这种通信是基于以前发表在病毒学报17在开放存取模式下的结果, 根据创作共用归属许可 (<a href="http://creativecommons.org/licenses/by/2.0">http://creativecommons.org/licenses/by/2.0</a>) 的条款, 以生物中心为人.作者衷心感谢在研究 "药物和慢性传染病" ("DRUCK 研究") 的框架内进行了卓有成效的合作, 其中有马库斯、w. 蔡、w. 张和 r. 齐默尔曼 (德国柏林)感谢 s Moyrer 在图 1中收集的照片, 以及 d.f Whybrew, 博士, (哥廷根, 德国) 为纠正手稿。他们还表示赞赏向阿克曼、Bayrambasi、U. Büttner、s Dziubek、i. Jakobsche、b. Krellenberg、克罗恩、p. Kusimski、梅特卡夫、j Piejek、s 萨尔、m. Schröter 和 k 赛的技术援助。这项研究部分得到了德国卫生部向全国丙肝中心提供的资助。

由于该议定书的目的是用于医疗诊断, 它的应用必须遵循《赫尔辛基宣言》19的原则。在这份来文中提出的结果的第一部分, 病人的单独协议和道德委员会的批准似乎是可有可无的, 原因有两个: (1) "入院后" 到埃森大学医院, "每个病人都提供书面同意所有必要的生物化学, 细菌学和病毒学调查。17 (2) "在评估 DBS 测试过程中使用的所有样本都被送往病毒学研究所进行常规临床诊断。因此, 没有一个标本是专门为研究目的收集的, 没有一个额外的静脉穿刺进行, 没有任何的材料是测试的任何参数以外的医生在正常的过程中要求诊断检查。17研究 "药物和慢性传染病" (DRUCK 研究)18, 其中 DBS 测试最终用于德国城市柏林和埃森, 以评估积极注射药物的人, 由联邦数据专员批准信息的保护和自由 (柏林, 德国) 并且由医学大学的道德委员会 charit (柏林, 德国)。
1. 采血
<ol><li>静脉穿刺14、15、20、21<ol>
<li>戴上一次性乳胶橡胶手套, 并清洁预期穿刺部位 (最好是 antecubital 窝的中位肘静脉) 与适当的消毒剂,例如, 70% 异丙醇。</li>
		<li>应用止血带4–6英寸以上的假定穿刺部位扩张静脉。引导针进入病人的静脉, 一旦到位, 轻轻地将连接的血管填满, 其中含有 EDTA 作为抗凝剂。</li>
		<li>当静脉穿刺完成后, 松开止血带并取出针头。然后, 在穿刺部位按一个干纱布垫, 随后可以用绷带在地方举行。</li>
	</ol>
</li>
	<li>皮肤穿刺 (图 1A)13-15, 20, 22, 23<ol>
<li>戴上一副一次性乳胶橡胶手套。</li>
		<li>在皮肤穿刺之前, 病人应该把手放暖和。手指被按摩 anterogradely 丰富的血流向穿刺部位。</li>
		<li>用合适的消毒剂 (例如, 70% 异丙醇) 清洁 non-writing 手第三或第四手指远端指骨的手掌侧皮肤。用一次性的安全柳叶刀刺穿皮肤。手指应保持在这样的位置, 重力促进指尖上的血液收集。</li>
		<li>通过皮肤穿刺收集毛细血管血是完整的, 在指尖贴上绷带。</li>
	</ol>
</li>
</ol>2. 血斑的制备
<ol><li>静脉穿刺采血的制备15<ol>
<li>尽快将收集到的 anti-coagulated (EDTA) 全静脉血放在滤卡上。静脉穿刺后不要准备24小时以上的干血斑。</li>
		<li>将所需的所有信息用于筛选器卡上的病人识别。一张卡片应该只用一个单独的血液被发现。</li>
		<li>戴上一次性乳胶橡胶手套。</li>
		<li>轻轻倒置采血管2–4次, 并随后打开塞子仔细。</li>
		<li>吸入50µl 的整个静脉血使用吸管与一次性小费。将血液转移到一个圆圈的中心, 而不用用吸管的尖端直接接触滤纸。尝试完全饱和的圈子。</li>
		<li>重复此过程以填充卡的所有必需的圆。</li>
	</ol>
</li>
	<li>从皮肤穿刺采集的血液制备 (图 1B和1C)13-16, 23<ol>
<li>用纱布垫擦拭第一滴血, 因为它可能含有过量的组织液。再按摩手指以增加穿刺部位的血流量。将下面的水滴转移到过滤卡的一个圆圈中, 而不直接用指尖接触表面。允许血液被浸泡到过滤器的质地仅由毛细管力量。</li>
		<li>让下一大滴毛细血管血在指尖上形成, 并在下一圈收集。继续这个过程, 直到所有必要的圈子被填满或血流停止。</li>
		<li>如果血流不足以填满所有所需的滤卡圈, 请勿过度挤压或 "挤奶" 手指。如果血流停止在指尖贴绷带。如果检查需要更多的血, 在另一个手指上进行第二次皮肤穿刺。</li>
	</ol>
</li>
</ol>3. 血液斑点的干燥
<ol><li>要干燥的血液斑点, 把过滤卡在一个干净的纸巾在一个生物危害安全柜, 让他们干, 最好是 O/N (但至少4小时), 在 RT 在没有任何外部热源。当干燥过程完成时, 血液斑点有一个均匀地深褐色颜色, 并且没有红色区域再是可看见的 (图 1D)13, 15, 16。</li>
</ol>4. 贮存及运送干血点 (DBS)
注意: 干燥后的血斑可以被打断。现在可以将筛选卡存储为13-16、23。
<ol><li>对于存储, 把滤纸卡放在一个单一的, 不透气的拉链袋, 包含1至2干燥剂包, 以保护标本的水分 (图 1E)。(可选) 添加湿度指示器卡。</li>
	<li>把这个包转到一个温度为-20 ° c 或更低的冰箱里, 尽快。如果在野外条件下没有冷藏库, 在-4 ° c 或常温下的贮存是可行的, 可长达14天。</li>
	<li>在干冰上运送冰冻的 DBS 标本。对于最初保存在环境温度下的滤卡, 使用三重包装系统, 其中包括拉链袋 (s) 作为内部容器 (s) 以及内部和外部信封。在外部信封上不需要通过常规邮件发送任何内容标记, 但必须将国际生物危害符号贴在主内部容器16上。</li>
	<li>如果干燥剂包和/或附加湿度指示器卡更改为粉红色, 则排除筛选卡的进一步处理。</li>
</ol>5. 洗脱干血斑点13、15、16、23
<ol><li>从903级过滤器卡 (图 1F) 的每个血液浸泡的圆圈中, 用一次性的 6 mm 设备打出一个点。将所有穿孔的干血斑从一个病人转移到12井板的一个井中。</li>
	<li>用含有0.05% 吐温20和0.08% 的叠氮化钠的磷酸盐缓冲盐水填充井 (图 1G)。调整所增加的缓冲液的体积, 使其对用于随后分析干血斑点的溶的测定的最低要求。</li>
	<li>重复这些步骤, 获得第二系列干血斑溶, 以进行分子分析。</li>
	<li>将细胞培养板放在实验室的摇床上, 让穿孔的干血点轻轻洗至少4小时, 或者最好是 O/N (图 1H)。</li>
	<li>第二天, 这些斑点几乎没有血液, 溶血性清已经形成 (图 1I)。将这些溶转移到离心管。然后, 让他们离心2分钟, 在 10500 x g (图 1J) 中释放清, 使其免受洗脱过程中形成的任何碎片 (图 1K)。</li>
</ol>6. 溶分析
<ol><li>离心溶现在已经准备好用于预期的分析。使用商用套件调查乙肝病毒、丙肝病毒和 HIV 感染标志物的溶, 并仔细按照各自制造商的指示操作 (图 1L)。</li>
</ol><img alt="Figure 1" src="/files/ftp_upload/52619/52619fig1.jpg"> 图 1.此通信中建议的用于制备和处理用于免疫和分子技术的干血斑点的协议的图形摘要通过柳叶刀 (a) 穿刺或刺穿皮肤获得的全静脉血和毛细血管血可以作为干血斑点分析的标本。在将血液转移到903级过滤卡 (B, C) 的圆圈后, 在生物危害安全柜中, 在环境温度下最好将样品烘干, 以形成均匀棕褐色的斑点, 而不点缀红色区域 (D)。当干燥的血液斑点是完整的, 储存是必要的, 过滤卡可以包装在不透气的拉链袋含有1–2干燥剂 (E)。星展集团的进一步实验室处理包括由一个 6 mm 的单一使用装置从圆周中心 (F) 产生冲床, G), 将 PBS-based 缓冲器中的冲洗液最小为4小时, 或者最好是在振动筛上的 O/N (H), 恢复溶 (I), 最后是实验室杯 (J) 的离心, 以释放星展溶在洗脱过程中可能产生的任何碎片 (K)。随后, DBS 溶已经准备好进行分析, 这是由一个全自动平台 (L) 进行的, 目的是分别检测 HBV、HCV 和 HIV 感染的血清学标记。<a href="https://www.jove.com/files/ftp_upload/52619/52619fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

在过去, 比雅获得了一个酬金, 为他的科学工作提供讲座和财政支持, 从 Abbott 诊断。NG 和 OM 宣称他们没有利益冲突。

DBS 已经使用了100年2 , 但令人惊讶的是, 在它们的制备和处理方面仍然没有普遍的共识。到目前为止, 这一重要的分析阶段的一个充分的标准化仅在新生儿筛查14领域实现, 而对于 DBS 测试的所有其他应用则存在各种不同的协议5、16、23。为了克服这一显著的异质性, 本文介绍了在免疫和分子技术中用于制备和处理 DBS 的综合 step-by 步指令, 并对其有效性进行了评估。用于检测乙肝病毒、HCV 和 HIV 感染的标志物。以下讨论的重点主要放在所建议的议定书的不同步骤上。
在 DBS 测试的历史中, 许多不同的滤纸卡已经使用了2 , 但是今天只有两个商业来源被 FDA 批准为第二类医疗器械, 用于采血5, 16。这些滤卡系统具有高度的均匀性, 并且具有非常相似的吸收特性, 因此从毛细管血液中制备的分析结果不会因4% – 5%28而不同。毫不奇怪, Masciotra 和同事29因此在从不同来源的血液收集卡洗脱后, 检测出 HIV-1 RNA 同样很好的定性化验. 鉴于这些数据, 它可以被实际排除, 在测试耦合血清/DBS 对乙肝病毒, HCV 和 HIV 病毒感染的标志物的任何差异,17是由单独选择的过滤卡造成的。然而, 当准确量化分析物是必不可少的, source-to-source 变异可能不再是可忽略和更复杂的技术,例如, 穿孔 DBS (pdb) 作为方法为 microsampling30或准备干血清斑点 (DSS)31可能需要应用, 而不是传统的 DBS 测试10。
因为只有少量的血液用于 DBS 测试 (一滴毛细血管血包括大约50µl)5, 16, 样本量的变化是至关重要的, 而且, 诚然, 血清学结果之间的差异至少有一些在 "DRUCK 研究" 的试验过程中, 参与者自我报告的 HBV 和 HCV 的状态是归因于这个变量的。最重要的因素, 最大限度地减少 "波动" 的样本量无疑是一个正确的技术, 毛细血管采血, 这只能通过仔细和持续培训技术人员22。此外, 作为质量控制的一项措施, 所有与星展集团合作的实验室都应该已经启动了识别的程序, 并随后排除了那些必须被认为是不满意或无效的16的 dbs 样本。
主要在艾滋病毒的背景下进行的研究32和新生儿筛查33表明, 高湿度可能导致分析的降解, 但迄今尚未就 DBS 应风干多久的问题达成共识.在此通信中建议至少4小时或最好是 O/N 的间隔, 因此采用了在所有相关出版物中的绝大部分使用的条件32, 34存储 DBS 的数据, 随后用于 HBV 和 HCV 检测相互冲突。在 1981年, 别墅和同事35, 谁应用当代分析技术, 报告说, 在 RT 的存储没有影响乙肝病毒分析的结果, 在整个观察期间的180天, 如果抗体滴是 > 1/1000, 但 DBS在15天后, 当血清中的滴只有1/100 时, 就成了边缘阳性甚至是阴性。存贮在-20 ° c 或4° c 没有导致坚固改善。相比之下, 在三十年后的一项研究中,36测试了 HBV 阳性样本的复制, 作者发现抗 HBc 和抗 HBs 抗体在 RT 时稳定了183天, 而 HBsAg 在同样的条件下成为假阴性已经在63天以后。关于从 DBS 溶的乙肝病毒 DNA 检测, 病毒核酸的浓度在37° c (37的至少七天内是稳定的, 或者在 RT 中被证明是 "耐" 的, 可达三周38。使用两个商用的第三代免疫39对抗 HCV 抗体进行检测, 在117天内提供准确的结果, 使用 DBS 样品分别储存在-20 ° c、2–8° c 和20–25° c。然而, 在-20 ° c 的存储, 导致了光学密度的最低变化。应用第四代丙型肝炎病毒 ELISA,即, Monolisa hcv-Ag-Ab 超, 在 DBS 测试的背景下, Larrat et al.40在 RT 中存储 DBS 标本超过三天后, 观察到分析特异性急剧下降。另一方面, 在不同的条件下 (-20 ° c, 2 –8° c, 22 –26° c) 中, 由 Brandao 和同事41, 使用相同的试剂盒获得的样品在60天内得到了精确的测试结果。在不同的存储条件下, DBS 中 HCV RNA 浓度下降的观察范围从 RT 到四周后的10年内没有显著的变化, 在室温下为42 , 在环境温度下为43。考虑到关于乙肝病毒和 HCV 抗原、核酸和抗体稳定性的这一相当矛盾的数据, 在拟议的议定书中收回一项协商一致意见似乎是合理的, 因为早先已界定了将 DBS 标本用于储存HIV 检测15, 30: 用于短期沉积 (最多两周) 抗原, 病毒核酸, 和抗体被认为是稳定的 RT, 而最佳存储长期处于冰冻状态。
一般来说, 在设计洗脱协议时应考虑三参数: (1) 洗脱缓冲器;(2) 洗脱的持续时间和温度;和 (3) 洗脱体积23。在所有相关刊物中, 绝大多数的磷酸盐缓冲盐水 (PBS) 被用于洗脱的 DBS, 大多数作者都添加了一种蛋白质,例如, 牛血清白蛋白 (BSA) 或吐温 20, 一种表面活性剂, 为了通过稳定蛋白质, 当它们进入溶液时, 同时阻断非特异性的绑定站点23。只有少数报告, 直接比较了不同的洗脱缓冲在 DBS 测试的背景下, 是可用的。比利亚尔et al.36,例如, 记录了所有使用的缓冲区的几乎等同的洗脱能力, 但是 PBS/BSA 0.5% 导致了非特异性反应的最低水平。一个非常相似的观察是由 Croom 和同事44在应用遗传学系统的标本稀释剂 rLAV EIA 为从 DBS 的抗 HCV 抗体的洗脱. 由于在制备 DBS (见上图) 后的第一个小时内, 样品降解的风险非常低, 因此决定在室温下孵育斑点, 并通过温和的涵管端混合来支持洗脱过程。这种方法的优势是, 前一天穿孔的标本可以直接转移到第二天早上的例行诊断23.洗脱缓冲液的体积应适应于随后的分析所用的最小各自的要求, 以便使稀释系数尽可能低。然而, 由于在 "DRUCK 研究"17期间必须在较短的时间内保证高样本吞吐量, 因此在前面的评估中不可能对每一个分析物的洗脱条件进行仔细的优化。因此, 为了完成整个洗脱过程的所有参数在两个单独的操作中, 使用了1000µl 的 PBS-based 缓冲器的相当不利的体积。
这一方法一方面失败 "完全在反 HBc/反 HBs 系统为那些个人感染艾滋病毒, 由于低抗体浓度;在乙肝病毒 DNA 和丙型肝炎病毒 RNA 检测方面, 它需要具有最佳分析灵敏度的分子生物学程序。17另一方面, 在整个评估过程中使用的试剂盒的高洗脱量对乙肝表面抗原、抗 HCV 和抗 HIV 的测定绝不不利。"检测乙肝表面抗原阳性材料从整体血液溶成功以敏感性98.6% 到相似地高程度和在早先研究, 部分使用了100µl, 250 µl, 或600µl, 或500µl" 17 的一个更小的洗脱容量,36、45、46。97.8% 测定179血清/DBS 对抗 HCV 抗体的灵敏度与现有报告24、44、47、48、49的结果相符, 这项研究的洗脱量低于5至10倍。"此外, 通过规定自己的截止点"17、24、48、49或通过将样本量从20µl 增加到100µl, 对反 HCV 检测的协议进行了适当的优化。17, 49最后, 为抗 HIV 检测而建立的分析特异性和灵敏度 (100% 个) 均等于或优于其他免疫的性能特征, 具体适应于 DBS 测试的50、51或逐步过程与多个抗 HIV 测试的联合使用 "17, 52。"事实上, 它们也超过了在 dbs 溶中专门开发和优化的检测抗 hiv 抗体的化验的性能记录 (Q-预防艾滋病毒 1 + 2 dbs 套件)。17, 53
综合起来, 这一通信中提出的全面的 step-by 步骤协议证明是一个可行的和 user-friendly 的工具, 准备和处理 DBS, 从而可以可靠地用于诊断病毒。它允许使用自动化54的方法, 并由于其优异的性能特性, 有潜力作为一种基础-石头为未来的普遍接受的协商一致的协议在全球 DBS 测试领域的实验室医学.

使用由纤维素制成的纸卡收集血液的想法被归因于埃法尔基督徒轰隆 (1869-1918), 现代临床微量分析的父亲1, 2。在 1913年, 砰的血糖测定从溶的干血斑点 (DBS)3 , 并随后, 也执行氮测量使用的凯氏定法与此滤纸技术2。随后, 几名调查员报告了使用 DBS 进行血清学测试诊断梅毒2。早在 1924年, 查普曼总结了 DBS 测试的优势, 当他特别强调四项目, 这仍然是有效的今天: (1) 与常规静脉穿刺相比, 需要较少的血量, 这一事实是最重要的儿科诊断;(2) 采血简单, 无创, 价格低廉;(3) 细菌污染或溶血的风险极小;和 (4) DBS 可以保存很长的时间, 几乎没有恶化的分析2, 4。除了在梅毒检测中的应用, 在 1953年2中, 星展集团技术的进一步早期使用包括,例如, 检测抗麻疹、腮腺炎、脊髓灰质炎病毒、流感和呼吸道合胞病毒的抗体,粪便中志贺氏菌在滤纸上的鉴定, 并由印尼的普通邮件运往荷兰莱顿, 以及在疫区拍摄的血吸虫病抗体检测, 并分析了超过三月后的5.在 1963年, 在轰隆的原始的通信的五十年以后2, 6, 他最后出版了他的著名方法为诊断苯丙酮尿症从从新生儿的脚跟刺获得了7, 8。
虽然从那时起, DBS 被认为是一种普遍适用的收集、储存、运输和分析各种人体体液的方法5, 但它们在诊断中的应用仍然主要集中在诊断感染, 特别是在资源有限的设置和系统筛选新生儿遗传性代谢紊乱几十年来9, 10。然而, 自2005年以来, 各种新的和创新的 DBS 应用已经开始出现。因此, 目前星展集团的有关科学刊物的数目, 每年约有50至近450个。在新兴的应用领域包括 toxico 和药代动力学研究, 代谢分析, 治疗药物监测, 法医毒理学, 或环境污染控制10, 11。
因此, 在过去的100年中, DBS 测试通过临床实验室诊断进行了一次胜利的游行2。在临床化学中12, 然而, 在这3月分析变量没有充分考虑多年。事实上, 即使在今天, 在 CDC 的过滤纸评估项目13或在新生儿筛查的框架内制定了一项关于过滤器纸的血液收集国家标准, 分析阶段在大多数其他领域仍然被低估了, 其中 DBS 测试应用了5, 10。
在这一背景下, 建议在免疫和分子技术中使用一个全面的 step-by 步骤协议14-16 , 其中包括所有必要步骤, 以便在以下通信中准备和处理 DBS: (1)采血;(2) 血斑的制备;(3) 血斑干燥;(4) 储运;(5) DBS 的洗脱;最后 (6) 分析了 DBS 溶。该协议的有效性首先评估了1762耦合血清/DBS 对检测乙肝病毒 (hbv) 表面抗原 (HBsAg), 抗体的 hbv 核心抗原 (抗 HBc), 抗体的乙肝表面抗原 (反 HBs), 乙肝病毒的 DNA, 抗体丙型肝炎病毒 (hcv), hcv RNA, 和人体免疫缺陷病毒 (hiv) 1-p24 抗原/抗 hiv 1/2 使用一个全自动平台或敏感定性核酸测试。17. 第二步, 该议定书在 "药物和慢性传染病" ("DRUCK 研究") 的试点研究中得到使用, 这是由罗伯特-科赫研究所与全国丙型肝炎参考中心密切合作进行的。吸毒者在柏林和埃森的德国城市18。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Latex rubber gloves</td>
			<td>Hartmann</td>
			<td>#4049500041515</td>
			<td></td>
		</tr>
		<tr>
			<td>70% isopropyl alcohol</td>
			<td>Bode Chemie</td>
			<td>#9768050</td>
			<td></td>
		</tr>
		<tr>
			<td>Mulifly safety cannula</td>
			<td>Sarstedt</td>
			<td>#85.1638</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA blood collection tube</td>
			<td>Sarstedt</td>
			<td>#02.1066.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive bandage</td>
			<td>Hartmann</td>
			<td>#2994163</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry gauze pad</td>
			<td>Hartmann</td>
			<td>#143213</td>
			<td></td>
		</tr>
		<tr>
			<td>Singel-use safety lancet</td>
			<td>Owen Mumford</td>
			<td>#3802421</td>
			<td></td>
		</tr>
		<tr>
			<td>Test Card</td>
			<td>Whatman/sigmaaldrich</td>
			<td>#10534612</td>
			<td>Filter paper card Grade 903</td>
		</tr>
		<tr>
			<td>Disposable pipette tips</td>
			<td>Starlab</td>
			<td>#S1126-7810, #S1120-8810</td>
			<td></td>
		</tr>
		<tr>
			<td>Zipper bag</td>
			<td>Flexico</td>
			<td>#20219</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini desiccant bag</td>
			<td>Tropack</td>
			<td>#-</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Punch</td>
			<td>pfmmedical</td>
			<td>#48601</td>
			<td>6.0 mm Disposable Biopsy Punch</td>
		</tr>
		<tr>
			<td>Disposable Forceps</td>
			<td>Servoprax</td>
			<td>#H7301</td>
			<td></td>
		</tr>
		<tr>
			<td>12 Well Cell Culture Plate</td>
			<td>Greiner</td>
			<td>#665180</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Buffer</td>
			<td>Gibco</td>
			<td>#14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Applichem</td>
			<td>#A1389.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Merck</td>
			<td>#66880250</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>#30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>ARCHITECT HBsAg (Qunatitative)</td>
			<td>Abbott</td>
			<td>#6C3642</td>
			<td></td>
		</tr>
		<tr>
			<td>ARCHITECT anti-HBc II</td>
			<td>Abbott</td>
			<td>#8L4425</td>
			<td></td>
		</tr>
		<tr>
			<td>ARCHITECT anti-HBs</td>
			<td>Abbott</td>
			<td>#7C1825</td>
			<td></td>
		</tr>
		<tr>
			<td>ARCHITECT anti-HCV</td>
			<td>Abbott</td>
			<td>#6C3725</td>
			<td></td>
		</tr>
		<tr>
			<td>ARCHITECT HIV Ag/Ab Combo</td>
			<td>Abbott</td>
			<td>#4J2727</td>
			<td></td>
		</tr>
		<tr>
			<td>MagNA Pure 96 DNA and Viral NA Large Volume Kit</td>
			<td>Roche</td>
			<td>#6374891001</td>
			<td></td>
		</tr>
		<tr>
			<td>artus HBV LC PCR Kit</td>
			<td>Qiagen</td>
			<td>#4506063 (24 tests), #4506065 (96 tests)</td>
			<td></td>
		</tr>
		<tr>
			<td>VERSANT HCV TMA Assay IVD</td>
			<td>Siemens Healthcare</td>
			<td>#2554311</td>
			<td></td>
		</tr>
	</tbody>
</table>

dried blood spots - preparing and processing for use in immunoassays and in molecular techniques

一个世纪前, 在一张纸卡上采集血液, 然后使用干血点 (DBS) 进行诊断的想法。从那时起, 星展集团在数十年来的测试主要集中在对传染性疾病的诊断上, 特别是在资源有限的环境中, 或者对遗传性代谢紊乱的新生儿进行系统的筛查, 而且最近才有各种新的和创新的 DBS 应用开始涌现。多年来, 分析变量只是在 dbs 测试领域被不适当地考虑, 甚至在今天, 除了新生儿筛查, 整个分析阶段, 其中包括为其准备和处理 dbs最终分析尚未标准化。在此背景下, 提出了一个涵盖基本阶段的全面的 step-by 步骤协议,即即采集血液;血斑的制备;血斑干燥;星展集团的仓储和运输;dbs 的洗脱, 最后分析 dbs 溶。该协议的有效性首次评估了1762耦合血清/DBS 对检测标志的乙肝病毒, 丙肝病毒, 和人体免疫缺陷病毒感染的自动化分析平台。在第二步中, 该议定书是在一项试验性研究中使用的, 该报告是针对德国柏林和埃森市的活跃吸毒者进行的。

通过分析1762种血清/dbs 对 HBV、HCV 和 HIV 感染的标志物, 首次评估了为 DBS 的制备和处理所建议的协议的有效性。17. 为此, DBS 从100µl 全静脉血 (cf 2.1.1-2.1.6) 制备, 并洗1000µl 的 PBS-based 缓冲, 每一个, (cf. 5.1 –5.5 的上述协议) 完成过程用于两个单独操作中的所有七参数。这种方法如果不仔细优化每个单一分析物的洗脱条件, 似乎是不可避免的, 因为在即将进行的实地研究中, 预计在较短的时间内将有相当高的样品吞吐量, "药物和慢性传染性疾病 "(" DRUCK 研究 ")。
在 DBS 分析过程中, 所有的测量都遵循了制造商的建议, 但必须对乙肝表面抗原和反 HBs 的测定进行修改, 以补偿溶血或稀释结果的影响。当与血清样本 (图 2A) 相比, DBS 溶的乙肝表面抗原检测导致的假阳性率为 14.7% (52 出 354)。接收机操作特性 (ROC) 分析数据的25, 26表明, 将阈值增加到0.15 毫升会导致乙肝表面抗原阳性的理想分离 (0.986 [灵敏度], 0.000 [1-特异性],图 2B)。另一方面, 由于对抗 HBs 抗体进行量化的10个单位/升的截止率, 记录了 14.2% (47 出 331) 的假阴性测量速率 (图 2C)。因此, 在 ROC 分析之后, 这一截降降低到1.5 个/l, 以达到最佳的值判别 (0.917 [灵敏度], 0.007 [1-特异性],图 2D)。
<img alt="Figure 2" src="/files/ftp_upload/52619/52619fig2.jpg"> 图 2. DBS 溶中的 HBsAg 和反 HBs 测试 (修改自17).从0.05 毫升 (a) 到0.15 毫升 (B) 将 HBsAg 定量化的截止值的增加, 将假阳性结果从14.7% 降至0%。同样, 中华民国反 HBs 浓度的分析表明, 从10人/升 (C) 到1.5 人/升 (D) 的阈值减少, 从而完全消除假阴性, 最初占14.2% 的所有决定。<a href="https://www.jove.com/files/ftp_upload/52619/52619fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
表 117中总结了血清/DBS 分析的结果。在 DBS 测试过程中, 没有异性七分析的记录。
当研究 DBS 溶在乙肝表面抗原的存在, 即急性和慢性感染 HBV 的血清学关键参数, 确定了98.6% 的分析灵敏度。两种假阴性血清的 HBsAg 浓度分别为749毫升和6毫升。176 204 (i. e 86.3%) 中成功检测到抗 HBc 抗体星展溶二十二的28标本中, 没有检测到的测量来自个人合并与艾滋病毒。因此, 如果将 HIV 阳性个体排除在计算之外, 就能从洗干全静脉血中提取抗 HBc 抗体, 达到97.1% 的灵敏度。对于抗 HBs 抗体的测定, 也有类似的观察。即使将测定的截止值调整到1.5 个/升, 九差异血清 DBS 结果发生。在未与 HIV 合并的患者中, 发现血清抗 HBs 浓度为11– 26, 其含量过低, 不能在干血洗脱后进行检测。
抗 HCV 抗体表明是急性或慢性或解决感染。只有四 (即 2.2%) 假阴性反 HCV 的结果是由 DBS 溶, 进一步的血清学和分子分析清楚地表明, 各自的血清很可能取自患者, 其感染早已解决.
全自动化系统在 DBS 溶中检测抗 HIV 抗体是一个完全平滑的过程, 其分析灵敏度为100%。
"全血溶的平均 HBV dna 浓度是100例 (平均 dna 浓度: 1573898 毫升, 范围: < 357-> 1786万/毫升), 并产生了93.0% 的值。因此, 在409至3643间血清 HBV DNA 浓度低的七样本, DBS 测试没有检测到。100血清已证明是 hcv rna 阳性 (平均丙型肝炎病毒 rna 浓度: 1415944/毫升, 范围: 2479-> 769.2万/毫升), 相应的全血等分可用。比较调查结果的分析灵敏度为100% 的 HCV RNA 测定从 DBS 溶。17
为了测试在野外条件下准备和处理 DBS 的协议, 在研究 "药物和慢性传染病" 的试验阶段, 它与罗伯特科赫研究所 (德国柏林) 密切合作, 这是在柏林和埃森的德国城市进行活跃的药物使用者18。所有534名参加者 (433 男性和101妇女) 接受毛细血管采血, 详见前一项议定书2.2.1 节的 2.2.3, 而星展集团再次洗了1000µl 的 PBS-based 缓冲器。
两个人被诊断为慢性乙肝病毒感染, 39 显示的血清模式的解决 hbv 感染。此外, 十五名参加者对反 HBs (免疫接种后的状况) 持积极态度, 八人被发现对反 HBc 是积极的。抗 HCV 流行在参加者之中是 57.3% (n = 193) 在柏林和 73.0% (n = 144) 在埃森。从抗体阳性样本 65% (n = 125) 和 62% (n = 89) 也 viremic。二十五人中有534人对反艾滋病毒进行了阳性测试。十九的感染已经知道, 其中24的人是合并与 HCV。
与研究参与者的性数据有关的自我报告的 HBV 和丙型肝炎病毒的状态得到了仔细比较。这一对比与测试结果结合血清/DBS 配对导致的测试算法的主要阶段的 "DRUCK 研究", 这将注册活跃的药物使用者在六额外的大德国城市: "个人感染艾滋病毒的人将总是检测乙肝病毒 DNA 的存在。溶为反 HBc 或反 hbs 积极的参与者, 在第二次会诊时应接受静脉穿刺, 以明确其反 HBc/反 hbs 的地位, 最后, 所有 dbs 溶将被筛选为 HCV RNA无论反 HCV 检测结果如何。17
<table border="1" cellpadding="0" cellspacing="0"><tbody>
<tr>
<td>参数</td>
			<td>耦合血清/DBS 溶 (N)</td>
			<td>不一致的结果 (N)</td>
			<td>检测血清与 DBS 溶差异的原因</td>
		</tr>
<tr>
<td>hbv</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>抗原</td>
			<td>299</td>
			<td>2</td>
			<td>两例慢性乙肝病毒感染。两人都受到抗病毒治疗, 其中一人合并艾滋病毒。血清 HBsAg 浓度分别为749毫升和6毫升。</td>
		</tr>
<tr>
<td>反 HBc</td>
			<td>305</td>
			<td>28</td>
			<td>在血清中, 二十二例患者的 HBV 感染已得到解决。六人显示血清学发现 "仅抗 HBc"。二十二的患者合并了 HIV。从 "乙肝解决程序", 二十被评估为 "单独的反 HBs 阳性" 由 DBS 测试, 因此, 被错误地归类为 "条件后接种"。</td>
		</tr>
<tr>
<td>反 HBs</td>
			<td>310</td>
			<td>9</td>
			<td>四例未合并的 HIV 患者血清抗 HBs 浓度为11‑ 26, 其过低, 不允许在洗脱后的干血斑点进行抗体检测。</td>
		</tr>
<tr>
<td>HBV DNA</td>
			<td>150</td>
			<td>7</td>
			<td>从七例低血清 HBV DNA 浓度的患者中获得了各自的血清, 从409人/毫升到3643毫升不等。</td>
		</tr>
<tr>
<td>hcv</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>抗 HCV</td>
			<td>339</td>
			<td>4</td>
			<td>这四的血清已经从病人身上取出, 自此之后很久就已经解决了 HCV 感染。</td>
		</tr>
<tr>
<td>HCV RNA</td>
			<td>150</td>
			<td>0</td>
			<td>–</td>
		</tr>
<tr>
<td>艾滋病</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>hiv 1-p24/抗 hiv 1/2</td>
			<td>209</td>
			<td>0</td>
			<td>–</td>
		</tr>
</tbody></table>表 1:结果从 1762 DBS, 这是准备和处理从整个静脉血后, 该协议提出的这一通信, 与研究结果的耦合血清样本作为参考。

Watch this Scientific Journal Video about Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques at JoVE.com

Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

在大多数诊断应用中, 对干血点 (DBS) 进行最终分析的准备和处理仍不规范。为了克服这一缺点, 提出了一个全面的 step-by 步协议, 并随后对其检测病毒感染标志的有效性进行了评估。

干血斑点. 免疫和分子技术中使用的制备和加工

The idea of collecting blood on a paper card and subsequently using the dried blood spots (DBS) for diagnostic purposes originated a century ago. Since ...

dried-blood-spots-preparing-processing-for-use-immunoassays-molecular

Research

JoVE Journal

Biology

39.4K 次观看.  University of Duisburg-Essen. 在大多数诊断应用中, 对干血点 (DBS) 进行最终分析的准备和处理仍不规范。为了克服这一缺点, 提出了一个全面的 step-by 步协议, 并随后对其检测病毒感染标志的有效性进行了评估。

视频: Dried Blood Spots - Preparing and Processing for Use in Immunoassays and in Molecular Techniques

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

科研

生物学

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

微探针：操作和使用的局部表面处理

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Non-Terminal Blood Sampling Techniques in Guinea Pigs

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6. All the described methods have been thoroughly tested and applied for repeated in vivo blood sampling in studies within our research facility.

Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.

豚鼠非终端采血技术

Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3. However, the use of guinea pigs currently ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

重复采血车在成年斑马鱼验血

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和“剪切环”中的应用：一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

小鱼肾注射液循环系统中微粒的简易有效管理与可视化

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...