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The prognostic significance of a positive B and/or T cell crossmatch test before transplantation was analyzed in 174 cadaver kidney transplants. Thirty-one B cell-positive cross-matches were observed. In 15 of these cases, T cell crossmatches were also found to be positive retrospectively (long incubation assays). In 16 patients with a positive B cell crossmatch, an anti-HLA-DR antibody was present, as judged by platelet absorption studies, whereas in the other patients the B cell activity of the sera was attributable to weak anti-HLA-A,B,C antibodies or to cold autoantibodies. The success rate at 3 months of positive B cell crossmatch transplants because of anti-HLA-A,B,C, or anti-HLA-DR antibodies was 84 whereas that of grafts with negative B and T cells crossmatches was 79. Patients with B cell autoantibodies had excellent graft survival.

Allogeneic bone marrow donors can be incompatible at different levels. Even HLA-identical pairs will be still incompatible for numerous minor histocompatibility antigens (mHag). Nevertheless, some incompatibilities are found to be associated with an increased risk of graft-versus-host disease (GVHD), which could be related to the way the immune system recognizes these antigens.

The lymphocyte-dependent antibody (LDA) assay has been used in parallel with the microlymphocytotoxicity test detecting complement-dependent antibody (CDA) in 100 cadaver kidney transplant recipients, in order to assess the role of the LDA phenomenon in the rejectin of human allografts. In 15 cases, acid eluates were prepared from the kidney graft after rejection or at autopsy and tested in parallel with pre- and post-transplantation sera. The results indicate a marked increase in sensitivity of LDA assay compared with the standard microlymphocytotoxicity test. In 12 patients the pretransplant serum was found retrospectively LDA positive, but CDA negative, using the kidney donor lymphocytes or allogeneic lymphocytes bearing the same incompatibel antigen(s) for the recipient as targets. Ten of them lost their graft, mostly during the first month after transplantation. Graft survival was also poor in the 11 patients having both LDA and CDA activity in their pretransplant serum, even when the specificity of the serum did not appear to be directed against antigens shared by the donor. Although it is not clear yet, whether the two assays detect the same or different antibody molecules, it appears that the detection of even a low level of LDA activity before transplantation is associated with a high risk of early transplant failure.

In renal transplantation a good HLA-DR match is associated with a higher success rate of graft outcome. However, due to a number of technical problems, reliable serological DR typing cannot always be obtained, and the very large number of HLA-DR alleles now discovered renders such DR matching more difficult. In view of the medical importance of HLA class II polymorphism in transplantation immunology, we have developed a simple HLA-DR oligotyping procedure on PCR-amplified DNA, by hybridization with 14 synthetic oligonucleotide probes able to recognize all major HLA-DR specificities. In particular, the probes used in this study allow the unambiguous discrimination of the DRw11, w12, w13, w14-Dw9 specificities or of rare alleles such as DR-Br or DRw13-DwHAG, which are very often difficult or impossible to identify by serology. In order to explore the potential of this methodology, we have analyzed by oligotyping 110 kidney transplant patients with doubtful or unreliable serological assignment, or with DR blank alleles. Comparison between serology and oligotyping shows that in 66.3 of the patients we observed an excellent correlation. About half these patients are homozygotes, as ultimately verified by RFLP typing. In 26.4 of the patients however, at least one HLA-DR antigen was discrepant, and in 7.3 of the cases oligotyping resolved uninterpretable serology. Almost all of the discrepancies were due to errors in allele assignment within the DRw52 group, mostly in the case of DRw13 alleles. This study confirms the expected qualitative advantage of the oligotyping technique and its simplicity as compared with the RFLP DNA typing procedure. Large scale application of the oligotyping methodology will therefore be beneficial to optimize HLA-DR matching in organ transplantation, particularly in high responders with first kidney graft rejection.

Since pretransplant blood transfusions have been shown to prolong the survival of kidney grafts, a new transfusion policy has been started in the frame of Swisstransplant. Before surgery all patients receive at least two and, if possible, five transfusions (whole blood or packed red blood cells). The present study includes 101 recipients of primary cadaver grafts. Of these, 41 were transfused regularly according to the new protocol, 46 had irregular transfusions because of therapeutic necessity, and 14 had no transfusion before grafting. The 1-year survival rate in pretransfused patients was over 70 as compared to 45 in the nontransfused group. There was no significant association with the number of transfusions, but a slight improvement in graft survival was seen in patients deliberately transfused when compared with those transfused because of severe anaemia. A delay of more than 3 months between the last transfusion and transplantation significantly decreased graft survival at 6 months (84 versus 58; P< 0.02). The occurrence of cytotoxic antibodies, both antiperipheral blood lymphocytes (PEL) and anti-B cell antibodies, was investigated in relation to the number of transfusions received. Broad-spectrum anti-PBL antibodies (>50 of random panel) were found in 5 of 74 patients transfused according to the protocol (7) and in 15 of 93 patients transfused for severe anaemia (16; P, not significant). Of 71 recipients followed up for 6 months, 15 (21) produced anti-PBL antibodies with limited specificity (<50), and 4 (6) produced broad-spectrum antibodies. Anti-B cell antibodies (<50) were produced in 21 of 64 patients (33). Six patients (9) had broad-spectrum activity. The occurrence of these antibodies was not associated with the number of transfusions received and did not significantly influence the graft survival at 6 months.

Blood transfusions administered before renal allografts are known to enhance graft survival. Among alternative hypotheses proposed to explain this effect, one of the most attractive is the possible induction of antiidiotypic antibodies directed against the specific antigen-binding site of donor-specific antibodies. In order to determine if such blocking antibodies are generated after blood transfusions, serial serum samples obtained before transplantation from 44 kidney recipients were analyzed for the development of HLA-DR alloantisera inhibitory activity by a microcytotoxicity inhibition assay.

In vitro and in vivo immunologic parameters were determined in 26 patients treated continuously with cyclosporine to prevent graft-versus-host-disease (GVHD) after allogeneic bone marrow transplantation (BMT) for acute and chronic leukemia and for aplastic anemia. A group of 18 patients was tested 6 months after BMT and another group of 10 patients was tested after one year. At 6 months after BMT, 94 of the patients had normal serum IgG and IgM levels, whereas at one year 29 of them had low IgA levels. The proportion of patients with normal lymphocyte responses in vitro at 6 months after BMT was 69 and 76 for the responses to concanavalin A and to soluble antigens; 75 and 53 for the responses to allogeneic cells and pokeweed mitogen, respectively; and 89 for the response to phytohemagglutinin. All but one were able to generate suppressor cells upon con A stimulation. At one year after the graft, only one patient had demonstrable multiple abnormalities in in vitro tests. Skin test reactivity at one year was comparable to pre- graft reactivity. After BMT a lymphopenia persisted for 6 months. The rate of infectious complications was high during the first 3 months after BMT, and it diminished progressively as immune functions returned to normal. Infection was the cause of death in two cases (one disseminated cytomegalovirus infection and one septicemia). GHVD occurred in 12 patients; in nine of them the disease was transient and mild, only 1 patient developed severe chronic GVHD. Acute GVHD did not influence the tempo of immunologic reconstitution. In comparison to other studies, it seems that cyclosporine does not delay immune restoration, or increase morbidity from infection, while preventing GVHD and its complications efficiently.

Transplantation with bone marrow from other than genotypically HLA-identical donors is associated with an increased incidence and severity of graft-versus-host disease (GvHD). The precise influence of HLA incompatibilities is not easy to analyze as even perfectly matched, HLA-identical unrelated donors might still express HLA differences that remain undetected by conventional typing.

We used a simple HLA-B35 PCR/SSO-oligotyping procedure, combined with exon 3 direct sequencing for the analysis of B35 subtype frequencies in our population, and for the evaluation of the degree of B35-subtype compatibility in serologically matched unrelated bone marrow and kidney transplant pairs. B3501 was the most frequent allele (0.6), followed by B3503 (0.19), B3502 (0.13), B3508 (0.07), and B3505 (<0.01). HLA-B35-subtype matching of donors and recipients was strongly dependent on the stringency of ABDRB1 matching. Among 10 kidney donor/recipient pairs, only 30 were B35-subtype-matched. Due to the lack of ABDRB1 haplotype matching, this low degree of matching was not better than what would be expected on the basis of the subtype frequency distribution in the population. In contrast, HLA-B35 subtype compatibility was higher in unrelated bone marrow donor/recipient pairs that were serologically ABDR-matched: 30 of the 62 (48.4) B35-positive combinations tested were B35-subtype-compatible. When all patient/donor plus donor/donor combinations (n=160) were taken into account, 46 of the ABDR-matched pairs were B35-subtype-compatible. When only pairs that were DRBl/DRB3/DRB5-subtype-matched by oligotyping (n=62) were considered, 71 were B35-sub-type-compatible. The fact that a significant percent of patient/donor pairs matched by conventional HLA-typing are found incompatible, as shown here for B35, explains the difficulties in assessing the beneficial effect of HLA matching in transplantation.

After the progressive improvement in the results of organ transplantation we now face the challenge of shortage in organ supply. The decreasing number of organ retrievals performed in 1990 at our hospital has raised questions concerning loss of potential organ donors and opposition to donation by the next of kin.

Serum of 68 patients with aplastic anemia was tested for the presence of autolymphocytotoxins (auto-LTs). Prior to specific disease treatment, 16 patients (24) displayed antibodies cytotoxic to their own lumphocytes. These antibodies had the characteristics of cold-reactive lymphocytotoxins. Their detection in patients' sera was found unrelated to a viral or toxic cause of th disease or the patients' HLA genotype. Broadly reactive anti-HLA a ntibodies were less frequent in pretreatment sera containing auto-LTs, suggesting that these autoantibodies could modulate alloantibodyu production. However, after specific disease treatment, the alloantibody frequency was comparable in patients with or without auto-LTs. We found no significant difeference in response to antilymphocyte serum or bone marrow graft outcome in the patients in ralationto the presence or absence of pretreatment auto-LTs. This observation suggests that the detection of these autoantibodies in aplastic anemia has no clinical relevance.