The correlation of results of panel reactive antibody,identification, and single antigen beads in detection of anti-HLA antibodies: Istanbul Faculty of Medicine,tissue typing laboratory experience

BackgroundWe have performed a retrospective analysis of anti-HLA class I MHC and class II MHC antibodies measured using a single antigen bead (SAB) assay and a panel reactive antibody (PRA) assay.Material and methodsA group of 256 patients with end-stage renal disease (ESRD) was tested for anti-HLA antibodies in the tissue typing laboratory between 2017 and 2020. In the cohort, the serum samples of patients waiting for transplantation were tested. Both the PRA and SAB tests of these patients were analyzed using the Luminex (Immucor) method. The threshold of positivity was accepted as median fluorescence intensities (MFI) ≥1000 for PRA screening and MFI ≥750 for SAB screening.ResultsOverall, antibodies to HLA antigens were detected in 202 (78.9%) out of 256 patients in the PRA study. Antibodies against both class I/II antigens were detected only in 15.6% of these patients, whereas antibodies against only against class I HLA in 31.3% and only against class II HLA in 32.0%. By comparison, the SAB study found that 66.8% of patients were positive for HLA antigens. Furthermore, donor-specific antibodies (DSA) were detected in 52.0% of PRA-positive patients and 52.6% of SAB-positive patients. It was shown that 168 patients (83.2%) out of 202 PRA-positive patients were found to be SAB-positive. In addition, 51 patients negative in the SAB assay (94.4%) were also negative in the PRA assay. Statistical analysis established a significant correlation between the PRA and SAB positivity (p > 0.001).It was also shown that MFI ≥3000 PRA positivity for class I HLA antigens (p = 0.049) and MFI ≥5000 PRA positivity for class II antigens (p < 0.001) correlated with the SAB positivity in patients.ConclusionOur results showed the importance of both PRA and SAB assays to define the status of sensitization in patients.     

Algorithm to manage highly sensitized kidney transplant recipients in Poland

Objective

Programs for immunized transplant recipients are essential to achieve graft survivals comparable to those of non-immunized recipients. The threshold in Poland is a PRA by the complement-dependent cytotoxicity (CDC) method greater than 50%, which includes approximately 3.8% of the patients. At the same time the United Network for Organ Sharing there recipients represent approximately 16% of the waiting list in the United Network for Organ Sharing (UNOS). The underestimation of the immunized group in Poland may be due to differences in laboratory techniques to assess alloantibodies.

Materials and methods

This study investigated 55 potential recipients with a PRA by CDC > 50%. We used the following algorithm to assess their immunization: Luminex screening test for an HLA antibody; specificity assessed with Luminex Single Antigen, vPRA (evaluation of immunization of the patient); and analysis of acceptable HLA incompatibilities (HLAMatchmaker).

Results

All recipients were positive class I anti-HLA antibodies and 94.5% were positive for class II. For the groups of subjects with PRA-CDC from 50% to 79% versus those greater than 80%, the average values of PRA-CDC were 62.2% and 89.5%, respectively. The virtual PRA results for these groups were 95.7% and 97.2%, respectively. In addition, anti-HLA-Cw, anti-DQ and anti-DP antibodies were detected in 77%, 84%, and 51% of recipients, respectively. Immunized recipients reported to the next transplant were characterized by the antibodies against mismatch only in 68%. For all potential recipients, additional acceptable non-compliance was determined with HLAMatchmaker: 152 specificity for locus A and 252 for locus B.

Conclusions

Evaluation of immunization status of recipient candidates should be routinely performed using tests to assess class and specificity as well as level of alloantibodies to enable determination of a safe potential donor. As a routine test, PRA-CDC underestimates the number of highly immunized patients. Exclusion from the list of patients with repeated non-compliance is a simplification, which reduces their chance for transplantation.     

Effect of panel-reactive antibody in predicting crossmatch selection of cadaveric kidney recipients

AIM: The influence of panel-reactive antibody level (%PRA) on crossmatch results was evaluated among 866 patients on the waiting list for cadaveric renal allografting from January 2001 to August 2005. We evaluated the results for 124 potential donors for a kidney, including 2008 crossmatches. Four hundred eighteen patients were tested against only 1 donor. METHODS: Serum samples were screened for anti-HLA antibodies using immunoglobulin (Ig)G enzyme-linked immunosorbent assay (ELISA) PRA kit and the %PRA of the most reactive sample (peak) was used for patient stratification, according to sensitization level. Crossmatches were performed on fresh donor T lymphocytes from peripheral lymph nodes, using classical and anti-human-globulin enhanced complement-dependent cytotoxicity (CDC-T) methods. The tests were performed using peak and current patient sera before and after dithiothreitol treatment. The crossmatch was assumed to be negative when no reactivity was observed in all tests. RESULTS: The incidences of positive crossmatch were as follows: 72.3%, 14.6%, and 7.2%, among patients with PRA >50%, PRA 相似文献   

Donor‐specific anti‐HLA antibodies detected by Luminex: predictive for short‐term but not long‐term survival after heart transplantation

In heart transplantation, the clinical significance of pretransplant donor‐specific antibodies (DSA) detected by solid phase assay (SPA), which is more sensitive than the conventional complement‐dependent cytotoxicity (CDC) assays, is unclear. The aim was to evaluate SPA performed on pretransplant sera for survival after heart transplantation. Pretransplant sera of 272 heart transplant recipients were screened for anti‐HLA antibodies using CDC and SPA. For determination of pretransplant DSA, a single‐antigen bead assay was performed. The presence of anti‐HLA antibodies was correlated with survival. Secondary outcome parameters were acute cellular rejection, graft coronary vasculopathy and ejection fraction. In Kaplan–Meier analysis, SPA‐screening did not predict survival (P = 0.494), this in contrast to CDC screening (P = 0.002). However, the presence of pretransplant DSA against HLA class I was associated with decreased short‐term survival compared to non‐DSA (P = 0.038). ROC curve analysis showed a sensitivity of 76% and specificity of 73% at a cutoff of 2000 MFI. In contrast, the presence of anti‐HLA antibodies had no influence on long‐term survival, rejection incidence, and graft function. Thus, detection of DSA class I in pretransplant serum is a strong predictor of short‐term, but not long‐term survival and may help in the early management of heart transplant patients.     

Association of HLA phenotypes of end-stage renal disease patients preparing for first transplantation with anti-HLA antibody status

Patients with pre-transplantation high levels of panel reactive antibody (PRA) have an increased risk of graft failure, and renal transplantation in sensitized patients remains a highly significant challenge worldwide. The influence of anti-human leukocyte antigen (HLA) antibodies on the development of rejection episodes depends on patient-specific clinical factors and differs from patient to patient. The HLA typing of the recipient might influence the development of anti-HLA antibodies. Some HLA antigens appear to be more immunogenic than others. The aim of this study is to demonstrate the distribution of HLA phenotypes in PRA-positive and PRA-negative end-stage renal disease (ESRD) patients on the basis of having sensitizing events or not. Our study included 642 (mean age: 41.54; female/male: 310/332) ESRD patients preparing for the first transplantation and who are on the cadaveric kidney transplantation waiting list of Istanbul Medical Faculty in 2008–2009. Class I HLA-A,B typing was performed by complement-dependent cytotoxicity (CDC) method, whereas class II HLA-DRB1 typing was performed by low-resolution polymerase chain reaction (PCR)-sequence-specific primer (SSP). All serum samples were screened for the presence of IgG type of anti-HLA class I- and II-specific antibodies by enzyme-linked-immunosorbent assay (ELISA). PRA-negative group consisted of 558 (86.9%) and PRA-positive group included 84 (13.1%) patients. We have found statistically significant frequency of HLA-A3 (p = 0.018), HLA-A66 (p = 0.04), and HLA-B18 (p = 0.006) antigens in PRA-positive patients and DRB1*07 (p = 0.02) having the highest frequency in patients with sensitizing event history but no anti-HLA development suggesting that DRB1*07 might be associated with low risk of anti-HLA antibody formation.     

Detection and analysis of HLA class I and class II specific alloantibodies in the sera of dialysis recipients waiting for a renal retransplantation

The objective of this study was to evaluate the specificities of HLA class I (-A,-B) and class II (-DR,-DQ) antibodies (Ab) detected in the sera of alloimmunized patients waiting for a subsequent renal transplantation. The study group consisted of 62 dialysis patients (42 men and 20 women, mean age: 43 +/- 18 yr) on waiting list for a subsequent kidney transplant (52 for a second and 10 for a third transplant) at S. Martino Hospital Transplant Centre in Genoa/Italy, who were enrolled from 2002 to 2004 for HLA antibody screening. Complement dependent cytotoxicity (CDC) technique was used firstly to select anti-HLA class I sensitized patients; indeed sera from 50 individuals out of 62 (80.6%) were found to display persistent HLA class I PRA (panel reactive antibody) values >4% (range: 20-100). ELISA technique was subsequently adopted to analyze HLA class I Ab positive sera for the presence also of HLA class II Ab and to characterize class I and class II Ab specificities. Anti-class I immunized patients were divided in three groups according to the type of class I Ab specificities, that were classified as private, public, and multispecific. The first group included 35 patients (70% of the total number of positive patients) showing only antibodies directed against private HLA class I specificities, represented in 33 cases by those expressed by graft donors (first or second transplant). In this group anti-class I PRA% values ranged from 20% to 60%. HLA class II Ab, with an heterogeneous specificity pattern (private, public or multispecific), were present in 25 (78.1%) out of the 32 patients, whose sera were also available for this analysis. The second group comprised 12 patients (24%) who displayed antibodies directed against class I public epitopes belonging to CREGs (Cross reactive Groups) or an association of anti-private and anti-public antibodies. In this group PRA values ranged from 25% to 90%. Five patients (46.7%) were positive for HLA class II Ab, whose specificity pattern appeared also heterogeneous (private or multispecific). The third group was represented by three patients (6%) displaying multispecific antibodies with PRA values > or = 90%. No multispecific class II Ab were found in this group, where only two patients had class II Ab showing anti-private or anti-private plus public specificities. Globally, 74% of anti-class I Ab positive patients, having at least one HLA class II antigen mismatch, appeared also positive for class II Ab. These results indicate that: (i) a large proportion of patients, waiting for a kidney retransplantation, display in their sera alloantibodies specific for graft mismatched HLA class I (80.6%) and class II antigens (54.2); (ii) the immunogenic determinants, mainly involved in HLA class I and II specific Ab production, were, in a significant rate, private specificities of mismatched HLA antigens (70% for class I and 59.4% for class II), and in a lesser percentage by public (CREG) epitopes (24% for class I and 34.3% for class II). In a few patients only no HLA class I and class II Ab specificities could be determined, as they displayed multispecific antibodies (6% for class I and 6.2% for class II). These findings may have important implications to improve donor-recipient matching in dialysis recipients waiting for a subsequent renal transplantation.     

Effect of implementing anti-HLA antibody detection by Luminex in the kidney transplant program in Chile

The development of new highly sensitive, specific technologies to detect HLA antibodies has allowed a better definition of the profile of non-permitted antigens for patients awaiting kidney transplantation. The use of calculated or virtual panel reactive antibodies (CPRA or vPRA) seeks to improve the prediction of positive crossmatches (XM), but increases the proportion of sensitized patients on the waiting list. In 2008-2009, we implemented detection of antibodies using Luminex technology and applied vPRA since 2009. The objective of this study was to evaluate the impact of these innovations in defecting patient sensitization on kidney transplant waiting lists for deceased donors and among transplanted patients. We analyzed the waiting list for 2007 through 2009 and the first semester of 2010, including the patients transplanted in these periods and the XM with deceased donors. We observed an increase in the mean peak PRA of transplanted patients from 7.2% in 2007 to 17.1% in 2010 (P = .001), and in the proportion of patients transplanted with a peak PRA > 50% from 2.8% in 2007 to 15.7% in 2010 (P = .0001), with no increase in the proportion of this population on the waiting lists. There was a concurrent decrease in positive XM among patients with a peak PRA > 50%. The use of vPRA and Luminex permitted a greater number of transplants of patients with peak PRA > 50% and was a good predictor of a positive XM.     

Influence of Test Technique on Sensitization Status of Patients on the Kidney Transplant Waiting List

The exquisitely sensitive single antigen bead (SAB) technique was shown to detect human leukocyte antigen (HLA) antibodies in sera of healthy male blood donors. Such false reactions can have an impact on critical decisions, especially with respect to the determination of unacceptable HLA‐antigen mismatches in patients awaiting a kidney transplant. We tested pretransplant sera of 534 patients on the kidney waiting list using complement‐dependent cytotoxicity (CDC), enzyme‐linked immunosorbent assay (ELISA) and SAB in parallel. Evidence of HLA antibodies was obtained in 5% of patients using CDC, 14% using ELISA, and 81% using SAB. Among patients without history of an immunizing event, 77% showed evidence of HLA antibodies in SAB. In contrast 98% of these patients were negative in ELISA and CDC. In patients without an immunizing event, SAB‐detected antibodies reacted not always weakly but with mean fluorescence intensity (MFI) values as high as 14 440. High‐MFI‐value antibodies were found in some of these patients with HLA specificities that are rather common in general population, consideration of which would lead to unjustified exclusion of potential kidney donors. False SAB reactions can be unveiled by testing with additional antibody assays. Denial of donor kidneys to recipients based on HLA‐antibody specificities detected exclusively in the SAB assay is not advisable.     

Panel reactive antibody positivity and associated HLA antibodies in Turkish renal transplant candidates

Pre- and post-renal transplantation panel reactive antibody (PRA) screening is associated with increased incidence of hyperacute or acute graft rejection and graft loss. This study was designed to find any relationship PRA sensitization and associated human leukocyte antigen (HLA)-specific antibodies in Turkish renal transplant candidates. We included 340 patients who were in the renal transplantation waiting list in the study. We determined PRA sensitization ratio and the associated anti-HLA IgG antibody distribution of the patient group. The PRA testing was currently performed and levels above 30% were accepted to be positive. The PRA class I positivity was determined in 24 (7%) and class II in 34 (10%) of the patients. The most frequent HLA antibodies for class I were B56, A2, A34, A1, A23, A24 and B61; and for class II were DR11, DR14, DQ7, DR10, DQ5, DR1 and DR7, respectively. From these, the increase of the numbers of anti-HLA class II antibodies was significantly correlated with the increase of PRA sensitization ratio. In conclusion, the identification of the associated HLA-specific antibodies and correlation with the Turkish population HLA antigen distribution will identify the high-risk patients who are candidates for transplantation.     

Anti-perlecan antibodies and acute humoral rejection in hypersensitized patients without forbidden HLA specificities after kidney transplantation

BackgroundThe improvement in the definition of serum anti-HLA antibodies (HLA-Abs) profiles after Luminex-assay implementation in transplant patients follow-up is clear. This success has permitted the development of hypersensitized-recipient allocation and donor-paired exchange programs improving the access to transplantation. However, non-HLA Abs have been described in transplanted patients but their effect in hypersensitized transplanted recipients is unclear.MethodsTwenty-seven HLA hypersensitized patients awaiting for kidney transplantation (KT) were studied and 11 of them were followed after KT. The HLA Abs profile was confirmed in serum by Single Antigen Luminex assay and panel reactive of antigens >98% was achieved in all patients. Subsequently, the ability to fix complement by C1q test was also assessed. Serum non-HLA Abs before and 1 month after transplantation were measured in the 11 hypersensitized recipients.Results95.2% of the hypersensitized on waiting list had concomitant serum anti-HLA and non-HLA Abs. The more frequent specificity in non-HLA Abs were found against Glutathione S-transferase theta-1 (GSST-1) (in 62%) and C-terminal fragment of perlecan (LG3) (in 52%). Four out of 11 transplanted patients presented early antibody-mediated rejection (ABMR) confirmed by biopsy and had serum anti-LG3 antibodies, two of them with concomitant anti-anti-angiotensin II type I receptor. Only one patient developed de novo-donor specific HLA antibodies.ConclusionsThe incidence of non-HLA antibodies in patients in the waiting list is largely underestimated. The concomitance anti-HLA and non-HLA Abs in hypersensitized patients is very common and the detection of non-HLA Abs in this population could allow to identify patients with an increased risk of humoral rejection.     

Novel insights into pretransplant allosensitization in heart transplant recipients in the contemporary era of immunosuppression and rejection surveillance

Solid‐phase assays (SPA) have facilitated detection and definition of antibodies to human leukocyte antigens (HLA) and major histocompatibility complex class I chain‐related antigen A (MICA). However, clinical consequences of pretransplant SPA results in heart transplantation have been studied insufficiently in the current era of immunosuppression and rejection surveillance. Pretransplant sera, panel‐reactive antibodies (PRA), pretransplant crossmatch, and clinical data were retrospectively analyzed in 264 adult heart transplant recipients. The specificity of HLA and MICA antibodies and C1q‐binding activity of donor‐specific antibodies (DSA) were defined using SPA. Pretransplant HLA antibodies were detected in 57 (22%) individuals, in 28 individuals (11%); these antibodies were DSA after transplant. Preformed DSA and elevated peak PRA were independent predictors of pathologic AMR, which occurred in 19 individuals (7%). The increasing number of DSA and the cumulative mean fluorescence intensity of DSA were associated with AMR. C1q‐binding assay was a suboptimal predictor of AMR in our cohort. Pretransplant allosensitization and MICA antibodies were related neither to impaired graft survival nor to other adverse clinical events during a median follow‐up of 39 months. Identification of preformed DSA by SPA, in addition to PRA monitoring, may predict AMR in the contemporary era of heart transplantation.     

Mastering the Risk of HLA Antibodies in Kidney Transplantation: An Algorithm Based on Pretransplant Single‐Antigen Flow Bead Techniques

The utilization of sensitive techniques of detection of HLA antibodies to define and measure sensitization has greatly evolved in recent years. We present here an approach to minimize the risk of HLA antibodies in kidney transplantation based on the evaluation of graft accessibility of sensitized patients by calculated PRA (cPRA) and estimation of potential matched donors (PMD) using a national simulation software program. This study included all registered patients on our waiting list (WL) for deceased donor (DD) kidney transplants. All patients were screened by single‐antigen flow bead (SAFB) techniques. Of the 502 registered patients, 174 (34.7%) were sensitized. Among these, 48.3% (84 pts) had a cPRA>85%. For 75.3% of sensitized patients (90 pts with cPRA≤85% and 41 pts with cPRA>85%), the flow of PMD was considered sufficient to allow a transplant avoiding all unacceptable antigens. The 41 patients with a cPRA>85% (48.8%) had a satisfactory donor flow in the framework of the national prioritization program for highly sensitized patients. Finally, 43 sensitized patients (24.7%) were deemed eligible for a strategy of higher immunological risk through desensitization protocols or transplantation against HLA‐DSAs. This approach provides a logical and systematic strategy to rationalize the access of sensitized patients to kidney transplantation minimizing the risk of HLA antibodies.     

The new French rules for the attribution of renal transplants

The French rules for the attribution of a kidney transplant are regularly evaluated and modified according to scientific progress, evolution of the waiting list and of health policies. Modifications, initiated by the Transplantation Commission of the French-speaking Society of Nephrology, have been introduced in 2004 by the Etablissement fran?ais des Greffes and aim at decreasing the number of patients on the waiting list having difficult access to transplantation because of their immunogenetic characteristics (rare ABO or HLA group, HLA immunization). Four points are concerned: 1/ better definition of hyperimmunisation; 2/ introduction of a program based on "acceptable mismatches" as a new priority for hyperimmunized patients; 3/ suppression of the full-match priority to non-immunized patients; 4/ attribution to immunized patients (anti-HLA antibodies=5-80%) who have difficult access to a transplant, of priorities similar to those followed for hyperimmunized patients. This article presents the new rules for the allocation of a kidney transplant and the rationale for the current modifications.     

Synthesis of new and memory HLA antibodies from acute and chronic rejections versus pregnancies and blood transfusions

BACKGROUND: This study compared HLA antibody (abs) production following acute (AR) and chronic (CR) rejection with HLA abs production following blood transfusions (TF) or pregnancies (P) among kidney and kidney pancreas transplant candidates. METHODS: Serum samples from 145 patients were screened for anti-HLA abs by flow cytometry using beads coated with purified HLA molecules. Among these patients, nine had lost their graft due to AR; 13, CR; six had been immunized by a single P; 20 by no more than two TF; 28 had no documented immunization; and 69 had multiple immunizations. A Fisher exact test was used for the statistical analysis. RESULTS: Neither a single P nor a couple of TF by themselves immunized primary transplant recipients (P = .17, and P = .42, respectively). However, multiple P and TF together provided a potent immunogenic stimulus (P = .00001). In contrast, a single allograft loss induced a strong anti-HLA response (P = 1.03 x 10(-8)). Following AR, anti-class I and II abs (P = .001), and following CR, anti-class II abs developed (P = .03). Waitlisted female candidates had significantly high PRA values than men (P = .00005), possibly because women had received significantly more immunizations than men (P = 4.0 x 10(-10)). SUMMARY: Patients are likely to produce both class I and II HLA abs following AR and only class II abs following CR. A couple of TF or a P may not be immunogenic; however, together they have great potential to sensitize female candidates. Perhaps that is why waitlisted women are far more immunized than waitlisted men.     

Donor-Specific Anti-HLA Antibodies After Bone-Graft Transplantation. Impact on a Subsequent Renal Transplantation: A Case Report

Immunological evaluation by panel-reactive antibody (PRA) and determination of anti-HLA specificity are important phases in the evaluation of patients awaiting kidney transplantation. The main causes of immunization are previous solid organ transplantation, hemotransfusion, and pregnancy. It is also possible that immunogenicity can be triggered by vascularized tissue grafts. Immune induction by cryopreserved bone prostheses is not yet understood. A 19-year-old patient with osteosarcoma had undergone resection of the left proximal tibia with reconstruction using human bone in 1997. The donor HLA typing was as follows: A3, A29 (19); B44 (12), Bw4; DR13 (6), DR7, DR52, DR53. The patient was subsequently enrolled onto the waiting list for cadaveric donor kidney transplantation due to chronic kidney failure caused by cisplatin toxicity. Pretransplantation immunological screening using the complement-dependent cytotoxicity (CDC) technique revealed a PRA of 63%. IgG antibody specificities were detected against class I and class II donor antigens, specifically anti-A3, B44, DR7 antibodies, using flow cytometry (Tepnel Luminex). Further immunological studies using single HLA specificity analysis (LSA Class I°-II°, Tepnel-Luminex) showed direct antibodies against all donor antigen specificities. This case showed immune induction after the implantation of bone prosthesis in a kidney transplant candidate, underlining the importance of the availability of HLA typing data of donors of a human prosthesis.     

Flow cytometric detection of HLA-specific antibodies as a predictor of heart allograft rejection

BACKGROUND: Historically, panel reactive antibody (PRA) analysis to detect HLA antibodies has been performed using cell-based complement-dependent cytotoxicity (CDC) techniques. Recently, a flow cytometric procedure (FlowPRA) was introduced as an alternative approach to detect HLA antibodies. The flow methodology, using a solid phase matrix to which soluble HLA class I or class II antigens are attached is significantly more sensitive than CDC assays. However, the clinical relevance of antibodies detected exclusively by FlowPRAhas not been established. In this study of cardiac allograft recipients, FlowPRA was performed on pretransplant sera with no detectable PRA activity as assessed by CDC assays. FlowPRA antibody activity was then correlated with clinical outcome. METHODS: PRA analysis by anti-human globulin enhanced (AHG) CDC and FlowPRA was performed on sera corresponding to final cross-match specimens from 219 cardiac allograft recipients. In addition, sera collected 3-6 months posttransplant from 91 patients were evaluated. The presence or absence of antibodies was correlated with episodes of rejection and patient survival. A rejection episode was considered to have occurred based on treatment with antirejection medication and/or histology. RESULTS: By CDC, 12 patients (5.5%) had pretransplant PRA >10%. In contrast, 72 patients (32.9%) had pretransplant anti-HLA antibodies detectable by FlowPRA (34 patients with only class I antibodies; 7 patients with only class II antibodies; 31 patients with both class I and class II antibodies). A highly significant association (P<0.001) was observed between pretransplant HLA antibodies detected by FlowPRA and episodes of rejection that occurred during the first posttransplant year. Fifteen patients died within the first year posttransplant. Of nine retrospective flow cytometric cross-matches that were performed, two were in recipients who had no pretransplant antibodies detectable by FlowPRA. Both of these cross-matches were negative. In contrast, five of seven cross-matches were positive among recipients who had FlowPRA detectable pretransplant antibodies. Posttransplant serum specimens from 91 patients were also assessed for antibodies by FlowPRA. Among this group, 58 patients had FlowPRA antibodies and there was a trend (although not statistically significant) for a biopsy documented episode of rejection to have occurred among patients with these antibodies. CONCLUSIONS: Collectively, our data suggest that pre- and posttransplant HLA antibodies detectable by FlowPRA and not AHG-CDC identify cardiac allograft recipients at risk for rejection. Furthermore, a positive donor reactive flow cytometric cross-match is significantly associated with graft loss. Thus, we believe that detection and identification of HLA-specific antibodies can be used to stratify patients into high and low risk categories. An important observation of this study is that in the majority of donor:recipient pairs, pretransplant HLA antibodies were not directed against donor antigens. We speculate that these non-donor-directed antibodies are surrogate markers that correspond to previous T cell activation. Thus, the rejection episodes that occur in these patients are in response to donor-derived MHC peptides that share cryptic determinants with the HLA antigens that initially sensitized the patient.     

Corrected Panel-Reactive Antibody Positivity Rates for Hypersensitized Patients in Turkish Population With Calculated Panel-Reactive Antibody Software

Introduction

High rates of panel-reactive antibody (PRA) may decrease the chance of kidney transplantation and may result in long waiting periods before transplantation. The calculated PRA (cPRA) is performed based on unacceptable HLA antigens. These antigens are identified by a program that was created based on the antibodies that developed against the HLA antigens circulating in serum and on the risk of binding of these antibodies to antigens. The antigen profile of the population and antigen frequencies can be measured, and more realistic cPRA positivity rates may be obtained using this method.

Sensitisation to Swine Leukocyte Antigens in Patients with Broadly Reactive HLA Specific Antibodies

We have previously shown that IgG HLA specific antibodies in the sera of highly sensitised patients awaiting renal transplantation can cross-react with swine leukocyte antigens (SLA). In this study we determined the frequency of patient serum IgG HLA specific antibody binding to a porcine lymphocyte panel and the likelihood of locating a cross-match negative pig donor for sensitised patients. Serum samples (n = 82) were obtained from 35 sensitised [current IgG panel reactive antibodies (PRA) > 10%] and seven nonsensitised patients awaiting renal transplantation at Addenbrooke's Hospital, Cambridge, UK. Fifty sera had IgG HLA specific PRA of 11-84%, 20 had IgG PRA of >84% and 12 had 0% PRA (negative controls). Sera were absorbed with porcine erythrocytes to remove xenoreactive natural antibodies and tested for cross-reactive IgG HLA specific antibody binding by flow cytometry against a panel of porcine lymphocytes obtained from 23 human decay accelerating factor (hDAF) transgenic pigs. A total of 1,884 cross-match combinations were tested and 369 (20%) gave a positive porcine lymphocyte cross-match. For sera from sensitised patients with IgG PRA (11-64%), only 6 of 805 (0.75%) cross-match tests were positive. In contrast, for sera from patients with high IgG PRA (>64%), 363 of 805 (45%) cross-match tests were positive (p < 0.0001). There was no difference in the frequency of positive cross-matches between patient sera with IgG PRA 65-84% and highly sensitised patient sera with IgG PRA 85-100% [156/345 (45%) vs. 207/460 (45%)]. This study demonstrates that only patient sera with broadly reactive IgG HLA specific PRA (>64%) cross-react with porcine lymphocytes. If future clinical trials of xenotransplantation are undertaken, it may be of value to select a cross-match-negative pig organ donor for such patients.     

Flow cytometry beads rather than the antihuman globulin method should be used to detect HLA Class I IgG antibody (PRA) in cadaveric renal regraft candidates

Abstract: HLA Class I antibody screening can be performed by flow cytometry using a mixture of 30 distinct bead populations each coated with the Class I antigen phenotype derived from different cell lines. In this study we compared the efficacy of Class I antibody screens done by flow cytometry beads with the antihuman globulin (AHG) method for patients awaiting cadaveric renal retransplantation. Class I panel reactive antibody (PRA) screening by flow cytometric beads of 21 regraft serum samples that had all been found to be negative by AHG DTT Class I PRA, revealed that 57.1% (12 of 21) had a flow Class I PRA of ≥ 10%. Furthermore, when five regraft sera with an intermediate PRA were screened (mean AHG DTT PRA = 33.2 ± 13%) the mean flow Class I PRA almost doubled (mean flow PRA = 72.4 ± 10.2%) ( p  < 0.01). When active UNOS waiting list regraft candidates, after several months of screening the Class I PRA by flow beads, were divided into the three PRA categories based on their peak flow Class I PRA value (0−20%, 21−79% and ≥ 80%), the incidence of a positive flow cross-match was 0%, 72% and 85% and the incidence of retransplantation was 60%, 22% and 10%, in each of these groups, respectively. These data provided our histocompatibility laboratory with the rationale to stop performing the AHG PRA and perform only the flow Class I PRA method for regraft candidates.     

Does human leukocyte antigens sensitization matter for xenotransplantation?

The major histocompatibility complex class I and class II human leukocyte antigens (HLA) play a central role in adaptive immunity but are also the dominant polymorphic proteins targeted in allograft rejection. Sensitized patients with high levels of panel‐reactive anti‐HLA antibody (PRA) are at risk of early allograft injury, rejection, reduced allograft survival and often experience prolonged waiting times prior to transplantation. Xenotransplantation, using genetically modified porcine organs, offers a unique source of donor organs for these highly sensitized patients if the anti‐HLA antibody, which places the allograft at risk, does not also enhance anti‐pig antibody reactivity responsible for xenograft rejection. Recent improvements in xenotransplantation efficacy have occurred due to improved immune suppression, identification of additional xenogeneic glycans, and continued improvements in donor pig genetic modification. Genetically engineered pig cells, devoid of the known xenogeneic glycans, minimize human antibody reactivity in 90% of human serum samples. For waitlisted patients, early comparisons of patient PRA and anti‐pig antibody reactivity found no correlation suggesting that patients with high PRA levels were not at increased risk of xenograft rejection. Subsequent studies have found that some, but not all, highly sensitized patients express anti‐HLA class I antibody which cross‐reacts with swine leukocyte antigen (SLA) class I proteins. Recent detailed antigen‐specific analysis suggests that porcine‐specific anti‐SLA antibody from sensitized patients binds cross‐reactive groups present in a limited subset of HLA antigens. This suggests that using modern genetic methods, a program to eliminate specific SLA alleles through donor genetic engineering or stringent donor selection is possible to minimize recipient antibody reactivity even for highly sensitized individuals.