treated patients or added in vitro. In vitro treated sera produced ATG concentration-dependent positive results for T/B CDC, FXM, and flow bead testing for HLA I/II, while the ELISA-based assay was unaffected. In vivo treated sera from ATG-treated patients produced positive test results for T CDC and T/B FXM, while the B-cell CDC crossmatch remained negative. Solid phase assays were minimally affected using in vivo treated sera. After ATG extraction, all tests became negative. CONCLUSION: We conclude that ATG produces positive results in anti-HLA antibody testing, and treatment to remove ATG abolishes this effect. This treatment allows ATG-treated patients to be monitored for anti-HLA antibodies.     

Detection of anti-HLA antibody by flow cytometry in patients with a left ventricular assist device is associated with early rejection following heart transplantation

BACKGROUND: Patients with a left ventricular assist device (LVAD) as a bridge to heart transplantation (HT) often have elevated levels of panel reactive antibodies (PRA). The clinical significance of anti-human histocompatibility leukocyte antigen (HLA) antibodies detected by flow cytometry in PRA negative patients remains unclear. METHODS: Eighteen patients who underwent LVAD placement as a successful bridge to HT had standard anti-human globulin complement-dependent cytotoxicity and retrospective flow cytometry assays performed to detect class I anti-HLA antibodies. A positive flow result was defined as a fluorescent ratio of 23:1 versus a negative control. RESULTS: Six patients had anti-HLA antibodies detected by flow cytometry. Univariate analysis demonstrated more moderate-severe rejection episodes (ISHLT > or = IIIA) at 2 months (0.83+/-0.75 vs. 0; P=0.04) and a trend toward decreased time to first rejection (61+/-17 vs. 225+/-62 days; P=0.06) in these patients. No differences were observed in donor-recipient HLA mismatch or 1 year Kaplan-Meier survival between patients with or without anti-HLA antibodies. CONCLUSION: Despite a negative PRA, LVAD patients with class I anti-HLA antibodies detected by flow cytometry have a greater incidence of moderate-severe rejection in the first 2 months after HT. Flow cytometry may be a useful clinical tool in screening PRA negative LVAD patients before transplantation. Patients with positive anti-HLA antibody screening by flow cytometry may require more intensive immunosuppression in the early post-HT period.     

Reappraisal of HLA Antibody Analysis and Crossmatching in Kidney Transplantation

Enzyme-linked immunosorbent assay (ELISA) and flow cytometric techniques have been introduced to overcome the limited sensitivity and specificity of the CDC assay. This retrospective study used lambda antigen tray-mixed screening and Luminex HLA class I and II specificity assays to re-examine: (1) the accuracy with which detection of HLA antibody and specificity by ELISA predicts pretransplantation National Institutes of Health (NIH)/Centers for Disease Control and Prevention (CDC) crossmatch; and (2) a comparison of Luminex and ELISA methods to detect HLA antibodies. Sera from 481 patients awaiting kidney transplantation were tested using the ELISA method lambda antigen tray-mixed and using NIH-CDC to determine how well HLA antibodies detected using ELISA predicted crossmatches using CDC. Pretransplantation sera from 48 patients with follow-up data were retested using both ELISA lambda antigen tray-mixed and Luminex to compare the efficacy of the 2 methods.     

C4d Fixing, Luminex Binding Antibodies—A New Tool for Prediction of Graft Failure After Heart Transplantation

The standard method to detect pretransplant antibodies has been the complement dependent cytotoxicity (CDC) test of donor leukocytes. Solid phase assays to detect HLA antibodies in pretransplant serum reveal a greater number of sensitized patients, but their clinical impact is less certain. Here we have developed a method of detecting C4d fixing HLA antibodies on Luminex beads. Pretransplant serum from 565 cardiac transplant patients was retrospectively tested for the presence of HLA antibodies using CDC, HLA coated Luminex beads and C4d deposition on Luminex beads, and the results correlated with graft survival. Whereas 5/565 patients had CDC positive donor specific antibodies (DSA) before their transplant, this number was increased by 19 using Luminex beads. The 1-year survival of CDC -ve/Luminex +ve patients with DSA (n = 19) was 42% compared with 77% for CDC -ve/Luminex +ve without DSA (n = 39, p = 0.0039). Fixation of C4d (22/67 Luminex positive sera) had a negative effect on graft outcome; 1-year graft survival was, C4d +ve/DSA +ve (n = 11) 20%, C4d +ve/DSA -ve (n = 11) 91%, C4d -ve DSA +ve (n = 13) 54%, C4d -ve DSA -ve (n = 32) 75%, compared with 75% for antibody-negative patients (p = 0.0002). In conclusion, detection of Luminex +ve DSA in pretransplant serum provides a powerful negative predictor of graft survival, especially if they bind C4d.     

Detection of antibodies against HLA-C epitopes in patients with rejected kidney transplants

BackgroundAlthough HLA-C matching is not considered in kidney transplantation, several reports have shown that anti-HLA-C antibodies are associated with rejection and graft failure. DNA-based typing methods can now accurately determine HLA-C compatibility and sensitive assays such as Luminex with single alleles can identify HLA-C antibodies. HLA-C displays considerable amino acid polymorphism that can be translated into a structurally defined epitope repertoire.MethodsWe have analyzed post-allograft nephrectomy sera from 45 HLA-C mismatched cases submitted by 15 laboratories worldwide participating in the 15th International Histocompatibility Workshop. All of them had HLA class I antibodies detected by a Luminex-based solid phase method using single-allele beads. This study addressed the determination of antibodies against donor HLA-C mismatches. Analysis of antibody reactivity patterns was performed using HLAMatchmaker, a structurally based matching program that considers 56 HLA-C eplets to define antibody-reactive epitopes. Many eplets shared by groups of HLA-C antigens, whereas others are also shared with HLA-A and/or HLA-B antigens.ResultsTwenty-seven patients (60%) had donor-specific HLA-C antibodies, significantly less than the donor-specific antibodies induced by HLA-A and HLA-B mismatches. HLA-C antibody responses correlated with the eplet loads of the HLA-C mismatches. There were 352 instances whereby a donor HLA-C eplet was mismatched and for 84 (24%) of them there was antibody reactivity with a particular eplet (69 instances) or an eplet pair (15 instances). The latter generally consisted of mismatched eplets paired with self-eplets shared between the immunizing HLA-C alleles and HLA alleles of the patient. Several HLA-C eplets exhibited a relatively high immunogenicity as evidenced by their frequencies of specific antibodies.ConclusionThese findings demonstrate the importance of HLA-C mismatching in humoral sensitization and that HLA-C epitopes can induce specific antibodies. They illustrate the usefulness of HLAMatchmaker in understanding donor-specific antibody reactivity patterns and the determination of HLA mismatch acceptability when transplantation is considered.     

Predictive value of mixed antigen screen beads in pre-transplant assessment of HLA immunization in solid organ transplant recipients

Pre-transplant serum screening of anti-HLA antibodies is recommended for solid organ transplantations. Many laboratories use the less expensive bead-based screening assay as the main technique and, if positive, turn to single-antigen beads (SAB). We studied the correlations between these two immunoassays. We re-analyzed the raw data of the two assays in 3030 first organ transplant recipients, explored with the two tests. We performed a ROC curve analysis of the screening ratio to predict a positive SAB assay. The AUC were 0.72 and 0.64 for class I and class II. The optimal thresholds of screening ratios were 3.28 (class I) and 2.11 (class II). Whatever the class, the negative predictive value was low, around 40%, with 36% of discordant sera, as defined by negative screening and positive SAB. Testing class I discordant sera on acid-treated SAB showed that 54% of antibodies reacted against denatured HLA molecules. However, these screening-negative sera may contain donor-specific antibodies in 13.9% and 28.7% of cases for class I and class II, respectively, involved in antibody-mediated rejection with the same frequency as non-discordant sera. Given the low predictive value of screening, both assays should be performed at least once on the same serum before transplantation.     

Detection of donor-specific anti-HLA class I and II antibodies using antibody monitoring system

The antibody monitoring system (AMS, GTI Inc) is a solid enzyme-linked immunosorbent assay (ELISA) crossmatch test for the detection of immunoglobulin G (IgG) antibody to donor-specific solubilized HLA class I and class II antigens. The objective of this study was to compare the results of the AMS assay with donor-specific anti-HLA IgG antibodies (DS-HLA Abs), as determined by ELISA panel reactive antibody (PRA) and the flow cytometric crossmatch test (FCXM). A total of 107 sera were screened for the presence of HLA Abs by ELISA PRA (LAT-M, One-Lambda Inc), the DS-HLA Abs were determined in 34 serum samples (31.8%) by an ELISA panel (LAT class I and class II, One-Lambda Inc) and FCXM. The FCXM and AMS assays were performed with matched lymphocytes from 56 donors. There was a significant degree of concordance (89.7%) between the two tests (P < .001). The sensitivity, specificity, positive predictive value, and negative predictive value of AMS assay to detect DS-HLA Abs was 88.2%, 94.5%, 88.2%, and 94.5%, respectively. The AMS is a simple, objective test, which has several advantages over the cell-based crossmatch test, such as elimination of non-HLA antibody reactivity, elimination of non-donor-specific antibody reactivity, no need for viable cells, and preparation of the donor's HLA antigens in advance. In summary, this study suggested that AMS may be useful as a supportive crossmatch test or as a monitoring test after transplantation to detect class I or class II DS-HLA Abs.     

Prevalence of donor-specific anti-HLA antibodies during episodes of renal allograft rejection

BACKGROUND: Recent studies suggest that the appearance of anti-HLA antibodies in the early posttransplant period is associated with an increased incidence of acute and chronic rejection months later. However, very little is known about the prevalence of anti-HLA antibodies at the time that the rejection episodes are diagnosed. The purpose of this study was to analyze retrospectively 420 sera from 263 renal allograft recipients who were readmitted to the hospital for any reason between 1989 and 1998 in order to determine if a correlation existed between the presence of donor-specific anti-HLA antibodies and graft rejection. METHODS: Sera were assayed for IgG HLA class I and II antibodies by ELISA. The ELISA results were analyzed using contingency tables with Fisher's exact test and compared with mismatched antigens in the donor. RESULTS: Antibodies to donor HLA class I molecules in the posttransplant sera were extremely rare, occurring in only 6 of the 420 sera (1.4%) analyzed. Antibodies to donor class II antigens were slightly more common, occurring in 25 of the 420 sera (6%). In 21 of these 25 cases (84%), the presence of donor-specific HLA class II antibodies was associated with episodes of either acute (n=14) or chronic rejection (n=7). Five patients had antibodies to both class I and class II donor antigens, and all five of them lost their grafts to rejection. CONCLUSION: Although the presence of donor-specific HLA antibodies presented a significant risk for acute or chronic rejection, 77% of all acute and chronic rejections occurred in patients without detectable HLA antibodies.     

Luminex and antibody detection in kidney transplantation

Preformed anti-human leukocyte antigen (HLA) antibodies have a negative effect on kidney transplantation outcome with an increased rejection rate and reduction in survival. Posttransplantation production of donor-specific anti-HLA antibodies is indicative of an active immune response and risk of transplantation rejection. For many years the primary technique for anti-HLA antibody detection was complement-dependent cytotoxicity (CDC), which has been integrated by solid-phase assays as HLA antigen-coated bead methods (Luminex). This new technological approach has allowed identification of anti-HLA antibodies, not detectable using conventional CDC method, in patients awaiting kidney transplantation. Moreover, use of Luminex technology has enabled better definition of acceptable or unacceptable antigens favoring transplantation in highly immunized patients. However, there are still many unresolved issues, including the clinical relevance of antibodies detected with this system.     

Advances in pre- and posttransplant immunologic testing in kidney transplantation

Pre- and posttransplant risk estimation in kidney transplantation is important for the selection of appropriate treatment strategies. Recently, using new immunologic tests, we made observations within the framework of the Collaborative Transplant Study that may influence clinical practice. Complement-dependent lymphocytotoxic panel reactivity as a measure of anti-HLA sensitization, although criticized for its low sensitivity, is a useful indicator of an increased risk of rejection. Using the more sensitive complement-independent ELISA methodology, which utilizes solubilized HLA molecules instead of lymphocytes, we found that recipients with preformed complement-dependent anti-HLA antibodies showed a decreased graft survival only if their antibodies were directed against both HLA class I and class II, whereas isolated reactivity only against HLA class I or class II was of no clinical consequence. Pretransplant serum-soluble CD30 (sCD30) was found to be an independent and highly predictive factor of immunologic risk. The effects of sCD30 and anti-HLA antibodies were additive. Importantly, even patients without anti-HLA antibodies showed a strong HLA matching effect if their pretransplant serum contained high levels of sCD30. Although the role of anti-HLA antibody formation after transplantation remains uncertain, ELISA-detected sCD30 was shown to indicate impending graft rejection as early as on posttransplant days 3 to 5. Another very sensitive indicator of impending rejection is provided by posttransplant monitoring of the cytotoxic T-lymphocyte effector genes, perforin and granzyme B, in peripheral blood using real-time PCR.     

Eighteen-Year Follow-Up of a Retrospective Study of HLA Antibody on Kidney Graft Survival

An increasing number of studies have demonstrated adverse graft survival in patients who have anti-HLA antibodies, whether preformed or developed posttransplantation. This retrospective study used Lambda antigen tray-mixed (LAT-M) screening and Luminex HLA class I and II specificity assay to re-examine the impact of pretransplantation HLA antibody on long-term graft survival. In this study, pretransplantation sera from 288 renal patients were tested using the enzyme-linked immunosorbent assay (ELISA) method, LAT-M. Among the 234 of the patients who did not have pretransplantation antibodies, 85% enjoyed 5-year functional graft survival, 76% 10-year functional graft survival, and 56% 15-year functional graft survival. The corresponding functional graft survival for the 54 patients who tested HLA antibody-positive was 65%, 53%, and 28%, respectively (P = .0021).     

Identification of the antibodies involved in B-cell crossmatch positivity in renal transplantation

BACKGROUND: The significance of a positive B-cell crossmatch (BCM) in kidney transplantation has always been controversial in the evaluation of its implications on graft survival and specificity of the antibodies involved. METHODS: We have investigated the sera of 62 recipients of a kidney allograft transplanted across a positive BCM (T negative) for the presence of autoantibodies and anti-human leukocyte antigen (HLA) class I and II antibodies, using a combination of lymphocytotoxicity, enzyme-linked immunosorbent assay (ELISA), and flow cytometry tests. The controls were the 930 patients transplanted over the same period of time with a negative T and BCM. RESULTS: Autoantibodies were detected in 16%, and donor specific anti-HLA class II antibodies, mainly DQ, in 23% of the patients. None had antibodies against donor HLA class I. The target of the antibodies was not identified in 61%. Graft survival was comparable in the controls and in the +BCM patients, with nondonor-specific HLA reactivity. Patients with donor-specific anti-HLA class II antibodies had lower early graft survival and a higher incidence of vascular rejection. However, long-term allograft survival was similar to that of the other groups. CONCLUSION: These data suggest that in 77% of the patients, BCM positivity was not related with anti-HLA antibodies, and, in this case, graft survival was similar to that of the -BCM controls. In a minority of patients, anti-HLA class II antibodies were responsible for the +BCM, and their presence was associated with lower early, but not long-term, graft survival. Consequently, a +BCM should not systematically contraindicate kidney transplantation.     

Kidney graft failure and presensitization against HLA class I and class II antigens

BACKGROUND: It is well known that kidney transplant recipients with preformed lymphocytotoxic antibodies against HLA antigens have an increased graft rejection rate. However, the individual contribution of anti-HLA class I and class II antibodies to this phenomenon is poorly understood. We investigated the clinical relevance of preformed anti-HLA class I and class II antibodies on graft outcome in more than 4000 kidney recipients. METHODS: Pretransplant sera of 4136 cadaver kidney recipients from 28 transplant centers were tested in ELISA for IgG-anti-HLA class I and IgG-anti-HLA class II antibodies. The influence of antibody reactivity on graft survival was analyzed. RESULTS: Four hundred eighty of the anti-HLA class I antibody-positive recipients had a graft survival rate at 2 years of 77+/-2%, compared with an 84+/-1% rate in 3656 anti-HLA class I antibody-negative recipients (P<0.0001), and 770 anti-HLA class II-positive recipients had a graft survival rate of 79+/-2%, compared with an 84+/-1% rate in 3366 anti-HLA class II-negative patients (P<0.0001). Importantly, good 2-year graft survival rates of 85+/-3% and 84+/-2%, respectively, were observed in 206 anti-HLA class I-positive/class II-negative and 496 anti-HLA, class I-negative/class II-positive recipients. In contrast, the 274 recipients positive for both types of antibodies showed a poor graft survival rate of 71+/-3% (P<0.0001). Among 853 patients who received a well-matched kidney (0 or 1 HLA-A+B+DR mismatch), sensitization against either class I or class II, or both, had no deleterious effect. However, in 113 class I and class II antibody-positive patients who received a kidney with > or =3 HLA-A+B+DR mismatches, the 2-year graft survival rate was only 60+/-5%. CONCLUSION: Presensitization of first kidney transplant recipients against either HLA class I or class II is of no clinical consequence, whereas sensitization against both HLA class I and class II results in increased rejection of HLA mismatched grafts.     

Anti-idiotypic antibodies from highly sensitized patients stimulate B cells to produce anti-HLA antibodies

BACKGROUND: Sustained allosensitization increases waiting time for transplantation and increases the risk of rejection. The purpose of this study is to examine the effect of anti-idiotypic antibodies on B-cell responses and to define their role in alloantibody production. METHODS: The Immunoglobulin G (IgG) fraction, or the sera of 19 highly sensitized (HS) patients was absorbed to remove anticlass I antibody and was incubated with B cells. The culture supernatant was assayed for antihistocompatibility leukocyte antigen (HLA) antibody and tested for reactivity against a panel of normal lymphocytes. Similar studies were performed in 5 of the 19 patients who had a fall in alloantibody levels. RESULTS: The IgG (HS) fraction induced anti-HLA antibody from normal and autologous B cells in all 19 HS patients studied. The reactivity to HLA antigens in the culture supernatant was similar to the sera for each patient studied. The in vitro generated anti-HLA antibody bound to the IgG fraction used to stimulate the B cells. The in vitro production of anti-HLA antibodies was absent in the serum of all five patients who became nonsensitized. CONCLUSIONS: All patients who have high levels of alloantibody have anti-idiotypic antibodies in their sera that stimulate B cells to produce anti-HLA class I antibody similar in reactivity to that of their own sera. In the patients who have nondetectable alloantibodies in their sera, the stimulating anti-idiotypes are not measurable. Anti-idiotypic antibodies may act as a vaccine and cause sustained levels of alloantibody production.     

C4d binding correlated with strong HLA antibodies involved in graft failures

Capillary C4d was an established marker of antibody-mediated rejection in kidney transplantation. Recently, C4d fixation to antibodies against HLA-coated beads were found to be a novel way to test for complement-fixing antibodies in the serum. To find differences between complement-fixing and nonfixing antibody assays, we tested HLA antibodies and C4d in 102 sera of graft-failure patients using the LABScreen and FlowPRA bead methods. HLA antibodies were observed in the sera of patients who subsequently failed and who continued to function. C4d fixing antibodies were present in the sera of patients who subsequently experienced graft failures and those with high fluorescence intensity of HLA antibodies by FlowPRA. In antibody dilution studies with LABScreen, we noted that C4d fixation occurred only at high antibody titers, and fluorescence was lost as sera were diluted. In conclusion, the fluorescence levels of antibodies were more important that the C4d content. Quantitative aspects of antibody testing using the fluorescence value appears to be a valuable factor when examining anti-HLA antibodies posttransplantation.     

Effect of high-dose intravenous immunoglobulin on anti-HLA antibodies in sensitized kidney transplant candidates

Limited data exist on the effect of intravenous immunoglobulin (IVIg) on anti-HLA antibodies as determined by solid-phase assays. We reviewed our experience treating sensitized wait-listed kidney transplant recipients with IVIg as a method for desensitization and report our results utilizing Luminex single antigen (LSA) bead assay to quantify antibody reactivity (MFI). Fifteen patients with a cPRA > 40% received 2 g/kg IVIg per month for four months or until transplanted. LSA testing was performed before and after IVIg. Median MFI for anti-class I antibodies fell in 11 (73%) and increased in 4 (27%) patients after IVIg. Similar significant changes in MFI for anti-class II antibodies were observed in 10 patients (66%). Administration of IVIg was associated with a modest decrease in reactivity to both class I and II HLA antigens (median MFI change 493 and 1110, respectively; p < 0.0001) but did not significantly alter mean cPRA (85% before IVIg vs. 80% after IVIg; p = 0.1). Our data suggest a smaller effect of IVIg on HLA antibody reactivity than previously described, leading us to question how best to measure the efficacy of a desensitization protocol in current practice.     

Assignment of C1q-binding HLA antibodies as unacceptable HLA antigens avoids positive CDC-crossmatches prior to transplantation of deceased donor organs

Soon, a virtual crossmatch shall replace the complement-dependent cytotoxicity (CDC) allocation crossmatch in the Eurotransplant region. To prevent positive CDC-crossmatches in the recipient centre, careful definition of unacceptable antigens is necessary. For highly sensitized patients, this is difficult by CDC alone. Assignment of all antibodies detected by sensitive assays, however, could prevent organ allocation. To assess the usefulness of the Luminex C1q-assay to prevent positive CDC-crossmatches, all CDC-crossmatches performed prior to deceased kidney transplantation in a 16-month-period were reviewed. Sera causing positive crossmatches were investigated by the C1q-assay. 31 out of 1432 crossmatches (2.2%) were positive. Sera involved in 26 positive crossmatches were available. C1q-binding donor-specific antibodies were detected in 19 sera (73.1%). The other sera were from recipients without any HLA antibodies detectable by CDC or common solid phase assays. Three patients had known Non-HLA antibodies causing positive CDC-results. Four crossmatches were only weak positive. Therefore, avoidance of donors with HLA antigens against whom C1q-binding antibodies were detected would have prevented all positive crossmatches due to HLA antibodies. Provided that all HLA specificities against which antibodies are detected by the Luminex C1q-assay are considered as unacceptable antigens, CDC-crossmatches prior to transplantation might safely be omitted in many patients. They should be maintained in highly immunized patients, however, for whom assignment of all C1q-positive antibodies as unacceptable antigens could lead to a significant delay or even prevention of transplantation.     

Sensitization of renal transplant candidates by cryopreserved cadaveric venous or arterial allografts

BACKGROUND: Cryopreserved cadaveric venous or arterial allografts are used in ESRD patients as an alternative to synthetic grafts for hemodialysis access. We evaluated the effect of these allografts on the PRA against HLA class I and II antigens in 11 ESRD patients awaiting a kidney transplant. METHODS: Flow Cytometry using purified antigen coated beads (Flow PRA Beads) was used to determine PRA against HLA class I and II antigens. RESULTS: Patients with no antibody prior to allograft implantation were all positive for HLA class I and II antibodies after implantation. Patients with anti-HLA class I and II antibodies prior to allografting, developed higher antibody titers. Of the 11 patients that received cryopreserved cadaveric allografts no deaths were reported. Two grafts were removed due to thrombosis consistent with rejection. CONCLUSION: Recipients of cadaveric venous or arterial allografts elicit an HLA antibody response attesting to the antigenicity of cryopreserved cadaveric allografts.