Avoiding false positive antigen detection by flow cytometry on blood cell derived microparticles: the importance of an appropriate negative control.
Avoiding false positive antigen detection by flow cytometry on blood cell derived microparticles: the importance of an appropriate negative control.
复制标题
DOI：
10.1371/journal.pone.0127209
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Stamatopoulos B
中科院分区：
文献类型：
--
作者：
Crompot E;Van Damme M;Duvillier H;Pieters K;Vermeesch M;Perez-Morga D;Meuleman N;Mineur P;Bron D;Lagneaux L;Stamatopoulos B
微粒（MP），也称为微泡（MV），是质膜衍生的碎片，大小范围为0.1至1μm。这些MP的表征通常通过流式细胞术进行，但对于可导致假阳性结果的适当阴性对照尚无共识。我们分析了来自血小板、B细胞、T细胞、NK细胞、单核细胞和慢性淋巴细胞白血病（CLL）B细胞的MP。通过正磁性分离纯化细胞并培养48小时。使用以下单克隆抗体表征细胞和MP（对于B细胞，CD 19，20;对于T细胞，CD 3，8，5，27;对于NK细胞，CD 16，56;对于单核细胞，CD 14，11 c;对于血小板，CD 41，61）。分离的MP用膜联蛋白-V-FITC染色，并在300 nm和900 nm之间设门。然后进行乳胶珠技术以便于检测MP。通过透射（TEM）和扫描电子显微镜（SEM）分析样品。在300- 900 nm的门内检测到膜联蛋白-V阳性事件，并将其定义为MP。我们的研究结果证实，在血小板衍生的MP上发现了特征性抗原CD 41/CD 61，验证了我们的技术。然而，对于来自其他细胞类型的MP，我们无法检测到任何抗原，尽管它们在MP产生细胞上清楚地表达，与文献中发表的几个数据相反。使用乳胶珠技术，我们证实了CD 41，61的检测。然而，其他抗原的表观表达（在几项研究中已经被认为是阳性的）被确定为假阳性，由阴性对照指示（在来自不同来源的MP上使用相同的标记）。我们观察到，母细胞抗原并不总是检测到相应的MP直接流式细胞术或乳胶珠细胞仪。我们的数据强调，由于抗体的非特异性，可能会产生假阳性结果，MP的表型表征是一个困难的领域，需要使用几个阴性对照。
Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results. We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM). Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins). We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.
登录
查看更多内容
影响因子：
5.2
作者：
Fonsato, Valentina;Collino, Federica;Herrera, Maria Beatriz;Cavallari, Claudia;Deregibus, Maria Chiara;Cisterna, Barbara;Bruno, Stefania;Romagnoli, Renato;Salizzoni, Mauro;Tetta, Ciro;Camussi, Giovanni
通讯作者：
Camussi, Giovanni
影响因子：
16
作者：
Crescitelli R;Lässer C;Szabó TG;Kittel A;Eldh M;Dianzani I;Buzás EI;Lötvall J
通讯作者：
Lötvall J
影响因子：
3.3
作者：
Budoni, Manuela;Fierabracci, Alessandra;Muraca, Maurizio
通讯作者：
Muraca, Maurizio
影响因子：
16
作者：
Gheldof, Damien;Hardij, Julie;Mullier, Francois
通讯作者：
Mullier, Francois
影响因子：
6.7
作者：
Connor, David Ewan;Exner, Thomas;Joseph, Joanne Emily
通讯作者：
Joseph, Joanne Emily