Name: measuring deformability and red cell heterogeneity in blood by ektacytometry
Uploaded: 2018-01-12T14:00:00
Description: Watch this Scientific Journal Video about Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry at JoVE.com

这项工作得到了国家糖尿病、消化和肾脏疾病研究所和国立卫生研究院国家心脏、肺脏和血液研究所的内部研究方案的支持。这里表达的意见是作者的唯一责任, 不一定代表国家卫生研究院的官方观点。

本研究的所有科目都根据《赫尔辛基宣言》和国家卫生研究院机构审查委员会批准的议定书, 给予书面知情同意。
1. 打开 ektacytometer
<ol><li>将清洗液的油管连接到低、高渗酮 (PVP) 解决方案。小心连接0渗管到低渗溶液和500渗管到高渗溶液。 注意:低渗 PVP 解决方案应该有一个渗透在35和 55 milliosmoles 每千克 (mOsm/千克), pH 7.25-7. 45 在25° c 和黏度在27.0 和33.0 厘 (cP) 之...

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	<li>Bessis, M., Mohandas, N., Feo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+ektacytometry:+a+new+method+of+measuring+red+cell+deformability+and+red+cell+indices.">Automated ektacytometry: a new method of measuring red cell deformability and red cell indices.</a> Blood Cells. 6 (3), 315-327 (1980).</li><li>Clark, M. R., Mohandas, N., Shohet, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmotic+gradient+ektacytometry:+comprehensive+characterization+of+red+cell+volume+and+surface+maintenance.">Osmotic gradient ektacytometry: comprehensive characterization of red cell volume and surface maintenance.</a> Blood. 61 (5), 899-910 (1983).</li><li>Da Costa, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+tool+for+red+blood+cell+membrane+disorders:+Assessment+of+a+new+generation+ektacytometer.">Diagnostic tool for red blood cell membrane disorders: Assessment of a new generation ektacytometer.</a> Blood Cells Mol Dis. 56 (1), 9-22 (2016).</li><li>Clark, M. R., Mohandas, N., Shohet, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformability+of+oxygenated+irreversibly+sickled+cells.">Deformability of oxygenated irreversibly sickled cells.</a> J Clin Invest. 65 (1), 189-196 (1980).</li><li>Rabai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformability+analysis+of+sickle+blood+using+ektacytometry.">Deformability analysis of sickle blood using ektacytometry.</a> Biorheology. 51 (2-3), 159-170 (2014).</li><li>Bessis, M., Mohandas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Diffraction+Patterns+of+Sickle+Cells+in+Fluid+Shear+Fields.">Laser Diffraction Patterns of Sickle Cells in Fluid Shear Fields.</a> Blood Cells. 3, 229-239 (1977).</li><li>Kim, Y., Kim, K., Park, Y., Moschandreou, T. 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所描述的 ektacytometry 技术简单明了, 自动化程度很好, 确保了有效和可重现的结果。尽管如此, 还是有一些关键的步骤存在。正确的血液温度控制是很重要的。室温贮存超过八小时可能影响 SS ½值。34确保机器的温度在37° c 时保持稳定也很重要, 因为悬浮介质的粘度是温度依赖性的。血液应充分含氧, 以避免因 non-oxygenated 或部分含氧样品而降低变形, 除非这些都是实验性的兴趣参数...

Ektacytometry 提供了一个方便的测量红细胞变形性的反应, 改变剪切应力 (测量在帕斯卡 (Pa)) 或悬浮介质渗透。红细胞变形性的相关参数包括最大伸长指数 (EI 最大值), 一种对红细胞最大变形性的测量, 以响应增加的剪应力, 剪切应力½ (SS ½), 所需的剪切应力达到半最大变形.1渗透梯度 ektacytometry 有几个信息参数。这些包括伸长指数最小值 (EI 最小值)、表面积比的测量和发生的渗透 (O min), 这是渗透脆性的量度。ei 最大和渗透的发生 (O (ei max)) 提供了有关膜的灵活性和细胞表面积的信息。渗透梯度高渗臂的半最大伸长率以 EI 超高为代表。EI 超和渗透, 它发生, O 超, 提供信息的细胞内粘度的红细胞, 这是由血红蛋白浓度确定。2,3测量异质血液中的变形性是复杂的, 因为刚性细胞, 如镰红细胞, 不能与流动方向 (如变形细胞) 相适应, 以增加剪切应力。刚性细胞不是产生一个特征性的椭圆形衍射图像, 而是产生一个球形图案, 当叠加在变形细胞产生的椭圆上时, 会形成菱形衍射图样。4,5,6通过对密度离心后分离的细胞片段进行 ektacytometry, 证明了球状模式与不可逆转的镰细胞相对应。6伸长率指数计算包括椭圆的长、短轴的测量;因此, 金刚石形状会通过增加短轴的宽度而产生明显的伸长率下降。7以前已经表明, 镰状细胞性贫血患者血液中的镰细胞百分比与镰状血红蛋白 (HbS) 的比例有关。5通过复杂的数学分析可以得到衍射图样失真的程度。8还可以通过调整 ektacytometer 上相机光圈的打开或拟合软件的灰度级别来改变衍射图案的高度。5但是, 有关如何调整灰度级别的详细信息没有得到很好的定义, 而且在最新的商业可用 ektacytometer 上, 照相机的光圈也不容易获得。为了绕过这些问题, 可以使用易于获取的相机增益来调整衍射图案的高度。9使用此方法来估计细胞异质性, 其衍射图样畸变程度可与镰状细胞性贫血患者血液中胎儿血红蛋白的百分比相关。10几种渗透梯度 ektacytometry 参数同样与镰状细胞贫血患者血液中胎儿或镰状血红蛋白的百分比相关。衍射图样畸变的相关性可能反映了血红蛋白组成对刚性、non-deformable 细胞百分率的贡献。额外的兴趣, 整个渗透梯度 ektacytometry 剖面经历的双相变化, 与血液中的致密细胞在镰状细胞危机期间的百分比。11
Ektacytometry 在其他一些疾病的研究中同样有用。渗透梯度 ektacytometry 是诊断遗传性红细胞膜疾病, 如遗传球形, 遗传性 elliptocytosis 和遗传性 pyropoikilocytosis。3,12,13,14减少了铁缺乏的变形性。15对血液的 "存储损害" 的定性应用了 ektacytometry 和未来的研究, 对病变的性质进行了调查, 并在储存血液时防止其形成的干预措施很可能得益于技术介绍。16降低红细胞变形能力也与糖尿病的微血管疾病相关。17最近有关高血糖、红细胞抗坏血酸浓度和渗透脆性的研究表明, 这些因素可能对微血管疾病的发展起到重要作用。18 Ektacytometry 研究目前正在调查这一假说 (Parrow 和莱文, 未发表的数据)。血阶段的疟疾感染是另一个有趣的途径红细胞变形性调查。恶性疟原虫的细胞变形性, 感染的红细胞在48小时内从环期到殖阶段的寄生虫的胞内成熟度急剧下降。证据表明, 这种降低变形性是逆转后, 寄生虫的成熟。逆转与被传染的红色细胞的发行相符到循环。降低变形性被认为是介导的疟原虫蛋白质, 促进螯合红细胞。19这些研究代表了在测量红细胞变形性和渗透梯度参数相关的临床重要条件的小样本。还有几个额外的研究领域存在。
测量红细胞变形性的替代技术包括光镊 (也称为激光阱), 它利用光子的物理性质在一个或多个方向上拉伸单个红细胞。20该技术具有测量单个红细胞变形性的优点, 但在力标定方面的某些不确定性在研究中产生了相当大的变异性21 , 数据分析可以 labor-intensive, 除非自动.22微吸入, 利用负压将红细胞吸入微, 也用于测量红细胞的变形性。7,23多项测量 (如吸入红细胞所需的压力) 是可能的, 每项测量都定义了红细胞的不同特征。23原子力显微镜是一种高分辨率的技术, 它通过量化激光束偏转作为红细胞表面沿悬臂偏转的指示器来测量膜的刚度。24这些技术提供了有关单个红细胞的信息, 不容易适应于测量红细胞数量的变化, 而且通常需要大量的技术专长。
同时想要对细胞的个体和群体进行取样, 这就导致了自动化和微和基于阵列的方法的发展。与 ektacytometry 一样, rheoscopy 测量变形性是剪切应力的函数, 而图像是通过显微镜直接获取的。25对于更高的通入分析, 采用自动细胞成像技术, 利用 rheoscope 产生变形分布。如果有健康控制对象的数据可用, 则可以通过此方法量化26细胞异质性。27微技术还允许对单个细胞进行高通量分析;多项设计使用了过滤的适应性,28细胞转运分析仪,29 , 它测量了红细胞流经微孔所需的时间, 以及衡量红细胞转运所需压力的替代品, 而已开发30的时间。对单个细胞进行高通入分析的另一个平台是单细胞 microchamber 阵列芯片, 它具有允许下游荧光细胞特性的额外优势。31尽管这些技术中的每一个都有潜在的用处, 并且可能优于特定的应用程序, 但 ektacytometry 的比较优势包括灵敏度、易用性和精确性。32最新生成的商用 ektacytometers 也具有相当多的通用性, 可以进行的化验数量。

<table border="0" cellpadding="0" cellspacing="0" width="878">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">LoRRca MaxSis standard version</td>
			<td style="width:115px;">Mechatronics</td>
			<td style="width:119px;">LORC109000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">LoRRca MaxSis Osmoscan</td>
			<td style="width:115px;">Mechatronics</td>
			<td style="width:119px;">LORC109001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Polyvinylpyrrolidone solution (PVP) 0mOsm</td>
			<td style="width:115px;">Mechatronics</td>
			<td style="width:119px;">QRR030910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Polyvinylpyrrolidone solution (PVP) 500mOsm</td>
			<td style="width:115px;">Mechatronics</td>
			<td style="width:119px;">QRR030930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Polyvinylpyrrolidone solution (PVP) 5mL vials</td>
			<td style="width:115px;">Mechatronics</td>
			<td style="width:119px;">QRR030901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">X clean</td>
			<td style="width:115px;">Mechatronics</td>
			<td style="width:119px;">QRR010946</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P1000&#160;</td>
			<td style="width:115px;">MilliporeSigma</td>
			<td>Z646555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">P200</td>
			<td style="width:115px;">MilliporeSigma</td>
			<td style="width:119px;">Z646547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">P200 filter tips</td>
			<td style="width:115px;">MidSci</td>
			<td style="width:119px;">AV200-H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">P1250 filter tips</td>
			<td style="width:115px;">MidSci</td>
			<td style="width:119px;">AV1250-H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">Kimwipes</td>
			<td style="width:115px;">MidSci</td>
			<td style="width:119px;">8091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">1.5 mL eppendorf tubes</td>
			<td style="width:115px;">MidSci</td>
			<td style="width:119px;">AVSS1700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:479px;">15 mL conical vial</td>
			<td style="width:115px;">MidSci</td>
			<td style="width:119px;">C15R</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring deformability and red cell heterogeneity in blood by ektacytometry

红细胞变形性降低是几种疾病的特点。在某些情况下, 缺陷变形的程度可以预测疾病的严重性或严重并发症的发生。Ektacytometry 使用激光衍射粘测量红细胞的变形性, 受剪切应力或渗透梯度的作用。然而, 在测量具有刚性和可变形红细胞的特征的异质血液时, 直接变形测量很难解释。这是由于刚性细胞不能适当地对齐, 以响应剪应力和结果在扭曲的衍射模式, 显着减少明显的变形。畸变程度的测量提供了血液中红细胞异质性的指标。在镰状细胞性贫血中, 这与刚性细胞的百分比相关, 这反映了红细胞的血红蛋白浓度和血红蛋白组成。除了测量变形性外, 渗透梯度 ektacytometry 还提供了有关红细胞渗透脆性和水合状态的信息。这些参数也反映了镰状细胞患者红细胞的血红蛋白组成。Ektacytometry 测量红细胞的变形性, 因此不提供单个红细胞的变形性或力学性质的信息。无论如何, 本文所描述的技术的目的是为测量血液的变形性和细胞非均质提供一种方便和可靠的方法。这些技术可能有助于监测时间变化, 以及疾病进展和治疗干预的反应, 在一些疾病。镰状细胞性贫血是一个良好的例子。其他可能的疾病, 如红细胞变形性和/或异质性的测量感兴趣的是血液储存, 糖尿病,疟原虫感染, 铁缺乏, 和溶血贫血由于膜缺陷。

本手稿中描述的 ektacytometry 结果可用于测量任何条件下的红细胞变形能力。ektacytometer 的一般设置示意图如图 1所示。红细胞的均匀种群将产生一个椭圆形的衍射模式, 以响应增加的剪切应力, 可用于计算伸长率指数, 如图 2所示。在异质血样中, 由于刚性红细胞与变形细胞不协调, 产生了一个球面图案, 覆盖椭圆, 形成菱形衍射图?...

Watch this Scientific Journal Video about Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry at JoVE.com

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Ektacytometry 血中红细胞异质性的测定

在这里, 我们提出的技术, 以测量红细胞变形性和细胞异质性的 ektacytometry。这些技术适用于红细胞变形性的一般调查和血液疾病的具体调查, 特点是刚性和变形红细胞在循环中存在, 如镰状细胞贫血。

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

Research

JoVE Journal

Immunology and Infection

Depletion of Specific Cell Populations by Complement Depletion

补消耗的特定细胞群的枯竭

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

科研

免疫与感染

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

体外红血细胞溶血含量的生物大分子药物的细胞内交货的评价的pH响应Endosomolytic代理

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

定量检测抗体诱导补体激活的方法对红血细胞

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

由成比例的荧光显微镜测量吞噬体pH值

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

人体血液中测粒及单核细胞吞噬功能和呼吸爆发活动

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

一个简单的红细胞溶解法对B淋巴母细胞系的建立

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...