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Summary. A radioimmunoassay (RIA) for the detection of platelets and platelet fragments was developed. A sandwich of two monoclonal antibodies directed against the platelet-specific glycoprotein complex IIb-IIIa (GP IIb-IIIa) was used in this assay. A discontinuous 7·5-20% (v/v) albumin gradient was applied to separate platelets and their fragments of various sizes. In platelet suspensions fractionated in this way, we observed that particles smaller than normal platelets still carried the GP IIb-IIIa antigens. This procedure enabled us to detect platelet-derived particles in platelet-rich plasma from thrombocytopenic patients.

The effect of high-dose intravenous gammaglobulin (IVG) therapy with a CLB preparation was studied in 42 patients: 8 patients had acute and 26 patients had chronic idiopathic thrombocytopenic purpura (ITP); 5 patients had thrombocytopenia accompanied by various diseases such as systemic lupus erythematosus, auto-immune haemolytic anaemia and neutropenia; 3 patients had hypoplastic anaemia and 1 patient had neutropenia and rheumatoid arthritis. After treatment, a rise in platelet count occurred in about 75% of the patients with ITP, although there was no sustained response in any of the patients. There was no correlation between the strength of platelet antibodies as detected by the direct immunofluorescence test before infusion and the pattern of response to the infusion. In most cases of ITP, no immune complexes, as measured by Clq-binding assay, were observed. Furthermore, we found no relationship between the amount of Clq-binding activity of patients' sera and the reaction pattern after infusion of IVG. Splenectomy of the patient had no influence on the outcome of IVG therapy.

Several variables that may affect accurate measurement of platelet-associated IgG (PA-IgG) were studied using a radioimmunoassay of the consumption type. The amount of PA-IgG of washed, unfixed normal donor platelets was 1.0 +/- 0.9 fg IgG/platelet (mean +/- 2 SD). Upon storage of washed platelets in a buffer containing EDTA, the amount decreased significantly to 0.2 +/- 0.2 fg IgG/platelet. Simultaneously, an increase in modal platelet volume was observed. Similar results were obtained when platelets were fixed with paraformaldehyde (PFA). We postulate that this decrease in PA-IgG is caused by the release of plasma IgG entrapped by the surface-connected canicular system of the platelet, when the platelets swell during storage in EDTA or fixation with PFA. This presence of varying amounts of entrapped plasma IgG may cause the wide discrepancies in PA-IgG found in normal donor platelets as well as platelets from ITP patients by other investigators. A good quantification of platelet-bound alloantibodies was possible with our assay when platelets were routinely fixed to diminish the amount of nonspecific PA-IgG. This was demonstrated with different anti-Zwa (= anti-PlA1), anti-Baka and anti-HLA sera. We also observed that fragments of platelets as well as fragments of cells of other types can cause aspecifically increased Pa-IgG values and can thus interfere with the proper measurement of platelet-bound antibodies in all kinds of immunoassays in general.

In persons with AIDS or at risk from AIDS, autoantibodies against platelets and granulocytes were frequently detected. Platelet-bound immunoglobulins were demonstrated by immunofluorescence in all 16 patients with AIDS, in five out of seven patients with AIDS-related complex/persistent generalized lymphadenopathy (ARC/PGL) and even in seven of 10 healthy sexually active homosexual men. Granulocyte-bound immunoglobulins were found by immunofluorescence in 12 of the 16 AIDS patients, five of the seven patients with ARC/PGL and two of the 10 symptomless men. Red cell bound immunoglobulins were not detected. All patients with AIDS and ARC/PGL and three of the symptomless men were seropositive for human immunodeficiency virus (HIV). The platelet- and granulocyte-bound immunoglobulins could be eluted in 93% and 67% of the cases, respectively. This indicates that specific autoantibodies, rather than circulating immune complexes, which were frequently increased, accounted for the findings. There was no relation between the serological findings and the platelet and granulocyte counts. We conclude that autoantibodies against platelets and granulocytes are common in patients with AIDS and those at risk.

The sensitivity and specificity of the platelet immunofluorescence test for the diagnosis of idiopathic thrombocytopenia (ITP) was studied in a series of 255 patients. Patients' platelets were tested directly. Diethyl-ether eluates of these platelets and patients' sera were tested indirectly with normal donor platelets. When all three tests were considered, positive results were obtained for 92.0% of the ITP patients with a platelet count of less than 150 X 10(9)/l and for 98.4% of the patients with a count of less than 100 X 10(9)/l. However, for many patients rather weak test results were obtained, with a score of 1/2-1 in 59.8% of the patients. Most patients (94.1%) with a positive direct test had a positive indirect test on the eluate. Thus, platelet-bound antibodies but not platelet-bound immune complexes were present in most, if not all, patients. Positive immunofluorescence tests were obtained for many patients with a diagnosis other than ITP. This resulted in a low specificity of the test for the diagnosis of ITP, evidently because autoimmune thrombocytopenia occurred together with many other diseases and also because antibodies against platelet cryptantigens (expressed by the action of EDTA or by platelet fixation) were present in many patients.

In persons with AIDS or at risk from AIDS, autoantibodies against platelets and granulocytes were frequently detected. Platelet-bound immunoglobulins were demonstrated by immunofluorescence in all 16 patients with AIDS, in five out of seven patients witn AIDS-related complex/persistent generalized lymphadenopathy (ARC/PGL) and even in seven of 10 healthy sexually active homosexual men. Granulocyte-bound immunoglobulins were found by immunofluorescence in 12 of the 16 AIDS patients, five of the seven patients with ARC/PGL and two of the 10 symptomless men. Red cell bound immunoglobulins were not detected. All patients with AIDS and ARC/PGL and three of the symptomless men were seropositive for human immunodeficiency virus (HIV). The platelet- and granulocyte-bound immunoglobulins could be eluted in 93% and 67% of the cases, respectively. This indicates that specific autoantibodies, rather than circulating immune complexes, which were frequently increased, accounted for the findings. There was no relation between the serological findings and the platelet and granulocyte counts. We conclude that autoantibodies against platelets and granulocytes are common in patients with AIDS and those at risk.