JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Tadução automática
Please note that all translations are automatically generated. Click here for the English version.
Imunologia e Infecção

使用数字高速睫状视频显微镜进行鼻刷采样和处理 – 适应 COVID-19 大流行

Published: November 07, 2020
doi:
10.3791/61949
, , , , , , , , ,
1Pneumology laboratory, I3 Group, GIGA Research Center,University of Liège, 2Department of ENT,University Hospital of Liège, 3Department for Occupational Safety and Health, Biosafety and Biosecurity Unit,University of Liège, 4Division of Respirology, Department of Pediatrics,University of British Columbia and British Columbia Children’s Hospital, 5Division of Cardiology, Department of Pediatrics,University Hospital of Liège, 6Department of Pneumology,University Hospital of Liège, 7Division of Respirology, Department of Pediatrics,University Hospital of Liège

Summary

为了保证PCD诊断的成功和高质量的睫状功能分析，一种精确而仔细的呼吸上皮取样和处理方法至关重要。为了在COVID-19大流行期间继续提供PCD诊断服务，睫状视频显微镜检查方案已更新，以包括适当的感染控制措施。

Abstract

原发性纤毛运动障碍（PCD）是一种遗传性活动性纤毛病，可导致严重的耳鼻肺疾病。由于不同诊断方式的挑战，PCD诊断经常被遗漏或延迟。睫状视频显微镜使用PCD诊断工具之一的数字高速视频显微镜（DHSV）被认为是进行睫状功能分析（CFA）的最佳方法，包括睫状搏动频率（CBF）和搏动模式（CBP）分析。然而，DHSV缺乏标准化的、公布的处理和分析样品的操作程序。它还使用活呼吸道上皮，这是 COVID-19 大流行期间的一个重要感染控制问题。为了在这场健康危机期间继续提供诊断服务，睫状视频显微镜检查方案已经过调整，以包括适当的感染控制措施。

在这里，我们描述了纤毛呼吸道样本采样和实验室处理的修订方案，重点介绍了为遵守COVID-19感染控制措施而进行的调整。描述了从16名健康受试者获得的鼻刷样品中CFA的代表性结果，根据该协议进行处理和分析。我们还说明了获得和处理最佳质量上皮纤毛条的重要性，因为不符合质量选择标准的样品现在确实允许CFA，这可能会降低该技术的诊断可靠性和效率。

Introduction

原发性纤毛运动障碍（PCD）是一种遗传性异质性活动性纤毛病，其中呼吸纤毛静止，缓慢或运动障碍，导致粘液纤毛清除受损和慢性耳窦肺疾病1，2，3，4。PCD的临床表现是婴儿早期开始的慢性湿咳和慢性鼻塞，复发性或慢性上呼吸道和下呼吸道感染导致支气管扩张，以及复发性或慢性中耳炎和鼻窦炎5，6，7。大约一半的PCD患者表现为器官偏侧性缺陷，例如位位逆位或位位模糊。一些患者还由于男性精子不动和女性输卵管不动纤毛而出现不孕问题1，2，8。PCD 很少见，但患病率难以定义，范围为 1：10，000 至 1：20，0009，10。然而，由于诊断困难和缺乏临床怀疑，PCD 的实际患病率被认为更高。PCD的症状与其他急性或慢性呼吸系统疾病的常见呼吸表现相似，确认诊断的诊断挑战是众所周知的，导致治疗和随访不足2，5，9，11。

使用数字高速视频显微镜（DHSV）的睫状视频显微镜是PCD4，8，12，13的诊断工具之一。DHSV被认为是进行睫状功能分析（CFA）的最佳方法，包括睫状搏动频率（CBF）和搏动模式（CBP）分析2，14，15，16。DHSV使用活的呼吸道上皮，通常通过鼻刷获得13。

鉴于当前的COVID-19爆发，PCD诊断的确认现在变得更加重要，因为有证据表明，潜在的呼吸道疾病可能导致COVID-19感染后更糟糕的结果17，18。与一般人群相比，在当前大流行期间安全有效的PCD诊断服务也将使确诊的PCD患者受益于额外的保护措施19。

COVID-19的传播主要通过飞沫传播20。鼻样本20中的高病毒载量表明无症状（或症状轻微）患者的传播可能性很高。此外，如果病毒颗粒被雾化，它们会在空气中停留至少 3 小时21。因此，呼吸系统医护人员在进行临床护理和诊断技术样本采集时会暴露在高病毒载量中22。此外，操纵活体呼吸道样本会使技术人员暴露于 COVID-19 污染中。虽然正在实施针对护理COVID-19患者的呼吸内科医生和耳鼻喉外科医生的最佳实践建议23，但缺乏在COVID-19大流行期间实施DHSV的建议。

为了继续提供PCD诊断服务，同时确保医护人员（进行样本采集）和技术人员（进行样本处理）的安全，必须在COVID-19大流行期间调整睫状视频显微镜方案。睫状视频显微镜技术目前仅限于研究服务和专业诊断中心，因为CFA需要广泛的培训和经验。此外，目前，使用DHSV4，13处理和分析样品缺乏标准化和精确的操作程序。

本文的目的是描述DHSV的标准操作程序，特别是感染控制措施和采样和处理活鼻上皮时的安全性。尽管目前COVID-19爆发，但这将使高质量的PCD诊断和护理得以继续。

Protocol

获得了列日医院-教师伦理委员会和大学卫生与健康保护部的批准。 1.呼吸道纤毛上皮取样 在采样前，确保受试者至少 4-6 周没有感染，并且没有鼻腔和吸入药物。 制备补充的M199制剂：用抗生素溶液（5mL链霉素/青霉素（5μg/ mL））和抗真菌溶液（5mL两性霉素B（2.5μg/ mL））补充细胞培养基199（M199）（500mL）。 准备 2 个（每个鼻孔一个）带盖的 15 mL 锥形?…

Representative Results

为了说明该技术的效率，我们在一系列16名健康成年志愿者（5名男性，年龄范围22-54岁）中展示了CFA的结果。 在总共16名志愿者中，来自14名（4名男性，年龄范围24-54岁）的鼻刷样本提供了足够合适的上皮边缘，满足进行CFA所需的选择标准。从这14个鼻刷样本中，共记录了242个纤毛边缘，212个边缘符合定义的纳入标准并进行了分析。所有这些边缘都记录在侧向剖面中（记录并分…

Discussion

本文旨在为使用鼻刷样本的CFA提供标准操作程序，并在COVID-19大流行期间根据适当的感染控制考虑进行调整。PCD诊断具有挑战性，根据国际建议，目前需要一组不同的诊断测试，包括鼻一氧化氮测量，使用DHSV的CFA，使用透射电子显微镜（TEM）的睫状超微结构分析，使用免疫荧光标记睫状蛋白，以及PCD致病基因4，37的基因检测。目前，没有单一的测试可?…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们要感谢Jean-François Papon，Bruno Louis，Estelle Escudier以及巴黎东部PCD诊断中心的所有团队成员在访问他们的PCD诊断中心期间的可用性和热烈的欢迎，以及众多的交流。我们还要感谢罗伯特·赫斯特和莱斯特PCD中心的所有团队成员的欢迎和时间，建议和专业知识。

Materials

15 mL conical tubes FisherScientific 352096 15 ml High-Clarity Polypropylene Conical Tube with lid
Amphotericin B LONZA 17-836E Antifungal solution
Blakesley-weil nasal forceps NOVO SURGICAL E7739-12 Used to hold the brush to perform the nasal brushing
Bronchial cytology brush CONMED 129 Used for nasal brushing
Cotton swab NUOVA APTACA 2150/SG Used for COVID-19 testing
Digitial high-speed videomicroscopy camera IDTeu Innovation in motion CrashCam Mini 1510
Glass slide ThermoScientific 12372098 Microscope slides used to create the visualization chamber
Heated Box IBIDI cells in focus 10918 Used to heat the sample
Inverted Light microscope Zeiss AXIO Vert.A1
Lens Heater TOKAI HIT TPiE-LH Used to heat the oil immersion lens
Medium 199 (M199), HEPES TermoFisher Scientific 12340030 Cell Culture Medium
Motion Studio X64 IDT Motion version 2.14.01 Software
Oil FischerScientific, Carl Zeiss 11825153
Rectangular cover slip VWR 631-0145 Used to cover the visualization chamber
Spacer (Ispacer) 0.25 mm Sunjinlab IS203 Used for the creation of the hermetic closed visualization chamber
Square cover slip VWR 631-0122 Used for the creation of lab-built open visualization chamber
Streptomycin/Penicillin FisherScientific, Gibco 11548876 Antiobiotics solution
DOWNLOAD MATERIALS LIST

Referências

  1. Chilvers, M. A., Rutman, A., O’Callaghan, C. Ciliary beat pattern is associated with specific ultrastructural defects in primary ciliary dyskinesia. Journal of Allergy Clinical Immunology. 112 (3), 518-524 (2003).
  2. Werner, C., Onnebrink, J. G., Omran, H. Diagnosis and management of primary ciliary dyskinesia. Cilia. , 1-9 (2015).
  3. Kempeneers, C., Chilvers, M. A. To beat, or not to beat, that is question! The spectrum of ciliopathies. Pediatric Pulmonology. 53 (8), 1122 (2018).
  4. Lucas, J. S., et al. European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia. The European Respiratory Journal. 49 (1), (2017).
  5. Knowles, M. R., Zariwala, M., Leigh, M. Primary Ciliary Dyskinesia. Clinics in chest medicine. 37 (3), 449-461 (2016).
  6. Shapiro, A. J., et al. Diagnosis, monitoring, and treatment of primary ciliary dyskinesia: PCD foundation consensus recommendations based on state of the art review. Pediatric Pulmonology. , (2016).
  7. Fitzgerald, D. A., Shapiro, A. J. When to suspect primary ciliary dyskinesia in children. Paediatric Respiratory Reviews. , (2016).
  8. Shoemark, A., Dell, S., Shapiro, A., Lucas, J. S. ERS and ATS diagnostic guidelines for primary ciliary dyskinesia: similarities and differences in approach to diagnosis. European Respiratory Journal. 54 (3), (2019).
  9. Mirra, V., Werner, C., Santamaria, F. Primary ciliary dyskinesia: An update on clinical aspects, genetics, diagnosis, and future treatment strategies. Frontiers in Pediatrics. 5, 1-13 (2017).
  10. Ardura-Garcia, C., et al. Registries and collaborative studies for primary ciliary dyskinesia in Europe. European Respiratory Journal Open Research. 6 (2), (2020).
  11. Leigh, M. W., et al. Clinical features and associated likelihood of primary ciliary dyskinesia in children and adolescents. Annals of the American Thoracic Society. , (2016).
  12. Chilvers, M. A., O’Callaghan, C. Analysis of ciliary beat pattern and beat frequency using digital high speed imaging: comparison with the photomultiplier and photodiode methods. Thorax. 55 (4), 314-317 (2000).
  13. Kempeneers, C., Seaton, C., Garcia Espinosa, B., Chilvers, M. A. Ciliary functional analysis: Beating a path towards standardization. Pediatric Pulmonology. 54 (10), 1627-1638 (2019).
  14. Barbato, A., et al. Primary ciliary dyskinesia: a consensus statement on diagnostic and treatment approaches in children. The European respiratory journal. 34 (6), 1264-1276 (2009).
  15. Raidt, J., et al. Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia. European Respiratory Journal. 44 (6), 1579-1588 (2014).
  16. Kempeneers, C., Seaton, C., Chilvers, M. A. Variation of Ciliary Beat Pattern in Three Different Beating Planes in Healthy Subjects. Chest. 151 (5), 993-1001 (2017).
  17. Götzinger, F., et al. COVID-19 in children and adolescents in Europe: a multinational, multicentre cohort study. The Lancet Child & Adolescent Health. , (2020).
  18. Yang, J., et al. Prevalence of comorbidities and its effects in coronavirus disease 2019 patients: A systematic review and meta-analysis. International Journal of Infectious Diseases. 94, 91-95 (2020).
  19. Brough, H. A., et al. Managing childhood allergies and immunodeficiencies during respiratory virus epidemics – The 2020 COVID-19 pandemic: A statement from the EAACI-section on pediatrics. Pediatric Allergy and Immunology. 31 (5), 442-448 (2020).
  20. Zou, L., et al. SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients. The New England journal of medicine. 382 (12), 1177-1179 (2020).
  21. van Doremalen, N., et al. Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1. The New England journal of medicine. 382 (16), 1564-1567 (2020).
  22. Tran, K., Cimon, K., Severn, M., Pessoa-Silva, C. L., Conly, J. Aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review. PloS one. 7 (4), 35797 (2012).
  23. Van Gerven, L., et al. Personal protection and delivery of rhinologic and endoscopic skull base procedures during the COVID-19 outbreak. Rhinology. 58 (3), 289-294 (2020).
  24. Marty, F. M., Chen, K., Verrill, K. A. How to Obtain a Nasopharyngeal Swab Specimen. New England Journal of Medicine. 382 (22), 76 (2020).
  25. Petruzzi, G., et al. COVID-19: Nasal and oropharyngeal swab. Head & Neck. 42, (2020).
  26. George, A., Prince, M., Coulson, C. Safe nasendoscopy assisted procedure in the post-COVID-19 pandemic era. Clinical Otolaryngology. , (2020).
  27. Hirst, R. A., et al. Culture of primary ciliary dyskinesia epithelial cells at air-liquid interface can alter ciliary phenotype but remains a robust and informative diagnostic aid. PLoS ONE. 9 (2), (2014).
  28. Jorissen, M., Willems, T., Van der Schueren, B. Ciliary function analysis for the diagnosis of primary ciliary dyskinesia: advantages of ciliogenesis in culture. Acta oto-laryngologica. 120 (2), 291-295 (2000).
  29. Thomas, B., Rutman, A., O’Callaghan, C. Disrupted ciliated epithelium shows slower ciliary beat frequency and increased dyskinesia. European Respiratory Journal. 34 (2), 401-404 (2009).
  30. Chilvers, M. A., Rutman, A., O’Callaghan, C. Functional analysis of cilia and ciliated epithelial ultrastructure in healthy children and young adults. Thorax. 58 (4), 333-338 (2003).
  31. Stannard, W. A., Chilvers, M. A., Rutman, A. R., Williams, C. D., O’Callaghan, C. Diagnostic testing of patients suspected of primary ciliary dyskinesia. American Journal of Respiratory and Critical Care Medicine. 181 (4), 307-314 (2010).
  32. Boon, M., et al. Primary ciliary dyskinesia: critical evaluation of clinical symptoms and diagnosis in patients with normal and abnormal ultrastructure. Orphanet Journal of Rare Diseases. 9 (1), 11 (2014).
  33. Armengot, M., Milara, J., Mata, M., Carda, C., Cortijo, J. Cilia motility and structure in primary and secondary ciliary dyskinesia. American Journal of Rhinology & Allergy. 24 (3), 175-180 (2010).
  34. Papon, J. F., et al. Quantitative analysis of ciliary beating in primary ciliary dyskinesia: a pilot study. Orphanet Journal of Rare Diseases. 7 (1), 78 (2012).
  35. Wallmeier, J., et al. Mutations in CCNO and MCIDAS lead to a mucociliary clearance disorder due to reduced generation of multiple motile cilia. Molecular and Cellular Pediatrics. 2, 15 (2015).
  36. Boon, M., et al. MCIDAS mutations result in a mucociliary clearance disorder with reduced generation of multiple motile cilia. Nature Communications. 5 (6), 4418 (2014).
  37. Shapiro, A. J., et al. Diagnosis of Primary Ciliary Dyskinesia. An Official American Thoracic Society Clinical Practice Guideline. American Journal of Respiratory and Critical Care Medicine. 197 (12), 24-39 (2018).
  38. Rubbo, B., et al. Accuracy of high-speed video analysis to diagnose primary ciliary dyskinesia. Chest. (19), 30205 (2019).
  39. Horani, A., Ferkol, T. W. Advances in the Genetics of Primary Ciliary Dyskinesia. Chest. 154 (3), 645-652 (2018).
  40. MacCormick, J., Robb, I., Kovesi, T., Carpenter, B. Optimal biopsy techniques in the diagnosis of primary ciliary dyskinesia. The Journal of Otolaryngology. 31 (1), 13-17 (2002).
  41. Jackson, C. L., et al. Accuracy of diagnostic testing in primary ciliary dyskinesia. European Respiratory Journal. 47 (3), 837-848 (2016).
  42. Jackson, C. L., Goggin, P. M., Lucas, J. S. Ciliary Beat Pattern Analysis Below 37°C May Increase Risk of Primary Ciliary Dyskinesia Misdiagnosis. Chest. 142 (2), 543-544 (2012).
  43. Green, A., Smallman, L. A., Logan, A. C., Drake-Lee, A. B. The effect of temperature on nasal ciliary beat frequency. Clinical otolaryngology and allied sciences. 20 (2), 178-180 (1995).
  44. Clary-Meinesz, C. F., Cosson, J., Huitorel, P., Blaive, B. Temperature effect on the ciliary beat frequency of human nasal and tracheal ciliated cells. Biology of the Cell. 76 (3), 335-338 (1992).
  45. Smith, C. M., et al. ciliaFA: a research tool for automated, high-throughput measurement of ciliary beat frequency using freely available software. Cilia. 1 (1), 14 (2012).
  46. Sisson, J. H., Stoner, J. a., Ammons, B. a., Wyatt, T. a. All-digital image capture and whole-field analysis of ciliary beat frequency. Journal of Microscopy. 211, 103-111 (2003).
  47. Blanchon, S., et al. Deep phenotyping, including quantitative ciliary beating parameters, and extensive genotyping in primary ciliary dyskinesia. Journal of Medical Genetics. , (2019).
  48. Feriani, L., et al. Assessing the Collective Dynamics of Motile Cilia in Cultures of Human Airway Cells by Multiscale DDM. Biophysical Journal. 113 (1), 109-119 (2017).
  49. Sears, P. R., Thompson, K., Knowles, M. R., Davis, C. W. Human airway ciliary dynamics. American Journal of Physiology – Lung Cellular and Molecular Physiology. 304 (3), 170-183 (2013).
  50. Quinn, S. P., et al. Automated identification of abnormal respiratory ciliary motion in nasal biopsies. Science translational medicine. 7 (299), (2015).

Tags

Keywords: Nasal BrushingDigital High-speed Ciliary VideomicroscopyPrimary Ciliary DyskinesiaCOVID-19Diagnostic TestSamplingProcessingInfection ControlSensitivitySpecificityElectron MicroscopyGenetic TestingWeil-Blakesley Nasal ForcepsEpithelial StripsGlass SlideSpacer
check_url/pt/61949?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Bricmont, N., Benchimol, L., Poirrier, A., Grignet, C., Seaton, C., Chilvers, M. A., Seghaye, M., Louis, R., Lefebvre, P., Kempeneers, C. Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy – Adaptation for the COVID-19 Pandemic. J. Vis. Exp. (165), e61949, doi:10.3791/61949 (2020).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image