我们感谢献血者和加拿大血液服务网络应用开发中心的人员, 用于收集和生产本研究中使用的血小板单位。我们承认加拿大创新基金会和迈克尔. 史密斯基金会在不列颠哥伦比亚大学血液研究中心提供基础设施的健康研究。该出版物的资金由 ThromboLUX 制造商 LightIntegra 技术 Inc. 提供。

按照加拿大血液服务 (CBS) 指南执行了以下协议。志愿者们同意他们的捐赠可以用来进行这些研究。根据 CBS 标准操作程序制备血小板浓缩物。这里描述的所有性能测试都是在加拿大温哥华的不列颠哥伦比亚大学血液研究中心进行的, 并获得了机构研究伦理委员会的批准。
1. 质量控制检查
<ol><li>在每个测试日至少执行一次质量控制检查, 以验证 dl 系统的正确操作。测量提供的控制珠 (50 nm 半径) 按照制造商的指示使用。</li>
	<li>从冷库中回收控制瓶, 并允许15分钟至室温温度。在使用前, 珠子必须平衡到室温。</li>
	<li>在准备样品前打开 dl 系统, 以便在第一个样品准备就绪时完成15分钟激光预热周期。</li>
	<li>一旦控制台启动, 按照触摸屏上的说明进行登录, 并选择系统测试选项来测量控制样本。</li>
	<li>漩涡混合瓶十年代, 以确保一个代表性的样品。</li>
	<li>用100µL 固定容积的吸管、吸管尖端和毛细管从测试套件中填充毛细管与控制珠。</li>
	<li>测试完成后, 按照 dl 系统屏幕上的说明进行操作。 注: 控制珠测试结果将自动与控制珠条码标签中存储的信息进行比较, 并在测试结束时显示在 dl 系统屏幕上的传递或失败通知以及示例信息。</li>
</ol>2. 从血小板浓缩物中提取样本
注: 这些步骤描述了非侵入性取样的过程, 从血小板输血到 dl 测试毛细管的常规血小板库存管理。概述如图 1所示。所需配件: 管封口机, 手动管剥离, 剪刀, 飞溅盾。
<ol><li>从未经检验的库存中取出血小板浓缩物。</li>
	<li>从未使用的取样袋 (进入步骤 2.3) 或油管段获得血小板产品样本 (如果为空或步骤 2.5, 如果不为空, 则继续执行步骤 2.4)。 断开邮袋油管或油管段使用管封口机。要操作管封口机, 请遵照制造商的指示。简单地, 钳夹在两个加热颌骨之间的精确位置密封油管。在手持设备中, 将熔化的油管压在一起并在高压下冷却, 同时按下拉杆 (持续时间由绿灯指示), 从而产生永久性的防漏密封。</li>
	<li>
从邮袋取样
	<ol>
<li>将血小板袋的含量与5秒的平缓水平运动混合均匀 (从端到端到五次)。</li>
		<li>打开夹子到邮袋。邮袋被疏散, 将自行填补。这是没有必要填写完整的邮袋, 因为只有100µL 样品将需要 dl 测试。</li>
		<li>通过热密封连接油管与管封口机, 从袋子上拔下邮袋。继续执行步骤3。</li>
	</ol>
</li>
	<li>
从空油管取样
	<ol>
<li>验证血小板袋管是否已被储存, 油管块仍然到位。目视检查油管, 以验证是否有大量的血小板集中在油管。如果油管不是空的继续与步骤2.5。</li>
		<li>关闭油管剥离管, 尽可能靠近油管块, 通过压缩手柄挤压油管之间的轧辊。</li>
		<li>将包垂直挂起, 并继续压缩脱模手柄。</li>
		<li>在保持剥离的同时, 松开油管块。</li>
		<li>慢慢地把汽提拉下来的空油管, 让油管慢慢填补后面的脱衣舞女。继续, 直到脱模剂下面的部分完全充气或直到脱模剂在油管末端的1英寸以内。</li>
		<li>一旦停止点到达与剥离, 使用管封口机热密封油管1英寸以上的脱衣舞女。</li>
		<li>松开手动管剥离手柄。</li>
		<li>热密封再次1至2英寸以上的前密封创建测试段。</li>
		<li>从血小板单元中切断测试段。此段将用于 dl 测试。</li>
	</ol>
</li>
	<li>
从非空管取样
	<ol>
<li>垂直挂袋, 并释放油管块, 如果到位。</li>
		<li>目视检查油管, 以确定是否有任何重要的固体团簇。如果存在固体团块, 密封在它们上面, 这样它们就不能被剥离到袋子里。</li>
		<li>关闭导管上的管剥离, 尽可能靠近油管的密封端。</li>
		<li>通过压缩手柄, 在滚筒之间挤压油管, 并在保持夹紧力的同时, 将管子的管板移到袋子上, 从而将油管中的管道剥离。</li>
		<li>从吊钩上取下血小板袋, 并将其从端到端五次, 轻轻地将包放在 5 s 上, 同时保持剥离。</li>
		<li>在保持脱衣舞女的同时, 垂直挂袋。</li>
		<li>慢慢地把汽提拉下来的空油管, 允许油管慢慢填补后, 脱衣舞女。继续, 直到脱模剂下面的部分完全充气或直到脱模剂在油管末端的1英寸以内。</li>
		<li>一旦停止点到达与剥离, 使用管封口机热密封油管1英寸以上的脱衣舞女。</li>
		<li>松开脱衣舞娘</li>
		<li>热密封再次1至2英寸以上的前密封创建测试段。</li>
		<li>切断和丢弃的最后一部分的油管。</li>
		<li>从血小板单元中切断测试段。此段将用于 dl 测试。</li>
	</ol>
</li>
</ol>3. 将样品装入 dl 测试毛细管
<ol><li>使用飞溅盾牌和清洁, 干剪刀, 削减邮袋油管或测试段的一端。 注: 用于 dl 测试的毛细管应立即填充。</li>
	<li>使用取样工具 (组装100µL 固定容积的吸管、吸管尖端和毛细管) 将毛细管直接从打开的邮袋或导管段中抽取到毛细管中。</li>
	<li>将填充毛细管的底部轻轻地推入毛细管密封胶, 同时施加温和的扭曲和对托盘的压力。</li>
	<li>断开100µL 固定容积的吸管和针尖从毛细管, 确保毛细管仍然牢牢地嵌入毛细管密封胶。</li>
	<li>从毛细管密封胶盘中取出毛细管, 用异丙醇垫擦拭, 并确保没有气泡被轻轻地轻拂底部毛细管。</li>
	<li>将毛细管放入 dl 系统。</li>
</ol>4. 执行 dl 测试
<ol><li>选择 MP 测试选项以启动新的 dl 测试, 用于测量血小板样本的微粒含量。</li>
	<li>使用条码扫描仪或手动输入示例信息 (捐赠标识号 (ISBT)、收集/生产过期日期和产品代码)。</li>
	<li>如果没有产品代码可供 dl 系统提取流体介质信息, 请手动选择适当的流体介质 (对于大多数血小板浓缩物选择血浆, 但对于含有血小板添加剂溶液的样品, 请输入残余血浆百分比)。 注意: 从标称65% 的 PAS 百分比中的微小偏差将导致粘度 (由 dl 系统自动计算) 的一个小错误, 它最小地影响计算出的 MP 半径, 但不%MP。</li>
	<li>当指示和启动测试时, 将毛细管放入 dl 系统。</li>
	<li>测试完成后, 将样品从毛细管夹中取出, 并根据设施指南适当地处理消耗品。将样品从毛细管夹中取出, 并根据设施指南适当地处理消耗品。</li>
	<li>标记与结果对应的颜色的血小板袋, 例如橙色为非激活 (同类) 与%MP 相等或在15% 和桃红色为激活 (异质) 与%MP 在15% 之上。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56893/56893fig1.jpg"> 图 1: 方法概述.医院血库常规血小板库存管理应采取的步骤概述: 从血小板集中获取样本, 将样品加载到毛细管中进行 dl 测量, 执行 dl 测试以识别微粒使用所报告的微粒含量来识别活化的血小板。<a href="//cloudflare.jove.com/files/ftp_upload/56893/56893fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

EMS 是 LightIntegra 技术 Inc. 的创始人, 它是一家医疗器械公司, 开发了 ThromboLUX 微粒和血小板质量检测系统。所有其他作者都是 LightIntegra 技术的员工。这篇文章的制作和免费访问是由 LightIntegra 技术 Inc. 赞助的。

本协议描述了一种动态光散射方法, 用于对生物样品中的高颗粒浓度 (如血小板浓缩物) 进行优化的微粒筛选。dl 的方法本质上是标准化的, 以精确测量大小。如果已知血小板浓度, 以血小板峰值区为参考峰值19, 则微粒的相对浓度可转化为绝对浓度。由于血小板浓度通常是通过血液分析仪或血流 cytometers, 这些方法可以被认为是辅助技术的 dl。
dl 系统的功能是通过定期运行控制珠来保险的。蒸馏水可以测量, 以验证背景噪音是最小的。商业可用的血小板标准可以被分析为 mp 阴性控制, 并且, 在加法125毫微米半径小珠以后, 作为 mp 正面控制。在 MP 的生物学范围内的兴趣, 这些程序是切实可行的, 并迅速执行的一部分, 血库例行。
与流式细胞术相比, 这种方法不是基于粒子的散射强度, 而是以其布朗运动的速度。因此, 体也可以检测, 尽管它们的体积较小, 并与 MP 分开报告。
作为一种筛选工具, 这种方法的局限性在于它不能区分不同类型的微粒。如果使用额外的隔离步骤, 可能有改进的余地;样品可以通过抗体耦合磁珠捕获, 在特定的微粒去除之前和之后进行测试。此外, 不能假定所有检测到的微粒都是细胞衍生的, 因为乳糜在高脂血症中形成的47,48和小细菌或病毒6也将在微粒范围内报告。然而, 在血液供应的其他保障措施, 以避免高度 lipemic 或受污染的血小板进入医院血库库存。
选择抗凝剂在样品中影响血小板活化的程度, 因此 MP 内容49。为了比较不同的产品, 需要考虑这个因素。此外, 与 mp 自由悬浮介质 (如 PAS) 交换等离子体将影响 mp 含量和确定异质性的阈值--如果只有大约三分之一的原 mp 内容留在浓缩物的残余等离子体中因此, 较低的 MP 含量阈值将表明与100% 血浆中的血小板活化水平相同。mp 百分比是相对于血小板的 mp 含量。据先前报道, PAS 产品的平均血小板计数较低, 平均%MP 仍为 9.5%19。美国许可的 PAS 血小板的%MP 阈值目前已设置为10%。
虽然血小板浓缩物中 mp 的主要来源是捐献者, 但对血小板产生压力的过程会根据血小板的易感性而增加 mp 水平--如果血小板已经高度活化, 小的压力源, 如延长货架期、病原体灭活、洗涤、辐照或长途运输可能导致 MP 含量显著增加。这些压力源都没有表现出明显的影响同质的, 非活化的血小板19。此外, 如果没有在准备后立即测试 (完成本议定书的步骤 3), 则应注意毛细血管内样本成分变化的可能性。
本议定书的重点是确定在血小板输注中存在的微粒组成和使用微粒作为血小板活化的生物标志物。血小板输注被标记为非活化 (橙色) 或活化 (粉红色) 基于微粒百分比阈值为15%。在100% 血浆中, 15% MP 的血小板阈值是由多个站点的平均 66th百分比确定的。
以常规微粒筛查为基础的血小板库存管理的目的是通过预防非免疫性血小板的顽固性, 提高患者的护理效率和降低成本。实施的 dl 系统筛选血小板袋将使用户引导非活化血小板的病人群体中最有风险的发展血小板的耐火。

对微粒的兴趣主要围绕在细胞与细胞的通讯和生物过程中。1,2,3最近, 微粒还吸引了人们对自身免疫和心血管疾病潜在的早期诊断标志的兴趣。4,5微粒, 也被称为胞外泡或体, 已被广泛研究的流式细胞仪。不幸的是, 尽管努力规范常规流式细胞术协议, 但对于使用的最佳协议却没有达成共识。6,7,8,9,10
虽然传统的流式细胞仪可以表征特定的 MP 亚群, 但11有几个报告的限制。11,12,13,14这些限制中的一些已经通过在所谓的 "小粒子选项"、15、16中使用更高功率的激光器和探测器来解决, 以及在 15-°前向散射角和改进的鞘压力检测.17,18然而, 使用微粒作为早期诊断标记仍然需要一种可以与这些复杂方法相结合的快速易用的筛选方法。在正常献血者中, 大约三分之一的人可以检测到高浓度的微粒19 , 并可能表明亚临床条件。因此, 捐血产品中的高微粒含量可能与易受感染的人不相容。20
当提供血小板输注时, 有一种非免疫性顽固性的风险--一种情况, 病人的身体拒绝连续输血, 不允许大量的血小板循环。21,22非免疫难治可导致输血浪费、患者的健康并发症和延长住院时间。23含有大量微粒的血小板输注可以成为易受伤害的血液肿瘤患者非免疫性难治的原因之一。20可以根据血小板输血的成分, 通过筛选微粒来管理血小板清单, 从而减少易受伤害患者的风险。然而, 大多数微粒测试需要分离微粒从血小板5,12或其他非常劳动密集型24,25 , 因此不能在医院中例行实施血库
这里描述的技术使用动态光散射 (dl)-也称为光子相关光谱或准弹性光散射。几十年来, 动态光散射已广泛应用于制药行业, 以表征脂质体药物配方或乳液的颗粒大小在亚微米范围。26,27但是, 为这些应用程序开发的工具没有经过优化以筛选血液产品。开发了一个新的 dl 系统, 以克服技术上的限制, 并使动态光散射有用的筛选血小板输血。28
动态光散射测量是通过对悬浮粒子进行激光照射, 并分析散射光强度的时间变化, 这是悬浮粒子运动的结果。此外, 该方法利用粒子速度与粒径之间的反比关系--小颗粒移动速度快, 大颗粒缓慢移动--以提供有关样品组分的大小分布和相对浓度的信息。利用 dl, 可以对微粒组分的平均微粒含量和平均半径进行量化。在血液制品中微粒的含量是以%MP 为基础的, 根据测量的直方图中的面积, 从 50-550 nm 的粒子半径。当有半径低于 50 nm 的粒子被 dl 检测并由 dl 系统报告时, 它们不包括在%MP 中。而不是从血小板中分离微粒, 根据样品中微粒和血小板的相对含量确定血小板输注的异质性。事实上, 当血小板计数已知时, 血小板和微粒峰的比值可以用来计算绝对 MP 浓度。19
dl 系统为卫生保健专业人员提供了关于从人体血液或血液制品中提取的样品中的微粒的定性和定量信息。所描述的 dl 技术的主要优势, 替代技术, 如流式细胞仪, 电子显微镜,29纳米粒子跟踪分析30或可调谐电阻脉冲传感31是样品准备:血小板浓缩物的等分可以直接测量, 而不需要将 MP 与血小板分离, 样品稀释或其他修饰。9,17此外, dl 是一种绝对大小的方法, 不受缺少适当折射率的校准珠子的影响。14,32
血小板活化与 mp 含量之间的相关性先前已通过显微镜观察33,20 , 并可从病理条件中 MP 内容的增加推断出15,34,35,36和体外条件下已知激活血小板。19,37,38然而, 需要进一步的研究来充分理解 dl 测量的微粒含量和血小板活化的关系。根据我们目前的知识, 活化的血小板含有大量的微粒, 它们是最好的治疗积极出血患者39, 而血液肿瘤患者受益于非激活无或低微粒水平的血小板20。最近有报道说,体外捐献血小板的反应对这些血小板在单个输血中的恢复和存活没有显著的影响, 而对稳定的, 大多数是非出血性血液肿瘤患者,40.从这一发现可以推断, 高 MP 含量的血小板活化在预防性治疗中也起不到重要作用。然而, 由于患者的选择范围狭窄, 这项研究并没有考虑到供体因素对发热 (不包括研究) 的复杂患者的影响, 不稳定, 而且接受的不仅仅是一个血小板输血。如何减少这些病人病例的复杂性的问题--它持有减少顽固性的承诺--仍然没有得到解答。
微粒是炎症的早期标记41,42,43,44和血小板活化45 , 因此在许多正常的捐赠者中被检测到。19因此, 血小板捐献中存在活化的血小板和微粒。有理由推测, 发烧的病人, 即一个完全活化的先天免疫系统, 不能容忍额外的挑战, 输血的活化血小板。然而, 需要研究来证明这个假说。微粒筛选可以减轻目前对血小板输注含量的不确定性, 降低患者治疗的复杂性。
在医院血库中, 活化到非活化血小板的比例主要依赖于捐献者的数量, 而在运输、辐照、病原体失活和其他可能增加血小板活化的过程中, 其程度要低得多集中.19来自美国主要医院血库的数据显示, 血小板清单的平均成份为49% 活化, 51% 非活化 (活化血小板范围:38-62%, 个人通讯)。如果血液产品提供者或医院血库想知道他们生产或接受了多少活化和非活化的血小板集中, 并且想要管理他们的存货基于微粒内容表明的血小板活化作用, 这协议可能适合他们。
根据血小板组成, 医院血库将能够指导非活化、均质血小板的预防性使用和活化、异种血小板的治疗用途。血小板筛查可使医院最大限度地利用现有库存, 从而改善病人护理并降低成本。本协议适用于熟悉血液产品基本操作和操作的实验室人员。
本文介绍了一种用于血小板输注中微粒的筛选方法, 可用于管理医院血库库存, 在该库中选择基于微粒含量的产品是可取的。本议定书的目的是概述用于筛查捐献血小板的 dl 的实施和评价。所描述的协议解决了对样本进行无侵入访问的常见问题, 将测试集成到血库工作流程中, 以及性能特征。

<table><tbody><tr><td>ThromboLUX-M</td><td>LightIntegra Technology Inc.&amp;nbsp;</td><td>LPN120</td><td>DLS System</td></tr><tr><td>ThromboSight Software</td><td>LightIntegra Technology Inc.&amp;nbsp;</td><td>LPN124</td><td>DLS analysis software</td></tr><tr><td>Capillaries</td><td>LightIntegra Technology Inc. (included in test kit, vendor Optinova MLE)</td><td>LPN065</td><td>Custom extruded non-activating, non-birefringent plastic tube, inspected and irradiated</td></tr><tr><td>MiniPet&amp;nbsp;</td><td>LightIntegra Technology Inc. (included in test kit, vendor TriContinent Scientific)</td><td>LPN082</td><td>100 &amp;micro;L fixed volume pipette for sampling&amp;nbsp;&amp;nbsp;</td></tr><tr><td>MicroFlex 1-200 &amp;micro;L Pipette tips</td><td>LightIntegra Technology Inc. (included in test kit, vendor ESBE Scientific)</td><td>LPN080</td><td>Pipette tips for sampling</td></tr><tr><td>Critoseal&amp;nbsp;</td><td>LightIntegra Technology Inc. (included in test kit, vendor Fisher Scientific)</td><td>LPN075</td><td>Capillary Tube Sealant</td></tr><tr><td>Dukal Alcohol Pad or equivalent</td><td>LightIntegra Technology Inc. (included in test kit, vendor Dukal Corp.)</td><td>LPN085</td><td>Isopropyl pad (saturated with 70% isopropyl alcohol)</td></tr><tr><td>Control beads, 50 nm radius</td><td>LightIntegra Technology Inc. (included in test kit, vendor PolySciences Inc.)</td><td>LPN128</td><td>Control beads</td></tr><tr><td>Microbead NIST Traceable Particle Size Standard, 3 &amp;micro;m</td><td>PolySciences Inc.</td><td>64060</td><td>Standard microbeads: 1.5 &amp;micro;m radius Polystyrene beads; range 1.43-1.58 &amp;micro;m; 1% solids suspensions in de-ionized water</td></tr><tr><td>Nanobead NIST Traceable Particle Size Standard, 250 nm</td><td>PolySciences Inc.</td><td>64014-15</td><td>Standard nanobeads: 125 nm radius Polystyrene beads; range 120 - 130 nm; 1% solids suspensions in de-ionized water</td></tr><tr><td>Genesis BPS RapidSeal II or equivalent</td><td>Genesis BPS</td><td>428-SE640</td><td>Tube sealer</td></tr><tr><td>Fresenius Kabi Tube Stripper or equivalent</td><td>Fresenius Kabi</td><td>6R4452</td><td>Manual tube stripper</td></tr><tr><td>Biohazard Shield - Cell Counter or equivalent</td><td>CardinalHealth</td><td>S1389-75</td><td>Splash Shield</td></tr><tr><td>Corning LSE Vortex Mixer or equivalent</td><td>Corning Incorporated</td><td>6775</td><td>Vortex mixer</td></tr><tr><td>FACSCanto II Flow cytometer&amp;nbsp; with FACSDiva software version 6.1.3 or equivalent</td><td>BD Biosciences</td><td>338960</td><td>Flow cytometer</td></tr></tbody></table>

routine screening method for microparticles in platelet transfusions

血小板集中筛查微粒含量是医院血液库质量改善的新举措。当它们被压力时, 细胞就会脱离微粒 (MP)。血液和血液成分可能含有来自各种细胞的细胞片段, 最显著的是来自活化的血小板。当在凝血和止血中扮演先天免疫细胞和主要角色时, 血小板会改变形状并产生微粒。以动态光散射 (dl) 为基础的微粒检测, 有可能区分活化 (高微粒) 从非活化 (低微粒) 血小板在输血, 并优化使用这种稀缺的血液产品。先前的研究表明, 提供非活化血小板预防性使用血液肿瘤患者可以降低其成为难治性和改善病人护理的风险。这种筛选方法的目的是定期区分激活的非活化血小板。此处描述的方法概述了在医院血库中常规血小板库存管理的步骤: 从血小板输血中获取样本, 将样本加载到毛细管中进行 dl 测量, 执行 dl 测试以识别微粒, 并使用所报告的微粒含量来识别活化的血小板。

平均准备时间 在图 1中总结了使用 dl 系统进行血小板筛选的过程。血小板在从血液供应商收到的时候使用 dl 系统进行测试。如表 1中所述, 经过培训的用户的平均准备时间为 2 min 二十三年代, 而清理和后测试工作次数分别为十四年代和四十六年代。总体而言, 每个测试的用户平均需要3分钟和二十三年代的时间。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>活动</td>
			<td>活动时间</td>
			<td>离开测试时间</td>
			<td>总</td>
		</tr>
<tr>
<td>准备 dl 系统</td>
			<td>十四年代</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>装配取样工具</td>
			<td>二十八年代</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>获取段</td>
			<td>五十二年代</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>填充毛细管并开始测试</td>
			<td>四十九年代</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td>5分钟</td>
			<td></td>
		</tr>
<tr>
<td>清理</td>
			<td>十四年代</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>标签和库存血小板袋</td>
			<td>四十六年代</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>每个示例所需的总时间</td>
			<td>3 min 二十三年代</td>
			<td>5分钟</td>
			<td>8 min 二十三年代</td>
		</tr>
</tbody></table>表 1: 活动测试时间故障.测试本身是一个步行距离测试, 平均持续时间为5分钟。一般用户需要3分钟的二十三年代, 准备 dl 系统的测试, 获取和测试一个样本后, 该协议, 并标记血小板袋。
精度 对 dl 系统的精确度进行了三微粒水平、0-7%、12-25% 和28-75% 的评估。%MP 的临床相关范围为 3-75%。两个操作员在两个 dl 系统上并行测试了16工作日的低、中、高控制样本。样品的测试是重复的, 但在每个测试日的随机顺序。
表 2总结了与血小板相对的微粒 (%MP) 的 dl 测量的内部设备精度。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td colspan="3">微粒含量</td>
		</tr>
<tr>
<td></td>
			<td>低</td>
			<td>中</td>
			<td>高</td>
		</tr>
<tr>
<td>平均%MP (%)</td>
			<td>4。4</td>
			<td>19。5</td>
			<td>53。8</td>
		</tr>
<tr>
<td>标准偏差 (%)</td>
			<td>1。8</td>
			<td>2。6</td>
			<td>5。8</td>
		</tr>
<tr>
<td>CV (%)</td>
			<td>40。4</td>
			<td>13。2</td>
			<td>10。6</td>
		</tr>
</tbody></table>表 2: 在设备内精度百分比微粒 (%MP).在非常低的微粒含量小血小板可能导致%MP 导致增加的可变性为低微粒含量样品。
表 3显示了在 50-550 nm 之间的平均微粒半径的 dl 测量的内部设备精度。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td colspan="3">微粒含量</td>
		</tr>
<tr>
<td></td>
			<td>低</td>
			<td>中</td>
			<td>高</td>
		</tr>
<tr>
<td>平均半径 (nm)</td>
			<td>331</td>
			<td>161</td>
			<td>188</td>
		</tr>
<tr>
<td>标准偏差 (mm)</td>
			<td>133</td>
			<td>41</td>
			<td>25</td>
		</tr>
<tr>
<td>CV (%)</td>
			<td>40。1</td>
			<td>25。2</td>
			<td>13。5</td>
		</tr>
</tbody></table>表 3: 微粒半径内设备精度.在极低的微粒含量下, 小血小板可能会导致微粒含量 (%MP) 的增加, 从而导致低微粒样品的变异。
表 4显示了对百分比微粒 (%MP) 的 dl 度量的重现性。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td colspan="3">微粒含量</td>
		</tr>
<tr>
<td></td>
			<td>低</td>
			<td>中</td>
			<td>高</td>
		</tr>
<tr>
<td>平均%MP (%)</td>
			<td>4。4</td>
			<td>29。2</td>
			<td>53。6</td>
		</tr>
<tr>
<td>再现性 (%)</td>
			<td>1。5</td>
			<td>2。3</td>
			<td>5</td>
		</tr>
<tr>
<td>CV (%)</td>
			<td>35</td>
			<td>11。8</td>
			<td>9。4</td>
		</tr>
</tbody></table>表 4: 微粒 (%MP) 的动态光散射 (dl) 测量的重现性。
线性 图 2显示了 dl 结果是线性的,即, 它适合于与样本的赋值有关的一条直线。用不同的微粒含量制备了七样品。制备了高 (mp7) 和低 (mp1) 微粒含量和匹配血小板浓度的样品, 并以不同比例混合生成中间样品 (mpx)。样品进行了流式细胞仪测试, 如前所述19 , 每个浓度的 dl 和%MP 结果绘制为输入和输出。测定系数为0.985。低和高微粒含量的 dl 直方图示例如图 3A所示。流式细胞仪 (图 3B) 确认了 dl 结果。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56893/56893fig2.jpg"> 图 2: 流式细胞仪和 dl 结果的比较.用流式细胞术 (输入) 和 dl (输出) 确定的%MP 之间的线性关系, 由打开的符号0标记的两个样本中的原始 dl 数据显示在图 3中。<a href="//cloudflare.jove.com/files/ftp_upload/56893/56893fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56893/56893fig3.jpg"> 图 3: 微粒含量来区分活化和非活化的血小板。(A)激活血小板中 MP 含量 (虚线) 的结果与非活化血小板 (实线) 的4% 相比, 为57%。测试是在37° c 的测量温度下进行的, 等离子体粘度设置为 1.06 x10-3 Pa·s, 以及200-600 赫之间的总强度设置。(B)流式细胞仪结果 (如前所述, 得到的19) 与 (A) 中所示的样本相同;前向散射直方图 P1 和 P2 分别代表 MP 和血小板门;对于活化血小板 (左) 76% 的事件下降到 MP 门相比, 6% 的非活化血小板 (右)。线性回归线表明, 流式细胞术对%MP 的背景噪声具有恒定的相对贡献, 导致持续的更高的结果。<a href="//cloudflare.jove.com/files/ftp_upload/56893/56893fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
特异性/干扰 国际标准化组织 (ISO) 定义了分析的特异性作为测量过程的能力检测或仅测量测量, 当有其他数量存在于样品。微粒筛选的分析特异性可能受红细胞 (红细胞) 的影响。dl 测量 MP 含量相对于血小板含量。rbc 可能会干扰, 因为红细胞的散射贡献包括在血小板的散射贡献中, 减少了 MP 的相对贡献。血液制品中允许红细胞含量的调节限值存在;AABB 建议的阈值保守转换为血小板 concentrates-2 毫升的红细胞浓度在一个单位的血小板-导致 8.0 x1010细胞/l (假设: 红细胞体积是 8.5 x10-14l,红细胞压积为 68%, 血小板单位容积为200毫升)。报告的残留红细胞浓度在不同的产品中远远低于这个阈值46。
三不同的捐献者在两天内捐献了血小板和红细胞 (红细胞), 作为合格的三独立实验。血小板浓缩物中的初始红细胞含量 (参考样品) 为 0.05-0.15 x109细胞/L, 例测定。五个额外的样本是通过峰值-在已知数量的红细胞进入等分的血小板集中;这些样本中的目标红细胞水平分别为1.0、5.0、10、40和 80 x109单元格/L。
红细胞的干扰阈值约为 1.0 x1010细胞/L (图 4), 这也与红细胞在视觉上明显可见的水平 (图 5) 相关。在这一水平之上,%MP 被低估, 这意味着, 在视觉上的红色样本-其中含有红细胞而不是血红蛋白-报告的微粒含量将过低。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56893/56893fig4.jpg"> 图 4:%MP (dl) 与红细胞浓度 (细胞/L) 之间的线性关系.红细胞浓度增加 0.1 x109, 1.0 x109, 5.0 x109, 1.0 x1010, 4.0 x1010, 和 8.0 x1010单元格/L (从左到右) 从3独立实验 (0 实验 1, 实验 2,-实验3, 每个实验都显示线性回归线。1.0 x1010 RBC/L 导致对%MP 的低估。包含示例的 RBC 的外观显示在图 5中。<a href="//cloudflare.jove.com/files/ftp_upload/56893/56893fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56893/56893fig5.jpg"> 图 5: 包含样本的 RBC 的外观.含红细胞浓度 0.1 x109、1.0 x109、5.0 x109、1.0 x1010、4.0 x1010和 8.0 x1010单元格 (从左到右) 的发红。<a href="//cloudflare.jove.com/files/ftp_upload/56893/56893fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
精度 精确度定义为单个测量结果和分配给样本的真实数量值之间的差异。该测量误差包括由测量偏差估计的系统分量和由标准偏差估计的随机分量。因此, 测量结果的准确度是真实性和精确度的结合。
用磁珠标准测定了 dl 测试的准确度。准确性评估在两个浓度的临床相关范围内的 3-75% MP 的化验。参考珠混合物被用来准备样品已知的浓度, 因为参考珠有已知的大小和浓度。因此, 参考珠可以混合, 以获得样品与期望的颗粒大小和浓度。
标准聚苯乙烯珠与 125 nm 半径被用来代表微粒和珠与1.5 µm 半径被用来代表血小板。用大约20% 和 50% MP 含量的微珠混合物对微粒测定的准确度进行了评估。被测量的微粒半径的准确性被比较了被记录的微粒半径在分析的证明为参考小珠如显示在表 5。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td colspan="3">125 nm 珠</td>
			<td colspan="3">1.5 µm 珠</td>
		</tr>
<tr>
<td rowspan="2"></td>
			<td>分析证书</td>
			<td colspan="2"></td>
			<td>分析证书</td>
			<td colspan="2"></td>
		</tr>
<tr>
<td></td>
			<td>20% MP</td>
			<td>50% MP</td>
			<td></td>
			<td>20% MP</td>
			<td>50% MP</td>
		</tr>
<tr>
<td>平均半径</td>
			<td>122。0</td>
			<td>113。8</td>
			<td>124。4</td>
			<td>1。5</td>
			<td>1。6</td>
			<td>1.60</td>
		</tr>
<tr>
<td>Std 开发</td>
			<td>4。5</td>
			<td>2.83</td>
			<td>3。6</td>
			<td>0.035</td>
			<td>0.06</td>
			<td>0.07</td>
		</tr>
<tr>
<td>简历</td>
			<td>3.60%</td>
			<td>2.48%</td>
			<td>2.89%</td>
			<td>2.20%</td>
			<td>3.74%</td>
			<td>4.05%</td>
		</tr>
<tr>
<td>精度</td>
			<td></td>
			<td>6.70%</td>
			<td>2.00%</td>
			<td></td>
			<td>6.50%</td>
			<td>6.30%</td>
		</tr>
</tbody></table>表 5: 动态光散射 (dl) 测量的准确度.dl 的大小比较结果与 125 nm 半径和1.5 µm 半径的珠子的分析证书的混合物。

Watch this Scientific Journal Video about Routine Screening Method for Microparticles in Platelet Transfusions at JoVE.com

Routine Screening Method for Microparticles in Platelet Transfusions

血小板集中筛查微粒含量是医院血液库质量改善的新举措。目的是区分活化的非活化血小板, 以优化血小板的使用。提供非活化血小板给血液学肿瘤患者可能会降低其高风险成为难治性。

血小板输血中微粒的常规筛选方法

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

routine-screening-method-for-microparticles-in-platelet-transfusions

Research

JoVE Journal

Medicine

15.9K 次观看.  LightIntegra Technology Inc.. 血小板集中筛查微粒含量是医院血液库质量改善的新举措。目的是区分活化的非活化血小板, 以优化血小板的使用。提供非活化血小板给血液学肿瘤患者可能会降低其高风险成为难治性。

视频: Routine Screening Method for Microparticles in Platelet Transfusions

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

一个简单的协议介导的血小板聚集<em&gt;恶性疟原虫</em&gt;受感染的红细胞在一个资源匮乏的设置

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

科研

免疫与感染

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

微流体流钱伯斯再造使用血止血建模和血小板输注<em&gt;体外</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

生物工程

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

人脐血巨分化与血小板形成的 CD34+单元格

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage and that carries factor VIII in the circulation. VWF is synthesized by endothelial cells and is either released constitutively into the plasma or is stored in specialized organelles, called Weibel-Palade bodies (WPBs), for on-demand release in response to hemostatic challenge. Procoagulant and proinflammatory stimuli can rapidly induce WPB exocytosis and VWF release. The majority of VWF released by endothelial cells circulates in the plasma; however, a proportion of VWF is anchored to the endothelial cell surface. Under conditions of physiological shear, endothelial-anchored VWF can bind to platelets, forming a VWF-platelet string that may represent the nidus of thrombus formation. A flow chamber system can be used to visually observe the release of VWF from endothelial cells and the subsequent platelet capture in a manner that is reproducible and relevant to the pathophysiology of VWF-mediated thrombus formation. Using this methodology, endothelial cells are cultured in a flow chamber and are subsequently stimulated with secretagogues to induce WPB exocytosis. Washed platelets are then perfused over the activated endothelium. The platelets are activated and subsequently bind to elongated VWF strings in the direction of fluid flow. Using extracellular histones as a procoagulant and proinflammatory stimulus, we observed increased VWF-platelet string formation on histone-treated endothelial cells compared to untreated endothelial cells. This protocol describes a quantitative, visual, and real-time assessment of the activation of VWF-platelet interactions in models of thrombosis and hemostasis.

In this paper, we describe a method to assess endothelial von Willebrand factor release and the subsequent platelet capture under fluid shear stress in response to inflammatory stimuli using an in vitro flow chamber system.

血管性血友病因子-血小板串形成的流室模型研究血管血友病因子的病理生理学

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage ...

医学

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%&#8211;20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

使用毛细血管电泳免疫测定来寻找人类血小板肌萎缩性侧索硬化的潜在生物标志物

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant ...

神经科学

<meta charset="utf8"></head><body>使用荧光成像和 EPR 检测血小板中的超氧阴离子

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ability to react with important biological molecules such as proteins, nucleic acids, and lipids. Superoxide anions are important ROS generated by the reduction of molecular oxygen reduction (i.e., acquisition of one electron). Despite their initial implication exclusively in aging, degenerative, and pathogenic processes, their participation in important physiological responses has recently become apparent. In the vascular system, superoxide anions have been shown to modulate the differentiation and function of vascular smooth muscle cells, the proliferation and migration of vascular endothelial cells in angiogenesis, the immune response, and the activation of platelets in hemostasis. The role of superoxide anions is particularly important in the dysregulation of platelets and the cardiovascular complications associated with a plethora of conditions, including cancer, infection, inflammation, diabetes, and obesity. It has, therefore, become extremely relevant in cardiovascular research to be able to effectively measure the generation of superoxide anions by&#160;human platelets,&#160;understand the redox-dependent mechanisms regulating the balance between hemostasis and thrombosis and, eventually,&#160;identify novel pharmacological tools for the modulation of platelet responses leading to thrombosis and cardiovascular complications. This study presents three experimental protocols successfully adopted for the detection of superoxide anions in platelets and the study of the redox-dependent mechanisms regulating hemostasis and thrombosis: 1) dihydroethidium (DHE)-based superoxide anion detection by flow cytometry; 2) DHE-based superoxide anion visualization and analysis by single platelet imaging; and 3) spin probe-based quantification of superoxide anion output in platelets by electron paramagnetic resonance (EPR).

<meta charset="utf8"></head><body>作者聚焦：ROS 检测的创新技术及其对血小板研究的影响

The generation of superoxide anion is essential for the stimulation of platelets and, if dysregulated, critical for thrombotic diseases. Here, we present three protocols for the selective detection of superoxide anions and the study of redox-dependent platelet regulation.

检测血小板中超氧阴离子生成的替代方法 

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

用螺旋粘度计对人血小板和细胞表面受体进行均匀剪切检测

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

生物学

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

小鼠血液中血小板的纯化

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...

血小板输血中微粒的常规筛选方法

摘要

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一个简单的协议介导的血小板聚集

微流体流钱伯斯再造使用血止血建模和血小板输注

人脐血巨分化与血小板形成的 CD34⁺单元格

血管性血友病因子-血小板串形成的流室模型研究血管血友病因子的病理生理学

使用毛细血管电泳免疫测定来寻找人类血小板肌萎缩性侧索硬化的潜在生物标志物

检测血小板中超氧阴离子生成的替代方法

检测血小板中超氧阴离子生成的替代方法

检测血小板中超氧阴离子生成的替代方法

用螺旋粘度计对人血小板和细胞表面受体进行均匀剪切检测

小鼠血液中血小板的纯化