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微颗粒的检测在出血性疾病、心血管疾病等方面都有着广泛的应用（见http://www.flowcyto.cn/bbs/thread-1776-1-1.html），这篇Cytometry Part A最新一期上的文章，提醒我们在检测和分析循环性微颗粒时，由于微颗粒的检测需要利用ANNEXIN V，而ANNEXIN V与PS的结合则需要钙离子的参与，这时钙离子与PBS中的磷酸盐就很可能结合成与微颗粒类似大小的微沉淀，从而干扰微颗粒的检测。作者建议，如可能，可改用非钙离子依赖性的PS检测试剂，如lactadherin等。
Calcium-Phosphate Microprecipitates Mimic Microparticles When Examined with Flow Cytometry
Michael C. Larson, Maia R. Luthi, Neil Hogg, Cheryl A. Hillery
Abstract
There are increased levels of circulating microparticles (MPs) in several disease states. Flow cytometry is a common method to examine MPs, but their small size necessitates the use of markers to distinguish specifically MPs from artifact. Annexin V, which binds phosphatidylserine, is a commonly used marker for MP detection. Annexin V requires millimolar calcium ion for optimum binding. Ca11 can precipitate with phosphate in phosphate-buffered saline (PBS). Calcium-phosphate microprecipitates were formed by titrating Ca11 into PBS and examined using flow cytometry. Calcium-phosphate microprecipitates were compared with MPs derived from aged donor blood units. Microprecipitates were 0.7–0.9 lm in diameter compared with standard beads of known size. The microprecipitates disappeared with the addition of Ca11 chelator. When we added fluorescently labeled antibodies to microprecipitates, the median fluorescent signal increased with increasing Ca11 concentration regardless of specificity of the antibody. When repeated with a biological sample, there was an apparent increase in the fluorescent signal that returned to baseline after Ca11 chelation. The flow cytometry signal of calcium-phosphate microprecipitates overlaps with the MP signal. Since Ca11 is essential for annexin V binding, it is essential to avoid artifacts from calciumphosphate microprecipitates when using any buffer or biological fluid containing phosphate. This also highlights the potential utility of flow cytometry for the analysis of crystals in biological fluids
不同浓度钙离子产生的微沉淀
不同浓度钙离子产生的微沉淀
钙离子对MP检测时荧光的影响，建议采用lactadherin
钙离子对MP检测时荧光的影响，建议采用lactadherin
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发表于 2012-11-14 21:37:16
发表于 2013-7-5 10:17:42