评价一种新的商业测定方法，用于测量从人血浆中分离的细胞外囊泡的组织因子活性

IF 3.4 3区医学 Q2 HEMATOLOGY

Research and Practice in Thrombosis and Haemostasis Pub Date : 2025-08-01 DOI:10.1016/j.rpth.2025.103007

Ana T.A. Sachetto , Alan E. Mast , Nigel Mackman

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引用次数: 0

摘要

组织因子（TF）阳性的细胞外囊泡（EVs）被释放到血液循环中，在脓毒症、病毒感染和癌症等多种疾病中激活凝血。目的：我们比较了一种名为“CY- quant MV-TF”（CY）的新型商业检测方法与用于测量EV TF活性的内部检测方法（Chapel Hill [CH]）。方法用脂多糖刺激4例健康供者柠檬酸人全血5小时，产生stf阳性ev。从低血小板血浆中分离EVs，在4°C下，20000 × g离心60分钟。结果CY法检测到的EV TF活性低于CH法（平均±SD， 0.54±0.30 pg/mL, n = 4 vs 1.59±0.43 pg/mL, n = 4, P = 0.01）。有趣的是，与CH法相比，CY法在分离EV时使用较低比例的血浆洗涤缓冲液。这导致与CH方案相比，CY方案分离的ev中TF途径抑制剂（TFPI）水平更高。重要的是，添加一种抑制抗tfpi抗体可提高两种检测方法的EV TF活性，消除两种检测方法之间的EV TF活性差异。结论CY法可检测血浆中分离的ev的TF活性。然而，使用CY EV分离方案的EV制剂中存在大量的TFPI，这抑制了TF活性，导致较低的EV TF水平。

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Evaluation of a new commercial assay for the measurement of tissue factor activity of extracellular vesicles isolated from human plasma

Background

Tissue factor (TF)-positive extracellular vesicles (EVs) are released into the circulation and activate coagulation in several diseases, such as sepsis, viral infections, and cancer.

Objectives

We compared a new commercial assay, called “CY-QUANT MV-TF” (CY), with an in-house assay (Chapel Hill [CH]) for the measurement of EV TF activity.

Methods

TF-positive EVs were generated by stimulating citrated human whole blood from 4 healthy donors with lipopolysaccharide for 5 hours. EVs were isolated from platelet-low plasma by centrifugation at 20,000 × g for 60 minutes at 4 °C.

Results

Lower levels of EV TF activity were detected in the samples using the CY assay compared with the CH assay (mean ± SD, 0.54 ± 0.30 pg/mL, n = 4 vs 1.59 ± 0.43 pg/mL, n = 4; P = .01). Interestingly, the CY assay used a lower ratio of plasma to wash buffer compared with the CH assay for EV isolation. This led to higher levels of TF pathway inhibitor (TFPI) in EVs isolated using the CY protocol compared with the CH protocol. Importantly, the addition of an inhibitory anti-TFPI antibody increased EV TF activity in both assays and eliminated the difference in EV TF activity between the 2 assays.

Conclusion

In this study, we found that the CY assay detected TF activity in EVs isolated from plasma. However, significant amounts of TFPI were present in the EV preparations using the CY EV isolation protocol, which inhibited TF activity, leading to lower apparent EV TF levels.

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