Dynamic challenges inhibiting optimal adoption of kidney paired donation: findings of a consensus conference.

While kidney paired donation (KPD) enables the utilization of living donor kidneys from healthy and willing donors incompatible with their intended recipients, the strategy poses complex challenges that have limited its adoption in United States and Canada. A consensus conference was convened March 29-30, 2012 to address the dynamic challenges and complexities of KPD that inhibit optimal implementation. Stakeholders considered donor evaluation and care, histocompatibility testing, allocation algorithms, financing, geographic challenges and implementation strategies with the goal to safely maximize KPD at every transplant center. Best practices, knowledge gaps and research goals were identified and summarized in this document.

Calculated PRA: initial results show benefits for sensitized patients and a reduction in positive crossmatches.

The calculated panel reactive antibody (CPRA), which is based upon unacceptable HLA antigens listed on the waitlist form for renal transplant candidates, replaced PRA as the measure of sensitization among US renal transplant candidates on October 1, 2009. An analysis of the impact of this change 6 months after its implementation shows an 83% reduction in the number of kidney offers declined nationwide because of a positive crossmatch. The increasing acceptance and utilization of unacceptable HLA antigens to avoid offers of predictably crossmatch-positive donor kidneys has increased the efficiency of kidney allocation, resulting in a significant increase in the percentage of transplants to broadly sensitized (80+% PRA/CPRA) patients from 7.3% during the period 07/01/2001-6/30/2002 to 15.8% of transplants between 10/1/09-3/31/10. The transplant rates per 1000 active patient-years on the waitlist also increased significantly for broadly sensitized patients after October 1, 2009. These preliminary results suggest that 'virtual' positive crossmatch prediction based on contemporary tools for identifying antibodies directed against HLA antigens is effective, increases allocation efficiency and improves access to transplants for sensitized patients awaiting kidney transplantation.

Proinflammatory events are associated with significant increases in breadth and strength of HLA-specific antibody.

Identification of factors responsible for an increase in the breadth or strength of HLA-specific antibody (HSA) is critical to the continued successful management and transplantation of sensitized patients. A retrospective review of our HLA registry identified 107 patients with known HSA and sufficient information in their electronic patient record to determine the presence or absence of a proinflammatory event. The patients were stratified according to transplant status [sensitized and on the transplant waitlist (n = 65); immunosuppressed recipients of a positive crossmatch (+XM) transplant (n = 42)]. Eighty-three percent of waitlist candidates and 55% of sensitized kidney transplant recipients with a documented proinflammatory event had an associated increase in HSA. Interestingly, among patients with a culture-proven infection, 97% of the waitlist patients and 54.8% of +XM recipients had an associated rise in HSA. Overall, proinflammatory events were associated with a greater increase among waitlist patients than +XM recipients, 5.3-fold [IRR 5.25, (95% CI 4.03-6.85), p < 0.001] versus 2.5-fold [IRR 2.54, (95% CI 1.64-3.95), p < 0.001] increase in HSA. Therefore, sensitized patients known to have an infection or undergoing surgery should be monitored for expansion of HSA.

Incidence of humoral sensitization in HLA partially mismatched hematopoietic stem cell transplantation.

As part of the 15th International Histocompatibility and Immunogenetics Workshop (IHIWS), seven centers participated in a collaborative project to determine whether any significant humoral sensitization occurred post-transplant among recipients of HLA partially mismatched hematopoietic cell transplants (HCTs). A total of 140 donor/recipient pairs were enrolled with a total of 367 pre-and post-transplant sera analyzed. The majority of the samples (69.1%) were obtained within 30-90 days post-HCT. HLA-specific antibodies were defined using single antigen bead assays on a Luminex platform with a positive cutoff value of 1000 normalized median fluorescence intensity (MFI). There was an overall incidence of post-HCT sensitization toward donor HLA mismatches of 5.7%; however, all cases were among recipients of one HLA haplotype-mismatched grafts under nonmyeloablative, pre-transplant conditioning. Among the one haplotype-mismatched recipients, 15.7% (8/51) developed donor HLA-specific antibodies and 29.4% also had antibodies directed toward third party HLA antigens. Among the donor-specific antibodies, 9.8% were directed toward HLA class I antigens; 7.8% were against class II antigens; and 2.0% had both class I and II specificity. The relative strength of post-transplant antibodies was low with no significant difference in the mean maximum MFI values between third party and donor-specific antibodies. Because only a small number (10.2%) of the post-transplant samples were obtained 180 days or more post-HCT, longer term study is needed to evaluate any clinical relevance of these low-to-moderate levels of donor-specific antibody in one haplotype-mismatched recipients, as well as to determine whether any other antibodies occur at later times.

Donor HLA-specific Abs: to BMT or not to BMT?

The engraftment failure associated with Abs to donor-specific HLA (DSA) limits options for sensitized BMT candidates. Fourteen of fifteen patients with no other viable donor options were desensitized and transplanted using a regimen of plasmapheresis and low-dose i.v. Ig modified to accommodate pre-BMT conditioning. DSA levels were assessed by solid-phase immunoassays and cell-based crossmatch tests. DSA levels were monitored throughout desensitization and on day -1 to determine if there was any DSA rebound that would require additional treatment. A mean reduction in DSA level of 64.4% was achieved at the end of desensitization, with a subsequent reduction of 85.5% after transplantation. DSA in 11 patients was reduced to levels considered negative post-BMT, whereas DSA in three patients remained at low levels. All 14 patients achieved donor engraftment by day +60; however, seven patients suffered disease relapses. Four patients experienced mild, grade 1 GVHD. Factors influencing the response to desensitization include initial DSA strength, number, specificity, DSA rebound and a mismatch repeated from a prior transplant. While desensitization should be reserved for patients with limited donor options, careful DSA assessment and monitoring can facilitate successful engraftment after BMT.

Reversal of skin test anergy during maintenance hemodialysis by protein and calorie supplementation.

The effect of protein and calorie supplementation on the immune function of two maintenance hemodialysis patients was assessed. Before nutritional supplementation, both patients were anergic to four skin test antigens and had low relative percentages and absolute number of T lymphocytes. After 3 months of nutritional supplements both patients responded to in vivo skin testing to at least two antigens and in both patients, the relative percentage and absolute number of T lymphocytes increased. These two cases illustrate that the defect in cell-mediated immunity and impaired delayed cutaneous hypersensitivity which is known to occur in hemodialysis patients may be a reversible manifestation of protein-calorie malnutrition.

The distribution of HLA antigens and phenotypes among donors and patients in the UNOS registry.

We have analyzed HLA data from the UNOS registry on 20,230 patients on the renal waiting list in 1991 and 18,708 donors from 1988-1992. Significant differences were found in the distribution of HLA antigens for comparisons of the total donor pool and the various racial groups of patients as well as for inter- and intraracial comparisons of donors and patients. Within a racial group, the frequencies of blanks and of broad antigens were usually higher in patients while those of splits were usually higher in donors. Comparisons between the total donor pool and the various racial groups of patients showed that the likelihood of mismatch was greater for African-Americans and Hispanics than for Caucasians but that the chance of mismatch is high for all groups and the average number of antigens mismatched will not vary greatly among the different races. Heterogeneity, as measured by the percentage of the population with different phenotypes, was higher in African-Americans (97.2-99.7%) and Hispanics (97.7-99.4%) than in Caucasians (83.3-86.5%) because of multiple occurrences of a few phenotypes, most containing A1, B8 and DR3, in Caucasians. However, the most common phenotypes of Caucasian donors differed from those of Caucasian patients. All phenotypes were rare (0.007-0.61%) and, with the exception of a small group of Caucasian patients, the likelihood of achieving a good match is low, regardless of race. These data explain the observations that, with the exception of the phenotypically identical match, HLA matching does not influence organ distribution significantly.

Effect of Bw4 and Bw6 epitope mismatches on antibody production, acute and chronic rejection, and graft survival in renal allografts.

BACKGROUND: Highly sensitized patients often have antibodies directed against the HLA Bw4 and Bw6 epitopes. Because of the high frequency of these epitopes, when present, these antibodies result in a high incidence of positive cross-matches. We sought to determine whether antibodies specific for Bw4 or Bw6 affected renal allograft outcome. METHODS: The effect of mismatches for the HLA class I public epitopes, Bw4 and Bw6, was examined in 72 recipients of one haplotype matched recipients of living, related donor renal allografts selected to control for degree of HLA mismatch. Analysis of the production of HLA-specific antibody was performed for 180 recipients of failed cadaveric allografts by complement-dependent cytotoxicity tests and by an enzyme-linked immunoadsorbent assay (ELISA). RESULTS: No significant difference was observed in the incidence of acute rejection, number of rejection episodes or 1-year allograft survival among Bw4/6 matched versus mismatched recipients of one haplotype matched allografts. Additionally, no significant difference in the development of chronic allograft nephropathy was noted among 56 recipients followed long-term (> or =3 years). In the recipients of failed cadaveric transplants, Bw4/6 mismatching was associated with the frequency and magnitude of production of HLA-specific antibody. However, the panel reactive antibodies correlated with the number of HLA-A and -B mismatches, and there was no additional impact of Bw4/6 mismatching. IgG, HLA-specific antibodies were found to be significantly increased among patients homozygous for Bw4 or Bw6, whether or not there was a Bw4/6 mismatch. CONCLUSIONS: Mismatching for Bw4 or Bw6 does not confer any independent, increased risk for humoral sensitization or renal allograft failure.

Strategies for determining HLA compatibility in related donor bone marrow transplantation.

BACKGROUND: Although HLA identity between donor and recipient is no longer an absolute requirement for bone marrow transplantation, knowledge of the degree of HLA compatibility is necessary for determining the induction and immunosuppression regimen to be used. In cases of related donor transplantation, HLA compatibility may be assessed by defining the HLA phenotypes at the allele level using high-resolution, DNA-based typing methods or by determining the genotypes of the patient and potential donor from the HLA phenotypes, ascertained by low-resolution typing, of their family members. METHODS: We developed an algorithm that can be used to assess the relative costs of these two approaches. We applied population frequencies for HLA-DR alleles to this algorithm to determine at what cost per test ratio for high-resolution:low-resolution testing the costs of the two approaches are equal. RESULTS: In transplants involving a sibling pair who have the same HLA-A, -B, and -DR antigens, these values are 1.16-1.83 for African-Americans and 1.23-1.97 for Caucasians, depending on the relatives available for testing. With a slight increase in the resolution level achieved with DR antigen testing, the range of values becomes 1.10-1.74. We also estimated that the probability that two antigenically identical siblings have identical HLA-DRB1 alleles is >99% for both African-Americans and Caucasians. A review of 615 cases from our transplant program showed that all of 192 pairs of antigenically identical patients and sibling donors were genotypically or allelically identical, indicating that this estimate is valid. CONCLUSIONS: Transplant programs can apply these algorithms to determine the most cost-effective scheme for histocompatibility testing.

Elimination of alloantibodies by immunoablative high-dose cyclophosphamide.

BACKGROUND: Alloimmunization is a major problem for patients being considered for solid organ transplantation and in patients who require blood transfusion support. We previously demonstrated that high-dose cyclophosphamide (200 mg/kg) without hematopoietic stem cell transplantation leads to durable complete remissions in aplastic anemia and other autoimmune disorders. We now examine the ability of high-dose cyclophosphamide to eliminate alloreactivity. METHODS: IgG-specific antibodies to HLA class I were assayed using enzyme-linked immunosorbent assays in 18 consecutive patients with severe aplastic anemia before and after treatment with high-dose cyclophosphamide. RESULTS: Anti-HLA antibodies were detected before or shortly after therapy in 5 of the 18 patients studied. Complete remission of aplastic anemia was achieved in four of these five patients. High-dose cyclophosphamide markedly reduced anti-HLA antibody titers in these four patients; they were completely eradicated in three patients. Only one patient did not achieve significant reduction in the alloantibody titer after high-dose cyclophosphamide. CONCLUSIONS: High-dose cyclophosphamide without stem cell transplantation can eradicate HLA-specific alloantibody.

Evaluation of HLA antibodies with the PRA-STAT test. An ELISA test using soluble HLA class I molecules.

HLA-specific antibody, present before or after transplantation, may adversely effect graft outcome. Antibody testing by cytotoxicity (CYT) is laborious, requires viable lymphocytes, does not differentiate non-HLA cytotoxic antibody, and cannot be used readily on specimens from patients being treated with cytotoxic antibodies. We have evaluated PRA-STAT, an antibody screening kit that uses an ELISA test with soluble HLA class I molecules as targets. We performed 219 tests on a variety of serum specimens, 128 of which were also tested by CYT. There was a highly significant correlation (r = 0.78, P < 0.001) between PRA-STAT (PS) and CYT for the detection of IgG antibodies. Of 66 sera reactive in both assays, 18% had identical specificities defined in both, 27% were more reactive in PS than in CYT, 8% were more reactive in CYT, and 47% had different specificities in the 2 assays, with overlap in slightly more than half the cases. Of 13 sera reactive only in PS, 2 were from non-transfused, nontransplanted males with no evidence of lymphocyte-reactive antibody by antiglobulin tests. PS uses an IgG-specific conjugate, therefore IgM class I-specific antibodies cannot be identified--however, their presence does affect test outcome. This, as well as the panel composition and interlot reproducibility, are areas we believe need to be addressed. The PRA-STAT system is rapid, does not require viable cells or complement, and can be automated in part. Resolution of the problems identified here and availability of an IgM-specific conjugate should make this test system a valuable tool in histocompatibility testing.

The frequencies of HLA alleles and haplotypes and their distribution among donors and renal patients in the UNOS registry.

HLA allele and haplotype frequencies are used in transplantation, anthropology, forensic medicine, and studies of the associations between HLA factors and the immune response. The cost of determining these frequencies through family studies can be avoided by estimating them from population data. We have utilized the data in the UNOS donor registry and kidney transplant waiting list to estimate allele and haplotype frequencies for the HLA-A, -B, and -DR(B1) loci and report the allele and a portion of the haplotype data here. Using programs written in A Program Language (APL) we were able to perform all analyses on a personal computer. We have found that the distribution of haplotype frequencies varies among the races, with Caucasians having a greater number of both more common and extremely rare haplotypes. Despite the sizes of the groups studied, only one-third to two-thirds of the haplotypes theoretically possible were actually observed. Although the data confirm the well-known fact that the distributions of alleles and haplotypes varies among races, they also reveal that certain common haplotypes are shared among all racial groups and represent an opportunity for well-matched transplants between donors and recipients of different races.

Local impact of 1995 changes in the renal transplant allocation system.

In 1995, changes to the United Network for Organ Sharing renal allocation system eliminated points for certain HLA matches, increased points for waiting time and for pediatric patients, and extended the mandatory share rule to include the zero-antigen mismatch. We analyzed data, from the period December 1993 to August 1996, on 393 donors, 348 kidney-only cadaveric transplants, and 615 patients ranked first or second on the allocation lists generated for each donor, to assess the effect of the changes in the point system. There was an appreciable (46%) but not significant increase in the frequency of transplants occurring under the mandatory share rule, with a greater relative increase seen in African-Americans than in Caucasians. Recipients of transplants not falling under the mandatory share rule had an increased average waiting time but no increase in sensitization or HLA mismatch, whereas patients ranked at the top of the allocation list had higher levels of sensitization but no increase in waiting time. There was an appreciable increase in the percentage of transplants occurring in African-Americans. Of the various changes we observed, only those involving the mandatory share rule could be attributed to changes in the allocation system, whereas others paralleled changes in the composition of our local waiting list.

Plasmapheresis and intravenous immune globulin provides effective rescue therapy for refractory humoral rejection and allows kidneys to be successfully transplanted into cross-match-positive recipients.

BACKGROUND: Hyperacute rejection (HAR) and acute humoral rejection (AHR) remain recalcitrant conditions without effective treatments, and usually result in graft loss. Plasmapheresis (PP) has been shown to remove HLA- specific antibody (Ab) in many different clinical settings. Intravenous gamma globulin (IVIG) has been used to suppress alloantibody and modulate immune responses. Our hypothesis was that a combination of PP and IVIG could effectively and durably remove donor-specific, anti-HLA antibody (Ab), rescuing patients with established AHR and preemptively desensitizing recipients who had positive crossmatches with a potential live donor. METHODS: The study patients consisted of seven live donor kidney transplant recipients who experienced AHR and had donor-specific Ab (DSA) for one or more mismatched donor HLA antigens. The patients segregated into two groups: three patients were treated for established AHR (rescue group) and four cross-match-positive patients received therapy before transplantation (preemptive group). RESULTS: Using PP/IVIG we have successfully reversed established AHR in three patients. Four patients who were cross-match-positive (3 by flow cytometry and 1 by cytotoxic assay) and had DSA before treatment underwent successful renal transplantation utilizing their live donor. The overall mean creatinine for both treatment groups is 1.4+/-0.8 with a mean follow up of 58+/-40 weeks (range 17-116 weeks). CONCLUSIONS: In this study, we present seven patients for whom the combined therapies of PP/IVIG were successful in reversing AHR mediated by Ab specific for donor HLA antigens. Furthermore, this protocol shows promise for eliminating DSA preemptively among patients with low-titer positive antihuman globulin-enhanced, complement-dependent cytotoxicity (AHG-CDC) cross-matches, allowing the successful transplantation of these patients using a live donor without any cases of HAR.

Characterization of HLA class I specific antibodies by ELISA using solubilized antigen targets: I. Evaluation of the GTI QuikID assay and analysis of antibody patterns.

The development of solid phase immunoassays using solubilized HLA molecules as targets has provided a means of detecting HLA-specific antibodies that overcomes many of the shortcomings of lymphocyte based assays. We have evaluated a commercially available assay, the GTI QuikID (QID), that uses solubilized class I molecules from 40 subjects selected for their HLA phenotype, to characterize HLA-specific antibodies. We tested 595 sera from 319 subjects and compared the results obtained with QID to those obtained with cytotoxicity (CYT) and with GTI QuikScreen (QS) as well as to historic data. The correlation of QID with CYT (r = 0.54) was comparable to that between QID and QS (r = 0.60). The majority of disparities between QS and QID were apparent false negatives with QID that could be overcome by analyzing QID data at lower cutoff values. In contrast, most of the disparities between QID and CYT were false negatives in CYT due to the relatively low sensitivity of that assay. As expected, the ELISA was more sensitive (97%) than CYT (78%) but had a somewhat lower specificity (87% vs. 92%) due, most likely, to selection of sera that excluded most sera that were known to be nonspecific by CYT. Determination of antibody specificity could be achieved quickly by manual analysis of the QID data because of the way the data are presented by the manufacturer's software. Interestingly, the frequencies of different antibodies detected by ELISA differed from those detected by CYT with ELISA identifying more sera containing antibodies to both A and B locus antigens.

Antigen, allele, and haplotype frequencies report of the ASHI minority antigens workshops: part 1, African-Americans.

HLA typing was performed on 977 African Americans residing throughout most of the United States. Class I and class II antigens and class II alleles were defined for all individuals and class I alleles were determined for a subset of individuals. The occurrence of 854 of the individuals in family groups permitted direct counting of allele and haplotype frequencies. The data were analyzed for antigen, allele, and haplotype frequencies; recombination frequencies; segregation distortion; distribution of haplotype frequencies; linkage disequilibria; and geographic distribution of DR antigens. Tables of the antigen, allele, the most common two and three point haplotypes, and 88 extended haplotypes that include class I and class II alleles are presented. Notable findings include a lower than expected frequency of recombination between the B and DR loci (theta= 0.0013), lower than expected frequency of inheritance (44.5% vs 54.5%) of the DRB1*1503; DQB1*0602 haplotype, lower than anticipated linkage disequilibrium values for DR; DQ haplotypes, and a skewed geographic distribution of DR antigens.

Characterization of HLA class I specific antibodies by ELISA using solubilized antigen targets: II. Clinical relevance.

Until recently, the nature of humoral sensitization to HLA has been characterized by data from lymphocyte-based assays, predominantly cytotoxicity tests. We have examined the characteristics, determined by enzyme-linked immunosorbent assay (ELISA), of sera from 191 subjects known to have produced HLA-specific antibody. We found that ELISA detected higher frequencies compared with cytotoxicity of many (74.5%), but not all, HLA-specific antibodies; in many cases (42.6%) the frequencies of these antibodies were higher than predicted from population frequencies whereas some antibodies (23.4%) occurred with lower than expected frequencies. Some of the increase in frequencies can be accounted for by crossreactivity, i.e., sensitization to epitopes shared among two or more allelic products. The presence of epitopes shared between a recipient's antigen and a mismatched antigen in a donor also tended to narrow the specificity of antibody produced. However the data also indicate differences in immunogenicity among different antigens suggesting that crossreactive group matching would be beneficial in some but not all cases. Finally, we present case studies to illustrate the value of ELISA in predicting humoral rejection episodes and in monitoring the efficacy of rejection therapies.

Sequence-based typing of HLA class I alleles in Alaskan Yupik Eskimo.

In comparison to South America, native North Americans tend to be less diverse in their repertoire of HLA class I alleles. Based upon this observation, we hypothesized that the Yupik Eskimo would exhibit a limited number of previously identified class I HLA alleles. To test this hypothesis, sequence-based typing was performed at the HLA-A, -B and -C loci for 99 Central Yupik individuals from southwestern Alaska. Two new class I alleles, A*2423 and Cw*0806, were identified. While A*2423 was observed in only one sample, Cw*0806 was present in 26 of the 99 individuals and all of the Cw*0806 samples contained B*4801. Allele Cw*0806 differs from Cw*0803 by a single nucleotide substitution such that Cw*0803 may be the progenitor of Cw*0806. Allele Cw*0803 was originally characterized as unique to South America, but detection of Cw*0803 in the Yupik indicates that Cw*0803 was a founding allele of the Americas. The presence of new alleles and previously unrecognized founding alleles in the Yupik population show that natives of North America are more diverse than previously envisioned.