[Histocompatibility testing of donors and receivers in keratoplasty (initial attempts with adapted tissue-typed transplants in bad risk cases)].
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A patient with an inborn cellular immunodeficiency syndrome developed a graft-versus-host reaction after a transfusion with packed red cells. This diagnosis was confirmed by skin biopsy and finally proved by tissue typing.
The question of whether or not HLA is important in transplantation can now be answered with a definite yes. The nationwide six-antigen-matching program, in which over 100 US transplantation centers have participated, has shown that a high (88% to 90%) 1-year graft survival rate can be achieved when kidneys are shipped to well-matched recipients. Long-term outcome (measured as half-life) is also markedly improved, from an average of 7 years for cadaveric donor transplants to as much as 19 years in the six-antigen-matched recipients. One of the major factors influencing long-term survival is histocompatibility matching, as shown by the survival differences among HLA-identical siblings (25-year half-life), one-haplotype-mismatched parental donors (12-year half-life), and two-haplotype-mismatched cadaveric donors (7-year half-life). For the past 25 years of kidney transplant experience, cadaveric donor half-life has remained stable at 7 years, despite many improvements in immunosuppression protocols. Histocompatibility matching unquestionably offers the best approach to prevention of chronic rejection.
We have performed class I HLA antigen testing in 42 women with recurrent habitual abortions and in their husbands. The main criterion for inclusion in this group was a frequency of more than two abortions without the known reasons for abortion and without a living child. 29 couples with a least two healthy children and no abortion in the clinical history served as a control group. The data were evaluated in respect to the association of HLA and disease as well as to the level of histocompatibility within the couples. In our results we did not find any significant association between HLA-A, B, C phenotypes and the habitual abortion. On the other hand, we observed a significantly higher level of histocompatibility in the couples with habitual abortions in comparison to the control couples. The frequency of identity in one and more histocompatibility antigens was in the patient group significantly higher than in the control group and as it could be expected by chance, calculated on the basis of antigen distribution within the loci A, B, C in the population in the southern part of the GDR. In no case we have revealed lymphocytotoxic antibodies as a possible cause for miscarriage. Our results are in favour of the hypothesis that a higher level of histocompatibility could be an immunological explanation for the habitual abortions. In addition, this hypothesis was supported by the successful immunotherapy with leukocytes derived from the husband or nonrelative donors.
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.
Recent progress in HLA typing involves new methods which allow typing for MLC (mixed lymphocyte culture) and Ia (immune associated) antigens. These antigens seem to play a major role in transplant rejection in HLA non-immunised patients. When the new typing methods eventually become applied in matching of kidneys it seems likely that the results of kidney transplantation will be significantly improved.
HLA variants that have lost expression of multiple cis-linked alleles as determined serologically and enzymatically were analyzed for expression of HLA-D PLT-stimulating (PL) determinants using the primed lymphocyte test. All 19 variants that had lost HLA-DRw3 expression simultaneously had lost expression of the HLA-D region-associated PL specificity. PLT cells made by priming to a variant that was hemizygous for HLA genes resulted in priming to HLA-D PL determinants encoded for by just one haplotype, analogous to priming to an HLA homozygous cell.
Forty high-risk patients awaiting corneal grafting were randomly selected from a 7-year cumulative list. Twenty were grafted with HLA-A and HLA-B antigen matched material. The other 20 patients were used as controls. Graft survival at two years was 60% (12/20) in the control group and 90% (18/20) in the study group; at five years it was 55% (11/20) in the control group and 85% (17/20) in the study group.
A new technical trial was carried out to detect polymorphism in HLA-DP genes, based on the diversity in electrophoretic mobility of single-stranded DNA (single-strand conformation polymorphism, SSCP). Genomic DNAs from 31 cell lines homozygous for 2 and 14 different DPA1 and DPB1 alleles, respectively, and from peripheral blood cells of a normal individual homozygous for another DPB1 allele were subjected to polymerase chain reaction (PCR) to amplify the polymorphic exon 2 of DPA1 or DPB1 genes. The PCR samples were denatured by heating in the presence of formamide to obtain single-stranded DNA, electrophoresed in a neutral polyacrylamide gel, and visualized by silver staining. Allelic differences were detected by the distinctive electrophoretic pattern of each single strand, depending on the sequence-specific conformation. Fifteen DPB1 alleles showed 11 distinct electrophoretic patterns, leaving four allelic combinations not distinguished. These four allelic combinations could be further distinguished by using another couple of primers in PCR, with which a part of the exon was amplified, and by subsequent SSCP analysis. The use of four pairs of primers in PCR allowed for discrimination of all the 15 DPB1 alleles tested. Two allelic differences in exon 2 of DPA1 gene could be clearly demonstrated. In addition, putative new alleles of DPA1 and DPB1 genes were detected by SSCP analyses. The PCR-SSCP analysis is simple and rapid, requires neither radioactive materials nor restriction enzymes, and is expected to be a useful tool for investigating the fine HLA-matching required for clinical transplantation of organs.
The human leukocyte antigen (HLA) region on chromosome 6p21.3 is the most polymorphic in the human genome. It encodes hundreds of genes, of which the class I and class II HLA alleles play a central role in the generation of an immune response, but at the same time represent a barrier to marrow and organ transplantation. There is overwhelming evidence that optimal HLA matching will allow for safer marrow transplants, however with more than 1,200 allelic variants, the need to accurately identify the precise alleles is a technically demanding challenge. Methods currently in use for HLA typing include microcytotoxity assay, sequence-specific oligonucleotide (SSO) probe, sequence-specific primer (SSP) amplification, and sequence-based typing (SBT). Ambiguous results may still arise, even with the best molecular typing methods but this problem can be resolved by using a combination of methods. It is clear that a HLA typing laboratory will have to provide allele level resolution of HLA data using the most appropriate means available and this will have the greatest impact in terms of offering the best matched donor for the benefit of patients.
A number of disease states have been found to have a positive association with certain HLA antigens. Weak associations have been described for a number of cancers. Seventy patients with transitional cell carcinoma of the bladder underwent serotyping for HLA, A, B, C, and DR antigens. There were 54 men and 16 women patients with a mean age of 65.3 years. Noninvasive lesions (Ta and T1) were present in 50 and 20 were invasive (T2, T3, and T4). Low grade tumors (G1 and G4) were found in 53 and 17 were moderately or poorly differentiated (G3 and G4). Gene frequencies for the HLA determinants in the cancer patients were compared to those for normal English caucasoids. No significant differences were found at either the A, B, C, or DR loci. DR4 was the commonest antigen expressed in bladder cancer patients with an allelic frequency of 39% compared to 28% in the normal population. The presence or absence of the DR4 antigen was not related to the stage or grade of the tumor.
The correlation between MLC reactivity (LD) and serological leukocyte typing (SD) was studied in a beagle colony. Disparity for a serologically defined non-DL-A lymphocyte antigen did not correlate with MLC reactivity. Lymphocytes of colony members with common ancestors and SD identical DL-A haplotypes did not stimulate each other in the MLC. This implies that LD typing in the beagle coolony can be generally predicted by DL-A SD typing. Consequently, lymphocytes of sibs homozygous for a given DL-A SD haplotype could be shown, with few exceptions, to be also homozygous for MLC determinants. Cells of these homozygous sibs can be used in MLC typing as reference cells for DL-A LD specificities. Two exceptions to the expected linkage between DL-A SD typing and MLC reactivity were found. These findings could not be explained by recombination with the DL-A region assuming a single major LD locus coding for MLC. Thus, suggestive evidence for more than one single LD locus has been obtained.
From January 1979 to August 1981, 76 primary renal transplants were performed at the University of Miami-Jackson Memorial Hospital. Fifty-five of these were from living, related (LR) donors and 21 were from cadaver (CAD) donors. Histocompatibility typing for HLA-A, -B, and -DR antigens and of mixed lymphocyte culture (MLC) were performed. In LR patients the ability to generate donor-specific and nonspecific suppressor cells in vitro was tested preoperatively. Serial posttransplant immunologic monitoring was used in all patients by determining total T-cell numbers (E rosette-forming cells) as well as helper and suppressor T-cell subsets by use of monoclonal antibodies. Overall patient and graft survival was 90% and 87%, respectively. In this study MLC reactivity did not correlate with DR typing in CAD recipients, most of whom had marked reaction despite one or two DR antigen matches. Graft survival was also independent of MLC reactivity in both LR and CAD patients. The ability of LR recipients to generate donor-specific suppressor cells in vitro before transplantation did correlate significantly with absence of rejection episodes. Immunologic monitoring with T-cell subsets had prognostic significance for the detection of patients with excessive immunosuppression and high risk for development of infection (P less than 0.001). Thus, the responder status of many transplant recipients seemed as important as the current assessment of histocompatibility in influencing graft survival.
Bone marrow transplantation has become the treatment of choice for many hematological malignancies. Approximately 30% of the patients lack an HLA-identical sibling donor, for them alternative donors have to be found. The chances of finding an unrelated donor can be estimated by consulting the Bone Marrow Donors Worldwide file and grouping the patients according to whether they have frequent or rare haplotypes. If the HLA-ABDR split antigens are taken into account, for less than 25% of the patients identical donors can be identified. In order to select the best matched donor for a patient, sensitive HLA typing techniques should be employed including DNA typing techniques, but for optimal selection of a suitable donor one should not only study genetics but also determine the immunological repertoire recognizing the histocompatibility differences between donor and recipient. Several promising cellular techniques such as the HTLp and CTLp test have recently become available and should also be used to select the best donor.
For a preliminary estimation of the prevalence and significance of HLA antigens, tests were carried out on the A and B loci in an unselected group of 107 patients with type 1 diabetes in Bucharest. Monospecific antisera furnished by NIH, Bethesda were used. For HLA-A the following data were obtained: A2 (20.3% of the total specificities); A1 (18.4%); A3 (14.0%); A28 (10.1%). Provisional estimations in the healthy population also indicated HLA-A2 as being more frequent than followed by A30/31, A1, A9, A3. For HLA-B: B7 (38.2%); B5, B12 and B14 (14.0% each); B8 (11.1%). In the healthy subjects, the order was B12, B35, B5, B8 the same as B18, then B7 (which did not exceed 11%). The most frequently encountered haplotypes in the diabetic patients were: A2/B7 (8.4% of the total haplotypes); A3/B7 (6.9%); A1/B7 (6.6%); A10/B7 (3.8%); A9/B7 and A11/B7 (3.6% each). An unexpectedly high frequency of the HLA-B7 antigen was found in group of diabetic patients investigated, contrasting with its assumed "protector" role in the Caucasian population. The frequency of antigen HLA-A3 and haplotype HLA-A3/B7 infringes their listing in the "resistance axis" to diabetes.
Immunological rejection is still the most important problem in clinical organ transplantation requiring the lifelong immuno-suppressive treatment of the allograft recipient. The clinical success rate however can be improved by a variety of diagnostic procedures: The reduction of the histocompatibility barrier between donor and recipient by HLA-typing and organ exchange in order to obtain the best possible match; posttransplant monitoring of donor specific immune responsiveness towards donor antigens; monitoring of T-cell subsets by monoclonal antibodies and cytofluometry. T-cell subset monitoring is a rapidly developing new technique with great potential. With the presently available first generation reagents already a detailed analysis of the major T-cell population in circulation is possible, which can markedly facilitate the diagnosis of rejection and viral infection. New monoclonal antibodies, defining certain activation stages of T-cells or permitting a more refined differentiation of functionally distinct subsets will lead to increased relevance of this diagnostic approach. A particularly interesting aspect of monoclonal antibodies against T-cell differentiation antigens is their potential therapeutic use as immunosuppressive drugs with a highly selective action on defined T-cell subsets.
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The transplantation of hematopoietic stem cells is sometimes the only treatment option for certain types of malignancies and hematological disorders. The best way to ensure a positive outcome from this type of procedure is to secure an identical human lymphocyte antigen-matched donor or use an autologous graft. This article reviews the indications for transplantation, the recipient and donor selection process, and posttransplant follow-up. The advantages of using haploidentical donors and the typing process also will be discussed.