背景:免疫性血小板减少症(ITP)是狗严重血小板减少症的常见原因。非关联的发病机理,原发性ITP(pITP)出现复杂,血小板生成反应不明确。
目的:开发一种免疫分析法来测量犬血浆血小板生成素(TPO)浓度并表征pITP犬的TPO浓度。
方法:41只健康狗,诱导ITP模型中的8只狗(3只对照,5ITP),和58只PITP狗。
方法:购买重组犬TPO(rcTPO),并通过质谱确认其身份。针对rcTPO产生单克隆抗体,并使用链霉亲和素-生物素检测来配置夹心ELISA。测定性能,变异系数,测定健康犬血浆TPO参考区间(RI),然后测定ITP样品。
结果:测定动态范围为15pg/mL(检测下限)至1000pg/mLTPO,定量限为62pg/mL。血浆TPORI为0至158pg/mL,35/41健康犬的血浆TPO<62pg/mL。所有具有诱导的ITP的狗均出现血浆TPO浓度的显著增加。峰值范围为515至>6000pg/mL。相比之下,只有2/58pITP犬的TPO值高于RI.
结论:对于大多数患有pITP的狗,血浆TPO浓度在诊断时是矛盾的低。这一发现表明无效的血小板生成有助于pITP犬的血小板减少症,并支持评估用于人pITP的TPO受体激动剂治疗。TPO分析提供了一种新的工具来研究狗的pITP和其他血小板减少综合征的血小板生成。
BACKGROUND: Immune thrombocytopenia (ITP) is a common cause of severe thrombocytopenia in dogs. The pathogenesis of nonassociative, primary ITP (pITP) appears complex, with ill-defined thrombopoietic response.
OBJECTIVE: Develop an immunoassay to measure plasma canine thrombopoietin (TPO) concentration and characterize TPO concentrations in dogs with pITP.
METHODS: Forty-one healthy dogs, 8 dogs in an induced ITP model (3 control, 5 ITP), and 58 pITP dogs.
METHODS: Recombinant canine TPO (rcTPO) was purchased and its identity confirmed by mass spectrometry. Monoclonal antibodies were raised to rcTPO and used to configure a sandwich ELISA using streptavidin-biotin detection. Assay performance, coefficients of variability, and healthy dog plasma TPO reference interval (RI) were determined, followed by assay of ITP samples.
RESULTS: Assay dynamic range was 15 pg/mL (lower limit of detection) to 1000 pg/mL TPO, with limit of quantitation of 62 pg/mL. Plasma TPO RI was 0 to 158 pg/mL, with plasma TPO <62 pg/mL for 35/41 healthy dogs. All dogs with induced ITP developed marked increases in plasma TPO concentration. Peak values ranged from 515 to >6000 pg/mL. In contrast, only 2/58 pITP dogs had TPO values above RI.
CONCLUSIONS: Plasma TPO concentration is paradoxically low at diagnosis for most dogs with pITP. This finding suggests that ineffective thrombopoiesis contributes to thrombocytopenia in pITP dogs and supports evaluating TPO receptor agonist treatment as used for pITP in humans. The TPO assay provides a new tool to study thrombopoiesis in pITP and other thrombocytopenic syndromes in dogs.