Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles
Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles
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DOI：
10.1111/j.1538-7836.2009.03654.x
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发表时间：
2010-02-01
影响因子：
10.4
通讯作者：
Osanto, S.
中科院分区：
文献类型：
--
作者：
Yuana, Y.;Oosterkamp, T. H.;Osanto, S.
背景：微颗粒（MPs）是从不同来源的细胞中释放出来的小泡，携带来自亲本细胞的表面抗原。据报道，心血管疾病和癌症患者血液中MPs水平升高。大多数MPs来源于血小板。目的：探讨原子力显微镜（AFM）能否用于检测血小板源性MPs并确定其大小分布。方法：在AFM成像之前，将7名献血者和3名癌症患者分离的血液MPs固定在涂有CD41抗体的改性云母表面上。采用液攻模式进行AFM检测cd41阳性MPs。同时，用流式细胞术检测cd41阳性MPs的数量。阴性对照采用小鼠IgG(1)同型对照。结果：AFM形貌测量cd41阳性MPs的数量是可重复的（变异系数为16%）。假设未结合的MPs呈球形，计算出献血者和癌症患者中cd41阳性MPs的直径（d(sph)）范围分别为10 ~ 475 nm（平均值：67.5 +/- 26.5 nm）和5 ~ 204 nm（平均值：51.4 +/- 14.9 nm）。cd41阳性MPs的数量比流式细胞术检测的多1000倍(3-702 × 10（9) L-1血浆vs. 11-626 × 10(6) L-1血浆）。分离的MPs经0.22 μ m过滤器过滤后，AFM仍可检测到cd41阳性MPs（平均d(sph): 37.2 +/- 11.6 nm），但流式细胞术无法检测到。结论：AFM提供了一种灵敏检测纳米范围内MPs定义亚群的新方法，远低于传统流式细胞术可以测量的下限。
Background: Microparticles (MPs) are small vesicles released from cells of different origin, bearing surface antigens from parental cells. Elevated numbers of blood MPs have been reported in (cardio) vascular disorders and cancer. Most of these MPs are derived from platelets. Objectives: To investigate whether atomic force microscopy (AFM) can be used to detect platelet-derived MPs and to define their size distribution. Methods: Blood MPs isolated from seven blood donors and three cancer patients were immobilized on a modified mica surface coated with an antibody against CD41 prior to AFM imaging. AFM was performed in liquid-tapping mode to detect CD41-positive MPs. In parallel, numbers of CD41-positive MPs were measured using flow cytometry. Mouse IgG(1) isotype control was used as a negative control. Results: AFM topography measurements of the number of CD41-positive MPs were reproducible (coefficient of variation = 16%). Assuming a spherical shape of unbound MPs, the calculated diameter of CD41-positive MPs (d(sph)) ranged from 10 to 475 nm (mean: 67.5 +/- 26.5 nm) and from 5 to 204 nm (mean: 51.4 +/- 14.9 nm) in blood donors and cancer patients, respectively. Numbers of CD41-positive MPs were 1000-fold higher than those measured by flow cytometry (3-702 x 10(9) L-1 plasma vs. 11-626 x 10(6) L-1 plasma). After filtration of isolated MPs through a 0.22-mu m filter, CD41-positive MPs were still detectable in the filtrate by AFM (mean d(sph): 37.2 +/- 11.6 nm), but not by flow cytometry. Conclusions: AFM provides a novel method for the sensitive detection of defined subsets of MPs in the nanosize range, far below the lower limit of what can be measured by conventional flow cytometry.