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.

Flow cytometric detection of platelet-reactive antibodies and application in platelet crossmatching.

BACKGROUND: Alloimmunization against HLA or platelet antigens can cause refractoriness to platelet transfusions in multiply transfused patients. Crossmatching of platelet concentrates is effective in overcoming this problem. STUDY DESIGN AND METHODS: A flow cytometric assay was used for simultaneous detection of lymphocyte-reactive and platelet-reactive antibodies in a single sample using fluorescein isothiocyanate-labeled anti-IgG. This assay was compared with the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay in selected sera containing HLA and platelet antibodies. In a further study, this assay was compared with lymphocytotoxicity test results from thrombocytopenic patients, for whom platelet concentrates were ordered. The results of both assays were then correlated with the 1-hour corrected count increment, with a corrected count increment greater then 7500 considered as an adequate transfusion response. RESULTS: The results of the MAIPA and flow cytometric assay in detecting platelet-reactive antibodies correlated well (p<0.0001, r = 0.84). The sensitivity and specificity of the flow cytometric assay in detecting platelet-reactive antibodies were 94.7 and 96.3 percent, when the MAIPA assay was taken as a reference. In unselected sera from patients, the sensitivity and specificity of the flow cytometric assays were, respectively, 72.7 and 91.7 percent in detecting lymphocyte-reactive antibodies and 70.6 and 77.7 percent in detecting platelet-reactive antibodies, when the lymphocytotoxicity test was used as a reference. With regard to an adequate transfusion response, the sensitivities and efficiencies were 20.0 and 82.1 percent, 33.3 and 84.3 percent, and 70.0 and 88.6 percent for the lymphocytotoxicity test and the lymphocyte-reactive and platelet-reactive flow cytometric assays, respectively. CONCLUSION: Flow cytometric crossmatching appears to be an effective method of detecting platelet-reactive antibodies that may affect the success of platelet transfusions. This procedure is well-suited for routine conditions and can be performed within 2 hours.

Risk of human immunodeficiency virus (HIV) transmission by anti-HIV-negative blood components in Germany and Austria.

In order to estimate the residual risk of transfusion-transmitted HIV infection we have analyzed the data from two transfusion centers in Austria (Vienna) and Germany (Göttingen) from 1985 to 1994. In Vienna, an incidence of 1:42,000 positive anti-HIV tests in repeat donors and a prevalence of 1:7000 in first-time donors were found in 1993. In Göttingen, the indicence was 1:67,000 and the prevalence 1:7900 from 1985 to 1993. Based on a mathematical model which takes (a) the window period and (b) the false-negative rate of anti-HIV tests, as well as (c) human and operational errors into consideration, we have calculated the residual risk of HIV infection. The residual risk (third generation anti-HIV test) was found to be 1:520,000 (95% confidence interval 1:1340,000-1:210,000), and 1:900,000 (95% confidence interval 1:2340,000-1:380,000) for Vienna and Göttingen, respectively, in 1993. Look-back studies from 1985 till 1994 revealed transfusion-transmitted HIV infections in three recipients (for 1.9 million donations in Vienna) and one recipient (for 160,000 donations in Göttingen) of blood components. Based on our model, as well as on prevalence and incidence rates of HIV infection, it is also possible to predict the efficacy of additional measures introduced to further decrease the risk of transfusion-transmitted HIV infection through blood components.

[Immunization with partner lymphocytes: improvement of pregnancy rate in sterility patients]. / Immunisierung mit Partner-Lymphozyten: Verbesserung der Schwangerschaftsrate bei Sterilitätspatienten.

In a two-centre prospective study 20 patients with a history of unsuccessful sterility treatment underwent immunization with paternal lymphocytes to improve the pregnancy rate in the subsequent therapeutic AIH or IVF/ET cycle. Unsuccessful sterility treatment was defined as no pregnancy after 8 properly monitored AIH cycles and at least one diagnostic IVF/ET or more than 3 IVF with transfer of 3 embryos. After successful immunization expressed by the induction of Fc-receptor blocking antibodies 10/20 patients became pregnant. Nine of these patients delivered healthy children, one patient experienced a first trimester abortion. A successful second pregnancy occurred in 6 of these patients. No significant correlation between the previous history and pregnancy success could be found, except a slight advantage for patients with a history other than tubal sterility. There were no differences in the anamnestic data as well as in the success rate between the two independent centres Göttingen and Leuven (10 patients each). These data suggest, that adjuvant immunotherapy might improve markedly the pregnancy rates in selected cases of sterility.

[Comparative analysis of various plasmapheresis methods--modern procedures of mechanical plasma collection compared with each other and with manual bag centrifugation procedures]. / Vergleichsanalyse verschiedener Plasmapheresemethoden--Moderne Verfahren apparativer Plasmagewinnung im Vergleich untereinander und mit dem manuellen Beutel-Zentrifugationsverfahren.

Four plasmapheresis procedures (manual blood bag centrifugation plasmapheresis, and the three plasmapheresis machines P.C.S./Haemonetics, Autopheresis-C/Baxter-Travenol, Plasmapur Monitor/Organon Teknika) were studied comparatively. The three machine procedures could be performed more easily and more rapidly and were well accepted by donors, autologous donors (patients) and staff. Compared with the traditional, well established manual procedure, a possible impairment of the plasma donors seems reduced rather than raised. Activation of the hemostatic system of the donors, measured with very sensitive methods, was found to be less pronounced when plasmapheresis was performed with the machines than when it was performed conventionally. The plasma product obtained by machine plasmapheresis was found to be of higher quality. All three systems showed less activation of the clotting system. Especially the plasma obtained by the P.C.S. showed a higher clotting factor yield. Plasma obtained by the Autopheresis-C and by the Plasmapur Monitor (both systems are equipped with filters) was markedly less contaminated with cells. The recently found low activation of the clotting system of plasma recipients, however, showed no differences when plasma obtained conventionally or by the Plasmapur Monitor was used. In summary, the new machine plasmapheresis devices offer a good alternative to the conventional blood bag centrifugation method and set new standards for the production of high quality plasma.

[Initial experiences with methylene blue virus inactivated fresh frozen plasma: results of a clinical and in vitro study]. / Erste Erfahrungen mit Methylenblau-Virus-inaktiviertem Fresh-Frozen-Plasma: Ergebnisse einer klinischen und einer In-vitro-Studie.

Methylene blue (MB) has recently been introduced to inactivate viruses in single donor fresh frozen plasma (FFP) units. In the first clinical study 519 units of MB-treated FFP were given to 103 patients. No specific clinical side effects with adverse reactions were recorded. However, after thawing, clots were observed in 9 of the first 110 units, although the medical staff had thawed the MB-FFP in exactly the same manner as the conventional FFP in which clots are extremely rare. The possible effects of methylene blue were investigated by subsequent in vitro experiments: Methylene blue together with light led to generation of fibrin(ogen) derivatives which have an increased tendency for aggregation but cannot be normally clotted by thrombin and even seem to interfere with normal fibrin polymerization. Inadequate conditions during plasma preparation such as long illumination or temperatures above 40 degrees C enhanced the generation of these fibrin(ogen)-derivatives. In addition, special requirements for producers and users of MB-FFP and further investigations are necessary in order to prevent complications.

[Immunotherapy for the prevention of abortion]. / Immuntherapie zur Abortprophylaxe.

It is now commonly assumed that a normal, successful pregnancy requires the specific recognition of the trophoblast by the immune system of the mother, resulting in production of protective or blocking factors (BF). In habitually aborting women, this recognition and/or BF production does not seem to occur. Immunisation of patients with leukocytes from the partner or from donors is postulated to induce these BF and to prevent fetal loss. After the exclusion of nonimmunological causes, 31 patients with at least three previous abortions and no pregnancies lasting longer than 16 weeks, were immunised with leukocytes from their partners. Eight women were already pregnant when treated, 15 became subsequently pregnant. Seventeen (= 74%) had uncomplicated pregnancies, six suffered again from early pregnancy loss. All 15 children, born so far had average birth weights and developed normally. The EAI-test (Power 1983) was of prognostic value: an increase of more than 50% in rosette inhibition activity in the serum of treated patients, correlated well with the successful outcome of their pregnancies; whereby the lack of this increase was associated with another early pregnancy loss. We could not confirm the reported of higher HLA-identity of couples with habitually aborting women, compared to fertile couples. HLA-typing (A, B, C, DR, and DQ) showed a higher proportion of identical allo-types in a group of 33 fertile couples than in a group of 44 couples with habitual abortions. This finding was significant (p less than 0.05).

Characterization and quantification of anti-T in human serum using a reliable hemolysis test.

The need of fresh-frozen donor plasma with a low level of anti-T has been emphasized recently. Anti-T, as administered by transfusion of fresh-frozen plasma, has been accused repeatedly of enhancing hemolysis in septic children with T transformation of red cells. Therefore, a new hemolysis test for the quantification of anti-T in human serum has been developed. With our test, anti-T-poor plasma donors can be found. Additional results raise substantial doubt as to the pathogenetic role of anti-T in the development of the hemolytic-uremic syndrome, found in septic children with red-cell T transformation. It is impossible to predict in vivo hemolysis induced by anti-T knowing the temperature characteristics and the ionic conditions causing this antibody to mediate hemolysis in vitro. Obviously, T transformation itself plays the major pathogenetic role in these patients, and not the presence of anti-T. In the case of disseminated intravascular coagulation, a content of anti-T cannot be construed as prohibiting transfusion of fresh-frozen plasma to such patients.

[Comparison of 4 plasmapheresis procedures]. / Vergleich von vier Verfahren zur Plasmapherese.

In recent years the collection of plasma has been increasingly carried out by apparative plasmapheresis. In the University Hospitals of Göttingen and Würzburg the plasmapheresis machines PCS from Haemonetics, Autopheresis-C from Baxter, as well as Plasmapur-Monitor from Organon-Teknika, were compared with each other and with the conventional centrifugation of blood bags; experiences in routine use were complemented by specific studies. Altogether, the apparative methods distinguished themselves through a high quality of retransfused blood and collected plasma. Furthermore, practicability, donor compatibility and donor safety resulted in good acceptance by both donors and staff.

[Hemolytic-uremic syndrome in an adult with T-cryptantigen liberation]. / Hämolytisch-urämisches Syndrom des Erwachsenen mit T-Kryptantigen-Freilegung.

Haemolytic uraemic syndrome was diagnosed in a 36-year-old woman with acute renal failure (creatinine 10.5 mg/dl), haemolytic anaemia (haemoglobin 9.7 g/dl, lactate dehydrogenase 1926 U/l) and thrombopenia (98,000/microliters). After initial plasmaphereses and high doses of furosemide all symptoms disappeared within three weeks. The lectin tests demonstrated that the illness was connected with the liberation of T-crypt-antigen (Thomsen-Friedenreich antigen) on the erythrocytes. This special form of the haemolytic uraemic syndrome (neuraminidase-induced haemolytic uraemic syndrome) has previously been observed almost exclusively in children. However, for diagnosis and differentiation of haemolytic uraemic syndromes the presence of liberated T-antigen on erythrocytes should also be tested for in adults.

[Comparison of 4 procedures in plasmapheresis: studies of modifying the hemostasis potential]. / Vergleich von 4 Verfahren der Plasmapherese: Untersuchungen zur Beeinflussung des Hämostase-Potentials.

The separation of plasma using the systems "Plasmapur Monitor" (Fa. Organon), "Autopheresis-C" (Fa. Baxter) and "PCS" (Fa. Haemonetics) was compared with the conventional blood bag centrifugation. In 16 apheresis per method, several parameters with the main focus on blood coagulation were examined in addition to other criteria. Compared to conventional centrifugation of blood bags, the plasma separation machines led to an only slight activation of the coagulation system, which seems to be negligible for donors as well as recipients. Furthermore, a decrease of coagulation factors and inhibitors in the collected plasma was most pronounced using bag centrifugation. Beyond this, particularly in apheresis systems with plasma filtration the low number of remaining cells meets the aim to reach a high quality of transfused plasma.

Patients with anti-Vel--immunohematological characterization and transfusion management.

As reported by other authors we can confirm that the frequency of the blood group Vel negative in Lower Saxony is 1:4000. We report on a patient whose serological characteristics (IgM- and IgA-anti-Vel) made the transfusion of Vel positive blood impossible. Since it was not possible to obtain sufficient Vel negative red cell units in time, the patient was convinced to donate blood for autologous transfusions. This should be the procedure of first choice in the transfusion management of such patients.

T and Tk transformation in hemolytic uremic syndromes.

In two cases of hemolytic uremic syndrome (HUS) the bacterial pathogenesis of the disease could be elucidated by lectin red cell agglutination tests. The possible role of anti-T and anti-Tk antibodies is discussed. Transfusions of fresh plasma had no adverse effects. The fatal outcome in one case was caused by disseminated intravascular coagulation (DIC).

Pregnancy-maintaining antibodies: workshop report (Giessen, 1988).

To analyse the nature of antibodies which are purported to be essential for the maintenance of normal human pregnancy, six centers participated in a workshop of "blind" tests on 19 allosera. Fc-receptor dependent assays detected antibodies with specificity only for HLA. In addition to cytotoxic antibodies, the Fc-receptor dependent immune phagocytosis inhibition test revealed two non-cytotoxic alloantibodies with HLA specificity. These antibodies had high titers and may, therefore, be essentially non-cytotoxic. Murine monoclonal antibodies to HLA-A, B, C or DR (W6/32 and 2MC3) were used to evaluate the methods. These antibodies inhibited immune rosette formation as well as immune phagocytosis. Diluted to concentrations below the threshold of complement-dependent cytotoxicity, the monoclonal antibodies still inhibited the mixed lymphocyte reaction and the immune phagocytosis. A human monoclonal immunoglobulin M with specificity for monomorphic non-HLA lymphocyte antigens inhibited the mixed lymphocyte reaction. The immune rosette inhibition test exhibited several false positive reactions, e.g. three out of four with a serum that did not contain alloantibodies to blood cells. Non-cytotoxic antibodies were therefore rare in the selected sera of the workshop and they exhibited HLA specificity only. No participant was able to identify pregnancy-maintaining non-HLA-antibodies.

[Immunotherapy of couples with habitual abortion]. / Immuntherapie bei habituell abortierenden Paaren.

After habitual abortions and the exclusion of nonimmunological causes of miscarriage, 26 patients were treated with lymphocytes of their partners of prophylaxis of abortion. The first treatment was done during early pregnancy in eight patients. Of the remaining 18, 12 have become pregnant up to now. Five pregnancies have gone to term, one is in the 13th week at the moment, and five are in the 20th week. Two patients lost their baby after the 20th week due to nonimmunological causes. Again seven patients had an early spontaneous abortion. Thus, the success rate of the immunological therapy is 63.2% at the moment.

[Prevention of habitual abortion by buffycoat transfusions]. / Zur Prävention habitueller Aborte durch Buffycoat-Transfusionen.

From an immunological point of view the product of pregnancy may be regarded as a haplo-different allotransplant. A system possibly closely linked to the HLA-region is postulated to lead to the immunological recognition of the fetus by the mother and, paradoxically, to a take of the "transplant". The postulated system apparently codes for antigens present on both trophoblast and adult lymphocytes (TLX = trophoblast-lymphocyte-crossreacting). The prevention of rejection is thought to be effected by blocking factors (BF) present in the serum or plasma of the mother. There may be different kinds of BF: a specific BF (detectable only in an autologous assay system), appearing late in pregnancy, which inhibits several lymphocyte-dependent reactions (e.g. production of MIF, MLC). This BF has been identified as an IgG-class antibody. a nonspecific BF, appearing early in pregnancy which inhibits the MLC in vitro. c) may be a third BF, also specific, which is found only in plasma but not in serum. All described BF-activities were absent in women with habitual abortions. HLA-identity or partial identity could imply TLX-identity. The consequence of such an identity could be: non-detection of the trophoblast by the immune system of the mother, no production of BF, abortion. However several investigators could not find any HLA-identity of the partners with habitual abortions. A protective effect on the fetus has been seen when pregnant women were immunised with adult leukocytes, using either buffycoats from various HLA-different but bloodgroup-compatible donors or isolated leukocytes from the spouse.(ABSTRACT TRUNCATED AT 250 WORDS)