[Determination of activated platelets: evaluation of methodology and application for patients with idiopathic thrombocytopenic purpura].

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.

Characterization of elevated neutrophil-associated IgG in various autoimmune disorders: not anti-neutrophil autoantibodies, but possibly immune complexes, bind to neutrophils.

Neutropenia is frequently observed in a variety of autoimmune disorders. As the mechanism of neutropenia in these disorders, the destruction of neutrophils by anti-neutrophil autoantibodies has been believed since elevated levels of neutrophil-associated IgG (NAIgG) have been described. However, no data exists to characterize the nature of NAIgG and show NAIgG is an anti-neutrophil autoantibodies. We investigated whether the elevated NAIgG in these patients consists of anti-neutrophil autoantibodies. The NAIgGs of 91 patients with autoimmune disorders including 50 patients with idiopathic thrombocytopenic purpura, 13 patients with systemic lupus erythematosus, 11 patients with Hashimoto's thyroiditis and 10 patients with Graves' disease were analyzed. The level of NAIgG was high in 36 of 91 patients. Elution studies were performed to determine whether NAIgG has a nature of autoantibodies. In model experiments, the ether eluate from neutrophils sensitized with neutrophil-specific alloantibody (anti-NA2) reacted with donor neutrophils, whereas the eluates from those with model immune complexes (ICs) failed. These data indicated that the ether elution technique is useful to determine whether NAIgG consists of anti-neutrophil autoantibodies. The NAIgG on patient's neutrophils was eluted with ether and the reactivity of the eluate with normal neutrophils was investigated. The eluates from 34 of 36 patients with various autoimmune disorders with elevated NAIgG level failed to react with donor neutrophils. These data indicated that the elevated NAIgG in the majority of these patients did not consist of anti-neutrophil autoantibodies, but possibly of ICs.

A simple solid-phase method for the identification of red cell-bound antibodies without elution.

The authors have developed a solid-phase direct hemadherence assay (SPDHA) to identify red cell-bound antibody without elution. The procedure of SPDHA is as follows: (1) commercially available panel cells were immobilized on the well of microplane; (2) 22% polymerized albumin and 0.3% test red cells were added, and the plate was centrifuged at low speed, and incubated; (3) finally the plate was centrifuged, and the results were read macroscopically. SPDHA could detect antibodies against D, C, c, E, e, Fya, Fyb, K, k, A and B antigens. The sensitivity of SPDHA was high in Rh antibodies as compared with that in the other antibodies. SPDHA failed to detect anti-Jka, -Jkb, -S, -s and -Dia antibodies. In cases of suspected hemolytic disease of newborn, Rh antibodies could be identified using very small volume of red cells. In conclusion, SPDHA is a useful and simple method to identify red cell-bound antibodies, especially when only a small volume of red cell sample is available, such as the sample from fetus or newborn.

[Analysis of immunoglobulin classes of five anti-phospholipid antibodies in sera from patients with SLE, ITP and recurrent fetal abortion by enzyme linked immunosorbent assay].

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.

[Detection of anti-neutrophil antibodies by enzyme linked immunosorbent assay--comparison of results obtained by ELISA with those by leukocyte agglutination test and granulocyte cytotoxicity test].

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.

[Evaluation of IgM anti human cytomegalovirus antibody].

An anti-human cytomegalovirus (HCMV) antibody in 330 sera from patients and normal subjects was examined by ELISA. The test results were compared with the results of CF test and there was 99.1% agreement. Specific anti-HCMV IgM antibody was searched for and only 4 sera were positive. This makes it unlikely that screening for IgM anti-HCMV will effectively prevent posttransfusion HCMV infections.

Analysis of antigen involved in circulating immune complexes in patients with idiopathic thrombocytopenic purpura.

Circulating immune complexes (CIC) were frequently observed in patients with idiopathic thrombocytopenic purpura (ITP). To analyse the pathogenic role of CIC, we studied the correlation between the disease activity and the CIC level, and whether platelet antigens were involved in CIC from ITP sera. Elevated CIC levels, measured by Clq, anti-C3 and spermatozoa micro-enzyme-linked immunosorbent assay, were found in 37%, 54% and 52% of the patients, respectively. However, there were no significant correlations (r = -0.11, -0.03 and -0.04, respectively) between the platelet count and the CIC level. Platelet antigens in CIC were detected with rabbit anti-human platelet serum. The CIC from 18%, 25% and 25%, respectively, of ITP sera contained platelet antigens, but the CIC from only 3%, 9% and 6% of SLE sera and from 3%, 2% and 5% of the sera of alloimmunized patients, respectively, contained these antigens. There were significant correlations (r = 0.51, 0.80 and 0.65, respectively) between the amount of platelet antigens and the CIC level in ITP sera. However, there were no correlations (r = -0.26, -0.29 and 0.08, respectively) between the platelet count and the amount of platelet antigens in the CIC. We detected platelet antigens in CIC from ITP sera, but surmise that these CIC perhaps do not play an important role in platelet destruction.

[Production and localization of human prolactin in the tissues associated with pregnancy (Report I) (author's transl)].

In the present paper, the authors studied the production and localization of human prolactin from normal human chorionic tissue and decidua in early pregnancy (from the 7th to the 9th week after last menstruation) obtained either by curettage or hysterectomy. Immunohistological investigation by the fluorescent antibody technique using human prolactin specific antiserum prepared by the immunization of rabbits revealed that syncytial trophoblast was a production site of prolactin. Though prolactin was recognized in compact layers of parietal decidua, it was concerned not with production but solely with deposition in intercellular space which was widened edematously with the existence of a collagen-like substance. By double staining method of the fluorescent antibody technique, prolactin could be differentiated with both hCG and hPL in syncytial trophoblast.

Radioimmunoassay method for baboon plasma gonadotropins.

Double antibody radioimmunoassay methods were developed for the determination of baboon luteinizing hormone (bLH) and follicle stimulating hormone (bFSH). The bLH radioimmunoassay employs a unique anti-ovine LH serum (GDN-15) and ovine LH (LER-1056-C2) for radioiodination, while the bFSH radioimmunoassay employs an heterologous system, i.e., an anti-ovine FSH serum (H-31) and purified human FSH for radioiodination. The reference standard used in both assays is a crude rhesus pituitary extract (LER-1909-2). Elevated endogeneous baboon plasma TSH and prolactin induced by the intravenous administration of 500 microgram of TRH had no influence on the levels of LH and FSH, whereas simultaneous intravenous administration of 100 microgram LHRH and 500 microgram TRH raised the levels of LH and FSH in plasma. hPRL, hCG, hTSH and hGH did not cross react with either the bLH or the bFSH assay system. The determination of plasma LH and FSH concentrations in daily samples from 6 mature female baboons throughout ovulatory menstrual cycles revealed patterns qualitatively similar to those of the rhesus monkey and human females.