Glycoprotein (GP)-specific platelet antibodies can cause allo-immune and auto-immune thrombocytopenia. The specific detection of relevant antibodies is a prerequisite for diagnosis and treatment. Here, we describe an improved method based on simultaneous detection of various platelet-specific immunoglobulin G (IgG) and IgM antibodies. Bead populations with distinct fluorescence intensities, coated with monoclonal antibodies specific for mouse heavy chain isotypes, were used for the simultaneous immobilization of platelet-GP [IIb/IIIa, Ib/IX, human leucocyte antigen (HLA) class I, or Ia/IIa, CD32, GPIV or CD109, Ib/IX, HLA class I]. In order to detect human IgG and IgM antibodies simultaneously, phycoerythrin- and fluorescein isotiocyanate-conjugated goat anti-human IgG and IgM were added. On this basis, the abundance of six distinct antibodies (three anti-GP, each with subclasses IgG and IgM) were simultaneously analysed without cross-reaction by flow cytometry. For evaluation, sera and platelets from 169 patients with platelet-binding and/or platelet-associated antibodies were investigated. The monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay was performed in parallel as reference test. The simultaneous analysis of platelet-specific antibodies (SASPA) assay was able to detect all platelet-specific IgG and IgM that were also recognized by MAIPA with a comparable sensitivity. SASPA proved to be a rapid and reliable assay that required less platelets than other methods. This method has the potential to pave the way for new investigations of platelet-specific antibodies.

A novel method for simultaneous analysis of specific platelet antibodies: SASPA.

Autoimmune thrombocytopenia is a disorder characterized by antibody-mediated accelerated platelet destruction. Despite its clinical importance, the diagnosis of which is one of exclusion, thus inevitably associated with potential difficulties. Current methods used to determine antigen-specific antibodies including MAIPA and the radioactive immunobead assay, are not routinely used due to methodological and practical limitations. To facilitate diagnosis, flow cytometric methods have been developed, suitable for testing a single or multiple samples. The feasible flow cytometric methods with their high sensitivity and specificity should facilitate the routine use of diagnostic methods for autoimmune thrombocytopenia and permit follow-up to determine immune remission.

Autoimmune thrombocytopenia: Determination of platelet‐specific autoantibodies by flow cytometry

Background: Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder caused by antiplatelet autoantibodies. In this study, we compared 2 methods for screening serum platelet antibodies in patients with ITP. Methods: A total of 44 adult patients were clinically classified with ITP. We used 2 indirect tests to detect human leukocyte antigen antibodies and/or platelet-specific antibodies in their sera. In method I, we used solid phase red cell adherence (SPRCA) assay. In method II, by flow cytometry, platelets from plateletpheresis components were used as target cells, and fluorescein isothiocyanate-conjugated sheep anti-human IgG Fc was used as the staining reagent. Positive results were defined as any test with the percentage of fluorescence exceeding the reference range by 3% or more in method II. Direct tests detecting platelet-associated IgG on platelets of patients with ITP were done by flow cytometry. Results: Serum specimens from 44 adult patients with ITP (28 female, 16 male) were tested. SPRCA assay could only detect platelet antibodies in 22 patients (50%). By method II, 31 serum specimens (70.5%) yielded positive results. There was a difference between the results of the SPRCA test and method II, with a high degree of significance (p < 0.001) by the McNemar test. No significant difference in platelet counts was observed for patients with and without discernible platelet antibodies by SPRCA assay (p = 0.90). The direct test was positive in 12 patients (66.7%) out of 18 ITP patients tested. Conclusion: Flow cytometry is more sensitive than SPRCA assay for detecting platelet antibodies. Detection of platelet antibodies is useful in explaining the immune mechanism and platelet transfusion refractoriness in ITP. [J Chin Med Assoc 2006;69(12):569–574]

Screening for platelet antibodies in adult idiopathic thrombocytopenic purpura: a comparative study using solid phase red cell adherence assay and flow cytometry.

EDITOR,—Duclos-Vallee et al have reported that infection with hepatitis C virus may have a role in triggering Hashimoto's thyroiditis.1 Using enzyme linked immunosorbent assay (ELISA), they detected antibodies to hepatitis C virus in 12 of 50 patients, and a second generation recombinant immunoblot assay confirmed the finding in five of the 12 cases.

As we did not see such a high prevalence of hepatitis C in our outpatients we retested 118 consecutive patients with Hashimoto's thyroiditis for possible infection …

/pdf/prevalence-of-hepatitis-c-virus-infection-in-hashimoto-s-2u5rh9t50o.pdf

Prevalence of hepatitis C virus infection in Hashimoto's thyroiditis.

Sensitive detection of platelet-specific antibodies with a modified MAIPA using biotinylated antibodies and streptavidin-coated beads.

Detection and differentiation of platelet-specific antibodies by flow cytometry: the bead-mediated platelet assay.

BACKGROUND Hepatitis C virus (HCV) is responsible for the majority of post-transfusion hepatitis cases. We compared the correlation and reproducibility of different screening and confirmatory tests. MATERIAL AND METHODS 1,406 samples of voluntary blood donors were tested in parallel using 3 enzyme-linked immuno sorbent assays (ELISA) for HCV antibodies. Those samples that were positive in at least 1 of the 3 tests were additionally tested in a 3rd-generation ELISA as well as in 3 different confirmatory tests. RESULTS 13 samples (0.92%) were repeat reactive in at least 1 of the ELISAs with different results in the confirmatory tests. Only 3 samples (0.21%) with high sample/cutoff ratios in the ELISAs were positive in all 3 confirmatory tests. CONCLUSIONS The reproducibility of the tested ELISAs and the correlation with confirmatory tests were good only in samples with a high signal to cutoff ratio. Two different high-positive ELISA results can be regarded as confirmation.

[Possibilities and limits of anti-HCV antibody screening in the blood donation service].

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Detection and differentiation of platelet-specific antibodies by flow cytometry: the bead-mediated platelet assay.

[Possibilities and limits of anti-HCV antibody screening in the blood donation service].