Development of an assay based on capture ELISA to detect IgG anti-platelet in thrombocytopenia caused by Plasmodium vivax

Background: Thrombocytopenia is one of the most common haematological complication observed in malaria, it is fre¬quent in Plasmodium vivax malaria. The frequency of thrombocytopenia caused by Plasmodium infection has been described from 24% to 94%, and there have been reports proposing that antibody-associated thrombocy¬topenia. There is ev¬idence that platelet-associated IgG (PAIgG) is increased in malaria and is associated with thrombocytopenia, with IgG antibodies molecules on platelet surface. But, few studies have investigated antibody-mediated platelet destruction, including detection of specific antibodies in sera samples from malaria patients. Besides, detection of IgG anti-Platelet has proven difficult, primarily due to low and variable expression of the platelet antigens, also these antibodies are not detectable with standard techniques. Materials and Methods: We have standardized a protocol to obtained whole antigen from human platelet by sonication of these cells, and established an assay based in capture ELISA with sensitivity in detecting antibodies against human platelet antigens. Results: This approach using a simple procedure was possible after collected platelet from donors in blood bank of Pará state, Brasil, and obtained total platelet antigens to investigate whether antibodies anti-platelet can be detected by this assay. Initially, we characterized groups of infected patients (N=51) and exposed individuals who living in malaria transmission area (N=95). Thrombocytopenia was observed in 54.9% (28/51) infected individuals and there was negative correlation with parasite density (R2 = - 0.4210, p=0.0023). In infected group with thrombocytopenia (N=28), median platelet count was 77,500 platelets/µL (range: 34,000 - 137,000), and without thrombocytopenia (N=23) was 200,000 platelets/µL) (154,000 - 331,000), (p<0.05). In non-infected group with thrombocytopenia (N=17), median was 140,000 platelets/µL) (72,000- 152,000), and in non-infected without thrombocytopenia (N=78), 225,000 platelets/µL) (157,000 - 383,000), (p<0.05). There was not difference between infected and non-infected with thrombocytopenia. In next step, we will test these samples by a capture ELISA assay developed in our laboratory for detection of specific IgG against platelet antigens. Conclusion: Although serological studies for this are currently restricted, this appears to be the first report describing an approach to purify platelet antigens, and study whether specific antibodies participate in thrombocytopenia Plasmodium vivax malaria.