J. J. Van Rood

HLA GENE AND HAPLOTYPE FREQUENCIES IN BONE MARROW DONORS WORLDWIDE REGISTRIES

To calculate reliable HLA gene and haplotype frequencies of bone marrow donors in various regions in the world, we have analyzed the HLA-A, -B, and -DR phenotype frequencies of 18 bone marrow donor registries with a total of more than 300,000 HLA-A, -B-typed donors. These registries were included in the 22nd edition of Bone Marrow Donors Worldwide. Maximum likelihood gene frequencies, Hardy-Weinberg equilibrium fit, and 2- and 3-locus haplotype frequencies were calculated as well as deltas, relative deltas, and their significance. Remarkable gene and haplotype frequency differences exist between the registries. The genetic distances between the different registries were used to draw phylogenetic trees that clearly show that the degree of similarity between registries is related to their geographic locations. The resulting frequencies can be used for the estimation of the probability of finding a hyplotypically identical related or unrelated bone marrow donor for an individual patient. Phylogenetic trees are useful representations of the similarity between donor pools and can also aid in the selection of donors.

How to improve the search for an unrelated haematopoietic stem cell donor. Faster is better than more

Summary:Many patients do not reach haematopoietic stem cell transplantation. Shortage of unrelated donors (UDs) is still seen as the main cause. However, with a worldwide UD pool containing more than 8 million donors, it is possible that other impediments are becoming more important. We analysed 549 UD searches for Dutch patients, performed between 1987 and 2000, in order to find the reasons for failure or success to reach transplantation. Between 1996 and 2000, 59% of the patients of Northwest European origin received a graft from an UD with a median time span of 4.4 months from the start of the search. In all, 11% of the patients lacked a compatible donor, while 30% became medically unfit for transplantation. This is in contrast to the patients of non-Northwest European origin for whom UD shortage is still the most important impediment; only 32% were transplanted while 50% lacked a compatible donor. We conclude that the shortage of donors is no longer the biggest constraint in unrelated stem cell transplantation for patients of Northwest European origin. It may be more effective to optimize the chance on transplantation by making the search process more efficient.

Problems and possible solutions in finding an unrelated bone marrow donor. Results of consecutive searches for 240 Dutch patients.

To evaluate the efficiency of our protocol for finding an HLA matched unrelated bone marrow donor, search results obtained between 1990 and 1995 for 240 Dutch patients were analyzed. The percentage of patients for whom, according to information given by the registries, a fully split-HLA antigen matched donor is available, increased from 24% in 1990 to over 70% in 1995. As a result the percentage of patients transplanted rose from about 24% in 1990–1991 to 44% in 1994–1995. The median time between the start of the search and transplantation was about 6 months. The systematic use of Bone Marrow Donors Worldwide (BMDW) which comprises the HLA groups of all volunteer bone marrow donors in Europe, Israel, South Africa, North America, Canada, India, Australia and New Zealand has been essential in this context. While searching for a suitable donor several problems were encountered such as unavailability of donors (12%) and discordant typing results (8%; range <1% to >25%). Thus it is advisable to select several donors for a patient. For 86% of patients with at least one HLA identical donor on the serological level for HLA-A,-B,-DR,-DQ, an HLA-DRB1/3/4/5, and -DQB1 identical donor could be identified. As expected, patients with two frequent haplotypes in strong linkage disequilibrium had the best chance of obtaining an HLA matched donor. Unexpectedly, patients with only one such haplotype had an almost similar chance. It could be calculated that HLA-DR typing of HLA-A,-B identical donors was rarely cost-effective after 1992. Only 12 of the 75 transplanted patients (16%) typeable at DNA level for class II, turned out to be completely matched for HLA-A,-B,-C,-DRB1/3/4/5,-DQB1,-DPB1 and had a negative MLC test. In the group of patients transplanted with a fully matched donor and for whom a CTLp test was performed, only 7% (4/54) of the tests were negative. Search results for patients of non-European origin were dismal, with only four of 26 patients referred being transplanted. In summary, of the 240 patients for whom the Europdonor office searched for a donor, about one-third were transplanted, one-third had a potential donor but did not reach transplantation, while for the remaining one-third of patients no suitable donor could be found.

Nomenclature for factors of the HLA system 1984

This article presents the decisions of the nomenclature committee on leukocyte antigens, which met in Vienna on 16-17 May 1984.

BLOOD-TRANSFUSIONS INDUCE PROLONGED KIDNEY ALLOGRAFT SURVIVAL IN RHESUS MONKEYS

A prospective study has been carried out in rhesus monkeys to investigate the influence of blood-transfusions on kidney allograft survival. Unrelated animals matched for 2 or 3 antigens of the A and B locus of the major histocompatibility complex were given five consecutive blood-transfusions before transplantation and received conventional immunosuppressive treatment. Transfused recipients showed a four-fold increase in mean survival time compared with the non-transfused controls. These results may contribute to a change of policy regarding blood-transfusions in transplant patients.

Validation of haplotype frequency estimation methods

In order to investigate the performance of haplotype frequency estimation methods using unrelated individuals, we compared the results of three estimation methods with those from the haplotypes deduced from family pedigrees. To that end we used the HLA phenotypes of the parents of 1040 families as data for the estimation methods and the full pedigree information as data for the deductive method. We evaluated the results of the following estimation methods: the method using two by two tables described by Mattiuz et al., the maximum likelihood method described by Yasuda and Tsuji and a crude method that uses the information on homozygosity in the phenotypes. All estimation methods generate reliable haplotype frequencies for the more frequent haplotypes, but are unreliable for the less frequent haplotypes. The maximum likelihood estimation method shows the best overall correlation with the results of the deductive method.

Improved fusion technique. I. Human umbilical cord serum, a new and potent growth promoter, compared with other B cell and hybridoma activators

Accelerated proliferation of hybridoma cells was observed in the presence of human umbilical cord serum (HUCS). This had very strong growth-promoting activity, even at a concentration of 2%. A comparison was made between HUCS and other B cell growth promoters, such as lipopolysaccharide (LPS) and dextran sulfate (DxS), macrophage supernatant, and human endothelial culture supernatant (HECS). The growth-promoting effect of HUCS was superior. Using a microcytotoxicity assay, we found no significant differences in the number of antibody producing clones with the various culture media, except for fetal calf serum.

Permissible mismatches, acceptable mismatches, and tolerance: new trends in decision making.

The identification of permissible mismatches that do not impair graft survival might open exciting new possibilities which we should follow up. The data are preliminary, but if true, 50% of the patients in Eurotransplant could get a kidney with optimal graft survival. For sensitized patients we might consider adding a CTLp test, with and without CsA, to our serological crossmatch tests. This might be beneficial especially for interpreting noncurrent crossmatches. Information on the activation status of the donor-specific cytotoxic T cells on the day of transplantation (primed or not, CsA-resistant or not) provides additional information which could facilitate the decision of whether or not to accept a mismatched graft, even in case of donor-specific antibodies in historical sera. Under certain circumstances, haplotype-shared blood transfusions can induce transplantation tolerance. Time will tell whether the determination of the level of donor-derived soluble HLA antigens might be helpful in an early diagnosis of rejection. It might become a new device to monitor homograft reactivity and to identify the patients in whom immunosuppression could be decreased or even discontinued.

The T-cell repertoire is not dictated by self antigens alone

The influence of genetic and environmental factors on the functional cytotoxic T cell (CTL) allorepertoire was studied by comparing the CTL precursor frequencies against the same HLA alloantigens in 116 sibling pairs. A significantly different precursor frequency was found in 68%, 61% and 59% of siblings sharing 0, 1 and 2 HLA haplotypes, respectively. These data show that, although HLA is important in determining the T-cell repertoire, this is hardly reflected in the allorepertoire. Even 50% of the monozygotic twin pairs showed a significant disparity in their CTL allorepertoire, indicating that environmental factors play a role as well. The non-inherited maternal HLA antigens could be identified as one of the environmental factors shaping the CTL allorepertoire.

Complete withdrawal of immunosuppression in kidney allograft recipients: a prospective study in rhesus monkeys

BACKGROUND We previously reported the successful withdrawal of immunosuppression in kidney-allografted rhesus monkeys. Recipients had received pretransplant blood transfusions and cyclosporine (CsA) immunosuppression for 6 to 12 months. One animal is still alive at more than 15 years after transplantation. Our hypothesis was that the sharing of a single DR antigen between blood donor and recipient, and the sharing of the same DR antigen with the kidney donor, may be beneficial to allograft survival. We now report on the results from a prospective study. METHODS The animals received three pretransplant blood transfusions from a single donor sharing one DR antigen with the recipient. Subsequently, a life-supporting kidney from a donor sharing the same DR antigen was transplanted. CsA was given for at least 6 months after transplantation. RESULTS Two animals rejected their graft at 5-8 weeks after cessation of CsA treatment. One animal is still alive at 700 days after transplantation. This animal showed MLR nonreactivity to its kidney donor, similar to the animal at more than 15 years after transplantation. CONCLUSION These results demonstrate that withdrawal of immunosuppression may be a realistic option in kidney graft patients under careful immunological monitoring of donor-specific immunity.