Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.
Retham Technologies, Wauwatosa, WI.
Blood. 2022 Dec 22;140(25):2722-2729. doi: 10.1182/blood.2022017283.
Heparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital diagnosis.
肝素诱导的血小板减少症（HIT）的疑似病例远多于确诊病例。技术上简单的血小板因子 4（PF4）-多阴离子酶联免疫吸附测定（ELISA）虽然敏感但特异性不高。相比之下，准确的功能测试，如 5-羟色胺释放试验、肝素诱导的血小板活化试验和 PF4 依赖性 P-选择素表达试验，需要新鲜血小板且具有复杂的检测终点，这限制了它们在专门的参考实验室中的可用性。为了能够广泛应用功能检测，我们试图通过优化储存条件显著延长血小板的存活时间，并通过测量血小板α颗粒蛋白（TSP1）在 ELISA 格式中的释放来开发一个简单的功能检测终点。通过控制冷却速率来优化血小板的冷冻保存条件，以保持可激活性。用 PF4 或肝素处理保存数月的冷冻血小板，并在 TSP1 释放试验（TRA）中评估其被 HIT 和疫苗诱导的免疫性血栓性血小板减少症（VITT）抗体激活的能力。与缺乏血小板激活抗体的疑似 HIT 患者样本相比，HIT 和自发性 HIT 患者样本在 PF4 处理（PF4-TRA）和肝素处理的冷冻血小板中，TSP1 的释放明显更高。后者包括几个在 PF4-多阴离子 ELISA 中检测呈强阳性但不具有血小板激活作用的患者。在 TRA 中检测的 4 个 VITT 患者样本激活了 PF4 处理但未激活肝素处理的冷冻血小板，这与最近的数据一致，表明需要 PF4 处理的血小板来检测 VITT 抗体。这些发现有可能改变 HIT 和 VITT 的检测模式，使分散式、技术简单的功能检测可用于快速准确的院内诊断。
