Hui Henry, Fuller Kathryn, Erber Wendy N, Linden Matthew D
Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Perth, Australia; School of Pathology and Laboratory Medicine, University of Western Australia, Perth, Australia.
Cytometry A. 2015 Mar;87(3):273-8. doi: 10.1002/cyto.a.22587. Epub 2014 Dec 16.
Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets express P-Selectin (CD62P) on the cell membrane and bind to P-Selectin glycoprotein ligand 1 expressing monocytes, influencing them toward a pro-adhesive and inflammatory phenotype. It is well established that elevated circulating monocyte-platelet aggregates (MPAs) are linked to atherothrombosis in high risk patients. However, whole blood flow cytometry (FCM) has recently shown that circulating MPAs may also occur in the absence of platelet activation, particularly in healthy children. A potential limitation of conventional FCM is the potential for coincident events to resemble monocyte platelet aggregates. Here we report a novel imaging cytometry approach to further characterize monocyte-platelet aggregate formation by P-Selectin dependent and P-Selectin independent mechanisms and distinguish circulating MPAs from coincidental events. Monocytes were identified by expression of the lipopolysachharide receptor (CD14 BV421), while platelets were identified by expression of the glycoprotein Ib (CD42b APC). Differentiation of P-Selectin dependent and P-Selectin independent binding was achieved with AF488 labeled CD62P. Overall analysis of circulating and in vitro generated MPAs by conventional and imaging cytometry methods showed very strong correlation (r(2) = >0.99, P < 0.01). The Bland-Altman bias of -1.72 was not significantly different to zero. However, when measuring only P-Selectin negative MPAs, a lack of correlation (r(2) = 0.27, P = n.s.) likely reflects better discrimination of coincidence events using imaging cytometry. Our data demonstrate that IFC is more accurate in enumerating MPAs than conventional FCM, which over-estimates the number of MPAs due to the presence of coincident events.
血小板是亚细胞血液成分，在止血过程中发挥着既定作用。激活后，血小板在细胞膜上表达P-选择素（CD62P），并与表达P-选择素糖蛋白配体1的单核细胞结合，促使它们向促黏附性和炎症性表型转变。众所周知，循环单核细胞-血小板聚集体（MPA）水平升高与高危患者的动脉粥样硬化血栓形成有关。然而，全血流式细胞术（FCM）最近显示，即使在血小板未激活的情况下，循环MPA也可能出现，尤其是在健康儿童中。传统FCM的一个潜在局限性是，同时发生的事件可能类似于单核细胞-血小板聚集体。在此，我们报告一种新型成像细胞术方法，以进一步表征通过P-选择素依赖性和P-选择素非依赖性机制形成的单核细胞-血小板聚集体，并将循环MPA与同时发生的事件区分开来。通过脂多糖受体（CD14 BV421）的表达鉴定单核细胞，而通过糖蛋白Ib（CD42b APC）的表达鉴定血小板。使用AF488标记的CD62P实现了对P-选择素依赖性和P-选择素非依赖性结合的区分。通过传统和成像细胞术方法对循环和体外生成的MPA进行的总体分析显示出非常强的相关性（r(2) = >0.99，P < 0.01）。-1.72的布兰德-奥特曼偏差与零无显著差异。然而，仅测量P-选择素阴性MPA时，缺乏相关性（r(2) = 0.27，P = 无显著性差异），这可能反映出使用成像细胞术对同时发生的事件有更好的辨别能力。我们的数据表明，与传统FCM相比，成像流式细胞术（IFC）在计数MPA方面更准确，传统FCM由于同时发生的事件的存在而高估了MPA的数量。
