The detection of blocking antibody against plasma thrombopoietin in subjects with childhood idiopathic thrombocytopenic purpura

特发性血小板减少性紫癜(ITP)是一种相对常见的出血性疾病，其特征是孤立的血小板减少症，没有任何可识别的全身性疾病。虽然人们普遍认为自身抗体通过吞噬系统介导的血小板清除导致了血小板减少，但巨核细胞成熟和随后的血小板产生的改变也被证明是ITP的混合病理生理机制。我们先前曾报道，在ITP患者血清存在的情况下，CBMC的TPO依赖的巨核细胞生成显著减少，同时伴随着形态上被鉴定为巨核细胞的细胞数量的减少。为了进一步明确ITP的病理生理机制，我们建立了检测患者血清中抗TPO自身抗体的新的ELISA法。简而言之，用抗TPO多克隆抗体包被96孔板，然后加入饱和量的rhTPO。将ITP患者血清复制加入Well，用生物素标记的抗人免疫球蛋白抗体和抗生物素-生物素-过氧化物酶抗体检测结合的免疫球蛋白抗体。对研究方案进行解释，并获得所有受试者的知情同意。80例ITP患者(急性37例，慢性43例)，抗TPO抗体阳性3例。通过吸附试验分析了这些ELISA活性的特异性，均未显示出特异性抗体活性。根据这些数据，我们得出结论，固有的抗TPO抗体不太可能解释ITP患者巨核细胞生成受损的原因。

Idiopathic thrombocytopenic purpura (ITP) is a relatively common hermorrhagic disorder characterized by isolated thrombocytopenia without any identifiable systemic disease. Although it is widely believed that the autoantibody-mediated platelet clearance through phagocytic system contributes to thrombocytopenia, the alteration of megakaryocyte maturation and subsequent platelet production is also evidenced indicating "mixed" pathophysiology of ITP. We prebiously reported that marked decrease of TPO-dependent megikaryocytopoiesis of CBMC was noted concomitant with a drop in number of cells morphologically identified as megakaryocytes when cultured in the existence of sera from ITP patients. To further identify pathophysiology of ITP, we developed new ELISA system to detect anti-TPO autoantibodies in patients' sera. Briefly, 96-well plate was coated with polyclonal antibody against TPO followed by adding satulating amount of rhTPO. Sera from ITP patients were then added replicately to wells, and bound IgG antibody was detected by biotin-labeled monoclonal antibody against human IgG and avidin-biotin-peroxidase sy stem. Study protocol was explained and informed consent was obtained for all enrolled. Of 80 sera from patients with ITP (37 acute, 43 chronic) 3 had positive results for anti TPO antibody. Specificity of these ELISA activities was analized by adsorption test, none of which revealed specific antibody activity. From these data we concluded that intrinsic anti TPO antibody is not likely to account for impaired megakaryocytopoiesis in ITP patients.