Serology versus ARMS-PCR in prospective HLA-class I typing for bone marrow transplantation.

While HLA class II alleles identification by means of complement mediated lymphocytotoxicity (serology) is almost replaced by DNA typing techniques, serology is still widely used for routine class I typing. The aim of this prospective study was to compare PCR-based Amplification Refractory Mutation System with serology in clinical HLA class I alleles assignment in patients receiving marrow transplants and their potential donors. The total discrepancy rate in 114 consecutively typed individuals for HLA-A and HLA-C alleles was only in favor of ARMS-PCR, whereas HLA-B typing was discrepant also in favor of serology. The discrepancies were higher in patients, particularly in those with acute lymphoblastic leukaemia, than in healthy individuals. We conclude, that ARMS-PCR is clearly superior to serology in definition of class I alleles, which might be of clinical importance particularly for bone marrow transplantation.

Segregation of HLA genes in multicase narcolepsy families.

In the past 15 years, 411 sporadic narcolepsy patients have been diagnosed in the Hephata Klinik, Schwalmstadt, Germany. They were explored for presence or absence of excessive daytime sleepiness and narcolepsy in their relatives. A subset of 39 patients were explored for presence or absence of parasomnias. Six patients had more than one relative affected by narcolepsy-cataplexy. Forty-seven family members were investigated with the Stanford Center for Narcolepsy Sleep Inventory and a standardized parasomnia questionnaire. Twenty-four relatives had nocturnal polysomnographies and Multiple Sleep Latency Tests. HLA class I typing was performed in all sporadic and familial cases, class II and microsatellite typing was performed in all members of multicase families. Based on the Finnish prevalence study by Hublin et al., 1994, the relative risk for first degree relatives to develop narcolepsy-cataplexy was in our sample 16.5, 34.2 for excessive daytime sleepiness and 426.9 for parasomnias. Cataplexy, excessive daytime sleepiness and single narcoleptic symptoms in the multicase families segregate with the DRBI*1501, CARII:200, CARI: 103, DQBI*0602 haplotype. In two families, members with narcolepsy and isolated symptoms have inherited the DRBI*1501/DQBI*0602 haplotype from the nonaffected parent. The observed segregations in these two families may support the view that narcoleptic symptoms are expressed by DRBI*1501/DQBI*0602 carriers, independent of haplotype origin. Parasomnias do not segregate with a specific haplotype. The frequency of parasomnias in narcolepsy is much higher than in the general population. The empirical risk for first degree family members of narcolepsy patients to develop cataplexy seems to be low, whereas it is higher for EDS and highest for parasomnias.

Extensive HLA class II studies in 58 non-DRB1*15 (DR2) narcoleptic patients with cataplexy.

Narcolepsy is a sleep disorder that has been shown to be tightly associated with HLA DR15 (DR2). In this study, 58 non-DR15 patients with narcolepsy-cataplexy were typed at the HLA DRB1, DQA1 and DQB1 loci. Subjects included both sporadic cases and narcoleptic probands from multiplex families. Additional markers studied in the class II region were the promoters of the DQA1 and DQB1 genes, two CA repeat polymorphisms (DQCAR and DQCARII) located between the DQA1 and DQB1 genes, three CA repeat markers (G51152, T16CAR and G411624R) located between DQB1 and DQB3 and polymorphisms at the DQB2 locus. Twenty-one (36%) of these 58 non-DR15 narcoleptic patients were DQA1*0102 and DQB1*0602, a DQ1 subtype normally associated with DRB1*15 in DR2-positive narcoleptic subjects. Additional microsatellite and DQA1 promoter diversity was found in some of these non-DR15 but DQB1*0602-positive haplotypes but the known allele specific codons of DQA1*0102 and DQB1*0602 were maintained in all 21 cases. The 37 non-DQA1*0102/DQB1*0602 subjects did not share any particular HLA DR or DQ alleles. We conclude that HLA DQA1*0102 and DQB1*0602 are the most likely primary candidate susceptibility genes for narcolepsy in the HLA class II region.

Diagnostic and prognostic significance of anticardiolipin antibodies in patients with recurrent spontaneous abortions.

PROBLEM: The role of ACA in unexplained RSA is controversial. In the present study, diagnostic and prognostic aspects were investigated. METHOD: One hundred five nonpregnant patients with primary, 29 with secondary RSA, and 209 controls were investigated for IgG-ACA. Follow-up studies were done during pregnancy in 76 individuals. IgM-ACA were tested in a subset of patients. RESULTS: Elevated ACA levels were significantly more frequent in both patient groups (26 and 24%) than in controls (16%). However, there was no correlation of ACA with various parameters including pregnancy outcome. In ACA-positive patients with successful pregnancy a significant decrease of ACA values during pregnancy was observed, while ACA remained high in aborting patients. IgG- and IgM-ACA correlated well. CONCLUSIONS: Although the data from nonpregnant RSA patients does not allow diagnostic or prognostic conclusions to be drawn, sequential testing of ACA-positive individuals provides the possibility to foresee pregnancy outcome.

Immunotherapy with intravenous immunoglobulin for prevention of recurrent pregnancy loss: European experience.

PROBLEM: Due to its strong "immunomodulating" effect in several well established disorders, high-dose intravenous immunoglobulins (IVIG) has been proposed as an alternative for immunotherapy with allogeneic leucocytes in patients with unexplained recurrent spontaneous abortion. This paper is intended to provide an overview on the European experience in this field. METHOD: Five European pilot studies with a total of 172 patients as well as one controlled double-blind multicenter study including 64 patients were considered. In the latter, 5% human albumin was used as placebo. RESULTS: Success rates of the pilot studies varied from 68 to 87%. In the German controlled study, a significant specific effect of IVIG could not be verified. However, success rates for both IVIG and albumin were in the same range as for allogeneic leucocytes. CONCLUSION: At present, it is not sufficiently proven that IVIG is an appropriate tool for immunotherapy of recurrent spontaneous abortions. It is suggested that success rates of both IVIG and albumin are due to a placebo effect. However, we cannot exclude that albumin itself provides immunomodulating capacity.

Immunogenetic and serological investigations in nonpregnant and in pregnant women with a history of recurrent spontaneous abortions. German RSA/IVIG Study Group.

In the context of a controlled multicenter study on intravenous immunoglobulin (IVIG) treatment of patients with a history of unexplained recurrent spontaneous abortions (RSA), a number of controversial immunological parameters were evaluated prior to and during pregnancy with respect to their diagnostic and/or prognostic significance. A total of 390 serum samples from 52 patients were investigated. Sharing of 2 or more HLA (A, B, DR, DQ) antigens was significantly more frequent in RSA couples than in controls. The rate of cytotoxic or Fc-receptor (FcR)-blocking antibodies was not significantly lower in RSA patients than in individuals with normal pregnancies. Both tumor necrosis factor-alpha (TNF-alpha) levels and IgG anticardiolipin antibodies (IgG-ACA) were significantly increased in the patient group. While the occurrence of HLA sharing, cytotoxic/FcR-blocking antibodies and IgG-ACA did not correlate with the outcome of pregnancy, TNF-alpha levels were found to be significantly higher in patients with subsequent miscarriage than in those with successful pregnancy. IgG-ACA, if present, significantly decreased during the course of successful pregnancy but remained high in patients with subsequent abortion. It is concluded that the diagnostic and/or prognostic value of HLA sharing and cytotoxic/FcR-blocking antibodies has been overestimated while TNF-alpha and ACA levels are potential diagnostic markers and/or exhibit prognostic significance in subgroups of RSA patients.

Intravenous immune globulin in recurrent abortion.

A specific effect of intravenous immune globulin (IVIG) on the outcome of pregnancy in patients with a history of habitual abortion has been postulated as an alternative to immunotherapy with allogeneic leucocytes. The results of different pilot studies have been promising, demonstrating a successful outcome of pregnancy in approximately 80% of treated patients. However, the evaluation and interpretation of the study results has to take into account that the probability of a successful pregnancy in women with a history of three spontaneous abortions is about 60% without treatment. Specific pharmacological effects therefore have to be verified in controlled studies in order to rule out psychological (placebo) effects. A specific therapeutic effect could not be verified in a German randomized, double-blind, multicentre trial in comparison to human albumin 5% which was used as a placebo. The result of another controlled study currently underway in the USA is expected.

[Serologic detection of HLA antigens and HLA antibodies]. / Serologischer Nachweis von HLA-Antigenen und HLA-Antikörpern.

The discovery of the HLA system as well as the progress of knowledge over several decades were based on serological methods. Today, methods of molecular biology tend to replace serological tests for HLA typing, particularly for class II alleles. However, HLA serology is still indispensable in all domains where HLA antibodies play a role. In this contribution, classical and more recently developed methods of HLA serology are described, and problems and solutions concerning the interpretation of results are discussed.

HLA phenotypes and multiple basal cell carcinomas.

BACKGROUND: Previous investigators noted an association of multiple basal cell carcinomas (BCC) with certain HLA antigens; however, these findings were contradictory, and the associations were only weak. OBJECTIVE: The aim of the study was to objectify the previously found associations. METHODS: Serologic HLA typing for class I and class II antigens was performed in 49 unrelated patients with 5 or more BCCs. RESULTS: HLA-DR4 showed decreased frequencies in the patient group as compared with healthy controls (n = 716). Cw7 was found to be increased in the total group of patients as well as in a subgroup with multiple BCCs of the face (n = 24), while a subgroup with BCCs mainly on the trunk (n = 25) revealed increased frequencies of HLA-A11, -B17, -B22 and -Cw3. However, none of these deviations appeared significant after correction of p values. CONCLUSION: We conclude that, if at all, the HLA system plays only a minor role in the development of multiple BCCs.

[New possibilities of donor selection for bone marrow and organ transplantation by HLA typing and genomic DNA]. / Neue Möglichkeiten der Spenderauswahl für die Knochenmark- und Organtransplantation durch HLA-Typisierung aus genomischer DNA.

For one year we determined HLA class II antigens from all patients with renal and haematological disorders and their healthy family members both by using serological methods and DNA typing (SSO, SSP). The rate of discrepancies between the results of serological DR typing and DRB1 typing was 10.8% (13/120). We were able to demonstrate that DNA typing for class II antigens leads to definite results even for patients with a poor cell quality or with lymphocytopenia where serological typing is often impossible. Furthermore, DNA typing from patients with haematological disorders is very important, especially if a bone marrow transplantation is considered. In addition to MLC testing, DRB1, DQB1 and DPB1 typing should be carried out for the patient and the potential donor. DNA typing is also recommended for patients waiting for a kidney transplantation particularly if they were serologically typed as 'homozygous', as the chance to receive an HLA-compatible organ is much higher for a heterozygous individual.

HLA antigens in Peyronie's disease.

This study presents data on HLA phenotypes of 52 unrelated patients suffering from idiopathic Peyronie's disease. This first investigation on HLA class II antigens detected an association of HLA-DR3 and -DQw2 in this disorder. HLA typing was done from ACD-stabilized peripheral blood using the modified lymphocytotoxicity test. Antigen frequencies of the patient group were compared with those of healthy individuals of the local population. There were no deviations of frequencies for antigens of the B7 cross-reacting group as described in earlier studies. In addition none of the other class I antigens (HLA-A, -B, -C) showed any significant deviation in frequencies after correction of p values. Regarding class II antigens HLA-DR3 was detected in the patient group in 33.3% compared with 16.0% of the control population (corrected p < 0.05). The closely linked antigen DQw2 was found in 58.8 compared with 31.2% (corrected p < 0.005). Not only genetic factors can be stated by these findings. As HLA-DR3 and -DQw2 are known to be the typical associated antigens of organospecific autoimmune disorders, this suggests possible autoimmunological factors in this disorder of otherwise unknown etiology.

Quantitation of HLA class II antigens on B-cell lines.

By quantitation of elutable immunoglobulin G in a recently described ELISA, the number of HLA class I and II molecules on B-cell lines was determined using monomorphic monoclonal antibodies. An average number of 183,000 binding sites per cell for HLA class I-, 99,000 for HLA-DR-, 63,000 for DQ- and 42,000 for DP-specific monoclonal antibodies were determined. The small amount of HLA class II molecules can in part explain the difficulties observed in HLA class II typing or in detection of class II antibodies using the lymphocytotoxicity text.

A deletion in the second exon of an HLA-DRB1 allele found in a DR2-negative narcolepsy patient.

In this report, we describe a new allele of the HLA-DRB 1 gene carrying a form of mutation that has not been observed before. It appeared in an HLA-DR2-negative narcolepsy patient who, besides HLA-DR4, revealed a serologic HLA-DR blank segregating with HLA-DQ1. Oligotyping showed that the new allele belongs to the HLA-DR8 group. Restriction analysis and DNA sequencing revealed the deletion of 12 bp as well as the substitution of 9 flanking base pairs between codons 36 and 43. The expression of the mutated gene was demonstrated by the presence of its messenger RNA and a few positive reactions with DR8 sera. Without interrupting the reading frame, the mutation leads to a gene product composed of a modified amino acid sequence. We anticipate that the mutation influences the conformation of the molecule with possible consequences concerning immune response.

Heparin-associated thrombocytopenia: no association of immune response with HLA.

Heparin-associated thrombocytopenia (HAT) is a severe adverse reaction of heparin therapy. Patients with the immunologic type of HAT are at risk of developing arterial and venous thromboembolic complications caused by an antibody which activates platelets in the presence of heparin. Yet, there are no means to identify patients at risk of developing HAT before heparin administration. We investigated the frequency of HLA class-I and class-II antigens in 47 patients with the immunologic type of HAT verified by a positive two-point heparin-induced platelet activation assay. Compared to a control group of 629 healthy individuals HLA antigens B8 and DR3 were less frequent, whereas DR4 and DR53 had a higher frequency. However, none of these antigens proved to be statistically significant. This is another example of an immunologically mediated disease showing no discernible association with the HLA system.

Alloimmune neonatal neutropenia is a potential side effect of immunization with leukocytes in women with recurrent spontaneous abortions.

Alloimmunization of a mother against granulocytes causing alloimmune neonatal neutropenia (ANN) in her newborn was found likely to be attributed to previous intradermal injections of paternal lymphocytes. Immunotherapy with leukocytes which was performed for recurrent spontaneous abortions hence provides the possibility of granulocyte alloimmunization and increases the risk of the occurrence of ANN.

[Granulocyte-specific and HLA antibodies in pregnancy: incidence and clinical value]. / Granulozytenspezifische und HLA-Antikörper während der Schwangeschaft: Häufigkeit und klinische Bedeutung.

Postpartum sera of 1,016 unselected women were examined for granulocyte-specific and HLA antibodies. A total of 11 out of 1,016 sera (1.1%) were only reactive with neutrophils. Cytotoxic HLA antibodies were detected in 24%, noncytotoxic HLA antibodies in 4.8% of the sera. All antibodies belonged to the IgG 1 and IgG 3 subclasses. NA1 and NB1 specificity were each determined in one serum, two sera contained NA2-specific antibodies. After 1 year all antibodies were no more detectable. As none of the newborns from immunized mothers developed neutropenia, the incidence of alloimmune neonatal neutropenia seems to be lower than 0.1%.

Influence of treatment with mouse immunoglobulin on the rate of viable neonates in the CBA/J x DBA/2 J model.

In the CBA/J (H-2k) x DBA/2J (H-2d) murine model, the protective value of pooled murine immunoglobulin i.p. was compared with that of serum taken 7 days after termination of CBA/J (H-2k) x BALB/c (H-2d) pregnancy. A control group of CBA/J females received treatment with autologous virgin serum. CBA/J females at 7 weeks of age were injected 3 days before mating with DBA/2J males and 3 days after sighting of the vaginal plug. An effect of treatment can be shown following IgG therapy (P less than 0.05). The highest rate of viable fetuses was documented in the immunoglobulin-treated females, while the rate of viable offspring did not differ in the two serum-treated animal groups.

HLA association of idiopathic Peyronie's disease: an indication of autoimmune phenomena in etiopathogenesis?

HLA typing for class I and class II antigens was done in 52 unrelated patients suffering from idiopathic Peyronie's disease. The controversially discussed association with the HLA-B7 cross-reacting group could not be confirmed. Marked deviations of antigen frequencies were observed for HLA-A1, B8, Cw7, DR3 and DQw2 compared to healthy local controls. After correction of p-values, A1 (pc less than 0.05) and DQw2 (pc less than 0.01) remained significant. A possible association of Peyronie's disease with markers of the HLA-A1, B8, Cw7, DR3, DQw2 haplotype, as first described here, would suggest autoimmunological factors in this disorder of otherwise unknown etiopathogenesis.