Application of high-sensitivity flow cytometry in combination with low-voltage scanning electron microscopy for characterization of nanosized objects during platelet concentrate storage

Platelet concentrates are used in clinic for therapy and prophylaxis of conditions associated with platelet deficiency or malfunction. The characteristics of platelet concentrates gradually change during pretransfusion storage, affecting their clinical effectiveness and the risk of adverse transfusion reactions. The presence of platelet-derived membrane vesicles is an important characteristic of platelet concentrates. Due to their functionality, changes in the number and molecular compositions of platelet-derived vesicles have major effects on the clinical properties of platelet preparations. The existence of different subpopulations of membrane vesicles requires analytical methods capable of providing information at the individual vesicle level. Such methods include flow cytometry and electron microscopy. However, conventional flow cytometry has certain limitations, since the diameters of many platelet-derived membrane vesicles are smaller than its detection limit. The use of classical scanning electron microscopy is also limited due to the requirement for coating with a layer of conductive material, which impedes the detection of small extracellular vesicles. Here, a combination of high-sensitivity flow cytometry and low-voltage scanning electron microscopy was used to increase sensitivity and resolution in the detection of nanosized objects present in platelet concentrates during storage. Apheresis platelet concentrates from eight healthy adult donors were investigated on days 2 and 7 of storage. Fractions of nanosized objects were obtained by differential centrifugation. Fluorophore-conjugated antibodies were used to detect marker-positive vesicles derived from platelets (CD41), red blood cells (CD235a), leukocytes (CD45), and endothelial cells (VEGFR2). Near-spherical objects with diameters ranging from 25 to 700 nm were observed by low-voltage scanning electron microscopy in platelet concentrates and its fractions. On day 7 of storage, objects with diameters of less than 100 nm were attached to and clustered near the terminal ends of pseudopod-like projections. High-sensitivity flow cytometry showed that during storage numbers of CD41(pos) vesicles elevated more than fivefold and numbers of marker-negative nanosized objects, which did not carry any of the investigated cell type-specific markers elevated more than twofold. Major changes in both CD41(pos) vesicles and marker-negative nanosized objects abundances were observed for objects with diameters around 100 nm bead equivalents. Overall, these results emphasized the importance of application of high-sensitivity methods for monitoring the characteristics of cell-derived nanosized objects during platelet concentrate storage.血小板浓缩物（Platelet concentrates）临床用于治疗和预防与血小板减少或功能异常相关的疾病。在输血前储存过程中，血小板浓缩物的特性会逐渐改变，进而影响其临床有效性以及发生输血不良反应的风险。血小板源性膜囊泡（platelet-derived membrane vesicles）的存在是血小板浓缩物的一项重要特征。鉴于其功能特性，血小板源性囊泡的数量与分子组成变化会对血小板制剂的临床属性产生显著影响。由于膜囊泡存在不同亚群，因此需要能够获取单囊泡水平信息的分析方法，这类方法包括流式细胞术（flow cytometry）与电子显微镜。然而，传统流式细胞术存在一定局限：许多血小板源性膜囊泡的直径小于其检测下限。经典扫描电子显微镜（scanning electron microscopy）的应用也受到限制，因其需要使用导电材料进行包被，这会干扰小型细胞外囊泡（small extracellular vesicles）的检测。 本研究采用高灵敏度流式细胞术（high-sensitivity flow cytometry）与低电压扫描电子显微镜（low-voltage scanning electron microscopy）联用的方案，以提升血小板浓缩物储存过程中纳米级颗粒物检测的灵敏度与分辨率。研究纳入8名健康成年志愿者的单采血小板浓缩物（apheresis platelet concentrates），分别在储存第2天和第7天进行检测。通过差速离心法（differential centrifugation）分离得到纳米级颗粒物组分。使用荧光素标记抗体（fluorophore-conjugated antibodies）检测源自血小板（CD41）、红细胞（CD235a）、白细胞（CD45）及内皮细胞（VEGFR2）的标记物阳性囊泡。 通过低电压扫描电子显微镜观察到，在血小板浓缩物及其分离组分中存在直径介于25~700 nm的近球形颗粒物。在储存第7天时，直径小于100 nm的颗粒物会附着并聚集在伪足样突起（pseudopod-like projections）的末端附近。高灵敏度流式细胞术检测结果显示，储存过程中CD41阳性囊泡的数量升高超过5倍，未携带任何所检测细胞类型特异性标记物的标记阴性纳米级颗粒物的数量升高超过2倍。在直径约为100 nm（微球当量，bead equivalents）的颗粒物中，CD41阳性囊泡与标记阴性纳米级颗粒物的丰度均发生了显著变化。综上，本研究结果凸显了采用高灵敏度方法监测血小板浓缩物储存过程中细胞源性纳米级颗粒物特性的重要性。