Infection and graft-versus-host disease (GvHD) are the major causes for mortality and morbidity of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Plasma-derived extracellular vesicles (EVs) contain disease-related proteins, DNAs and RNAs, and have recently been suggested as potential biomarker candidates for transplantation complications. However, EV isolation from small plasma volumes in clinical biomarker studies using conventional methods is challenging. We therefore investigated if EVs isolated by novel automated acoustic trapping could be developed as potential biomarkers for allo-HSCT complications by performing a clinical proof-of-principle study. Plasma samples were collected from twenty consecutive patients with high-risk/relapsed hematologic malignancies undergoing allo-HSCT before transplantation and post-transplant up to 12 weeks. EVs were isolated from small plasma sample volumes (150 μl) by an automated, acoustofluidic-based particle trapping device, which utilizes a local λ/2 ultrasonic standing wave in a borosilicate glass capillary to capture plasma EVs among pre-seeded polystyrene microbeads through sound scatter interactions. We found that EVs could be reliably isolated from all plasma samples (n = 173) and that EV numbers increased more than 2-fold in the majority of patients after transplantation. Also, sufficient quantities of RNA for downstream microRNA (miRNA) analysis were obtained from all samples and EV miRNA profiles were found to differ from whole plasma profiles. As a proof of principle, expression of platelet-specific miR-142-3p in EVs was shown to correlate with platelet count kinetics after transplantation as expected. Importantly, we identified plasma EV miRNAs that were consistently positively correlated with infection and GvHD, respectively, as well as miRNAs that were consistently negatively correlated with these complications. This study demonstrates that acoustic enrichment of EVs in a clinical biomarker study setting is feasible and that downstream analysis of acoustically-enriched EVs presents a promising tool for biomarker development in allo-HSCT. Certainly, these findings warrant further exploration in larger studies, which will have significant implications not only for biomarker studies in transplantation but also for the broad field of EV-based biomarker discovery.

中文翻译：

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异源造血干细胞移植中用于生物标志物发现的声学隔离细胞外囊泡的开发

感染和移植物抗宿主病（GvHD）是同种异体造血干细胞移植（allo-HSCT）致死和发病的主要原因。血浆来源的细胞外囊泡（EV）包含与疾病相关的蛋白质，DNA和RNA，最近已被建议作为移植并发症的潜在生物标志物候选物。然而，在使用常规方法的临床生物标志物研究中，从小血浆中分离出EV具有挑战性。因此，我们通过进行临床原理验证研究，研究了通过新型自动声阱分离技术隔离的电动汽车是否可以发展为异基因造血干细胞移植并发症的潜在生物标记。从移植前和移植后长达12周的连续20例高危/复发性血液恶性肿瘤患者中收集血浆样品。通过自动的基于声流的颗粒捕集装置从少量血浆样品（150μl）中分离出EV，该装置利用硼硅酸盐玻璃毛细管中的局部λ/ 2超声驻波来通过声音捕获预接种的聚苯乙烯微珠中的血浆EV分散互动。我们发现，可以从所有血浆样本中可靠地分离出电动汽车（n = 173），并且大多数患者移植后的电动汽车数量增加了两倍以上。同样，从所有样品中获得了足够数量的用于下游microRNA（miRNA）分析的RNA，并且发现EV miRNA谱不同于整个血浆谱。作为原理的证明，如预期的那样，EV中血小板特异性miR-142-3p的表达与血小板计数动力学相关。重要的是，我们确定了血浆EV miRNA分别与感染和GvHD始终呈正相关，以及与这些并发症始终呈负相关的miRNA。这项研究表明，在临床生物标志物研究环境中进行电动汽车的声富集是可行的，并且对声学富集的电动汽车的下游分析为异源HSCT中生物标志物的开发提供了有希望的工具。当然，这些发现需要在更大的研究中进行进一步的探索，这不仅对移植中的生物标志物研究有重要意义，而且对基于EV的生物标志物发现的广泛领域都具有重要意义。EV中血小板特异性miR-142-3p的表达与移植后的血小板计数动力学相关。重要的是，我们分别确定了与感染和GvHD始终呈正相关的血浆EV miRNA，以及与这些并发症始终呈负相关的miRNA。这项研究表明，在临床生物标志物研究环境中进行电动汽车的声富集是可行的，并且对声学富集的电动汽车的下游分析为异源HSCT中生物标志物的开发提供了有希望的工具。当然，这些发现需要在更大的研究中进行进一步的探索，这不仅对移植中的生物标志物研究有重要意义，而且对基于EV的生物标志物发现的广泛领域都具有重要意义。EV中血小板特异性miR-142-3p的表达与移植后的血小板计数动力学相关。重要的是，我们确定了血浆EV miRNA分别与感染和GvHD始终呈正相关，以及与这些并发症始终呈负相关的miRNA。这项研究表明，在临床生物标志物研究环境中进行电动汽车的声富集是可行的，并且对声学富集的电动汽车的下游分析为异源HSCT中生物标志物的开发提供了有希望的工具。当然，这些发现需要在更大的研究中进行进一步的探索，这不仅对移植中的生物标志物研究有重要意义，而且对基于EV的生物标志物发现的广泛领域都具有重要意义。