Name: high-speed video microscopy analysis for first-line diagnosis of primary ciliary dyskinesia
Uploaded: 2022-01-19T14:00:00
Description: Watch this Scientific Journal Video about High-speed Video Microscopy Analysis for First-line Diagnosis of Primary Ciliary Dyskinesia at JoVE.com

我们要特别感谢儿科护士Johanna Juvankoski女士在刷牙方面的出色帮助。我们还要特别感谢Heymut Omran教授（UKM明斯特大学诊所）允许使用其网站上的正常睫状运动示意图。最后，我们要感谢PGCE的Alan Brown BA（Hons）先生校对手稿。

道德声明：
这项研究得到了当地伦理委员会（69/2017）的批准，并按照赫尔辛基宣言进行。
1. 呼吸道上皮细胞的收集和运输
<ol><li>刷牙<ol>
<li>刷牙前，给患者口服阿莫西林-克拉维酸两周疗程，以根除干扰纤毛功能的生物膜。确保在手术前2天终止抗生素。</li>
		<li>为了减少刷洗上皮细胞条上的粘液物质覆盖，请患者彻底擤鼻涕。请父母帮助?...

<ol>
	<li>Werner, C., Onnebrink, J. G., Omran, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+primary+ciliary+dyskinesia.">Diagnosis and management of primary ciliary dyskinesia.</a> Cilia. 4 (1), 2 (2015).</li><li>Mirra, V., Werner, C., Santamaria, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+ciliary+dyskinesia:+An+update+on+clinical+aspects,+genetics,+diagnosis+and+future+treatment+strategies.">Primary ciliary dyskinesia: An update on clinical aspects, genetics, diagnosis and future treatment strategies.</a> Frontiers in Pediatrics. 5, 135 (2017).</li><li>Schultz, R., Elenius, V., Lukkarinen, H., Saarela, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+novel+mutations+in+the+DNAH11+gene+in+primary+ciliary+dyskinesia+(CILD7)+with+considerable+variety+in+the+clinical+and+beating+cilia+phenotype.">Two novel mutations in the DNAH11 gene in primary ciliary dyskinesia (CILD7) with considerable variety in the clinical and beating cilia phenotype.</a> BMC Medical Genetics. 21 (1), 237 (2020).</li><li>Knowles, M. R., 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=Mutations+of+DNAH11+in+patients+with+primary+ciliary+dyskinesia+with+normal+ciliary+ultrastructure.">Mutations of DNAH11 in patients with primary ciliary dyskinesia with normal ciliary ultrastructure.</a> Thorax. 67 (5), 433-441 (2012).</li><li>Boon, 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=Primary+ciliary+dyskinesia:+critical+evaluation+of+clinical+symptoms+and+diagnosis+in+patients+with+normal+and+abnormal+ultrastructure.">Primary ciliary dyskinesia: critical evaluation of clinical symptoms and diagnosis in patients with normal and abnormal ultrastructure.</a> Orphanet Journal of Rare Diseases. 9, 11 (2014).</li><li>Kouis, P., 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=Prevalence+of+primary+ciliary+dyskinesia+in+consecutive+referrals+of+suspected+cases+and+the+transmission+electron+microscopy+detection+rate:+a+systematic+review+and+meta-analysis.">Prevalence of primary ciliary dyskinesia in consecutive referrals of suspected cases and the transmission electron microscopy detection rate: a systematic review and meta-analysis.</a> Pediatric Research. 81 (3), 398-405 (2017).</li><li>Marthin, J. K., Nielsen, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choice+of+nasal+nitric+oxide+technique+as+first-line+test+for+primary+ciliary+dyskinesia.">Choice of nasal nitric oxide technique as first-line test for primary ciliary dyskinesia.</a> European Respiratory Journal. 37 (3), 559-565 (2011).</li><li>Jackson, C. L., Behan, L., Collins, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+diagnostic+testing+in+primary+ciliary+dyskinesia.">Accuracy of diagnostic testing in primary ciliary dyskinesia.</a> European Respiratory Journal. 47 (3), 837-848 (2016).</li><li>Lucas, J. S., 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=European+Respiratory+Society+guidelines+for+the+diagnosis+of+primary+ciliary+dyskinesia.">European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia.</a> European Respiratory Journal. 49 (1), 1601090 (2017).</li><li>Shoemark, A., Dell, S., Shapiro, A., Lucas, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERS+and+ATS+diagnostic+guidelines+for+primary+ciliary+dyskinesia:+similarities+and+differences+in+approach+to+diagnosis.">ERS and ATS diagnostic guidelines for primary ciliary dyskinesia: similarities and differences in approach to diagnosis.</a> European Respiratory Journal. 54 (3), 1901066 (2019).</li><li>Kouis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effectiveness+analysis+of+three+algorithms+for+diagnosing+primary+ciliary+dyskinesia:+a+simulation+study.">Cost-effectiveness analysis of three algorithms for diagnosing primary ciliary dyskinesia: a simulation study.</a> Orphanet Journal of Rare Diseases. 14 (1), 142 (2019).</li><li>Rubbo, B., 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=Accuracy+of+high-speed+video+analysis+to+diagnose+primary+ciliary+dyskinesia.">Accuracy of high-speed video analysis to diagnose primary ciliary dyskinesia.</a> Chest. 155 (5), 1008-1017 (2019).</li><li>Friedman, N. R., Pachigolla, R., Deskin, R. W., Hawkins, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+technique+to+diagnose+primary+ciliary+dyskinesia.">Optimal technique to diagnose primary ciliary dyskinesia.</a> The Laryngoscope. 110 (9), 1548-1551 (2000).</li><li>Chilvers, M. A., Rutman, A., O&#180;Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+cilia+and+ciliated+epithelial+ultrastructure+in+healthy+children+and+young+adults.">Functional analysis of cilia and ciliated epithelial ultrastructure in healthy children and young adults.</a> Thorax. 58 (4), 333-338 (2003).</li><li>Lucas, J. S., Paff, T., Goggin, P., Haarman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+methods+in+primary+ciliary+dyskinesia.">Diagnostic methods in primary ciliary dyskinesia.</a> Paediatric Respiratory Reviews. 18, 8-17 (2016).</li><li>Ballenger, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+effect+of+cigarette+smoke+on+human+respiratory+cilia.">Experimental effect of cigarette smoke on human respiratory cilia.</a> New England Journal of Medicine. 263, 832-835 (1960).</li><li>Stanley, P. J., Wilson, R., Greenstone, M. A., Mac William, L., Cole, P. J. <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+cigarette+smoking+on+nasal+mucociliary+clearance+and+ciliary+beat+frequency.">Effect of cigarette smoking on nasal mucociliary clearance and ciliary beat frequency.</a> Thorax. 41 (7), 519-523 (1986).</li><li>Kobbernagel, H. E., 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=Efficacy+and+safety+of+azithromycin+maintenance+therapy+in+primary+cilia+dyskinesia+(BESTCILIA):+a+multicentre,+double-blind,+randomised,+placebo-controlled+phase+3+trial.">Efficacy and safety of azithromycin maintenance therapy in primary cilia dyskinesia (BESTCILIA): a multicentre, double-blind, randomised, placebo-controlled phase 3 trial.</a> Lancet Respiratory Medicine. 8 (5), 493-505 (2020).</li><li>Takeyama, K., Tamaoki, J., Chiyotani, A., Tagaya, E., Konno, 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+macrolide+antibiotics+on+ciliary+motility+in+rabbit+airway+epithelium+in-vitro.">Effect of macrolide antibiotics on ciliary motility in rabbit airway epithelium in-vitro.</a> Journal of Pharmacy and Pharmacology. 45 (8), 756-758 (1993).</li><li>Toskala, E., Haataja, J., Shirasaki, H., Rautliainen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+cells+harvested+with+nasal+brushing:+a+method+for+evaluating+ciliary+function.">Culture of cells harvested with nasal brushing: a method for evaluating ciliary function.</a> Rhinology. 43 (2), 121-124 (2005).</li><li>Pifferi, 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=Simplified+cell+culture+method+for+the+diagnosis+of+atypical+primary+ciliary+dyskinesia.">Simplified cell culture method for the diagnosis of atypical primary ciliary dyskinesia.</a> Thorax. 64 (12), 1077-1081 (2009).</li><li>Hirst, R. A., 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=Culture+of+primary+ciliary+dyskinesia+epithelial+cells+at+air+liquid+interface+can+alter+ciliary+phenotype+but+remains+a+robust+and+informative,+diagnostic+aid.">Culture of primary ciliary dyskinesia epithelial cells at air liquid interface can alter ciliary phenotype but remains a robust and informative, diagnostic aid.</a> PLoS One. 9 (2), 89675 (2014).</li><li>Papon, J. 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=Quantitative+analysis+of+ciliary+beating+in+primary+ciliary+dyskinesia:+a+pilot+study.">Quantitative analysis of ciliary beating in primary ciliary dyskinesia: a pilot study.</a> Orphanet Journal of Rare Diseases. 7 (10), 78 (2012).</li><li>Smith, C. 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=Cilia+FA:+a+research+tool+for+automated,+high-throughput+measurement+of+ciliary+beat+frequency+using+freely+available+software.">Cilia FA: a research tool for automated, high-throughput measurement of ciliary beat frequency using freely available software.</a> Cilia. 1 (8), 14 (2012).</li><li>Sampaio, P., 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=Ciliar+Move:+new+software+for+evaluating+ciliary+beat+frequency+helps+find+novel+mutations+by+a+Portuguese+multidisciplinary+team+on+primary+ciliary+dyskinesia.">Ciliar Move: new software for evaluating ciliary beat frequency helps find novel mutations by a Portuguese multidisciplinary team on primary ciliary dyskinesia.</a> European Respiratory Journal Open Research. 7 (1), 00792 (2021).</li><li>Mullowney, T., 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=Primary+ciliary+dyskinesia+and+neonatal+respiratory+distress.">Primary ciliary dyskinesia and neonatal respiratory distress.</a> Pediatrics. 134 (6), 1160-1166 (2014).</li><li>Leigh, M. W., 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=Standardizing+nasal+nitric+oxide+measurement+as+a+test+for+primary+ciliary+dyskinesia.">Standardizing nasal nitric oxide measurement as a test for primary ciliary dyskinesia.</a> Annals of the American Thoracic Society. 10 (6), 574-581 (2013).</li></ol>

在这里，根据一线诊断来描述和使用HSVMA的PCD的诊断过程。尽管HSVMA相对容易建立，成本效益11，并且在有经验的手中是一种可靠的方法12，但HSVMA并不是一种没有陷阱的诊断措施。异常CBF和CBP可能是由于继发感染，导致支气管 - 呼吸道上皮炎症15，并且出于同样的原因，吸烟个体可能有异常的心跳频率16，...

原发性纤毛运动障碍（PCD）是一种罕见的遗传性遗传性疾病，可引起跳动纤毛运动的紊乱。如果未确诊，由于MCC的严重损害，它会导致晚年严重的肺损伤。过去，其患病率估计在1：4，000至50，000之间。由于诊断方法的稳步改善和对该病症的认识不断提高，关于PCD患病率的最新情况表明，它可能更为常见，可能在1：4，000至20，000的范围内，而不是1，2。然而，PCD患者仍然被低估或诊断得太晚1，3。因此，应怀疑患有先天性坐姿与异型或围产期鼻漏、新生儿呼吸窘迫、鼻塞和喂养困难的婴儿。在晚年，慢性中耳炎、复发性肺炎、鼻-鼻窦炎和由 MCC 受损引起的慢性典型湿性咳嗽是 PCD 的标志性症状，与支气管扩张症和肺功能受损相结合，持续到成年期2. 
怀疑患有PCD的患者可以通过使用不同的诊断工具进行诊断。过去，TEM被认为是一线诊断的黄金标准。然而，高达30%的PCD病例没有显示异常的超微结构1，3，4，5，6，需要不同的诊断方法。因此，越来越多的中心和欧洲呼吸学会 （ERS） 指南建议将鼻一氧化氮 （nNO） 和 HSVMA 联合作为一线诊断方法 1，7，9，10。HSVMA 和 nNO 也是识别 PCD11 患者的最具成本效益的选择。然而，即使基因检测包含在诊断中，也必须记住，目前没有独立的检测或组合检测可以100%确定性排除PCD8，9，10。 
在现有的诊断选项中，HSVMA 是唯一一种专注于活的纤毛包被呼吸道细胞并评估纤毛搏动模式 （CBP） 和纤毛搏动频率 （CBF） 的检查。与TEM相反，HSVMA的结果通常通常在测试当天快速获得，而TEM的结果可能在标本采集后数月到达。HSVMA可以适用于所有年龄组，而nNO要求高度合规;5岁以下尝试使用它通常不成功10.在有经验的人手中，HSVMA具有出色的敏感性和特异性，可分别诊断PCD 100%和96%，12。
本文描述了执行HSVMA的分步程序，包括从鼻子的下鼻甲中收获纤毛包被的呼吸道细胞，将收获的细胞保存在细胞滋养培养基中以运输到研究部位，以及微观视频分析以确定CBF和CBP的过程。此外，还显示了一些来自患者的视频剪辑，将正常的CBP和CBF与异常纤毛功能（视频3， 视频4， 视频5， 视频6， 视频7和 视频8）进行比较。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amoxiciline-clavulanic acid</td>
			<td>Orion Oyj</td>
			<td></td>
			<td>40 mg/kg divided in 2 doses/day, for adults 875/125 mg 1 tablet x2/day</td>
		</tr>
		<tr>
			<td>Camera Software</td>
			<td>Hamamatsu</td>
			<td></td>
			<td>HCI Image</td>
		</tr>
		<tr>
			<td>Cold pack</td>
			<td>any</td>
			<td></td>
			<td>for preservation and transport</td>
		</tr>
		<tr>
			<td>Differential interference microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Inverted, cell observer microscope</td>
		</tr>
		<tr>
			<td>Digitial High Speed Video Camera</td>
			<td>Hamamatsu</td>
			<td></td>
			<td>Orca Flash 4.0, digital camera type C11440</td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Modified Eagle Medium</td>
			<td>Thermo Fisher</td>
			<td>10565018</td>
			<td>basal cell culture medium</td>
		</tr>
		<tr>
			<td>Eppendorf tube</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td>1.5 mL tube</td>
		</tr>
		<tr>
			<td>Glass-bottom microwell dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td>cuvette for microscopy</td>
		</tr>
		<tr>
			<td>Heating Unit</td>
			<td>Carl Zeiss/PeCon</td>
			<td>810-450001</td>
			<td>Carl Zeiss incubation elements with PeCon TempModule S1 temperature control</td>
		</tr>
		<tr>
			<td>Interdental brush 0.6 mm</td>
			<td>Doft</td>
			<td>872267</td>
			<td>Interdental brush on a long wire with a reusable handle and cap in zipbag</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>100x/1.46, &#945; Plan-Apochromat DIC objective</td>
		</tr>
		<tr>
			<td>Small polystyrene box with lid</td>
			<td>any</td>
			<td></td>
			<td>for transport</td>
		</tr>
	</tbody>
</table>

high-speed video microscopy analysis for first-line diagnosis of primary ciliary dyskinesia

原发性睫状体运动障碍（PCD）是一种先天性疾病，主要遗传于常染色体隐性遗传性状。该疾病导致纤毛运动紊乱，导致黏膜纤毛清除率（MCC）的严重损害。如果未确诊或诊断得太晚，该病症会导致支气管扩张症的发展，并在以后的生活中对肺部造成严重损害。大多数诊断PCD的方法都很耗时，需要大量的经济资源来建立它们。高速视频显微镜分析 （HSVMA） 是 体外可视化和分析纤毛跳动的活呼吸道细胞的唯一诊断工具。它快速，经济高效，并且在经验丰富的手中，作为PCD的诊断工具非常可靠。此外，由于不存在形态学变化，透射电子显微镜（TEM）等经典诊断措施不适用于某些突变。 
本文介绍了收集呼吸道上皮细胞的过程，标本的进一步制备以及HSVMA的过程。我们还描述了如何在诊所不具备执行HSVMA的设备的情况下，通过将刷毛细胞保存在滋养介质中以储存并运输到调查地点，从而成功地保持不受伤害和跳动。还显示了来自动力蛋白臂重链11基因（DNAH11）突变的患者的病理性跳动模式的视频，该基因不能被诊断为TEM;由于上气道感染导致的 HSVMA 不确定的结果，以及刷牙与红细胞叠加不成功。通过本文，我们希望鼓励每个处理肺病患者和罕见肺部疾病的单位进行HSVMA，作为其PCD日常常规诊断的一部分，或将标本送到专门从事HSVMA的中心。

视频 1 和 视频 2 显示了 CBF 和 CBP 处于正常范围的正常控制（参见 图 1）。 视频3， 视频4， 视频5和 视频6 代表了两例PCD患者在 DNAH11 基因中纯合突变（c.2341G>A; p. Glu781Lys）3。之所以选择这些具有代表性的视频，是因为 DNAH11 基因突变的表型值得注意，因?...

Watch this Scientific Journal Video about High-speed Video Microscopy Analysis for First-line Diagnosis of Primary Ciliary Dyskinesia at JoVE.com

High-speed Video Microscopy Analysis for First-line Diagnosis of Primary Ciliary Dyskinesia

高速视频显微镜分析是一种相对容易执行、快速、经济高效，而且在有经验的人手中，它是原发性睫状体运动障碍一线诊断的相当可靠的工具，每个参与诊断和治疗严重肺部疾病的中心都应该提供这种工具。

高速视频显微镜分析在原发性睫状体运动障碍一线诊断中的应用

Primary ciliary dyskinesia (PCD) is a congenital disorder predominantly inherited in an autosomal recessive trait. The disorder causes disturbance in the ...

高速视频显微镜分析是原发性睫状体运动障碍一线诊断的可靠工具。这种技术相对容易执行，快速，具有成本效益，并且在经验丰富的人手中是一种非常可靠的工具。适用于所有年龄组的患者。视频显微镜检查还显示继发性纤毛功能。例如，由于细菌或病毒感染。但它主要用于诊断原发性纤毛运动障碍。科学家也使用这种方法来可视化活细胞中的细胞内过程或生物功能。协助我完成这一程序的将是我们的实验室技术员Tina Peromaa女士。在收集呼吸道上皮细胞之前，请患者彻底擤鼻涕。当患者准备好时，用一只手固定患者的头部，另一只手使用0.6毫米大小的齿间刷刷刷鼻孔的下鼻甲，以收集足够的上皮细胞条。将收获的细胞条放入含有细胞滋养DMEM的1.5毫升微量离心管中。关闭微量离心管的盖子后，将管保持在光源上并摇动管以发现收获的细胞条和砾岩。之后，将盖子正确关闭的微量离心管放在聚苯乙烯盒中。在关闭盒子之前，请确保管子固定在盒子内。然后在盒子中加入冷敷，同时避免冻结试样。从聚苯乙烯盒中取出含有样品的微量离心管后，将一管置于显微镜加热单元的罩下，以升温至37摄氏度，以进行高速视频显微镜分析或HSVMA。接下来，启动计算机上的摄像机和视频单元的软件。使用移液器取一小块样品，并将两滴样品放入比色皿中。用盖子盖住比色皿，然后将其置于显微镜下。使用放大倍率为100倍的油浸透镜，并将一滴浸油滴在光学元件的表面上。当用显微镜镜片接近培养皿的底部时，寻找没有红细胞且粘液含量低的细胞簇。在找到具有代表性的兴趣区域或ROI后，将重点放在一组具有最大纤毛运动的跳动纤毛上，并录制视频序列。记录细胞与跳动纤毛侧明智和从上面。代表性分析显示，从侧面和上方看到的正常纤毛运动。原发性睫状体运动障碍或PCD患者DNAH11基因突变的样本显示，睫状动脉搏动的典型僵硬最小移动模式。从侧面和上方观看时。在另一个具有相同突变的PCD患者中，观察到跳动纤毛的不同运动过度但低效的模式。在复发性上气道感染的患者中也可见病理性的睫状动脉搏动模式和纤毛搏动频率。如果严格执行刷牙程序，则无法进行HSVMA分析，因为纤毛上皮细胞被红细胞覆盖。该程序中最重要的一步是从刷牙中获得足够的呼吸细胞，并保持这些材料保存完好，以便进行视频分析。剩余的材料可用于进行电子显微镜或免疫荧光显微镜检查，以检测纤毛中可能的结构缺陷或揭示缺陷和睫状蛋白的合成。

Research

JoVE Journal

Medicine

FISH for Pre-implantation Genetic Diagnosis

鱼的胚胎植入前基因诊断

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

科研

医学

Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer

里维斯的蒙特雅克鹿怀孕诊断的跨性别超声波

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

视网膜血流小鼠活体视频显微镜测量

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

一种计算机辅助心血管分析诊断平台的研究

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

石蜡内嵌组织中人与鼠中性粒细胞外陷阱的免疫荧光标记

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

Testing Acetylcholine Followed by Adenosine for Invasive Diagnosis of Coronary Vasomotor Disorders

检测乙酰胆碱，然后使用腺苷进行冠状动脉血管舒缩疾病的侵入性诊断

More than 50% of patients with signs and symptoms of myocardial ischemia undergoing coronary angiography have unobstructed coronary arteries. Coronary ...

Measurement of Myocardial Lactate Production for Diagnosis of Coronary Microvascular Spasm

测量心肌乳酸生成用于诊断冠状动脉微血管痉挛

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina ...

Basophil Activation Test for Allergy Diagnosis

嗜碱性粒细胞活化试验用于过敏诊断

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

原代人鼻上皮细胞模型的收集、扩增和分化，用于定量纤毛搏动频率

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...