Second German consensus on immunogenetic donor search for allotransplantation of hematopoietic stem cells.

The present paper summarizes the results of the second German consensus meeting on immunogenetic donor search for allotransplantation of hematopoietic stem cells held in Essen in November 1999 under the auspices of the German Society for Immunogenetics (DGI) and the German Working Party for Blood and Marrow Transplantation (DAG-KBT). Immunogeneticists and transplant physicians from all over the country agreed to update the national standards for: (1) search strategy including the role of unrelated and extended family donor search after unsuccessful core family donor search, (2) histocompatibility loci to be typed, (3) histocompatibility typing techniques to be used (HLA serology vs DNA-based HLA typing, cellular tests, serum cross-match), and (4) acceptable HLA mismatches in the context of a defined underlying disease, donor type, and conditioning regimen.

Cord blood as a source of autologous RBCs for transfusion to preterm infants.

BACKGROUND: This prospective study was conducted to gain experience as to whether it is technically possible to produce autologous RBCs in additive solution from cord blood (CB), to optimize the blood supply for preterm infants. STUDY DESIGN AND METHODS: CB was collected from 47 infants with a mean (+/- SD) birth weight of 1717 (+/- 699) g. Whenever possible, RBC components were prepared by standard centrifugation using a six-bag system. All samples were put in sterility testing quarantine for 5 days, and a maximum storage of 14 days from collection to transfusion was specified. The babies were given either the autologous RBCs or standard allogeneic RBC concentrates, if autologous blood was not available. RESULTS: In 81 percent of the samples, autologous RBC components could be processed (vol, 7-87 mL; Hct, 31-82%). But within the group of extremely low birth weight infants (body weight <1000 g), a mean CB net volume of only 37 mL was collected, and the RBC preparation was successful only in exceptional cases. Three CB samples (8.6%) tested positive in sterility testing. Of the 47 infants, 21 were treated with a total of 62 allogeneic and 4 autologous RBC transfusions. Most infants with a body weight over 1400 g did not need any RBC transfusion. CONCLUSION: The preparation of autologous RBCs from the CB of preterm infants is technically possible in principle. However, major concerns must be raised as to whether such preparations are of benefit in ensuring safe care of neonates with blood components, with respect to the high rate of bacterial contamination and the limited availability in babies with low birth weight.

Identification of the novel allele HLA-B*1546 which belongs to the serological B72 type: implications for bone marrow transplantation.

The identification of the novel allele HLA-B*1546, which has serological B50 and B72 reactivity, was found in two members of a family of Turkish origin. In the sequence analysis the new allele differs from B*1501 by four nucleotides in exon 2. Its structure suggests that it may have originated by gene conversion with B*40, B*41, B*44, B*4501, B*47, B*4901 or B*50. At the protein level, the new allele has three amino acid differences compared to B*1501. Sequence alignment demonstrates that amino acid residue 46 is crucial for serological B72 reactivity. Due to substantial differences with other B*15 variants a possible mismatch may impair clinical outcome of bone marrow transplantation.

Micro-column affinity test and gel test: comparative study of two techniques for red cell antibody screening.

BACKGROUND AND OBJECTIVES: We describe the results of a comparative evaluation of a gel test (ID Micro Typing) and a micro-column affinity test (MCAT, Cellbind Screen) for red cell antibody screening and identification under routine conditions. MATERIALS AND METHODS: 3,000 serum samples of patients from the Mannheim University Hospital were tested in parallel by means of the gel test and the MCAT, using the low-ionic-strength-saline indirect antiglobulin test and the protein G affinity technique, respectively. Test cells used were the same in all tests. In addition, we performed titration studies with all detected antibodies as well as with 59 frozen sera containing antibodies of known specificity. RESULTS: A total of 154 antibodies (5.1%) were detected, 149 by gel test and 147 by MCAT. The overall sensitivity and specificity of the gel test was 96.8 and 96.5% and of the MCAT 95.5 and 97.2%. No significant differences between the gel test and MCAT were found when the titer scores of all 213 (fresh and frozen) antibodies were used to check the results. The mean scores for the gel test and the MCAT were 26.8 and 28.5, respectively. For anti-Fy(a) and anti-Kell, a significantly higher titration score could be obtained in the MCAT, whereas anti-Lu(a) showed a significantly higher score with the gel test. CONCLUSION: For the screening of unexpected red blood cell antibodies, the MCAT is as sensitive as the gel indirect antiglobulin test. The sensitivity and specificity of the two systems are more or less the same although it seems that IgM antibodies are better detected by the gel test.

Recognition of HLA-DR1/DRB1*0101 molecules presenting HLA-A2 derived peptides by a human recombinant antibody, Fab-5 A1.

MHC molecules present peptides in their binding groove to T-cell receptors inducing proliferation or cytotoxicity of alloreactive T cells. A previously generated human monoclonal antibody (mAb) UL-5 A1, recognizing a conformational epitope formed by HLA DR1/DRB1*0101 molecules and HLA-A2 derived peptides, demonstrates T-cell-like recognition of the peptide/MHC complex (PMC). To study the genes of the antigen binding region, the nucleotide sequences of the rearranged genes in the variable regions of UL-5 A1 were determined and the V-gene usage (VH3, V lambda 2) was identified by comparison with published germlines. The genes encoding heavy (Fd) and light (L) chains of UL-5 A1 were linked and expressed in a bacterial system. Specificity of the recombinant Fab-5 A1 was determined with HLA-typed LCLs by flow cytometric analysis. As demonstrated in competitive inhibition assays, UL-5 A1 and Fab-5 A1 recognize the same PMC epitope on HLA-A2+, -DR1/DRB1*0101+ typed LCLs. Additionally, mAb UL-5 A1 and Fab-5 A1 both recognize HLA-A2-, -DR1/DRB1*0101+ LCLs exogenously loaded with HLA-A2 peptides (105-117, 103-117). UL-5 A1-like antibodies against peptide/MHC complexes could prove valuable tools for research on T-cell recognition and MHC function.

HLA and alveolar echinococcosis.

Evidence in animal intermediate hosts that susceptibility to larval infection with Echinococcus multilocularis is restricted to individual host factors prompted us to investigate the susceptibility markers in humans. Because antigens of the extracellular parasite E. multilocularis are possibly presented by MHC molecules in a restricted way, we speculated that MHC polymorphism may influence resistance of the host towards infection and course of disease. We studied HLA-A, -B, -DRB1, -DQB1 and -DPB1 polymorphism in 151 patients with alveolar echinococcosis. Patients with an observation period of more than 2 years were grouped according to the clinical follow-up into cured (no recurrence following surgery) patients and patients with regressive or progressive forms of disease during benzimidazole chemotherapy. By comparing phenotypic frequency between patients with alveolar echinococcosis and healthy controls, HLA-DRB1*11 was associated with a reduced risk for disease development (odds ratio=0.55, 95% confidence interval=0.34-0.88; P=0.01). HLA-DQB1*02 was more frequent in patients with progressive disease when compared with patients with regressive disease (54.3% vs 28.3%, P=0.02). The result suggests that HLA-DRB1*11 might confer protection against alveolar echinococcosis and that HLA-DQB1*02 may indicate a risk for progressive disease development. The findings may facilitate the search for immunodominant T-cell epitopes of E. multilocularis.

5% Me2SO is sufficient to preserve stem cells derived from cord blood.

On the basis of historical findings, 10% dimethylsulfoxide is still used to cryopreserve stem cells. We studied a final concentration of 5% dimethylsulfoxide in cryopreservation of cord blood-derived stem cells in autologous plasma. In our opinion, a final concentration of 5% dimethylsulfoxide in autologous plasma without further additives is sufficient for cryopreservation of cord blood stem cells in a banking routine.

Human monoclonal antibody with T-cell-like specificity recognizes MHC class I self-peptide presented by HLA-DR1 on activated cells.

Alloreactive T cells recognize peptides presented in the binding groove of major histocompatibility complex molecules (MHCs), whereas B cells mainly recognize the MHCs independent of bound peptides. Here, we demonstrate that the human B-cell repertoire comprises B cells which can be stimulated during pregnancy to produce antibodies reacting with MHCs in a way similar to T cells. The human monoclonal antibody UL-5A1 recognizes DR1(DRA/DRB1*0101) molecules on lymphoblastoid cell lines only if they co-express HLA-A2 or if they have been loaded with HLA-A2-derived peptides. The effect of the HLA-A2 peptide 105-117 on UL-5A1 reactivity was specific, time and dose-dependent. Reactivity increased when naturally processed peptides were removed from DR1 molecules before the HLA-A2 peptide 105-117 was loaded. UL-5A1 reacted specifically with cells that had been activated. The results imply a role of activation of cells in peptide processing and/or loading.

Photometric evaluation of the solid-phase antiglobulin test using length measurement of the absorption curve.

BACKGROUND AND OBJECTIVES: The very sensitive solid-phase antiglobulin test is used widely in blood group serology. However, a disadvantage of the test is the sedimentation variability of indicator cells during centrifugation, which causes errors in the photometric evaluation. MATERIALS AND METHODS: To solve this problem a technique was developed which depends on the 'length measurement of the absorption curve' of the sedimented indicator cells. RESULTS: 9,075 sera were tested for the presence of alloantibodies on the basis of the visual type of measurement and the newly developed technique. The agreement between the two evaluation systems was 99.94%. No false-negative results were observed. CONCLUSION: The new method allows the complete automation of the solid-phase antiglobulin test, enabling the analysis and evaluation of the solid-phase microtiter plate assay to be more discriminating and safe.

German consensus on immunogenetic donor search for transplantation of allogeneic bone marrow and peripheral blood stem cells.

In Germany allotransplantation of bone marrow or peripheral blood stem cells is presently performed by 34 different teams operating more or less independently. Thus, strategies of immunogenetic donor search, use of the various tissue typing techniques and policy on acceptable HLA mismatches in related and unrelated settings may vary considerably from one transplant centre to another. This paper summarises the results of the first German consensus meeting on immunogenetic donor search for bone marrow/peripheral blood stem cell grafting. The main goal of the participating transplant physicians and immunogeneticists was to define national standards for the above issues.

Characterization of two human IgM monoclonal antibodies reactive with HLA-B27.

We describe here the generation and characterization of two human monoclonal IgM antibodies (UL-4F11 and UL-F6) reactive with HLA-B27. The monoclonal antibody (mAb) UL-4F11 is cytotoxic for peripheral mononuclear cells and, therefore, useful as typing reagent for HLA-B27 and HLA-B38. Protein chemistry showed that the mAb UL-4F11 precipitates HLA-B27 molecules. Epitope mapping analysis suggests that the amino acids 45, 67, 82 and 83 (alpha-1 domain) of the HLA-B27 sequence are necessary for mAb UL-4F11 reactivity. The mAb UL-F6 is suitable for complement dependent lysis of lymphoblastoid cell lines and stimulated peripheral blood mononuclear cells with HLA-B27 (B*2701, B*2702, B*2703, B*2705, B*2707), B13, B40 (60,61), B47 and B48 specificities. Its reactivity indicates that the amino acid valine in position 152 and glutamic acid in position 163 of the alpha-2 domain are crucial for the binding epitope.

Mild course of fetal RhD haemolytic disease due to maternal alloimmunisation to paternal HLA class I and II antigens.

The patient is a pregnant women of African origin with a prior history of spontaneous abortion and newborn dystrophy. The investigation showed an anti-Rh D antibody (IgG isotypes 1 and 3) with an indirect antiglobulin tube test (IAT) titre of 1:512. The monocyte monolayer assay (MMA) proved clearly the interaction of Fc receptors with the maternal anti-D, and so a clinical significance was expected. In spite of this, no signs of severe haemolysis in the Rh-D-positive and direct antiglobulin test-positive fetus could be observed. Furthermore, two HLA class I and II alloantibodies (anti-A10, anti-DR13) directed against paternal and fetal antigens were detected in the serum of the gravida. Both antibodies showed an inhibitory effect on the in vitro phagocytosis capacity of mononuclear cells expressing at least one of the corresponding HLA antigens (immunophagocytosis inhibition (IPI) test). Thus, the mild course of haemolytic disease may be explained by an effective inhibition of the fetal mononuclear phagocyte system by maternal HLA class I and/or class II antibodies resulting in a diminished destruction of anti-D-coated fetal red blood cells.

Development of a batch-processed, passive latex agglutination test for cytomegalovirus.

BACKGROUND: The passive latex agglutination test is commonly used for the identification of cytomegalovirus IgG and IgM antibodies. This test is used because of its sensitivity, specificity, speed, and ease of performance, but it is unsuitable for large numbers of samples or for batch processing. STUDY DESIGN AND METHODS: To solve this problem, comparative studies to assess the cytomegalovirus passive latex agglutination test on microtiter plates were done with a new photometric particle agglutination method (PPAM) and an enzyme-linked immunosorbent assay as a control. RESULTS: A total of 3430 sera were tested using both the PPAM and enzyme-linked immunosorbent assay. A high degree (97.6%) of correspondence between the results of the tests was observed. The new PPAM was easier and faster to use (96 wells in 25 min). CONCLUSION: These results, as well as the possibility of adapting this method to a fully automated system, suggest that the PPAM could be an important contribution to the field of infection serology.

Generation and characterization of a human monoclonal IgG antibody specific for HLA-B12.

We describe here the generation and characterization of a human monoclonal IgG antibody (UL/F14) specific for HLA-B12. The antibody is suitable for complement-dependent lysis on lymphoblastoid cell lines and stimulated peripheral blood mononuclear cells. UL/F14 competes for antigen binding with an HLA-B12 human monoclonal IgM antibody and with a specific alloantiserum. Protein chemistry shows that the monoclonal antibody UL/F14 can precipitate solubilized HLA-B12 molecules.

[Comparison of HLA typing and retyping data in patients with bone marrow transplantation]. / Vergleich der HLA-Typisierungs- und Retypisierungsdaten bei Patienten mit Knochenmarktransplantation.

Comparing HLA data from 851 patients with serological HLA-A, -B, -DR and/or PCR-SSO HLA-DRB, -DQB reevaluated HLA types produced 107 (12.5%) different results. The high discrepancy of HLA data from patients confirm the demand for a serological HLA-A, -B and a DNA HLA-DRB, -DQB reevaluation before starting an unrelated donor search for bone marrow transplantation. The reliability of serological HLA-DRB, -DQB typing was evaluated as 97.8%, whereas DNA HLA-DRB, -DQB typing was 99.5%.

TAP2 gene polymorphism segregates with DR-DQ in DR/DP recombinant siblings.

RING 11, a second transport-associated gene (TAP2), has been recently identified in the DR-DP interval of the human class II region. Two predominant alleles, TAP2A and TAP2B, differing by 17 amino acids at the C-terminus of the ATP-binding domain are present in the Caucasoid population at frequencies of 79% and 21%, respectively. In the rat, polymorphism of the TAP genes were found to influence peptide loading of MHC class I molecules and, in humans, it was speculated that variation in peptide loading of HLA-B27 molecules might be also linked to factors altering antigen presentation presumably encoded in the HLA region. To determine whether TAP2 gene polymorphism may be relevant to peptide loading in humans, we typed 41 HLA-ABC, DR-identical pairs for TAP2A and TAP2B by PCR-SSO hybridization or direct genomic sequencing. In eight cases, GLO-different and, in six cases, DP-different recombinant siblings were included. Allele frequencies for TAP2A and TAP2B were as previously reported (74% and 26%, respectively). In all pairs, TAP2 gene polymorphism segregated with the DR-DQ type, mapping the TAP2 gene telomeric to the recombination hot spot in the DR-DP interval of the human class II region. We conclude that, in HLA-identical siblings, TAP2 gene differences are very unlikely to occur. Thus, in HLA-identical siblings, minor histocompatibility antigenic differences cannot be attributed to variant peptide loading due to TAP2 gene polymorphism.