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) 之间在37.0 ±0.5 ° c。高渗 PVP 解决方案应该有一个渗透在764和 804 mOsm/千克之间, pH 值为 7.25-7. 45 在25° c 和黏度措施 27.0-33.0 cP 在37.0 °0.5 ° c。</li>
	<li>确保鲍勃被完全调到杯子里。启动软件和素数的机器 (硬件检查 |仪器 IO)。允许仪器完成启动周期。一旦循环完成, 把鲍勃从杯中取出, 并完全干燥的鲍勃和杯与低皮棉清洁组织。 注意:残余水会溶解红细胞, 产生干扰。</li>
</ol>2. 测量变形性作为增加剪切应力的函数
<ol><li>获得血液 (少于1毫升是足够的执行这些技术与复制) 在瓶中含有适当的抗凝剂。 注意:EDTA 优于肝素, 因为它对血液流变学参数影响较小。如果在6小时的采血中进行测量, 则33在室温下保持血液。</li>
	<li>在测试前, 要轻轻地将整个血样混合, 将瓶子多次倒置。用移将25µL 的全血加到5毫升的 iso 渗 PVP 溶液中, 将瓶子盖上, 然后轻轻地用反转几次。iso-渗 PVP 解决方案应该有一个渗透之间的284和 304 mOsm/千克, pH 值 7.3-7.4 在25° c 和粘度 27.0-33.0 cP 在37.0 ±0.5 ° c。</li>
	<li>在软件上从主菜单中选择变形。创建新的分析并添加实验细节 (变形 |添加所需的详细信息 |好的)。<ol>
<li>将盖子升至 ektacytometer, 确认鲍勃已完全放进杯中, 杯子正在转动。</li>
		<li>加入1毫升的 PVP 血液溶液进入杯和鲍勃之间的空间, 由移。</li>
		<li>稍微提起鲍勃, 把样品拿下来。等到所有的气泡都移出解决方案, 然后关闭盖子的 ektacytometer。如果需要, 按下 "抽吸" 按钮以帮助消除气泡。</li>
	</ol>
</li>
	<li>通过移动软件上滚动条上的箭头将增益调整到 200 (请参阅注释)。当温度稳定在37° c 和衍射图像是稳定的, 按开始 (开始)。 注意:对于许多研究, 一个良好的衍射图像可以获得从健康的血液 (血红蛋白浓度 > 12.0 克/dl, 平均红细胞体积 80-96 fL 和平均红细胞血红蛋白浓度33-36 克/dl) 与相机增益设置为200。为了研究镰状细胞贫血的血液, 我们建议调整相机增益以生成4.5 厘米衍射图像, 以此作为默认设置, 以便对研究和实验室的结果进行比较。9
</li>
	<li>观察衍射图样作为数据获取的进展, 以确保它们保持圆形, 椭圆形或菱形。完成数据获取后, 保存或打印报表 (文件 |保存或文件 |打印)。EI 最大和 SS ½值将自动报告, 随着延伸指数对应于用户指定或缺省剪应力。软件也将自动保存数据。</li>
	<li>最后, 按 "计算机监视器" 上对话框上的 "清除" 选项 (清除)。样品吸气后, 通过将去离子水喷入太空, 在仪器保持清洁循环的同时, 冲洗杯与鲍勃之间的空隙。一旦清洁循环完成后, 将 bob 从杯中取出, 并将 bob 和杯中的清洁剂完全晾干 (关键: 剩余的水会溶解红细胞, 产生干扰)。</li>
	<li>单击软件上的主菜单按钮返回主页 (主菜单)。</li>
</ol>3. 测量细胞异质性
<ol><li>在测试前, 要轻轻地将整个血样混合, 将瓶子多次倒置。吸管25µL 的全血到一个新的5毫升的 iso 渗 PVP 溶液, 瓶盖瓶和混合轻轻地倒置, 直到混合物是均匀的。</li>
	<li>从主菜单中选择变形。创建新的分析并添加实验细节 (变形 |添加所需的详细信息 |好的)。<ol>
<li>将盖子升至 ektacytometer, 确认鲍勃已完全放进杯中, 杯子正在转动。</li>
		<li>吸管1毫升的 PVP 血液溶液进入杯和鲍勃之间的空间。</li>
		<li>等到所有的气泡都移出解决方案, 然后关闭盖子的 ektacytometer。</li>
		<li>确保屏幕上存在稳定的衍射图像。通过在软件上沿滚动条移动箭头来调整相机增益, 直到产生3.8 厘米的衍射高度。使用标尺验证计算机屏幕上图像的高度。</li>
	</ol>
</li>
	<li>当温度稳定在37° c 和衍射图像是稳定的, 按开始 (开始)。</li>
	<li>观察衍射图样, 作为数据获取的进展, 以确保它们保持圆形, 椭圆形或菱形。完成数据获取后, 保存或打印报表 (文件 |保存或文件 |打印)。</li>
	<li>最后, 在 "计算机监视器" (干净) 上的对话框上按 "干净" 选项。在样品被吸气后, 用喷水从瓶子中喷射去离子, 然后在清洁的循环中, 冲洗杯子和鲍勃之间的空隙。一旦清洁循环完成后, 将 bob 从杯中取出, 并将 bob 和杯中的清洁剂完全晾干 (关键: 剩余的水会溶解红细胞, 产生干扰)。</li>
	<li>重复步骤 2.1-2.5, 并调整相机增益, 以获得4.5 厘米衍射图案高度 (步骤 2.2)。</li>
	<li>重复步骤 2.1-2.5, 并调整相机增益, 以获得5.4 厘米衍射图案高度 (步骤 2.2)。</li>
	<li>最后, 单击主菜单按钮返回到软件的主页 (主菜单)。</li>
	<li>为了确定以 EI Max 为百分比的衍射图样失真度, 请使用以下等式 (如果需要, 可以用 4.5 cm 衍射图样高度的数据执行相同的公式): <img alt="Equation 1" src="/files/ftp_upload/56910/56910eq1v2.jpg">
</li>
	<li>同样, 要确定基于 SS1/2 的衍射图样失真度, 使用与报告的 SS1/2 值相同的方程: <img alt="Equation 2" src="/files/ftp_upload/56910/56910eq2v2.jpg">
</li>
</ol>4. 渗透梯度 ektacytometry
<ol><li>获取示例, 如1.1 所述。在测试前, 轻轻地混合整个血液样本。将250µL 的全血加到5毫升的渗 PVP 瓶中, 由移, 盖上瓶子, 然后轻轻地混合, 直到混合物是均匀的。</li>
	<li>从主菜单 (osmoscan) 中选择 osmoscan。将血液 PVP 溶液放在机器 left-hand 一侧的针头下面。把针放下, 直到它触及瓶子的底部。确保油管正确连接到低和高渗解决方案的梯度生产。关闭 ektacytometer 的盖子, 打开下半部分的门, 这样你就可以看到血液进入油管。</li>
	<li>按新的分析和类型在实验细节 (Osmoscan |最新分析 |输入所需的详细信息 |好的)。调整相机增益到 200, 通过移动箭头控制它的软件, 并允许机器运行, 直到血液被看到进入杯从油管下的仪器。<ol>
<li>一旦血液进入了杯子和一个稳定的衍射图案图像在计算机屏幕上, 通过按下对话框上的 "立即开始" 按钮 (现在开始) 来开始数据采集。</li>
	</ol>
</li>
	<li>允许 ektacytometer 获得大约 500 mOsm/千克的数据, 然后停止仪器。保存或打印报表 (文件 |保存或文件 |打印)。数据也将自动保存。</li>
	<li>取出旧的 PVP-血瓶。用含有去离子水的干净的瓶子代替它。把它放在针下面, 把针放下, 使它触及瓶子的底部, 然后按下对话框中的冲洗按钮来冲洗渐变系统 (冲洗)。</li>
	<li>冲洗完成后, 在 "计算机监视器" 上的对话框上按 "清除" 选项 (清除)。一旦清洁循环完成后, 将 bob 从杯中取出, 并将 bob 和杯中的清洁剂完全晾干 (关键: 剩余的水会溶解红细胞, 产生干扰)。 注意:osmoscan 报告提供贯穿渗透梯度的伸长指数。报告中自动生成 ei 极小、o (ei min)、ei 最大、o (ei max)、ei 超和 o 超高。从9健康志愿者血液中获得的渗透梯度 ektacytometry 参数的范围是: EI Min 0.12-0. 196 任意单位 (a.u);O 最小 117-144 mOsm/千克;EI 最大 0.551-0. 573 a.u;O (EI Max) 272-312 mOsm/千克;EI 超级 0.278-0. 286;O 超高 454-505 mOsm/千克。</li>
</ol>5. 关闭 ektacytometer
<ol><li>在关闭仪器之前, 应将其正确清洗。<ol>
<li>要做到这一点, 连接的油管从低和高渗解决方案的 y 适配器导致清洁解决方案。将装有清洁液的小瓶放在机器 left-hand 一侧的针下面, 然后把针头放下, 直到它触及瓶子的底部。确保鲍勃被完全调到杯子里。</li>
		<li>关闭该软件, 然后在计算机监视器上的 "每天结束时按下开始" 对话框 (关闭 |启动)。允许仪器完全循环通过清洁。</li>
	</ol>
</li>
	<li>将油管与废瓶断开, 并将其从仪器中取出以丢弃废料。把鲍勃彻底干了。关掉机器</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+membrane+concept+applied+to+the+analysis+of+fluid+shear-+and+micropipette-deformed+red+blood+cells.">New membrane concept applied to the analysis of fluid shear- and micropipette-deformed red blood cells.</a> Biophys J. 13 (9), 941-954 (1973).</li><li>Chen, X., Feng, L., Jin, H., Feng, S., Yu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+erythrocyte+deformability+using+atomic+force+microscopy:+correlation+study+of+the+erythrocyte+deformability+with+atomic+force+microscopy+and+hemorheology.">Quantification of the erythrocyte deformability using atomic force microscopy: correlation study of the erythrocyte deformability with atomic force microscopy and hemorheology.</a> Clin Hemorheol Microcirc. 43 (3), 243-251 (2009).</li><li>Musielak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cell-deformability+measurement:+review+of+techniques.">Red blood cell-deformability measurement: review of techniques.</a> Clin Hemorheol Microcirc. 42 (1), 47-64 (2009).</li><li>Dobbe, J. G., Streekstra, G. J., Hardeman, M. R., Ince, C., Grimbergen, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+the+distribution+of+red+blood+cell+deformability+using+an+automated+rheoscope.">Measurement of the distribution of red blood cell deformability using an automated rheoscope.</a> Cytometry. 50 (6), 313-325 (2002).</li><li>Dobbe, J. G., 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=Analyzing+red+blood+cell-deformability+distributions.">Analyzing red blood cell-deformability distributions.</a> Blood Cells Mol Dis. 28 (3), 373-384 (2002).</li><li>Kikuchi, Y., Arai, T., Koyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+filtration+method+for+red+cell+deformability+measurement.">Improved filtration method for red cell deformability measurement.</a> Med Biol Eng Comput. 21 (3), 270-276 (1983).</li><li>Moessmer, G., Meiselman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+micropore+filtration+approach+to+the+analysis+of+white+cell+rheology.">A new micropore filtration approach to the analysis of white cell rheology.</a> Biorheology. 27 (6), 829-848 (1990).</li><li>Guo, Q., 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=Microfluidic+analysis+of+red+blood+cell+deformability.">Microfluidic analysis of red blood cell deformability.</a> J Biomech. 47 (8), 1767-1776 (2014).</li><li>Doh, I., Lee, W. C., Cho, Y. H., Pisano, A. P., Kuypers, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformation+measurement+of+individual+cells+in+large+populations+using+a+single-cell+microchamber+array+chip.">Deformation measurement of individual cells in large populations using a single-cell microchamber array chip.</a> Appl Phys Lett. 100 (17), 173702-173703 (2012).</li><li>Baskurt, O. K., 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=Comparison+of+three+commercially+available+ektacytometers+with+different+shearing+geometries.">Comparison of three commercially available ektacytometers with different shearing geometries.</a> Biorheology. 46 (3), 251-264 (2009).</li><li>Baskurt, O. K., 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=New+guidelines+for+hemorheological+laboratory+techniques.">New guidelines for hemorheological laboratory techniques.</a> Clin Hemorheol Microcirc. 42 (2), 75-97 (2009).</li><li>Uyuklu, 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=Effects+of+storage+duration+and+temperature+of+human+blood+on+red+cell+deformability+and+aggregation.">Effects of storage duration and temperature of human blood on red cell deformability and aggregation.</a> Clin Hemorheol Microcirc. 41 (4), 269-278 (2009).</li><li>Uyuklu, M., Meiselman, H. J., Baskurt, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hemoglobin+oxygenation+level+on+red+blood+cell+deformability+and+aggregation+parameters.">Effect of hemoglobin oxygenation level on red blood cell deformability and aggregation parameters.</a> Clin Hemorheol Microcirc. 41 (3), 179-188 (2009).</li><li>Embury, S. H., Clark, M. R., Monroy, G., 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=Concurrent+sickle+cell+anemia+and+alpha-thalassemia.+Effect+on+pathological+properties+of+sickle+erythrocytes.">Concurrent sickle cell anemia and alpha-thalassemia. Effect on pathological properties of sickle erythrocytes.</a> J Clin Invest. 73 (1), 116-123 (1984).</li><li>von Tempelhoff, G. F., 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=Correlation+between+blood+rheological+properties+and+red+blood+cell+indices(MCH,+MCV,+MCHC)+in+healthy+women.">Correlation between blood rheological properties and red blood cell indices(MCH, MCV, MCHC) in healthy women.</a> Clin Hemorheol Microcirc. 62 (1), 45-54 (2016).</li><li>Da Costa, L., Galimand, J., Fenneteau, O., 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=Hereditary+spherocytosis,+elliptocytosis,+and+other+red+cell+membrane+disorders.">Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders.</a> Blood Rev. 27 (4), 167-178 (2013).</li></ol>

作者没有什么可透露的。

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 ...

measuring-deformability-red-cell-heterogeneity-blood

Research

JoVE Journal

Immunology and Infection

14.4K 次观看.  Saint Louis University School of Medicine. 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。

视频: Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

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)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

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

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 anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

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

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. 
In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

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

A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.

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

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

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

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using 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

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

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

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

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

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

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

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 method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

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 ...