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Platelet activation causes a change in surface expression of several endogenous proteins, such as CD62P, CD63 and CD40L. Therefore, it is possible to analyze the functional in vivo status of the circulating platelet population directly by flow cytometry. In this study we developed the method to be suitable for use in clinical studies. We used EDTA-2K as anticoagulant since the sample anticoagulated with EDTA-2K, sodium citrate or ACD-A showed no difference in the data of activated platelets. We determined whether fixation of sample is necessary. The samples stained before or without fixation showed abnormally high level of activated platelets, indicating that fixation is necessary before staining. It is controversial whether activated platelets circulate in patients with idiopathic thrombocytopenic purpura(ITP). We measured activated platelets in patients with ITP using our optimised method. The percentages of CD62P, CD63 and CD40L positive platelets were significantly high in patients with ITP and 24%, 55% and 36% (respectively) of ITP patients showed elevated level of activated platelets. These data indicate that activated platelets circulate in ITP patients.

Using a panel of phospholipid(PL) antigens, we have established an enzyme linked immunosorbent assay (ELISA) for measuring both IgG and IgM type anti-phospholipid antibodies (APA) in sera from patients with systemic lupus erythematosus (SLE), idiopathic thrombocytopenic purpura (ITP) and recurrent fetal abortion (RFA). The percentage of anticardiolipin antibody (aCL) positive patients was increased in SLE (73% for IgG, 74% for IgM), ITP (24% for IgG, 8% for IgM) and RFA (20% for IgG, 52% for IgM) as compared with normal controls. The percentage of other APAs in each disease was significantly different from one another, suggesting the existence of disease-specific APA in these autoimmune disorders. We consider that it is important to analyze these APAs for the investigation of pathogenesis of autoimmune disorders.

We examined anti-neutrophil antibodies in 12 patients with various disorders using enzyme-linked immunosorbent assay (ELISA), and the results obtained by ELISA were compared with those obtained by leukocyte agglutination test (LAT) and granulocyte cytotoxicity test (GCT). IgG anti-neutrophil antibody was positive in 7 of 12 patients, and IgM type antibody was positive in 6 patients. There was a significant correlation between IgG and IgM anti-neutrophil antibodies. The results obtained by ELISA were not in accord with those obtained by LAT or GCT. In addition, we examined anti-neutrophil antibodies in serially collected serum samples from two patients with immune neutropenia. The results obtained by ELISA correlated with their neutrophil counts, suggesting that anti-neutrophil antibodies detected by ELISA have pathological relevance.

Dr Kira et al. (Lancet 1991, 338: 64-65) stress the high incidence of the co-infection with HTLV-I and HTLV-II in the HTLV-I associated myelopathy or tropical spastic paraparesis (HAM/TSP) detected by a polymerase chain reaction (PCR). They showed that 67% of HAM/TSP patients and 6% of healthy carriers had the co-infection. They suggested the co-infection might be important for the development of the myelopathy. Recently, we have analyzed the seropositivity of antibodies to HTLV-I and II in Adult T-cell Leukemia (ATL), HAM/TSP and carriers in Japan using SELECT-HTLV (IAF Biochem International Inc., Montreal, Canada). This system was a solid phase enzyme immunoassay utilizing synthetic peptides to differentiate between antibodies to type I and II. We examined 101 samples (31 ATL patients, 20 HAM/TSP and 50 carriers) which were all positive with the particle agglutination (PA, Fujirebio, Tokyo) and ELISA (Eisai, Tokyo). All samples were negative with HTLV-II and positive with HTLV-I. The results of our serological survey suggested that HTLV-II did not associate with the etiology of ATL and HAM/TSP in Japan.