Differential effects of donor-specific HLA antibodies in living versus deceased donor transplant.

The presence of donor-specific anti-HLA antibodies (DSAs) is associated with increased risk of graft failure after kidney transplant. We hypothesized that DSAs against HLA class I, class II, or both classes indicate a different risk for graft loss between deceased and living donor transplant. In this study, we investigated the impact of pretransplant DSAs, by using single antigen bead assays, on long-term graft survival in 3237 deceased and 1487 living donor kidney transplants with a negative complement-dependent crossmatch. In living donor transplants, we found a limited effect on graft survival of DSAs against class I or II antigens after transplant. Class I and II DSAs combined resulted in decreased 10-year graft survival (84% to 75%). In contrast, after deceased donor transplant, patients with class I or class II DSAs had a 10-year graft survival of 59% and 60%, respectively, both significantly lower than the survival for patients without DSAs (76%). The combination of class I and II DSAs resulted in a 10-year survival of 54% in deceased donor transplants. In conclusion, class I and II DSAs are a clear risk factor for graft loss in deceased donor transplants, while in living donor transplants, class I and II DSAs seem to be associated with an increased risk for graft failure, but this could not be assessed due to their low prevalence.

Underdiagnosing of antibody-mediated transfusion-related acute lung injury: evaluation of cellular-based versus bead-based techniques.

BACKGROUND AND OBJECTIVES: Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality. To support the diagnosis of antibody-mediated TRALI, HLA and HNA antibodies are tested in involved blood donors. Identification of antibody positive donors is important as exclusion of these donors is part of preventative strategies against TRALI. We compared cellular-based versus bead-based techniques for diagnosis of antibody-mediated TRALI. MATERIALS AND METHODS: All reported TRALI cases in the Netherlands during a 5-year period were evaluated. Donors were screened for the presence of HLA class I and class II antibodies using both cellular-based and bead-based techniques. RESULTS: In total, 100 TRALI cases were reported of which 91 were fully tested. In 113 donors, HLA antibodies were detected of which 84 were only detected by bead-based techniques, 12 only by cellular-based tests and 17 by both assays. Antibody-mediated TRALI was diagnosed in 44 of 91 reported cases. Twenty-one (48%) of these cases would not have been identified using only cellular-based assays. CONCLUSION: Bead-based techniques show a higher sensitivity for detecting incompatible donors in TRALI cases than cellular-based assays. These results suggest that the use of bead-based assays will result in a significant reduction of future TRALI reactions as more antibody positive donors will be excluded from future donations.

Identification of two new HLA class II alleles: DRB1*01:50 and DQB1*05:18.

Two new human leukocyte antigen (HLA) class II alleles were identified during routine sequence-based typing.

Minor H antigen matches and mismatches are equally distributed among recipients with or without complications after HLA identical sibling renal transplantation.

Studies of the effect of minor H antigen mismatching on the outcome of renal transplantation are scarce and concern mainly single center studies. The International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 16 laboratories of the IHIW, the role of 15 autosomal, 10 Y-chromosome encoded minor H antigens and 3 CD31 polymorphisms, was investigated in relation to the incidence of renal graft rejection and graft loss in 444 human leukocyte antigens (HLA)-identical sibling renal transplantations. Recipient and donor DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, C19Orf48, LB-ECGF-1, CTSH, LRH-1, LB-ADIR and HY. The correlation between minor H antigen mismatch and the primary outcome graft rejection or graft loss was statistically analyzed. The incidence of rejection was very low and no correlation was observed between one or more minor H antigen mismatch(es) and a rejection episode (n = 36), of which only eight resulted in graft loss. In summary, in our study cohort of 444 renal transplants, mismatching for neither autosomal nor HY minor H antigens correlate with rejection episodes or with graft loss.

Characterization of a new allele: HLA-A*03:134.

The new A*03:134 differs from A*03:01:01:01 by one amino acid change at codon 264.

Characterization of three new HLA-B alleles: B*35:05:03, B*52:29 and B*57:01:13.

Three new HLA-B alleles were identified during routine sequence-based typing.

Resistance to collagen-induced arthritis in a nonhuman primate species maps to the major histocompatibility complex class I region.

Type II collagen-induced arthritis (CIA) is an experimentally inducible autoimmune disorder that is, just like several forms of human arthritis, influenced by a genetic background. Immunization of young rhesus monkeys (Macaca mulatta) with type II collagen (CII) induced CIA in about 70% of the animals. One major histocompatibility complex (MHC) class I allele was present only in young animals resistant to CIA and absent in arthritic animals. This strong association suggests that the MHC class I allele itself, or a closely linked gene, determines resistance to CIA. The mechanism controlling the resistance to CIA becomes less efficient in aged animals since older rhesus monkeys, which were positive for the resistance marker, developed a mild form of arthritis. At the cellular level it is demonstrated that resistance to CIA is reflected by a low responsiveness of T cells to CII. This association between a specified MHC class I allele and resistance to an autoimmune disease points at the importance of the MHC class I region in the regulation of the immune response to an autoantigen.

High resolution definition of HLA-DRB haplotypes by a simplified microsatellite typing technique.

In humans, the region configurations DR1, DR8, DR51, DR52 and DR53 are known to display copy number as well as allelic variation, rendering high resolution typing of HLA-DRB haplotypes cumbersome. Advantage was taken of microsatellite D6S2878, present in all DRB genes/pseudogenes with an intact exon 2-intron 2 segment. This DRB-STR is highly polymorphic in composition and length. Recently, it was proven that all exon 2 sequences could be linked to a certain DRB-STR that segregates with the respective DRB allele. Because haplotypes show differential copy numbers and compositions of exon 2-positive DRB genes/pseudogenes, unique DRB-STR patterns could be described that appear to be specific for a particular DRB haplotype. The aim of this workshop project was to approve and to qualify this simple typing protocol in a larger panel covering different European populations. All participants succeeded in correctly defining the DRB-STR amplicons varying from 135 to 222 base pair (bp) lengths. The panel of 101 samples covered 50 DRB alleles distributed over 37 different haplotypes as defined by exon 2 sequence-based typing. These haplotypes could be refined into 105 haplotypes by DRB-STR typing. Thus, discrimination of exon 2-identical DRB alleles was feasible, as well as the exact description of three different crossing-over events that resulted in the generation of hybrid DR region configurations. This typing procedure appears to be a quick and highly robust technique that can easily be performed by different laboratories, even without experience in microsatellite typing; thus, it is suitable for a variety of researchers in diverse research areas.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments.

Influence of HLA-DRB1* incompatibility on the occurrence of rejection episodes and graft survival in serologically HLA-DR-matched renal transplant combinations.

BACKGROUND: The aim of the present study was to analyze the effect of HLA-DRB1* mismatches on graft function and graft survival in 92 patients who received serologically HLA-DR split antigen-matched cadaveric renal transplants. METHODS: The polymorphic second exon of the HLA-DRB1 alleles was typed using the sequence-specific oligonucleotides technique. RESULTS: The results show that in 26 of the 92 analyzed combinations, one or more HLA-DRB1* mismatches were found (28%). The analysis of the occurrence of treatable rejection episodes during the first 3 months after transplantation demonstrated a significantly higher incidence of rejection episodes in the HLA-DRB1*-mismatched group: 18 of 26 (69%) in the HLA-DRB1*-mismatched group against 23 of 66 (35%) in the HLA-DRB1*-matched group (P(uncorr)=0.0033). However, no effect of HLA-DRB1* mismatches on graft survival was found, although in general graft survival in the whole patient group was negatively influenced by the occurrence of rejection episodes during the first 3 months after transplantation (P(uncorr)=0.0008). In contrast, in the HLA-DR4-matched donor-recipient combinations (n=28), the effect of mismatching for the HLA-DRB1*04 alleles seemed to have a pronounced effect not only on the occurrence of rejection episodes but also in the form of diminished graft survival. CONCLUSIONS: Thus, this study indicates that the existence of HLA-DRB1* allele mismatches in renal transplant recipients, matched for the serologically defined HLA-DR split antigens, is not harmful for the transplant. The exception is the HLA-DRB1*04 mismatch, which seems to be deleterious for the grafted organ.

Fine specificity of the alloantiserum MSD-51: epitope mapping of HLA-DRw53 determinants.

HLA-DRw53 is a supertypic specificity expressed by HLA-DR4-, HLA-DR7-, and HLA-DR9-positive cells. In the present study, the fine specificity of an HLA-DRw53-specific alloantiserum (MSD-51) was analyzed in serology and by the isoelectric focusing technique. In serology, MSD-51 recognized HLA-DRw53-positive cells with the exception of cells expressing the HLA-DR7/DRw53/DQw9 haplotype. The immunoprecipitation studies and the use of the IgM-reducing agent dithiothreitol revealed that MSD-51 consisted of at least two antibodies: (1) an IgM antibody which reacted with the HLA-DRB4 gene product of HLA-DRw53-positive cells, except HLA-DR7/DRw53/DQw9-expressing cells, and (2) at least one IgG antibody which recognized a linear sequence or conformational structure formed by positions 67 to 70 on the HLA-DRB1 gene product of HLA-DR4- and HLA-DR9-positive cells. These findings demonstrate the complexity of the supertypic HLA-DRw53 antigen analyzed with a serologically well-defined HLA-DRw53-specific alloantiserum.

HLA-DRB4 gene encoded HLA-DR53 specificity segregating with the HLA-DR7, -DQ9 haplotype: unusual association.

HLA phenotyping of a leukemia patient of Caucasoid origin revealed the presence of the serological HLA-DR53 specificity. Comprehensive pedigree analysis demonstrated that the HLA-DR53 specificity segregated with the HLA-DR7, -DQ3 haplotype. High resolution PCR- SSP genotyping of the HLA class II genes revealed the presence of the HLA-DRB4*0101101 allele segregating together with the HLA-DRB1*0701, -DQA1*0201 and DQB1*03032 alleles. This finding is in contrast to known linkages in that thus far, the HLA-DR7, -DQ9 haplotype has only been described in association with the non-expressed HLA-DRB4*0103102N allele. The existence of this "novel" haplotype may be explained by a homologous recombinational event that occurred between the HLA-DR7, -DR53, -DQ2 and the HLA-DR7, -DQ9 haplotypes.

High-resolution histocompatibility testing of a group of sixteen B44-positive, ABDR serologically matched unrelated donor-recipient pairs. Analysis of serologically undisclosed incompatibilities by cellular techniques, isoelectrofocusing, and HLA oligotyping.

We have characterized HLA incompatibilities in a group of 16 B44-positive patients who were serologically ABDR matched with their 23 (unrelated) potential bone marrow donors. After analysis with a combination of cellular techniques, IEF for HLA-A/B and oligotyping for class II and HLA-B44, 44% of the patients revealed one or more HLA incompatibility with at least one of their potential donors. CTL activity was detected in 12 of the 22 combinations tested. CTL incompatibility occurred more frequently in DR subtype-mismatched combinations, but CTL reactivity was always directed against class I. To characterize these incompatibilities between matched unrelated individuals, we analyzed the specificity of T-cell clones from seven primary CTL cultures. In three combinations, CTL reactivity was directed against a subtype of B44. In two combinations, the CTL reactivity was directed against a non-B44 class I subtype. In two of seven combinations, the CTLs recognized an antigen that, though unconditionally associated with B4403, was expressed by 60% of the B4403+ cells only. Because all 12 of these B4403+ targets recognized could be typed for one HLA-C allele only (Cwl-Cw8), we believe that this alloreactivity might be directed against a serologically undefined Cw antigen.

Prenatal HLA-matching to determine suitability for allogeneic bone marrow transplantation.

For several haematological malignancies, allogeneic stem cell transplantation is the treatment of choice. In most cases an HLA-identical sibling is required. If the mother of a patient is pregnant, cord blood from a related donor, which can be used for stem cell transplantation, might be obtainable in the near future. For the patient, knowledge of the foetal HLA-type can be important since it might influence choice of treatment and timing of transplantation. If the foetus is HLA compatible, as would be the situation in 25% of cases, the delivery has to be arranged in such a way that cord stem cells can be collected. As a result, in the other 75% of cases (spontaneous) delivery can take place in the home/local setting. Here we report four cases in which amniocentesis was performed and HLA-typing influenced treatment of the patient and delivery of the sibling.

Antinuclear antibody and HLA-B27 positive uveitis: combination of two diseases?

AIMS/BACKGROUND: Anterior uveitis associated with juvenile chronic arthritis concerns two different clinical entities: firstly, antinuclear antibody (ANA) positive patients who have a chronic anterior uveitis with severe complications and often a poor visual prognosis; secondly, usually HLA-B27 positive children, predominantly boys, with unilateral recurrent anterior uveitis. Three patients are described who had a combination of clinical and laboratory features of both diseases. METHODS: Retrospective clinical and laboratory analysis of three patients. RESULTS: Ocular features in the three patients combined the clinical picture of ANA positive chronic anterior uveitis during early childhood with the clinical features of HLA-B27 unilateral acute anterior uveitis during adolescence. The patients fulfilled the diagnostic criteria of juvenile chronic arthritis, and they had no ankylosing spondylitis. All three patients had the HLA-B*2705 subtype. CONCLUSIONS: Whether the association of ANA positive chronic anterior uveitis and HLA-B27 unilateral acute anterior uveitis is a coincidence or represents a distinct clinical entity is not yet clear.

Seronegative spondyloarthropathies and HLA-B27 subtyes: a study in Asian Indians.

In this study, 60 HLA-B27+ve SSA patients and 17 healthy controls belonging to North India were analyzed to ascertain heterogeneity of the B27 molecule in this population. ID-IEF and PCR-SSOP technologies were used to analyze polymorphism in exon 2 and 3 of the HLA-B27 gene. Four different subtypes were encountered: B*2702,04,05 and 07. Other subtypes of B27 viz B*2701,03,06 and 08 were not encountered. B*2704 (common oriental subtype) and B*2705 (common Caucasian subtype) were the most common subtypes in the control and patient groups. B*2707 was less frequently encountered in both groups and B*2702 was found in only one AAU patient. B*2704 was the predominant subtype in the AS group (70.8%) compared to its frequency of 47% in healthy controls (RR = 2.73) while in the undiff SpA group, B*2705 occurred most frequently (73.1%, RR = 3.05). B27 subtypes segregated differently in males and females. 12 of the 17 male AS patients carried B*2704 as compared to 1 of 8 healthy males (X2 = 3.9, P < 0.05). On the other hand, in the undiff SpA, B*2705 was significantly raised in female patients (100%) as compared to healthy females (22.2%, X2 = 4.9, P < 0.05). Subtype distribution is indicative of racial admixture in the Asian Indian population.

Clinical features of spondyloarthropathy in Chinese and native Indonesians.

The prevalence of spondyloarthropathy (SpA) in Chinese Indonesians is much higher than in native Indonesians. This is due to HLA-B27 subtype differences. In the present study we re-examined the clinical features of SpA in Indonesians to see whether, besides the HLA-B27 subtype differences, other factors affect the frequency of SpA. Seventy two patients with SpA were re-examined. The patients came from two clinics for rheumatic diseases. The overall entry ratio of Chinese to native Indonesians was 1:2. Ankylosing spondylitis (AS) was more frequent among the Chinese (n = 32, 94% B27 positive) than among the native Indonesians (n = 5, 40% B27 positive). HLA-B27 subtyping was performed on 22 of the 37 HLA-B27-positive AS patients. Twenty Chinese were positive for B*2704 and two native Indonesians were B*2705 positive. The clinical features of AS and reactive arthritis (ReA) showed no differences between the two populations and were similar to the clinical descriptions in other parts of the world. In conclusion, it can be stated that in spite of HLA-B27 subtype differences the clinical features of SpA in Chinese and native Indonesians are fully comparable.

The PROCARE consortium: toward an improved allocation strategy for kidney allografts.

Kidney transplantation is the best treatment option for patients with end-stage renal failure. At present, approximately 800 Dutch patients are registered on the active waiting list of Eurotransplant. The waiting time in the Netherlands for a kidney from a deceased donor is on average between 3 and 4 years. During this period, patients are fully dependent on dialysis, which replaces only partly the renal function, whereas the quality of life is limited. Mortality among patients on the waiting list is high. In order to increase the number of kidney donors, several initiatives have been undertaken by the Dutch Kidney Foundation including national calls for donor registration and providing information on organ donation and kidney transplantation. The aim of the national PROCARE consortium is to develop improved matching algorithms that will lead to a prolonged survival of transplanted donor kidneys and a reduced HLA immunization. The latter will positively affect the waiting time for a retransplantation. The present algorithm for allocation is among others based on matching for HLA antigens, which were originally defined by antibodies using serological typing techniques. However, several studies suggest that this algorithm needs adaptation and that other immune parameters which are currently not included may assist in improving graft survival rates. We will employ a multicenter-based evaluation on 5429 patients transplanted between 1995 and 2005 in the Netherlands. The association between key clinical endpoints and selected laboratory defined parameters will be examined, including Luminex-defined HLA antibody specificities, T and B cell epitopes recognized on the mismatched HLA antigens, non-HLA antibodies, and also polymorphisms in complement and Fc receptors functionally associated with effector functions of anti-graft antibodies. From these data, key parameters determining the success of kidney transplantation will be identified which will lead to the identification of additional parameters to be included in future matching algorithms aiming to extend survival of transplanted kidneys and to diminish HLA immunization. Computer simulation studies will reveal the number of patients having a direct benefit from improved matching, the effect on shortening of the waiting list, and the decrease in waiting time.