The presence of messenger RNA for HLA class I in human platelets and its capability for protein biosynthesis

Summary. In order to determine whether platelets contain specific messenger RNA encoding for HLA class I molecules, polymerase chain reaction (PCR) was performed with RNA from different platelet donors. Two amplified 300 bp and 279 bp cDNA fragments were obtained which encompassed sequences from 321 to 620 and from 795 to 1073. The 300 bp fragment encodes exon 2 and exon 3, the 279 bp encodes a portion of exon 4, exon 5, exon 6 and a portion of exon 7. A 300 bp nested PCR product from one donor, that encoded for the highly polymorphic region α2, was cloned and sequenced. The resulting nucleotide sequences fitted to the expected sequence for HLA B*3801 of this donor. Sequence analysis of the 279 bp PCR product demonstrated the presence of exon 5 encoding for the 117 bp transmembrane domain.
In addition, de novo protein biosynthesis was studied by radioimmunoprecipitation of HLA class I molecules from ³⁵S-methionine metabolically labelled platelet lysates with a monoclonal antibody (mab) w6/32 specific for a monomorphic epitope on the heavy chain of HLA class I antigens. Analysis of the immunoprecipitates on SDS polyacrylamide gel electrophoresis showed a specific band with apparent molecular weight (M_r) of 44 kD corresponding to integral membrane HLA protein.
On the basis of these results, we conclude that platelets contain specific messenger RNA encoding for HLA class I molecules and have the capability to synthesize the integral HLA membrane protein.     

Autoantibodies and drug-or metabolite-dependent antibodies in patients with diclofenac-induced immune haemolysis

Two patients with acute immune haemolytic anaemia caused by diclofenac are described. Both patients had developed IgG drug-independent autoantibodies and drug-dependent antibodies. The drug-dependent antibodies in one patient reacted with red blood cells (RBC) only in the presence of urine from patients receiving diclofenac (ex vivo antigen) but not in the presence of the drug itself or its known metabolites. This antibody appeared to recognize a trace metabolite which has not yet been identified. The drug-dependent antibodies in the second patient reacted with RBC in the presence of urine as ex vivo antigen as well as in the presence of the drug itself and its main metabolites. Incorrect diagnosis of such cases often is due to the occurrence of autoantibodies and/or unusual drug metabolism and excretion.     

HPA-5b (Bra) neonatal alloimmune thrombocytopenia: clinical and immunological analysis of 39 cases

Maternal alloimmunization against fetal platelets can cause fetal and neonatal thrombocytopenia (NAIT). The HPA-1a (PIA1, Zwa) antigen is by far the most common antigen implicated in NAIT. However, today another antigen often linked with that affection is HPA-5b (Br(a)). This is a report of 39 cases of NAIT involving the HPA-5b antigen. Thrombocytopenia may be of grave consequence. Three infants developed intracerebral haemorrhages (ICH). Of these, one died presumably as a consequence of ICH. Central nervous system (CNS) sequelae in the neonatal period was observed in two children. The potential hazards of death or disabling neurologic sequelae following intracerebral haemorrhage call for rapid and reliable diagnosis and effective therapy. Because there is high risk that subsequent pregnancies might be also affected by NAIT, the mothers of a previously affected child should be managed similarly to the HPA-1b mothers (PIA2, Zwb). The antenatal diagnosis of thrombocytopenia should be made and if necessary the in utero therapy instituted.     

Zum Problem des Nachweises antithrombocytärer Autoantikörper

Zusammenfassung Blutproben von 161 Patienten (davon 63 Patienten mit einer Thrombocytopenie unter 90000/mm³ und 98 Patienten mit normalen Thrombocytenzahlen) wurden mit dem direkten und/oder indirekten Antiglobulin-Konsumptionstest (AGKT) und dem direkten und/oder indirekten Fluorescenz-Antiglobulintest (FT) under Verwendung von Thrombocyten untersucht. Ohne Berücksichtigung der klinischen Diagnose wurde gefunden, daß thrombocytopenische Patienten in 37% einen positiven dir. AGKT, in 18% einen positiven dir. FT, in 27% einen positiven indir. AGKT und in 28% einen positiven indir. FT aufwiesen. Bei Patienten ohne Thrombocytopenie betrug der Anteil positiver Ergebnisse für den dir. AGKT 30%, für den dir. FT 16%, für den indir. AGKT 7%, für den indir. FT 9%. — Mit Thrombocyten von gesunden Blutspendern waren die Ergebnisse fast ausnahmslos negativ. — Unsere Untersuchungen lassen vermuten, daß der Ausfall der Tests zum Nachweis von antithrombocytären Autoantikörpern stark von nicht-immunologischen Faktoren beeinflußt wird. Beziehungen zu den Plasmaeiweißverhältnissen spielen dabei wahrscheinlich eine wichtige Rolle.

---

Summary The blood of 161 patients with different internal diseases was investigated with the direct and/or the indirect antiglobulin consumption test (AGCT) and with the direct and/or indirect fluorescence antiglobulin test (FT) on platelets. 63 patients revealed thrombocytopenia (platelets less than 90000 per mm³) in the course of various diseases, the remainder had normal platelet counts.Irrespective of clinical diagnosis positive results were found in the thrombocytopenic group in 37% with the direct AGCT, in 18% with the direct FT, in 27% with the indirect AGCT and in 28% with the indirect FT. Patients with normal platelet numbers showed positive tests in 30% (direct AGCT), in 16% (direct FT), in 7% (indirect AGCT) and in 9% (indirect FT). Control examinations on thrombocytes of healthy blood donors were almost regularly negative under the same conditions.These results suggest that the tests used for the detection of antiplatelet autoantibodies are severely influenced by non-immunologic factors. There seem to exist correlations between the results of the tests and the level of gammaglobulins in the plasma.

     

Alternative Treatment to Lymphocyte Immunization for Treatment of Recurrent Spontaneous Abortion: 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.     

High dose gammaglobulin therapy in adults with idiopathic thrombocytopenic purpura (ITP) clinical effects

Summary This study of the effect of high-dose intravenous gammaglobulins with one or two courses of therapy in 18 adults with idiopathic thrombocytopenia purpura showed a platelet rise in thirteen patients. The highest response rates were seen in splenectomized adults. In chronic patients the response was transient only. If therapy was effective, increased values of platelet-associated IgG were reduced, while shortened platelet survival times were prolonged. There was no influence of high-dose gammaglobulins on platelet function. Different 7S-preparations such as -propiolactone modified Ig, pH 4 treated Ig and reduced and alkylated Ig have comparable effects.Supported by the Deutsche Forschungsgemeinschaft (Mu 277/9-4)     

Improved assay for detection of platelet-specific PlA1 antibodies in neonatal alloimmune thrombocytopenia

A technique is described which permits the detection of platelet-specific, noncomplement-fixing ("blocking') PlA1 antibodies in serologically negative sera of mothers from children suffering from neonatal alloimmune thrombocytopenia. The technique consists of (1) antibody enrichment in eluates prepared from maternal serum and PlA1-positive platelets, and (2) quantitation of antibodies in eluates by the platelet radioactive anti-IgG test. With this assay, PlA1 antibodies were demonstrated in 6 out of 7 maternal sera, and anti-A antibodies in one. It proved valuable also for longitudinal studies for periods up to 30 months after delivery.     

Reappraisal of the clinical and etiologic significance of immunoglobulin deviations in autoimmune hemolytic anemia (‘warm type’)

Summary 56 patients with autoimmune hemolytic anemia (warm type) (AIHA) were investigated for immunoglobulin deviations. Of these, 43 were repeatedly analyzed (mean 4 times). The mean observation time was 20 months. The immunoglobulin values were correlated with clinical (degree of hemolysis) and serological (immunoglobulin class of autoantibodies; strength of antiglobulin reaction) parameters and statistically evaluated by variance analysis. Although no significant deviations of immunoglobulins in AIHA were found as compared to a normal control group, the immuno-globulin disturbance most frequently seen was an elevation of IgM. This is inter-preted as a possible lack or functional impairment of immunoregulatory T cells in AIHA.Supported by the Deutsche Forschungsgemeinschaft (Mu 277/6).