Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis.
Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis.
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循环微泡的收集、分离和储存方法对流式细胞术分析的影响
DOI：
10.3892/etm.2015.2780
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发表时间：
2015-12
影响因子：
2.7
通讯作者：
Chen Z
中科院分区：
文献类型：
--
作者：
Kong F;Zhang L;Wang H;Yuan G;Guo A;Li Q;Chen Z
体液中的微泡参与了多种生理和病理过程，被认为是多种疾病的潜在生物标志物。流式细胞术（FCM）是MV检测中最常用的技术之一。然而，不同的处理方法必然会导致FCM测定的MV计数和大小的分析前变化。本研究的目的是研究离心、储存条件和抗凝剂对MV测量的影响。从13名健康供体获得血液样品，包括4名女性和9名男性。钙黄绿素-AM染色用于标记MV，并评估分析前制备（包括离心）和储存条件对使用FCM获得的MV测量值的影响。用于比较的因素范围包括：无血小板血浆（PFP）在− 80 ° C下储存1或4周; MV在4 ° C下储存3 - 4天或1周; MV在− 80 ° C下冷冻1或4周;抗凝剂，肝素或乙二胺四乙酸（EDTA）。在两种离心速度（16，000和20，500 × g）之间或研究的三种离心时间（15、30和60 min）之间未检测到MV计数的统计学显著差异。同样，两种检测的抗凝剂（肝素和EDTA）之间的MV计数也无显著差异。然而，在肝素抗凝血浆中储存PFP或MV的不同时间显著影响检测到的MV计数和尺寸分布。EDTA抗凝血浆中MV的数量和大小仅在MV在-80 °C冷冻4周时受到影响。总之，钙黄绿素-AM能够有效地从血浆中识别MV，并且可以替代Annexin V用于MV染色。在钙黄绿素-AM染色下，与肝素相比，EDTA更准确地保留了MV计数和大小。以16，000 × g离心15 min的PFP足以分离MV，从而能够对样品进行批量处理。PFP，而不是单独的MV，似乎是样品储存的优选模式，因为储存在PFP中的MV受储存温度和持续时间的影响较小。本研究提供了一种MV收集、储存和分离的方法，以促进MV作为疾病生物标志物的进一步研究。
Microvesicles (MVs) in body fluids participate in a variety of physical and pathological processes, and are regarded as potential biomarkers for numerous diseases. Flow cytometry (FCM) is among the most frequently used techniques for MV detection. However, different handling methods unavoidably cause pre-analytical variations in the counts and sizes of MVs determined by FCM. The aim of the present study was to investigate the effect of centrifugation, storage conditions and anticoagulant on MV measurements. Blood samples were obtained from 13 healthy donors, including 4 women and 9 men. Calcein-AM staining was used to label MVs and assess the impact of pre-analytical preparation, including centrifugation, and storage conditions on MV measurements obtained using FCM. The range of factors investigated for comparison included: Platelet-free plasma (PFP) stored at −80°C for 1 or 4 weeks; MVs stored at 4°C for 3–4 days or 1 week; MVs frozen at −80°C for 1 or 4 weeks; and anticoagulants, either heparin or ethylenediaminetetraacetic acid (EDTA). No statistically significant differences in MV counts were detected between the two centrifugation speeds (16,000 and 20,500 × g) or among the three centrifugation times (15, 30 and 60 min) investigated. Similarly, no significant differences were noted in MV counts between the two anticoagulants tested (heparin and EDTA). However, the storage of PFP or MVs in heparin-anticoagulated plasma for different periods markedly affected the detected MV counts and size distribution. The counts and sizes of MVs from EDTA-anticoagulated plasma were only affected when the MVs were frozen at −80°C for 4 weeks. In conclusion, calcein-AM is able to efficiently identify MVs from plasma and may be an alternative to Annexin V for MV staining. EDTA preserves the MV counts and size more accurately compared with heparin under calcein-AM staining. PFP centrifuged at 16,000 × g for 15 min is sufficient to isolate MVs, which enables the batch processing of samples. PFP, rather than MVs alone, appears to be the preferable mode of sample storage, as MVs stored in PFP were less affected by storage temperature and duration. The present study provides a methodology for MV collection, storage and isolation, to facilitate further investigation of MVs as biomarkers in disease.