Search

Background and Objectives: There are several cPRA websites based on large enough samples in Eurotransplant, the United Network for Organ Sharing (UNOS), and the Canadian Transplant Registry (CTR). On the other hand, those calculators can differ based on the ethnicity to which they are applied. We developed the Iranian PRA calculator and compared it with UNOS and CTR calculators., Methods: The allele and haplotype frequencies of the Iranian donor pool were estimated using the HLA typing of 523 deceased Iranian kidney donors. The Organ Procurement and Transplantation Network formula was used to generate cPRA (cPRA frequency). We also used a computer script to compare the undesirable antigens of patients with the human leukocyte antigen (HLA) phenotype of donors (cPRA filtering). A total of 100 anti-HLA antibody profiles were determined in 100 sensitized individuals on the waiting list, and cPRA was estimated using various PRA calculators., Results: Variable allelic frequencies were obtained from population heterogeneity in each calculator's donor panel. However, no significant changes in cPRA were identified between the Iranian calculator, UNOS, and the Canadian online calculators. Lin's concordance correlation coefficient of .98 showed that cPRA (freq) and cPRA (filter) values had almost perfect agreement., Interpretation and Conclusion: The cPRA values from the Iranian calculator are comparable to those from UNOS and CTR calculators. The donor filtering method was more useful because of factors like cost and flexibility. It also makes it easier to update cPRA on a regular basis., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: While mismatching between donor and recipient human leukocyte antigen (HLA) alleles has been associated with increased graft loss in pediatric heart recipients, it is actually the surface amino acid structures, termed eplets, which determine the antigenicity of each HLA molecule. We hypothesized that HLA eplet mismatch analysis is a better predictor of adverse outcomes after pediatric heart transplant than conventional allele mismatch comparison., Methods: A retrospective review of the Pediatric Heart Transplant Society database identified pediatric heart recipients (<18 years at listing) with complete donor and recipient HLA typing (A, B, and DR). Imputed high-resolution HLA genotypes were entered into HLAMatchmaker software which then calculated the number of eplet mismatches between each donor-recipient pair. Multivariable Cox regression analysis was used to examine associations between allele or eplet mismatching and adverse outcomes., Results: Compared to those with <20 HLA class I eplet mismatches, recipients with 20 or more HLA class I eplet mismatches had an increased risk of graft loss (HR 1.46 [1.01-2.12], p = .049). HLA class I eplet mismatching was also associated with rejection (>20 mismatches: HR 1.30 [1.03-1.65], p = .030), while HLA class II eplet mismatching was associated with specified antibody-mediated rejection (10-20 mismatches: HR 1.57 [1.06-2.34], p = .025; >20 mismatches: HR 3.14 [1.72-5.71], p < .001). Neither HLA class I nor class II allele mismatching was significantly associated with graft loss or rejection., Conclusion: Eplet mismatch analysis was more predictive of adverse post-transplant outcomes (including graft loss and rejection) than allele mismatch comparison. Further study, including prospective high-resolution HLA typing, is warranted., (© 2021 Wiley Periodicals LLC.)

Background: HLA epitope-based matching offers the potential to improve immunological risk prediction and management in children receiving renal allografts; however, studies demonstrating the association between systems for defining epitope mismatches and clinical end-points are lacking in this population., Methods: We conducted a pragmatic, retrospective, registry-based study of pediatric recipients of primary renal allografts in Victoria, Australia between 1990 and 2014 to determine the association between HLA EpMM and clinical outcomes including graft failure, re-transplantation and dnDSA formation., Results: A total of 196 patients were included in the analysis with a median age of 11 years. Median follow-up period was 15 years during which time 108 (55%) primary grafts failed and 72 patients were re-transplanted. HLA class I but not class II EpMM was a significant predictor of graft failure on univariate analysis but not in adjusted models. EpMM was associated with reduced likelihood of re-transplantation in univariate but not adjusted analysis. Within the limitations of the study, class-specific EpMM was a strong predictor of dnDSA formation. Associations were stronger when considering only the subset of antibody-verified EpMM., Conclusion: Associations between HLA EpMM and clinical outcomes in pediatric renal allograft recipients seen on univariate analysis were attenuated following adjustment for confounders. These findings are inconclusive but suggest that HLA EpMM may provide one tool for assessing long-term risk in this population while highlighting the need for further clinical studies., (© 2020 Wiley Periodicals LLC.)

Background: Assessment of human leukocyte antigen (HLA) matching by using high-resolution allele typing and knowledge of HLA molecule structure may lead to better prediction of de novo donor-specific antibody (dnDSA) development., Methods: We conducted a single-center cohort study among 150 non-sensitized first kidney transplant recipients to compare the association between antigenic (Ag), allelic (Al), eplet (Ep), amino acid (AAMS) HLA matching and electrostatic (EMS) and hydrophobic (HMS) mismatch scores, and the development of dnDSA., Results: After a mean follow-up time of 49.3 ± 17.7 months, 18 patients (12%) developed dnDSA. The number of HLA mismatches (MM) was significantly associated with the development of dnDSA. The optimal threshold, determined by Harrell's C-index, varied according to the method (5 MM for Ag, P = 0.006; 6 for Al, P = 0.009; 22 for Ep, P = 0.005; 42 for AAMS, P = 0.0007; 45 for EMS, P = 0.009 and 44 for HMS, P = 0.026). C-indices were similar for all matching approaches, suggesting a similar prediction of dnDSA development., Conclusion: In this cohort of low immunological risk transplant patients, the use of Al or Ep matching did not improve the prediction of dnDSA development in comparison with the traditional approach., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Type 1 diabetes (T1D) mellitus is an autoimmune disorder involving both complex genetic and environmental factors. The incidence rates are low in Asian countries, and the specific, explanatory genetic factors underlying this have been investigated. The aim of this study was to elucidate the association of human leukocyte antigen (HLA) alleles/haplotypes with T1D in Taiwan., Methods: We performed direct comprehensive genotyping of 6 classical HLA loci (HLA-A, -B, -C, -DPB1, -DQB1, and -DRB1) to 4-digit resolution in 104 unrelated T1D patients and 504 controls. Twenty-four of the 104 patients also exhibited thyroid autoimmunity., Results: Three major susceptibility haplotypes were identified: DRB1*03:01-DQB1*02:01 (odds ratio [OR] = 5.39 under the dominant model, P = 2.3 × 10 -13 ), DRB1*04:05-DQB1*04:01 (OR = 2.44, P = 5.0 × 10 -4 ), and DRB1*09:01-DQB1*03:03 (OR = 2.02, P = 1.4 × 10 -3 ); one protective haplotype was identified: DRB1*08:03-DQB1*06:01 (OR = 0.10, P = 1.6 × 10 -3 ). DRB1*03:01-DQB1*02:01, the major T1D susceptibility haplotype, was found at a lower frequency in T1D patients with thyroid autoimmunity. The T1D protective allele DRB1*12:02 was shown to be protective against Graves' disease in our previous report., Conclusion: In addition to clarifying the roles of several known T1D HLA alleles and haplotypes, we discovered that the DRB1*08:03-DQB1*06:01 haplotype is protective against T1D. The DRB1*12:02 allele protected against both T1D and Graves' disease., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The impact of human leukocyte antigen (HLA) compatibility and positive lymphocyte cross-match (LCM) on organ transplantation is well-recognized particularly in kidney and heart transplantation; however, it is still debatable in liver transplantation (LT). So, the aim of this study was to evaluate the impact of HLA mismatch and positive LCM on the outcome of LT., Methods: We retrospectively analyzed the data of all adult recipients who underwent living donor LT at our institute between January 2010 and July 2016. We excluded all ABO blood group incompatible LDLT patients and patients with incomplete data regarding HLA genotyping (n = 134). The type and degree of HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ mismatch and LCM were assessed in each donor-recipient pair and their relationship to the occurrence of rejection, CMV infection and graft survival was evaluated., Results: A higher percentage (>50%) of donor-recipient pairs had 1 HLA mismatch at each locus in the host-vs-graft direction and seventeen recipients (13%) had positive LCM. Human leukocyte antigen mismatch and positive LCM were not correlated with increased incidence of acute rejection (P = .37, P = .6, respectively), CMV infection post-transplant (P = .52, P = .76, respectively), or graft failure (HR 1.22, P = .68 and HR 1.73, P = .34, respectively)., Conclusion: Positive LCM and HLA mismatches did not affect the overall graft survival after adult-to-adult LDLT and should not be considered as contraindications for liver transplantation., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Complement-dependent cytotoxicity cross-match (CDCXM) is used for evaluation of preformed HLA-specific antibodies in patients undergoing heart transplantation. Flow cytometry cross-match (FCXM) is a more sensitive assay and used with increasing frequency. To determine the clinical relevance of a positive FCXM in the context of negative CDCXM in heart transplantation, the United Network for Organ Sharing (UNOS) database was analyzed. Kaplan-Meier analysis and Cox proportional hazard modeling were used to assess graft survival for three different patient cohorts defined by cross-match results: T-cell and B-cell CDCXM+ ("CDCXM+" cohort), CDCXM- but T-cell and/or B-cell FCXM+ ("FCXM+" cohort), and T-cell/B-cell CDCXM- and FCXM- ("XM-" cohort). During the study period, 2558 patients met inclusion criteria (10.7% CDCXM+, 18.8% FCXM+, 65.5% XM-). CDCXM+ patients had significantly decreased graft survival compared to FCXM+ and XM- cohorts (P = .003 and <.001, respectively). CDCXM- and FCXM+ patients did not have decreased graft survival compared to XM- patients (P = .09). In multivariate analysis, only CDCXM+ was associated with decreased graft survival (HR 1.22, 95% CI 1.01-1.49). In conclusion, positive FCXM in the context of negative CDCXM does not confer increased risk of graft failure. Further study is needed to understand implications of CDCXM and FCXM testing in heart transplant recipients., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Since the institution of the new kidney allocation system in December 2014, kidney transplant candidates with the highest calculated panel reactive antibodies (cPRA) of 99-100 have been transplanted at much higher rates. However, concerns have been raised that outcomes in these patients might be impaired due to higher immunological risk and longer cold ischemia times resulting from long-distance sharing of kidneys. Here, we compare outcomes at the University of Wisconsin between study patients with cPRA 99-100 and all other recipients of deceased donor kidneys transplanted between 12/04/2014 and 12/31/2015. All patients had at least 6 months post-transplant follow-up. The mean follow-up was 13.9±3 months in cPRA ≥99% and 12.3±3.5 months in cPRA ≤98%. There was a total of 152 transplants, 25 study patients, and 127 controls. No statistically significant differences were found between the two groups in delayed graft function, rejection, kidney function, graft and patient survival, or infections. We conclude that transplanting the most highly sensitized patients with kidneys shared outside their local donation service areas is associated with excellent short-term outcomes that are comparable to controls., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

It is now appreciated that more HLA-DR mismatching at the time of the first renal transplant is associated with higher degrees of sensitization, lower rates and longer times to retransplantation, and worse graft outcomes in children who are subsequently retransplanted. As such, our pediatric renal transplant program preferentially uses 0 or 1 HLA-DR-mismatched kidneys and reserves 2 DR-mismatched kidneys for recipients with an eminent need for a kidney. Based on a new HLA class II epitope matching strategy that is designed to minimize dnDSA production to DR and DQ antigens, we evaluated the prevalence of DR and DQ eplet mismatching for dd offers made to our pediatric wait-listed candidates. Each candidate/dd pair were HLA-DR (β1 and β3 and/or β5) and DQ (α1 and β1) allele typed by rSSO and were then evaluated for eplet mismatches by the HLAMatchmaker program. We evaluated 78 offers made to 16 children on our UNOS waiting list from 27 consecutive dd from 4/14/14 to 3/23/15. The data show that 40% (8/20) of the 1 DR-mismatched dd offers and 64% (37/58) of the 2 DR-mismatched offers were in the high-risk category for both DR and DQ dnDSA development. Whereas only 15% (3/20) of the 1 DR-mismatched offers and 5% (3/58) of the 2 DR-mismatched offers were in the low-risk category for both DR and DQ dnDSA development, 55% and 33% of the 1 DR- and 2 DR-mismatched offers, respectively, had a favorable DQ eplet mismatch threshold. In summary, HLA class II eplet mismatching is common in potential pediatric transplant recipient/donor pairs. Additional study will be necessary to validate the DR and DQ eplet threshold levels in children and to determine whether eplet mismatching strategies in donor selection result in improved transplant outcome and decreased dnDSA production., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: We sought to investigate temporal trends in the methodology of human leukocyte antibody assessment in heart transplantation., Methods: The United Network for Organ Sharing database was queried from June 2004 to March 2013 to obtain pre-heart transplantation human leukocyte antibody results. The % panel reactive antibody for class I and II antibodies was recorded along with the methodology of assessment. Allosensitization was defined as class I and/or II panel reactive antibody of ≥ 10%. The primary outcome measure was graft survival., Results: During the study period, 12,858 patients with available data underwent heart transplantation. The prevalence of allosensitization increased, with 16.8% in 2005-2006 sensitized at the time of transplantation compared to 23.1% in 2010-2011 (p < 0.001); this occurred in conjunction with an increase in the utilization of flow cytometry (77.2% in 2005-2006; 97.0% in 2010-2011, p < 0.001). Using multivariable analysis, a positive pre-heart transplantation panel reactive antibody by flow cytometry independently predicted graft loss., Conclusions: There has been a recent increase in flow cytometric assessment of human leukocyte antibodies prior to heart transplantation, which may be associated with an increase in the prevalence of pre-transplant patients being characterized as allosensitized. Flow cytometry may identify patients with the highest likelihood of graft loss., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Previous studies have shown poor outcomes in pediatric heart transplant recipients with a high PRA or a positive direct donor-recipient cross-match. This study describes outcomes in patients with a positive cross-match at a large pediatric program. Pediatric heart transplant patients at a large single center between January 1993 and July 2009 were reviewed; those with cross-match data were analyzed. Cross-match data were available in 242/262 (92.4%) patients. Indications for transplant were cardiomyopathy (58%), CHD (32%), and retransplant (7%). PRA was ≥10% in 31/213 (14.6%) patients. A retrospective cross-match was positive in 17/31 (55%) patients with PRA ≥10% and 0/182 with PRA <10%. In positive cross-match patients, rejection frequency in the first year post-transplant was higher than negative cross-match patients (1.69 vs. 0.96 episodes/pt year, p = 0.014). There was no difference in rejection frequency after the first year post-transplant (0.18 vs. 0.12 episodes/pt year, p = 0.14). Overall survival was not significantly different between the groups with a median follow-up time of 4.5 yr. Heart transplantation in patients with a positive cross-match may result in good medium-term survival but a higher frequency of early rejection. Further investigation is warranted to define which patients with a positive cross-match will do poorly., (© 2011 John Wiley & Sons A/S.)

The detection of donor-specific antibodies after organ transplantation might provide an incisive way to monitor allo-specific immunity and predict graft outcome. Still, the availability of new assays for these antibodies prompts us to pose some questions about results that might be observed. These questions include whether the antibodies detected in the blood are a sensitive measure of alloimmunity, whether the detected antibodies are truly specific for the donor and whether they are noxious for the graft. Here, we explain why answers to these questions might interest the basic scientist and clinician., (© 2011 John Wiley & Sons A/S.)

There is increasing evidence that DSA are associated with poor graft survival, although there are little data in children. We aimed to describe the incidence of DSA in this group and to determine correlation with graft survival. HLA antibodies were analysed in 59 paediatric cardiac transplant recipients. Mean age 10.4 (0.7-18.5) yr, mean time post-transplant 5.1 (0.3-17.3) yr. Antibody detection/identification was performed on the Luminex platform with subsequent identification using Lifescreen Identification kits/One-Lambda Single antigen kits. Forty patients (69%) had no HLA antibodies. DSA were found in four (7%). One had transient Class I antibodies and normal cardiac function. The other three had persistent Class II antibodies (two subsequently required re-transplantation, the third had cardiac failure due to CAV). Non-DSA were found in 15 (25%), all with normal graft function and without rejection. There was no difference in function or CAV prevalence between those with non-DSA and those without antibodies. HLA DSA is uncommon in paediatric cardiac allograft recipients but, if persistent, suggests poorer prognosis. In our series, antibodies to HLA class II on donor tissue were associated with increased graft loss. Routine screening and regular testing are recommended., (© 2010 John Wiley & Sons A/S.)

Allosensitization represents a major obstacle to successful re-transplantation since circulating antibodies can elicit antibody-mediated rejection episodes with subsequent graft failure. Since sensitization is primarily considered a contraindication to transplantation the duration for patients waiting for a suitable donor organ to become available is considerably prolonged. Herein, we report on the successful application of a Luminex-based virtual crossmatch approach to facilitate combined third-time cardiac and de novo renal transplantation in a sensitized patient with sustained antibody-mediated cardiac allograft rejection., (© 2009 John Wiley & Sons A/S.)

SAB assays have increased the sensitivity and specificity to detect HLA alloantibodies, but there is uncertainty about the clinical relevance of SAB-positive alloantibodies when the FCXM is negative. We performed a retrospective study to evaluate the clinical significance of SAB-detected DSA in 82 pediatric recipients of a first kidney transplant between January 2000 and December 2005 who had a negative pretransplant FCXM. Pretransplant sera were evaluated by SAB for DSA. Graft loss and rejection between patients with (DSA+) and without DSA (DSA-) were compared. DSA were detected in 13.9%. Eighty percent of DSA+ subjects were DD transplant recipients vs. 56.9% in the DSA- cohort. The RR of graft loss in DSA+ vs. DSA- was 3.3 (95% CI, 1.4-7.9) and in DD was 4.3 (95% CI 1.4-13.1). By Cox regression, the HR of graft loss in DSA+ vs. DSA- was 2.8 (95% CI 0.7-10.9; p = 0.14) and in DD was 5.1 (95% CI 1-25.6; p = 0.05). Acute rejection occurred in 60% in the DSA+ vs. 44.4% in DSA- (p = 0.5). SAB-detected DSA was associated with impaired renal allograft survival in pediatric renal transplant recipients. Impaired graft survival in pediatric renal transplant recipients with DSA detected by solid-phase assays.

Sensitization following renal transplant is a significant barrier to repeat transplantation in children. We report a successful DD renal transplant, with the use of PP, in an 11-yr-old girl who became highly sensitized following a prior failed transplant. She received PP treatments after failure of high-dose IVIg (Gamimune). We established the effectiveness of PP by attaining a 0% PRA and negative cross-matches after five PP treatments. Subsequently, our patient underwent a second round of scheduled PP. When the PRA was 0%, unacceptable antigens were removed from the UNOS wait list, PP was continued, and a kidney became available within 10 days. The final flow cytometry cross-match with the eventual donor was negative. This success demonstrates that coordination of desensitization by PP and advanced laboratory monitoring techniques with recent policies regarding allocation of organs to pediatric patients provides new opportunities for children awaiting transplantation. Since the transplant, our patient sustained a low-titer increase of anti-HLA antibodies. However, she has had no episodes of acute rejection and has maintained excellent graft function more than 17 months later.

Background: HLA antibody sensitization is a risk factor for morbidity and mortality following heart transplantation. We previously reported the results of our in vitro study demonstrating the predictive value of the Virtual Crossmatch (VXM) and have since applied it clinically for sensitized children listed for heart transplant at our center. The VXM utilizes the results of specific antibody screening via flow cytometry to predict acute incompatibility. This review examines the effect of the VXM on wait times and outcomes., Methods: The study population included all patients listed for heart transplantation at Children's Hospital of Wisconsin. Antibody sensitization was defined as PRA>10% and/or by the identification of HLA specific antibody using AHG-CDC or flow cytometry. Categorical data was analyzed via Fisher's exact test while continuous variables were compared via an unpaired t test., Results: There were a total of 111 listed patients between 7/91-9/06. The sensitization rate was 23% (25/111). 19 patients who went on to transplant, deterioration or death were divided into 3 groups depending on listing strategy; Group 1 were listed with a prospective crossmatch requirement, Group 2 with a VXM, and Group 3 with a retrospective cross match. 7/8 patients in group 1 died prior to transplant with median wait time of 119 days. 9/10 patients in group 2 were transplanted with 100% survival and median wait time of 65 days, Group 3 included 1 patient who received a graft after 54 days and died 3 months post transplant with humoral rejection. The VXM was highly concordant with the retrospective crossmatch., Conclusions: Use of a VXM can lead to shorter wait times and better outcomes as a listing strategy for sensitized children requiring cardiac transplantation. The VXM allows transplant physicians to risk stratify patients and consider an anticipated positive crossmatch as one additional factor in the risk-benefit analysis inherent to any donor offer. This experience supports use of the VXM as an alternative to prospective crossmatch requirements. One should recognize that other era dependent improvements such as updated criteria for patient and donor selection along with newer therapies and bridging options may have contributed to superior outcomes seen in the more recent cohort of patients treated with the VXM approach.

The use of intravenous immunoglobulin (IVIG) in sensitized transplant candidates has resulted in reduced HLA antibody levels and shorter transplant wait times. In addition, the HLAMatchmaker program has been used to identify acceptable mismatches to permit transplantation in highly sensitized patients. We used IVIG desensitization in conjunction with high resolution HLA allele typing and HLAMatchmaker grading of donor offers to facilitate successful transplantation in two highly sensitized children who were awaiting second renal transplants. Both patients lost their initial transplant in <10 days to accelerated acute rejection, and were on dialysis for an average of 50 months with high panel reactive antibody (PRA) levels. They were started on monthly IVIG infusions (2 g/kg/dose). Within one wk following their third and fifth IVIG doses, both patients received a crossmatch compatible, deceased donor renal transplant selected by HLAMatchmaker as a suitable donor offer. Both patients remain rejection free with excellent renal function 19 and 15 months post-transplant, respectively. In conclusion, combining IVIG therapy and donor selection by HLA humoral epitope matching permitted successful transplantation of two highly sensitized children. Further studies in larger numbers of patients with longer follow-up are needed to determine the individual role played by, and relative importance of each component of this combined strategy.

Introduction: The influence of a positive B-cell crossmatch on graft outcome in renal transplantation is controversial., Methods: We analyzed graft survival using Kaplan-Meier estimates for recipients of deceased donor kidneys who were either regraft transplant patients (n = 198) from 1990 to August 20, 2004, or primary transplant patients (n = 361) from December 15, 2000 to August 8, 2004, each of whom had a flow T- and B-cell IgG crossmatch performed before transplantation. The flow B-cell crossmatch (FBXM) was not used to decide whether or not to transplant. Graft survival was analyzed by whether the patient's FBXM was positive or negative. We also evaluated creatinine levels and graft survival of 131 transplant patients (June 1, 2004 to July 1, 2005) by their FBXM result and by their HLA class II flow-defined IgG PRA., Results: One- and three-yr graft survival for the primary transplant patient group with a positive FBXM (98% and 84%) was not significantly different from the group with a negative FBXM (96% and 93%) (log-rank = 0.9). Similarly, graft survival at one, five, and 10 yr for the regraft transplant group whose FBXM was positive (91%, 76%, and 61%) was not significantly different from the group whose FBXM was negative (91%, 79%, and 77%) (log-rank = 0.4). Creatinine levels in the group of patients whose FBXM was positive (1.4 +/- 0.4 mg/dL; n = 76) were not significantly different from the group with a negative FBXM (1.4 +/- 0.4 mg/dL; n = 42). Even in the presence of class II PRA, a positive FBXM did not impact a patient's creatinine levels or graft outcome., Conclusion: Neither short nor long-term graft survival of deceased donor kidneys is influenced by a positive flow B-cell IgG crossmatch, even when caused by HLA class II antibody.

The HLAMatchmaker program is based on the donor/recipient comparison of the polymorphic triplet amino-acid sequences of the antibody-accessible regions on the human leucocyte antigen (HLA) molecule. The previous reports on its predictive value for renal allograft outcomes are conflicting. We conducted a retrospective study in a predominantly African-American (AA) cohort (N = 101, 94% AA). HLA typing was performed by molecular methods and triplet matching using HLAMatchmaker. Study end points included graft survival and incidence of acute rejection. The relationship between the number of triplet mismatches (TMM) and the degree of HLA antigen MM was evaluated using Pearson's correlation coefficient. Logistic regression models were used to examine the association between triplet matching and the study end points. Kaplan-Meier and Cox proportional hazard models were used for graft survival analysis. The strongest relationship between the number of TMM and HLA antigen MM was observed for HLA-DQ (r = 0.88). The association between triplet matching at HLA-A, -B, -DR and -DRw HLA loci and the study end points was not statistically significant. However, after grouping, the unadjusted estimates of graft survival for those with more than 10 Class I TMM were significantly worse than the others (p = 0.03). Adjusting for the effect of donor source, recipient characteristics and the immunosuppressive regimen did not change this association (hazard ratio = 0.2, confidence interval = 0.04-1.1). We conclude that triplet matching using HLAMatchmaker can provide useful prognostic information in kidney transplantation and that more than 10 donor/recipient Class I HLA TMM is predictive of worse graft outcome.

Heart transplantation in the setting of human leukocyte antigen (HLA) sensitization is challenging, as a time-consuming prospective crossmatch (XM) may be required, severely limiting the number of potential donors. We evaluated a 'virtual XM', defining a positive virtual XM as the presence of recipient pre-formed anti-HLA antibodies to the prospective donor HLA type, and compared the virtual XM to a standard direct XM. Bead-based flow cytometric analysis was used to identify anti-HLA antibody (Ab) present in a child listed for heart transplantation. Using recipient serum, direct-flow cytometric T- and B-cell XM were run for potential donors against whose HLA type the recipient had specific antibodies (group 1, n = 7) and for potential donors with predicted compatible HLA types by virtual XM (group 2, n = 7). Results were expressed as median channel difference (MCD) between the control and recipient serum. A positive T-cell XM was defined as MCD > 50, whereas MCD > 100 constituted a positive B-cell result. The rate of T-cell reactivity was significantly less in group 2 than in group 1 (29% vs. 100%, p = 0.02); similarly, B-cell reactivity was also less for group 2 (14% vs. 100%, p = 0.005). The virtual XM was 100% sensitive in detecting positive flow cytometric XM results for T and B cells. Although only 72% specific in predicting a negative T-cell XM, and 86% specific for negative B-cell XM, the false negatives were weakly positive and would probably have been clinically acceptable. Currently, potentially suitable donor organs are often declined for lack of a prospective XM; these organs may ultimately be allocated to more distant recipients or perhaps not used at all. While further studies are needed, virtual XM has the potential to improve availability of organs for sensitized patients and improve the overall allocation process.

Refinements in human leukocyte antigen (HLA) typing techniques for hematopoietic cell transplantation (HCT) have permitted a more precise assessment of donor-recipient histocompatibility, which has impacted transplantation outcomes. More recently, differences in transplant outcomes associated with killer immunoglobulin-like receptor compatibility have emphasized the potential importance of natural killer (NK) cell typing in HCT. This article reviews the current state of the art for HCT donor selection based on both HLA and NK cell typing.

Background: Although there is an increasing body of evidence for a deleterious effect of mismatched donor HLA antigens on the outcome of human cardiac transplantation, the role of anti-HLA lymphocytotoxic antibodies remains controversial. Thus, their appearance after cardiac transplantation has been associated with poor outcome by some groups; whereas others have reported them to be of no clinical significance. Furthermore, their presence prior to cardiac transplantation has also been the subject of similarly conflicting reports. The deleterious effect of such pre-existing antibodies has been predicted by a positive lymphocyte cross-match (LCM), which, for most patients awaiting renal transplantation and in many requiring a cardiac allograft, leads to cancellation of the operation. The reason for undertaking the current study was to test the hypothesis that the constraints which a positive LCM result impose in preventing renal transplantation may not apply to orthotopic heart transplantation (OHT)., Patients and Methods: Four sensitized patients underwent OHT across a positive prospective LCM. Three were females, and one of those females also underwent cadaveric renal transplantation at the time of OHT. All four patients received aggressive early post-transplant immunosuppressive therapy, which included plasmapheresis, intravenous immunoglobulin (IVIg), antiproliferative agents (cyclophosphamide, basiliximab) and cytokine down-regulators (calcineurin inhibitors, muromonab-CD3) and anticell antibodies (OKT3, ATG). They also received standard immunosuppressive therapy which included corticosteroids. Complement-dependent cytotoxicity (CDC) was used for the identification of anti-HLA lymphocytotoxic antibodies. Reactivity of the latter against more than 10% of a panel of well-characterized T cells was considered sensitization, and required LCM to be performed prospectively, which test was also performed using the CDC technique., Results: Three of the patients exhibited evidence suggestive of acute or hyperacute rejection in endomyocardial biopsy specimens by postoperative day (POD) 7. Two of the three patients with rejection also exhibited haemodynamic instability (elevated filling pressures and reduced cardiac index) on POD 1, which improved with inotropic support. One patient sustained a cardiac arrest on POD 7, and was successfully resuscitated without sequelae. All patients are now doing well, postoperatively (follow-up: 17-57 months) post-transplant. Two patients have normal left ventricular function and one patient has mild left ventricular dysfunction. Two have no further evidence of sensitization (PRA < 10%)., Conclusions: Although the number of patients in this study is small, the long-term successful outcome of OHT following positive prospective cross-matches suggests that such a test result, in contrast to the restraints it imposes on renal transplantation, may not be a contra-indication to transplantation of the human heart. If OHT proceeds after the LCM is reported positive, aggressive immunotherapy should not only be initiated early, but should also be targeted at humoral-vascular rejection in particular.

Unlabelled: Minimizing the amount of cold ischaemia time (CIT) added to cadaveric kidneys before their transplantation is an important goal since longer CIT is associated with worse long-term graft outcome. Our organ procurement organization (OPO) and HLA laboratories have taken the approach of performing the histocompatibility testing, including the final cross-match, as early in the donor process as possible., Methods: The data in this study were collected from all consecutive final cross-matches done for cadaveric kidney (n = 113) and simultaneous pancreas + kidney (SPK) (n = 25) transplants done with organs recovered from donors in the Midwest Transplant Network OPO from 1 January 2001 to 9 May 2002. We evaluated the time the final cross-match was completed from when the kidneys from that donor were taken from the operating room (OR) and compared that time with CIT., Results: For kidney transplants, 72% of the final cross-matches were complete before the kidneys were taken from the OR. The CIT of that group (10.4 +/- 3.8 h) was significantly lower than that of the group of kidney transplant patients whose final cross-match was done after the kidneys were taken from the OR (15.5 +/- 5.8 h) (P < 0.001). Similarly, for SPK transplants, 88% of the final cross-matches were completed before the organs left the OR and the CIT of that group (10.2 +/- 3.4 h) was less than in the group whose final cross-match was done after the organs left the OR (14.3 +/- 4.8 h) (P > 0.1)., Conclusions: These data show that the practice of completing the final cross-match as early in the donor process as possible helps to minimize the amount of cold ischaemia time added to the kidneys and pancreata before transplantation. That should reduce the detrimental influence that longer CIT has on short- and long-term function in kidney as well as SPK transplantation.

We examined histological features of allograft biopsies in ABO minor-mismatched kidney transplantation. Forty-five patients who underwent ABO minor-mismatched kidney transplantation between September 1999 and December 2001 at our institute. The mean age was 32.6 years, with 28 males and 17 females. We divided them into five groups based on the donor and recipient ABO blood groups. Group 1, O renal allografts given to A patients (13 patients); Group 2, O to B (9), Group 3, O to AB (2); Group 4, A to AB (9); and Group 5, B to AB (12). From September 1999 to April 2002, we performed 127 allograft biopsies in these 45 ABO minor-mismatched kidney transplant recipients. Among a total of 127 biopsy specimens, 47 specimens were taken as 0- or 1-h biopsies and 6 were protocol biopsies. Pathological analysis of 74 episode biopsy specimens showed: acute humoral rejection (AHR) in 13 (18%); acute cellular rejection (ACR) in 17 (23%); combined AHR and ACR in eight (11%); borderline change in six (8%); chronic rejection in 10 (12%); cyclosporin or tacrolimus nephrotoxicity in seven (10%) and chronic allograft nephropathy in three (4%). In total, some form of acute rejection (AR) was seen histologically in 38 biopsy specimens (48%) from 19 patients (42%). When we investigated AR in two separate categories, i.e. AHR and ACR, AHR was diagnosed in 21 biopsy specimens (26%) from 15 patients (33%) and ACR was seen in 25 biopsy specimens (31%) from 13 patients (29%). We compared the incidence rate of acute rejection in the cases of ABO minor-mismatched renal transplantation with ABO-incompatible and ABO-compatible cases between January 1989 and December 1999. The incidence rate of AR in ABO minor-mismatched cases (42%) was statistically lower than that in ABO-incompatible cases (63%). There was no statistical difference in the incidence rate of AR between ABO minor-mismatched cases and ABO-compatible cases (49%). There was statistical difference in the incidence of AR among the donor and recipient ABO blood groups. Group 4 (A allografts given to AB patients) had the statistically highest rate of AR (89%), followed by Group 1 (54%), Group 5 (33%) and Group 2 (11%), and there was no AR case in Group 3 (O to AB). In conclusion, the incidence rate of AR in ABO minor-mismatched kidney transplantation is statistically lower than ABO-incompatible cases and is not statistically different from that in ABO-compatible cases. The incidence cases of AHR are slightly higher than that of ACR in ABO minor-mismatched kidney transplantation and this finding is similar to findings of ABO incompatible kidney transplantation. Finally, there is a statistical difference in AR incidence among the donor and recipient ABO blood groups.

We have analysed two Caucasian families in which recombinant individuals have been identified. In both families, initial low resolution typing of class I and II antigens of possible patients referred for bone marrow transplantation and their respective potential donors (based on inherited haplotypes analysis) revealed them to be HLA identical and supposedly inheriting-non-recombinant haplotypes. The mothers were found to be DRB1*04 generic allele homozygous, but possessing two DRB1*04 specific alleles, DRB1*0403 and DRB1*0404 (family A) and DRB1*0401 and DRB1*0402 (family B). In both cases the patients inherited a maternal haplotype that is the result of a recombination event between the mother's HLA-DRB1 and -B genes on their chromosomes. Based on linkage disequilibrium it is likely that the recombinant haplotypes are present in the patients rather than their brothers. In both families, the results of the MLC in terms of relative response was positive. Thus, these cases illustrate the importance of high resolution DNA class II typing when assignment of MHC antigens is of extreme importance (i.e. bone marrow transplantation).

Renal transplant recipients with positive flow cytometric crossmatches (FCXM) face greater risk of early rejection and graft failure. It is clear that the pharmacologic needs of this high risk group have not been identified. We retrospectively compared the impact of two drug regimens upon early rejection and 5 yr actuarial survival among 324 primary cadaveric transplant recipients with positive and negative FCXM. Patients received either Regimen I (OKT3 induction, cyclosporine and steroids) or Regimen II (mycophenolate mofetil with cyclosporine or Prograf). Recipient gender, age, disease etiology, ethnic distribution and cytotoxic panel reactive antibody (PRA) were equivalent between regimens (p=ns). With Regimen I, the incidence of rejection was greater for FCXM positive vs. FCXM negative patients (51 vs. 21%, p=0.001). In contrast, with Regimen II the incidence of rejection for FCXM positive and FCXM negative patients was equivalent (18 vs. 12%, p=ns) and lower than patients treated with Regimen I (p < 0.01). Ethnic variation was only observed with Regimen I in which African Americans with positive FCXM had more rejections than Caucasians (60 vs. 45%, p < 0.05). Five-year actuarial survival was lower for FCXM positive vs. FCXM negative patients treated with Regimen I (40 vs. 75%, p=0.0006) or Regimen 2 (60 vs. 90%, p=0.001). Allograft survival was equivalent (p=ns) among FCXM positive individuals receiving Regimen I or II. However, allograft survival among FCXM negative individuals improved with Regimen II (p < 0.05). Ethnic variation in survival was not observed with either regimen (p=ns).

Background: Acute allograft rejection (AR) in solid organ transplantation is generally regarded to develop through cell-mediated immune response following activation of helper T cells. Since production of antibodies is also mediated by helper T cells, humoral immunity may play some roles in AR. Although flow cytometry crossmatch (FCXM) is reported as a useful method for the detection of antibodies against donor antigen, specific role of T- or B-cell FCXM and its sensitivity for AR is controversial., Methods: T- and B-cell FCXM using fresh donor peripheral lymphocytes were performed before and after blood-type compatible living donor liver transplantation in 47 patients. IgM and IgG anti-donor antibodies were analyzed in relation to clinical AR., Results: Positive pre-transplant T-cell FCXM was associated with a high incidence of positive post-transplant T-cell FCXM (p=0.017). Four of five cases (80%) with positive pre-transplant T-cell FCXM experienced earlier AR (day 8.0+/-4.4, mean+/-SD) than 16 of 42 cases (31%) with negative pre-transplant T-cell FCXM (17.3+/-6.8; p=0.016). In addition, higher dose of steroids was given to treat AR episodes in cases with positive pre-transplant T-cell FCXM (79.9+/-10.3 mg/kg/month) than in those with negative pre-transplant T-cell FCXM (47.1+/-26.6; p=0.039). In the first month after transplantation, 13 episodes of positive post-transplant T-cell FCXM were all concomitant with or preceded clinical AR compared with seven ARs in T-cell FCXM-negative cases (p<0.0001). T-cell FCXM between positive sera and third parties revealed some crossreactions. In contrast, detection of antibodies by B-cell FCXM in pre- and post-transplant phases was scarcely associated with AR, and no correlation was found between T- and B-cell FCXM before and after transplantation., Conclusions: Positive T-cell FCXM is closely related with AR and that before transplantation is a predictor of early and refractory AR as well as post-transplant FCXM. In contrast, not a few detections of antibodies irrelevant to AR are observed in B-cell FCXM, suggesting its low specificity.

Methods to enhance natural microchimerism, which occurs after any successful organ transplant, are currently explored using unmodified donor bone marrow both in experimental and in clinical trials. Because of the potential immunomodulatory effects of donor bone marrow cells, we performed this study to evaluate the effect of single and multiple donor-specific bone marrow infusions (DSBMI) on chimerism and small bowel allograft survival in a fully histoincompatible rat model. Forty-five male DA rats and 45 female Lewis rats were used as donors and recipients, respectively, for a heterotopic small bowel transplant. Animals were separated into 10 groups according to the number of bone marrow infusions and immunosuppressive protocol used. Control groups (groups 1 and 2) did not receive any bone marrow infusion, groups 3 and 4 received one infusion at day 0 (150 x 10(6) cells), groups 5 and 6 received two infusions at days 0 and 4 (75 x 10(6) cells each), groups 7 and 8 received two infusions at days 4 and 10 (75 x 10(6) cells each), and groups 9 and 10 received five infusions at days 4, 10, 15, 20 and 25 (30 x 10(6) cells each). Animals in groups 1, 3, 5, 7 and 9 were immunosuppressed with 0.5 mg/kg FK506 while the remaining groups were immunosuppressed with 1 mg/kg FK506, from day 0 to 4 after transplant. Every 15 days, the chimeric state was determined by flow cytometry in order to detect cells expressing DA rat class I antigen, and small bowel biopsies were obtained from ileostomies. Animals in all groups showed minimal to moderate acute rejection at day 15 after transplant, however, vascular rejection (vasculitis, arteritis) was observed in only bone marrow groups (100% in 0.5 mg/kg and 42.1% in 1 mg/kg FK506 groups). On day 30, 58.3% of bone-marrow-infused animals and 66.6% of controls showed severe acute and early chronic rejection. The chimeric levels varied from 0 to 12% after transplant and were significantly higher in bone-marrow-infused groups compared with controls (p < 0.05). We conclude that modulation of immune response with short-course immunosuppression and a single or multiple DSBMI did not improve allograft or recipient survival. The inability to achieve a stable chimeric state did not allow us to determine the effect of chimerism on graft and recipient survival after small bowel transplantation.

Knowledge of DNA sequence variation may help us understand how genetic variability gives rise to functional variability and, in so doing, revolutionize the development of strategies to combat and prevent disease. Single nucleotide polymorphisms (SNPs) are stable, inherited, biallelic, single base pair differences which are present in the human genome at a density of 1 to 10 per 1,000 nucleotides. It is anticipated that SNPs will account for much of the functional heterogeneity in gene expression and protein activity exhibited in the human population. Susceptibility to or protection from a number of diseases, particularly those of autoimmune etiology, has been associated with specific alleles of the human leukocyte antigen (HLA) complex. Interestingly, the precise molecular defects in the HLA genes are unknown and the notion that non-HLA genes, within the same chromosomal region, are involved remains a formal possibility. We have determined the nucleotide sequence of a contiguous 2.2 Mbp segment of chromosome six that includes all of the HLA class I region, and have identified over 10,000 SNPs therein. Because of the derivative knowledge of gene and SNP content and position, the scientific community is now uniquely poised to identify disease-contributory SNPs that lie within the MHC.

The methodology and applications of oligonucleotide array technology are reviewed. An oligonucleotide array system for typing HLA-B alleles is described. Oligonucleotide probes representing all known polymorphisms in exons 2 and 3 of HLA-B are immobilized on glass slides. Exons 2 and 3 are amplified from human genomic DNA using fluorescent-labeled primers and subsequently allowed to hybridize to an oligonucleotide array on glass supports. The presence of positive hybridization is detected by fluorescence scanning. HLA-B alleles are assigned by quantitative analysis of hybridization patterns. Proof of principle has been established in blinded testing of heterozygous DNA samples.

DNA typing systems for alleles of the HLA class I loci A, B, C at intermediate (IR) and high resolution (HR) levels were developed. The approaches combine locus-specific amplification of genomic DNA by the polymerase chain reaction (PCR) and hybridization with sequence-specific oligonucleotide probes (SSOP). The SSOP were designed to match nucleotide sequences at all polymorphic sites of exons 2 and 3 at these loci. Alleles and genotypes for these loci are assigned by their unique hybridization patterns. Some genotypes with particular allele combinations resulted in the same hybridization pattern. These genotype ambiguities were resolved by performing additional group-specific amplifications with appropriate GSA primers and hybridization with informative SSOP. At intermediate resolution level, many groups of alleles of HLA-A and B with the same serologic equivalence resulted in the same hybridization pattern. Both HR and IR typing approaches required the design, validation and testing of locus- and group-specific primers and sequence-specific oligonucleotide probes (SSOP). Single locus-specific amplification and hybridization with sets of 67 SSOP for HLA-A, 99 for HLA-B and 57 for HLA-C allowed us to identify unequivocally the majority of A, B, C alleles at HR level. To resolve ambiguous genotypes at HLA-B, we performed 4 GSA with 5' primers at codon 45-46 and hybridization with selected sets of SSOP. About 22,415 high resolution typing results were obtained (4,953A, 6,621B, 10,841C). In these samples, 63 HLA-A, 170 HLA-B and 40 HLA-C alleles were observed. In the course of these studies, more than 30 new alleles have been identified. In IR testing, sets of 39 SSOP for HLA-A typing and 59 SSOP for HLA-B typing allowed us to obtain maximal resolution of the majority of common genotypes. To achieve IR level, the majority of SSOP selected were those that span codons encoding amino acid residues located in the alpha helical segments of the class I molecules. A total of 50,522 samples were typed for HLA-A and B at IR level. Approximately 2.0% of them carried ambiguous genotypes associated with alternative switches of Bw4/w6 related sequences. All these ambiguities were resolved by Bw4/Bw6 GSA by 2 primer pairs (77N-IALR-83/3B.1; 77S-DLRG-83/3B.1) and hybridization with 9 selected SSOP Testing was performed by individual hybridization of replicate dot blot membranes with the SSOP of the corresponding set. The approach was robust and cost-effective in large-scale HLA class I molecular typing. The resolution provided by HLA-A, B IR reached serologic split-level or higher. The description of primers and probes for HLA class I typing may be utilized as starting elements for development of second generation methods with a more rapid turn around time.

Sequence-specific amplification (SSP) is simply a form of polymerase chain reaction (PCR) which involves designing one or both primers so that they will or will not allow amplification (the 3'-mismatch principle). Its origins are probably legion, i.e. many people probably thought of it at the same time. For example, in 1988 a group from Guy's Hospital, London, described a form of SSP for HLA-DR4 detection and in the same year a group from Upjohn described its use at the American Society of Histocompatibility and Immunogenetics (ASHI). Both are published in abstract form (British Society of Rheumatology and ASHI). The 3'-mismatch principle can be used to identify virtually any single nucleotide point mutation (SNP) within one or two PCR-SSP reactions and the first peer-reviewed statements of this came in 1989 (1, 2). Thus, although the use of SSP probably began around 1990, it was 5 years before its popularity erupted, mainly due to the work of Olerup & Zetterquist (3, 4), who defined its potential for solid organ transplantation. It is now the method of choice for high resolution HLA typing in many laboratories. In addition, over a thousand applications for genes outside the MHC are in the literature.

Reference Strand Mediated Conformation Analysis (RSCA) is a novel conformational method that offers high resolution and high sample throughput for typing the HLA class I and class II genes. This technique differs from conventional sequence-based typing methodologies in that the HLA type is assigned on the basis of accurate measurement of conformation-dependent DNA mobility in polyacrylamide gel electrophoresis (AGE). RSCA utilises a fluorescent-labelled locus-specific reference DNA to selectively modify the molecular conformation of the tested DNA. The use of laser-based instrumentation and computer software, in addition to internal DNA markers for correction of gel variability, allows the discrimination of HLA alleles which differ by one nucleotide in a DNA fragment nearly as large as a kilobase in length. RSCA has been successfully applied in blind studies of HLA typing, demonstrating that is reproducible, able to identify new alleles and able to resolve ambiguous heterozygous combinations.

The last twenty years have seen an exponential growth in the application of DNA technology to the field of Histocompatibility and Immunogenetics (H&I). Initially, this was confined to a few research laboratories. However, the development and application of several different DNA methods by many laboratories has led to the situation whereby nearly every H&I laboratory performs some DNA typing for the detection of HLA alleles. It would not be unfair to say that H&I has shown diagnostic laboratories in other disciplines how useful the DNA techniques can be. This review attempts to summarise the history and application of DNA methods in the field of Histocompatibility (Table 1).

Transplantation has become the treatment of choice for end-stage organ failure in the four and a half decades since the first successful kidney transplant procedure was performed in 1954. The achievements of solid organ transplantation can be gauged by the progressive increase in the number of kidney, liver, heart, pancreas and lung transplants that are performed annually. Advances in surgical technique, immunotherapy, control of opportunistic infections and tissue typing have collectively contributed to current 1-year patient survival rates that exceed 95% and current 1-year graft survival rates in excess of 85%. This paper will review some of the major advances that have lead to the current state of transplant immunobiology.

Allogeneic bone marrow transplantation (BMT) from an HLA-matched sibling appears to improve survival and diminish some of the physiologic derangements seen in children with mucopolysaccharidosis (MPS)-I (Hurler Syndrome), an inherited metabolic storage disease resulting from the lack of alpha-L-iduronidase enzyme activity. Death is usually expected in the first decade of life. Unfortunately, most patients lack an HLA-matched sibling donor and alternative donors have been identified for transplant. This study reports on a five-year median follow-up (range: 985-2,355 days) in 11 Hurler Syndrome patients who underwent allogeneic BMT from partially mismatched related donors (PMRDs). The median age was 20 months (range: 11-44 months). The overall survival rate was 64% (95% CI 34-94%). The overall graft failure rate (36%) was higher than reported with matched sibling BMT. All patients with sustained engraftment experienced improvement in physical manifestations, such as corneal opacity, gum and tongue hypertrophy, hepatosplenomegaly and joint mobility. Skeletal abnormalities, such as dysostosis-multiplex, were stabilized but not reversed. Some patients have continued to show decline in neuropsychometric testing, while others appear to stabilize and one has demonstrated improvement. Until better methods for replacing enzyme activity are developed, BMT from a matched sibling of alternative donors can be considered a viable intervention for Hurler Syndrome patients to achieve partial improvement or stabilization from the deterioration caused by substrate storage, particularly in minimally affected patients early in life.

The use of immunomagnetic beads for STAT cadaveric crossmatching for renal transplantation reduces test cost by 50%, laboratory work-up time by 50% and cold ischemia time by 4 h as compared with the nylon wool method of T- and B-lymphocyte separation.

The purpose of this study is to investigate the impact of recipient race as well as HLA matching upon long-term survival following heart transplantation (HTx). The study also determines whether the degree of HLA matching between Caucasians and African Americans differs. This study was a retrospective analysis of 336 males (77% Caucasians and 23% African Americans) transplanted between 1983 and 1994, all having received cyclosporine-based immunosuppression. The results showed African Americans were transplanted at a significantly younger age than Caucasians (39.1 +/- 11.2 vs. 48.2 +/- 10 yr). The composition of end stage cardiac disease was startlingly different. Caucasians demonstrated 62% CAD, 31% IDCM and 7% other, whereas African Americans exhibited 24% CAD, 69% IDCM and 7% other. The ten-year survival rate for African American male recipients was inferior to Caucasian males. Survival at 1, 3, 5, 7 and 9 yr was 83%, 73%, 63%, 51%, 46% for Caucasians and 70%, 58%, 51%, 38% and 32% for African Americans. Furthermore, African Americans received more poorly matched organs compared to Caucasians. Among Caucasians for Class I, 46%, 53% and 1% received poor, moderate and well matched hearts, respectively. In contrast, African American were allocated 65%, 33% and 0% of poor, moderate, or well matched hearts. An analysis of Class II data revealed the same pattern (63%, 35% 2% of Caucasians and 78%, 18%, and 4% of African Americans received poor, moderate or well matched hearts). Survival for Caucasians improved when moderately versus poorly matched for Class I. Class II matching did not have an effect. African Americans showed a similar trend, although statistical significance was not reached. When comparing equivalent degrees of matching, African Americans had inferior survival rate when poorly matched for Class I relative to Caucasians. No statistical difference was observed for moderate matched Class I or for Class II analysis.

The purpose of presenting this case is to illustrate the importance of high-resolution DNA Class 2 typing when assignment of MHC antigens is of extreme importance (i.e. bone marrow transplantation); suggest this test as a substitute for the mixed lymphocyte culture (MLC), and provide evidence that DRB1 subtype mismatches may be clinically significant. Initial serological and monoclonal HLA Class 1 and low-resolution DNA-SSP Class 2 typing of a potential bone marrow transplant patient and two sisters revealed all to be HLA identical with apparent homozygosity for DRB1 x 04. High-resolution DNA-SSP Class 2 typing was then performed and revealed electrophoretic banding patterns which were not specific for any DRB1 x 04 subtype. One sister and the patient had identical patterns, while the other sister had a different pattern. Complete HLA Class 1 and high-resolution DNA-SSP Class 2 typing of the parents was performed. The mother was found to be heterozygous for Class 1 and Class 2 antigens and possess the DRB1 x 0404, 0405 subtypes. In contrast, the father was found to be homozygous for Class 1 antigens, heterozygous for Class 2 antigens and possess the DRB1 x 0404, 0407 subtypes. This led to the initial false assumption of HLA identity for the patient and her two sisters. However, assignment of haplotypes revealed one sister to be HLA identical with the patient and the other sister to be a one-haplotype five-antigen match with the patient, mismatching for one DRB1 allele. Bone-marrow transplantation was performed utilizing the latter sister, which resulted in intractable acute graft vs. host disease that resulted in the patient's demise.

The SEOPF high-grade match (HGM) algorithm for sharing kidneys was designed to increase utilization of kidneys for excellent HLA matches, decrease sharing for poor HLA matches, and improve the chances for highly sensitized patients to receive a kidney transplant through the use of ROP crossmatch trays. All retrieved kidneys from participating centers were offered to the system to be shared for high-grade HLA matches after six-antigen match obligations were satisfied with UNOS. Kidneys were shared according to HLA matches in the following priority: 1) 0-ABDR mismatch six - Antigen Matches. 2) 0-AB mismatch PRA > 40%, ROP tray negative crossmatch. 3) 0-BDR mismatch PRA > 40%, ROP tray negative crossmatch. 4) 0-BDR mismatch PRA < 40%. All patients with PRA's > 40% must be represented on the ROP tray to be considered for a shared kidney. Preliminary ROP tray crossmatch results must have been known for these patients in order to access the system. Here we report results of 126 shared kidneys using the HGM algorithm from January 1994 to September 1995. HLA typing concordance between the initial typing of cadaver donors by the donor center and repeat typing by recipient center was 94.7%. There were six (5.3%) typing discrepancies: HLA-A, no typing discrepancies; HLA-B, 2 (1.8%) typing discrepancies; and HLA-DR, 4 (3.5%) discrepancies. A total of 34 of 126 (27%) kidneys were shared for patients with high grade HLA matches, PRA's > 40% and a negative ROP tray. Twenty-eight of 34 (82.4%) shared kidneys were final crossmatch negative and transplanted into the intended recipient. Six of 34 (17.6%) shared kidneys were transplanted into backup recipients. All six of the intended recipients had positive final crossmatches using enhanced crossmatch procedures, such as antiglobulin and flow cytometry. In conclusion, the HLA concordance rate for shared kidneys within SEOPF was 94.7%. Kidney sharing using the HGM algorithm was very effective in transplanting highly sensitized patients when good HLA matching was combined with a negative ROP tray result.

The Southeastern Organ Procurement Foundation (SEOPF) data base from October 1987 to September 1995 included 12,536 first cadaveric kidney transplants, of which 39.5% were placed into non-white recipients (N-WR). Consequently, the SEOPF data offered an excellent sample in which to examine the effect of a number of conventional HLA-A, B versus public (crossreactive group, CREG) matching schemes on access and graft survival (GS). The standard applied was that 3-yr GS had to be greater than that of the mean GS of the racial group. Because of the excellent 1- and 3-yr GS of 0 A, B, DR mismatched kidneys in both white recipients (WR) = 87.9% and 82.8% and N-WR = 86.9% and 80.9%, these transplants were excluded from further analysis. Thereafter, the mean 1- and 3-yr GS of the remaining 7011 WR and 4862 N-WR, transplants used for all subsequent comparisons were 82.5% and 72.4% for WR versus 80.9% and 63.0% for N-WR. Four different matching schemes were compared: 1) Class I A, B versus CREG hierarchical effect disregarding DR; 2) 0, 1, 2B, DR versus 0-2 CREG, DR mismatching; 3) DR mismatching disregarding Class I A, B or CREG mismatching and 4) Class I A, B versus CREG mismatching stratified for DR. Two and possibly three CREG, DR mismatched groups were identified in which improved access and GS occurred in comparison with the 0, 1, and 2B, DR mismatched transplants currently receiving 7, 3 and 1 point in the UNOS sharing algorithm. The 1- and 3-yr GS in WR and N-WR of these groups were as follows: 0 CREG, 0 DR mismatches: 85.7% and 78.3% in WR (n = 385); 83.0% and 69.4% in N-WR (n = 157). 1-3 CREG, 0 DR mismatches: 84.5% and 74% in WR (n = 1550); 82.7% and 65.7% in N-WR (n = 1083). Based on this analysis, there may be a significant potential advantage of prospective Class I CREG over A, B matching for first cadaver kidney transplantation.

There is limited information regarding the role of flow cytometry crossmatching (FCXM) in primary cadaver kidney allografting and even less about B cell reactivity and graft survival (GS). Furthermore, there is little or no published data concerning reaction strength (cutoff value), the effect of historic sera reactions, and the usefulness of performing autologous crossmatches (XMs) on GS. These factors were examined retrospectively on 214 primary transplants performed from August 1991 to January 1994 with follow-up to July 1995. Three-color FCXMs were done on a 1024-channel BD-FACScan, and the shift in median channel fluorescence (MCF) over the negative control was calculated. All patients had a negative T cell (AHG) and warm B cell (2 was, extended incubation) cytotoxicity XM, and none was excluded in calculating GS. A quantitative effect was noted as stronger MCF shifts vs. T or B cells correlated with decreased GS (r = 0.98 and 0.92, respectively). Significant differences were seen with cutoff values of T = 50 and B = 110 which were 1.7-1.8 times the SD above the mean MCF of normal sera controls T neg patients (n = 198) and 1- and 3-yr actuarial GS of 86% and 79% compared to T pos patients (n = 16) of 75% and 49%, p = 0.008. B neg patients (n = 177) had 1- and 3-yr GS od 86% and 81% compared to B pos patients (n = 37) of 78% and 47%, p = 0.005. Most informative was the analysis of combined T and B cell FCXM results. Three years GS for T neg - B neg patients (n = 171) was 81% and for T pos - B neg patients (n = 6), it was 83%, p = 0.98. The 27 T neg - B pos group's GS was lower at 62% but did reach significance. Poorest GS was seen for T pos - B pos patients (n = 10) at 23%, p = 0.0001. Reaction patterns showed that T cells detected only HLA Class I antibodies, whereas B cells detected both Class I and II. Historic sera (> or = 1 month old) reactivity influenced GS. Patients with > or = 2 past sera positive but current serum negative reactions vs. T or T plus B cells (n = 7) had a poor 29% GS, while those historically positive only vs. B cells (n = 7) had 100% GS. On the other hand, patients positive only with the current serum (n = 16) had 2-yr GS of 100% (false positive test?), while patients whose current and historic sera reactions were positive (n = 21) had a 25-50% GS (true positive test?). About 1 in 15 patients (19%) displayed positive autologous FCXM reactions. Subtraction of autologous MCF shift values from those vs. the donor converted 17 patients to the T neg - B neg or T pos - B neg group whose 2-yr actual GS was not significantly different (p > 0.8) from those initially testing T neg B neg vs. their donors.

In a retrospective study of 84 primary cadaver donor orthotopic liver transplants (OLTs), we investigated the damaging effect of preformed donor-specific antibodies by a standard T-cell warm crossmatch test (TWXM) and a more sensitive flow cytometry crossmatch test (FCXM). The incidence of a positive crossmatch was 12% (10/84) by TWXM and 24% (20/84) by FCXM, respectively. The incidence of 1-month graft failure was 30% (3/10) for patients with a positive TWXM, compared with 23% (17/74) for a negative TWXM. The deleterious effect was more pronounced when the patients were examined by FCXM. Nine of 20 (45%) FCXM-positive recipients lost their grafts within 1 month (p = 0.01) and 8 of those 9 failures occurred within 2 weeks posttransplant. A comparison of graft survival rates at 6 months demonstrated that FCXM-positive patients had a 25% lower survival rate (p = 0.01) than those with a negative FCXM. The positive rate of FCXM was 4 times higher in sensitized patients (PRA 11-100). However, 9 patients in the non-sensitized group (PRA 0-10) had a positive FCXM, and 5 of 9 patients had graft failure within 1 month, suggesting that even a low level of antibodies or non-complement fixing antibodies may have a very damaging effect. This study shows that preformed donor-specific antibodies do have a deleterious effect on allografts. Although the effect is not necessarily expressed as hyperacute rejection, the antibody response is capable of damaging the allografted organs within 1 month. Consideration should be given to crossmatching OLT patients in order to avoid transplantation over a positive crossmatch.

The use of DNA fingerprinting with synthetic oligonucleotides is illustrated for practical applications familiar in clinical diagnostics: pre- and postnatal zygosity determination and the monitoring of bone marrow transplantation. A simple method for non-radioactive detection is described which may be interesting for many diagnostic laboratories.