We wish to express special thanks to the pediatric nurse Mrs. Johanna Juvankoski for her excellent help with the brushings. We would also like to express special gratitude to Professor Heymut Omran (University Clinic M&#252;nster, UKM) for granting permission to use the schematic figure of normal ciliary motion from their website. Finally, we would like to thank Mr. Alan Brown BA (Hons), PGCE, for proofreading the manuscript.

Ethics Statement:
This study was approved by the local ethics committee (69/2017) and was conducted in compliance with the declaration of Helsinki.
1. Collection and transport of respiratory epithelial cells
<ol>
	<li>Brushing
	<ol>
		<li>Before brushing, administer a two-week course of oral amoxicillin-clavulanic acid to the patient to eradicate biofilms interfering with cilia function. Ensure the antibiotic is terminated 2 days before the procedure.</li>
		<li>To diminish an overlay of mucous material on the brushed epithelial cell strips, ask the patient to blow their nose thoroughly. Ask parents to help their small children blow their noses.</li>
		<li>For brushing, use an interdental brush of 0.6 mm size (see the Table of Materials). Keep the head of the patient fixed with one hand, and with the other hand, brush the inferior turbinate of both nostrils to collect sufficient epithelial cell strips. 
		NOTE: A slight resistance is usually experienced when inserting the interdental brush deeply under the inferior turbinate. Nasal brushing is carried out by quick turning and picking for approximately 2 s. Longer and intense brushing might otherwise cause severe epithelial injury and consecutive epistaxis.</li>
		<li>If bronchoscopy is planned for reasons other than suspected PCD, collect samples during bronchoscopy by brushing the carina or bronchial epithelium or by biopsy13.</li>
		<li>Drop the harvested cell strips into a 1.5 mL microcentrifuge tube containing the cell-nourishing Dulbecco&#180;s Modified Eagle Medium (DMEM). 
		NOTE: Epithelial cell strips usually drop off the brush more easily by tossing and turning the brush against the tube wall.</li>
		<li>Close the lid of the microcentrifuge tube and hold the tube against a light source. Shake the tube to discover harvested cell strips and conglomerates.</li>
	</ol>
	</li>
	<li>Transport of the specimen
	<ol>
		<li>Place the microcentrifuge tubes in a tightly closed polystyrene box and ensure that the lid of the tubes is properly closed and the tubes are fixed inside the box.</li>
		<li>Add a cold pack; however, avoid freezing the specimen. 
		NOTE: The optimal temperature for preserving the specimen before investigation is approximately 4-8 &#176;C (39.2-46.4 &#176;F).</li>
		<li>Ensure that the preserved samples are analyzed during the next 24 h. 
		NOTE: In these experiments, the mean time between brushings and HSVMA was 3 h.</li>
	</ol>
	</li>
</ol>
2. High-speed video microscopy analysis (HSVMA)
<ol>
	<li>Video microscopy of the samples
	<ol>
		<li>After receiving the microcentrifuge tubes containing the samples, warm them up to 37 &#176;C (98.6 &#176;F) to mimic an optimal, in vivo-like environment.</li>
		<li>If the microscope is equipped with a heating unit, place the tubes under the hood and warm them up there. Alternatively, use an incubator to warm up the samples.</li>
		<li>Start the camera and the software of the video unit on the PC.</li>
		<li>Using a pipette, take a small amount of the sample and place two drops into a cuvette or glass-bottom dish (see the Table of Materials). Cover the cuvette or dish with a lid and place it under the microscope.</li>
		<li>For evaluation of the samples, use a differential-interference microscope equipped with a cold-light source and a video camera able to record at high speed (at least 200 frames/s). Use an oil immersion lens with a 100x magnification and put a drop of immersion oil onto the surface of the optic. 
		NOTE: Microscopes with a lens approaching from below are recommended.</li>
		<li>Approach the bottom of the dish with the microscope lens and search for cell clusters without red blood cells and with low mucus content. After finding a representative region of interest (ROI), focus on one specific group of beating cilia with the largest cilia movements and record a video sequence. Record cells with beating cilia sidewise and from above. After that, search for another representative cluster of cells and repeat the recording. 
		NOTE: The cilia coated cells must be investigated under a 1,000x magnification, and beating cilia should be recorded with a digital high-speed video (DHSV) camera set on a frame rate of 200/s or more (256/s in this protocol). Because the data obtained from the recorded video clips requires a lot of space on the hard drive, an external SSD hard drive is recommended.</li>
	</ol>
	</li>
	<li>Analysis of video sequences
	<ol>
		<li>To determine CBF and CBP, play the video clips back frame by frame.</li>
		<li>To determine CBF, set the frame rate into slow motion with 15 frames/s and count 10 consecutive beats.</li>
		<li>Record the number of frames passing by during a single cycle of 10 beats and insert the result into Eq (1). Determine the frequency by calculating the mean of all recorded cilia beat cycles and compare the result with the reference values for age (see Table 1)14. 
		<img alt="Equation 1" src="/files/ftp_upload/63292/63292eq01.jpg" /> (1) 
		Where X is the number of frames passing by during a cycle of 10 beats.</li>
		<li>For evaluating CBP, watch if the movement of the beating cilia is in full range (see Figure 1) and synchronized. Have two independent operators evaluate the CBP to prevent selection bias.</li>
		<li>Report the results of the HSVMA analysis to be either compatible, unlikely, or inconclusive with PCD.</li>
	</ol>
	</li>
</ol>

<ol><li>Werner, C., Onnebrink, J. G., Omran, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diagnosis+and+management+of+primary+ciliary+dyskinesia%5BTitle%5D)+AND+%22Cilia%22%5BJournal%5D)">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/pubmed?term=((Primary+ciliary+dyskinesia%3A+An+update+on+clinical+aspects%2C+genetics%2C+diagnosis+and+future+treatment+strategies%5BTitle%5D)+AND+%22Frontiers+in+Pediatrics%22%5BJournal%5D)">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/pubmed?term=((Two+novel+mutations+in+the+DNAH11+gene+in+primary+ciliary+dyskinesia+%28CILD7%29+with+considerable+variety+in+the+clinical+and+beating+cilia+phenotype%5BTitle%5D)+AND+%22BMC+Medical+Genetics%22%5BJournal%5D)">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/pubmed?term=((Mutations+of+DNAH11+in+patients+with+primary+ciliary+dyskinesia+with+normal+ciliary+ultrastructure%5BTitle%5D)+AND+%22Thorax%22%5BJournal%5D)">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/pubmed?term=((Primary+ciliary+dyskinesia%3A+critical+evaluation+of+clinical+symptoms+and+diagnosis+in+patients+with+normal+and+abnormal+ultrastructure%5BTitle%5D)+AND+%22Orphanet+Journal+of+Rare+Diseases%22%5BJournal%5D)">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/pubmed?term=((Prevalence+of+primary+ciliary+dyskinesia+in+consecutive+referrals+of+suspected+cases+and+the+transmission+electron+microscopy+detection+rate%3A+a+systematic+review+and+meta-analysis%5BTitle%5D)+AND+%22Pediatric+Research%22%5BJournal%5D)">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/pubmed?term=((Choice+of+nasal+nitric+oxide+technique+as+first-line+test+for+primary+ciliary+dyskinesia%5BTitle%5D)+AND+%22European+Respiratory+Journal%22%5BJournal%5D)">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/pubmed?term=((Accuracy+of+diagnostic+testing+in+primary+ciliary+dyskinesia%5BTitle%5D)+AND+%22European+Respiratory+Journal%22%5BJournal%5D)">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/pubmed?term=((European+Respiratory+Society+guidelines+for+the+diagnosis+of+primary+ciliary+dyskinesia%5BTitle%5D)+AND+%22European+Respiratory+Journal%22%5BJournal%5D)">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/pubmed?term=((ERS+and+ATS+diagnostic+guidelines+for+primary+ciliary+dyskinesia%3A+similarities+and+differences+in+approach+to+diagnosis%5BTitle%5D)+AND+%22European+Respiratory+Journal%22%5BJournal%5D)">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/pubmed?term=((Cost-effectiveness+analysis+of+three+algorithms+for+diagnosing+primary+ciliary+dyskinesia%3A+a+simulation+study%5BTitle%5D)+AND+%22Orphanet+Journal+of+Rare+Diseases%22%5BJournal%5D)">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/pubmed?term=((Accuracy+of+high-speed+video+analysis+to+diagnose+primary+ciliary+dyskinesia%5BTitle%5D)+AND+%22Chest%22%5BJournal%5D)">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/pubmed?term=((Optimal+technique+to+diagnose+primary+ciliary+dyskinesia%5BTitle%5D)+AND+%22The+Laryngoscope%22%5BJournal%5D)">Optimal technique to diagnose primary ciliary dyskinesia.</a> The Laryngoscope. 110 (9), 1548-1551 (2000).</li>
<li>Chilvers, M. A., Rutman, A., O´Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+analysis+of+cilia+and+ciliated+epithelial+ultrastructure+in+healthy+children+and+young+adults%5BTitle%5D)+AND+%22Thorax%22%5BJournal%5D)">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/pubmed?term=((Diagnostic+methods+in+primary+ciliary+dyskinesia%5BTitle%5D)+AND+%22Paediatric+Respiratory+Reviews%22%5BJournal%5D)">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/pubmed?term=((Experimental+effect+of+cigarette+smoke+on+human+respiratory+cilia%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">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/pubmed?term=((Effect+of+cigarette+smoking+on+nasal+mucociliary+clearance+and+ciliary+beat+frequency%5BTitle%5D)+AND+%22Thorax%22%5BJournal%5D)">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/pubmed?term=((Efficacy+and+safety+of+azithromycin+maintenance+therapy+in+primary+cilia+dyskinesia+%28BESTCILIA%29%3A+a+multicentre%2C+double-blind%2C+randomised%2C+placebo-controlled+phase+3+trial%5BTitle%5D)+AND+%22Lancet+Respiratory+Medicine%22%5BJournal%5D)">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/pubmed?term=((Effect+of+macrolide+antibiotics+on+ciliary+motility+in+rabbit+airway+epithelium+in-vitro%5BTitle%5D)+AND+%22Journal+of+Pharmacy+and+Pharmacology%22%5BJournal%5D)">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/pubmed?term=((Culture+of+cells+harvested+with+nasal+brushing%3A+a+method+for+evaluating+ciliary+function%5BTitle%5D)+AND+%22Rhinology%22%5BJournal%5D)">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/pubmed?term=((Simplified+cell+culture+method+for+the+diagnosis+of+atypical+primary+ciliary+dyskinesia%5BTitle%5D)+AND+%22Thorax%22%5BJournal%5D)">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/pubmed?term=((Culture+of+primary+ciliary+dyskinesia+epithelial+cells+at+air+liquid+interface+can+alter+ciliary+phenotype+but+remains+a+robust+and+informative%2C+diagnostic+aid%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Quantitative+analysis+of+ciliary+beating+in+primary+ciliary+dyskinesia%3A+a+pilot+study%5BTitle%5D)+AND+%22Orphanet+Journal+of+Rare+Diseases%22%5BJournal%5D)">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/pubmed?term=((Cilia+FA%3A+a+research+tool+for+automated%2C+high-throughput+measurement+of+ciliary+beat+frequency+using+freely+available+software%5BTitle%5D)+AND+%22Cilia%22%5BJournal%5D)">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/pubmed?term=((Ciliar+Move%3A+new+software+for+evaluating+ciliary+beat+frequency+helps+find+novel+mutations+by+a+Portuguese+multidisciplinary+team+on+primary+ciliary+dyskinesia%5BTitle%5D)+AND+%22European+Respiratory+Journal+Open+Research%22%5BJournal%5D)">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/pubmed?term=((Primary+ciliary+dyskinesia+and+neonatal+respiratory+distress%5BTitle%5D)+AND+%22Pediatrics%22%5BJournal%5D)">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/pubmed?term=((Standardizing+nasal+nitric+oxide+measurement+as+a+test+for+primary+ciliary+dyskinesia%5BTitle%5D)+AND+%22Annals+of+the+American+Thoracic+Society%22%5BJournal%5D)">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>

The authors have nothing to disclose.

Here, the diagnostic process for PCD using HSVMA is described and discussed in the light of first-line diagnostics. Despite being relatively easy to establish, cost-effective11, and a reliable method in experienced hands12, HSVMA is not a diagnostic measure without pitfalls. Abnormal CBF and CBP may be due to secondary infection, leading to inflammation of the broncho-respiratory epithelia15, and for the same reason, smoking individuals may have abno...

Primary ciliary dyskinesia (PCD) is a rare, hereditary genetic disorder, which causes disturbances in the movement of beating cilia. If undiagnosed, it leads to severe lung damage in later life due to severe impairment of MCC. In the past, its prevalence has been estimated to be in the range of 1:4,000 to 50,000. Due to steadily improving diagnostics and a growing awareness of the condition, updates on the prevalence of PCD suggest that it might be much more common and probably in the range of 1:4,000 to 20,000 instead1,2. However, patients with PCD are still underdiagnosed or diagnosed too late1,3. Therefore, infants with either congenital situs inversus and/or heterotaxy or perinatal rhinorrhea, neonatal respiratory distress, a blocked nose, and feeding difficulties should be suspects for PCD. In later life, chronic otitis, recurrent pneumonia, rhinosinusitis, and a chronic, typical wet cough due to impaired MCC are the hallmark symptoms of PCD, which in combination with bronchiectasis and impaired lung function, continue into adulthood2. 
Patients suspected of having PCD can be diagnosed by using different diagnostic tools. TEM has been considered the gold standard for first-line diagnostics in the past. However, up to 30% of PCD cases do not show abnormal ultrastructure1,3,4,5,6, demanding a different diagnostic approach. Therefore, a growing number of centers and the guidelines of the European Respiratory Society (ERS) suggest a combination of nasal nitric oxide (nNO) and HSVMA as first-line diagnostics1,7,9,10. HSVMA and nNO are also the most cost-effective options in identifying a patient with PCD11. However, even if genetic testing were included in the diagnostics, it must be kept in mind that there is currently no stand-alone test or combination of tests that can exclude PCD with 100% certainty8,9,10. 
Out of the available diagnostic options, HSVMA is the only test that focuses on living, cilia-coated respiratory cells and evaluates ciliary beat pattern (CBP) and ciliary beat frequency (CBF). In contrast to TEM, the results of HSVMA are available quickly, usually on the day of testing, whereas results of TEM might arrive months after the specimen has been taken. HSVMA can be applied for all age groups, whereas nNO demands a high degree of compliance; attempts to use it under the age of 5 years are usually unsuccessful10. In experienced hands, HSVMA has excellent sensitivity and specificity to diagnose PCD at 100% and 96%, respectively12.
This paper describes the step-by-step procedure to perform HSVMA, including the harvesting of cilia-coated respiratory cells from the inferior turbinate of the nose, the preservation of harvested cells in a cell-nourishing medium for transport to the site of investigation, and the process of microscopic video analysis to determine CBF and CBP. Additionally, some video clips from patients are shown, comparing normal CBPs and CBFs with abnormal cilia function (Video 3, Video 4, Video 5, Video 6, Video 7, and Video 8).

<table><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&amp;acute;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, &amp;alpha; 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

Primary ciliary dyskinesia (PCD) is a congenital disorder predominantly inherited in an autosomal recessive trait. The disorder causes disturbance in the motion of cilia, leading to severe impairment of mucociliary clearance (MCC). If undiagnosed or diagnosed too late, the condition leads to the development of bronchiectasis and serious damage to the lungs in later life. Most of the methods for diagnosing PCD are time-consuming and demand extensive economic resources to establish them. High-speed video microscopy analysis (HSVMA) is the only diagnostic tool to visualize and analyze living respiratory cells with beating cilia in vitro. It is fast, cost-effective, and, in experienced hands, very reliable as a diagnostic tool for PCD. In addition, classical diagnostic measures such as transmission electron microscopy (TEM) are not applicable for some mutations as morphological changes are absent. 
This paper describes the process of collecting respiratory epithelial cells, the further preparation of the specimen, and the process of HSVMA. We also describe how brushed cells can be successfully kept unharmed and beating by keeping them in a nourishing medium for storage and transport to the investigation site in cases where a clinic does not possess the equipment to perform HSVMA. Also shown are videos with pathologic beating patterns from patients with a mutation in the dynein arm heavy chain 11 gene (DNAH11), which cannot be diagnosed with TEM; the result of an inconclusive HSVMA due to infection of the upper airways, as well as an unsuccessful brushing with superimposition of red blood cells. With this article, we would like to encourage every unit dealing with pulmonology patients and rare lung diseases to perform HSVMA as part of their daily routine diagnostics for PCD or send the specimens over to a center specializing in performing HSVMA.

Video 1 and Video 2 show a normal control where CBF and CBP are in the normal range (see Figure 1). Video 3, Video 4, Video 5, and Video 6 represent two cases of PCD patients with a homozygous mutation in the DNAH11 gene (c.2341G &#62; A; p. Glu781Lys)3. These representative videos were chosen because phenotypes of mutations in the DNAH11...

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

High-speed video microscopy analysis is a relatively easy-to-perform, fast, cost-effective, and, in experienced hands, a considerably reliable tool for first-line diagnostics of primary ciliary dyskinesia, which should be available in every center involved in diagnostics and the treatment of severe lung diseases.

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

high-speed-video-microscopy-analysis-for-first-line-diagnosis-primary

Research

JoVE Journal

Medicine

4.2K Views.  Tampere University Hospital. Hoge snelheid Video microscopie analyse is een betrouwbaar hulpmiddel voor de eerste lijn diagnose van primaire ciliaire dyskinesie. Deze techniek is relatief eenvoudig uit te voeren, snel, kosteneffectief en in ervaren handen anders een zeer betrouwbaar hulpmiddel. En het is van toepassing op patiënten van alle leeftijdsgroepen.Videomicroscopie onthult ook de secundaire ciliaire functie. Zoals bijvoorbeeld door bacteriële of virale infecties. Maar het is voornamelijk gebruikt om pri...

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

FISH for Pre-implantation Genetic Diagnosis 

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

FISH for Pre-implantation Genetic Diagnosis

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

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

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Neuroscience

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Biology

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &ndash; Adaptation for the COVID-19 Pandemic

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed due to challenges with different diagnostic modalities. Ciliary videomicroscopy, using Digital High-Speed Videomicroscopy (DHSV), one of the diagnostic tools for PCD, is considered the optimal method to perform ciliary functional analysis (CFA), comprising of ciliary beat frequency (CBF) and beat pattern (CBP) analysis. However, DHSV lacks standardized, published operating procedure for processing and analyzing samples. It also uses living respiratory epithelium, a significant infection control issue during the COVID-19 pandemic. To continue providing a diagnostic service during this health crisis, the ciliary videomicroscopy protocol has been adapted to include adequate infection control measures.
Here, we describe a revised protocol for sampling and laboratory processing of ciliated respiratory samples, highlighting adaptations made to comply with COVID-19 infection control measures. Representative results of CFA from nasal brushing samples obtained from 16 healthy subjects, processed and analyzed according to this protocol, are described. We also illustrate the importance of obtaining and processing optimal quality epithelial ciliated strips, as samples not meeting quality selection criteria do now allow for CFA, potentially decreasing the diagnostic reliability and the efficiency of this technique.

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &#8211; Adaptation for the COVID-19 Pandemic

To guarantee a successful and high-quality ciliary functional analysis for PCD diagnosis, a precise and careful method for respiratory epithelium sampling and processing is essential. To continue providing PCD diagnostic service during the COVID-19 pandemic, the ciliary videomicroscopy protocol has been updated to include appropriate infection control measures.

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed ...

Immunology and Infection

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

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

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

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function critically in cell motility and signaling. Since cilia are incapable of synthesizing their own protein, nearly 200 unique ciliary proteins need to be trafficked between the cytosol and primary cilia. However, it is still a technical challenge to map three-dimensional (3D) locations of transport pathways for these proteins in live primary cilia due to the limitations of currently existing techniques. To conquer the challenge, recently we have developed and employed a high-speed virtual 3D super-resolution microscopy, termed single-point edge-excitation sub-diffraction (SPEED) microscopy, to determine the 3D spatial location of transport pathways for both cytosolic and membrane proteins in primary cilia of live cells. In this article, we will demonstrate the detailed setup of SPEED microscopy, the preparation of cells expressing fluorescence-protein-labeled ciliary proteins, the real-time single-molecule tracking of individual proteins in live cilium and the achievement of 3D spatial probability density maps of transport routes for ciliary proteins.

Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia.

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

In vivo Evaluation of Mucociliary Clearance in Mice

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a metachronal, synchronal fashion, these multiple, motile, actin-based organelles generate a cephalad fluid flow clearing the respiratory tract of inhaled pollutants and pathogens. With increasing environmental pollution, novel viral pathogens and emerging multi-drug resistant bacteria, cilia generated mucociliary clearance (MCC) is essential for maintaining lung health. MCC is also depressed in multiple congenital disorders like primary ciliary dyskinesia, cystic fibrosis as well as acquired disorders like chronic obstructive pulmonary disease. All these disorders have established, in some case multiple, mouse models. In this publication, we detail a method using a small amount of radioactivity and dual-modality SPECT/CT imaging to accurately and reproducibly measure MCC in mice in vivo. The method allows for recovery of mice after imaging, making serial measurements possible, and testing potential therapeutics longitudinally over time. The data in wild-type mice demonstrates the reproducibility of the MCC measurement as long as adequate attention to detail is paid, and the protocol strictly adhered to.

In this publication, we describe protocols for assessing airway mucociliary clearance (MCC) in mice in vivo utilizing dual-modality radionuclide imaging. This protocol is designed for a single photon emission computed tomography (SPECT) and computed tomography (CT) acquisition protocol using mouse whole body (MWB) collimators in a dual SPECT/CT system.

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a ...

Artificial Intelligence Approaches to Assessing Primary Cilia

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and co-localization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.

The use of artificial intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. ...

Assessing Biophysical Properties of Differentiated Airway Epithelium 

Determining Ciliary Function and Membrane Impermeability of the Pseudostratified Lung Airway Epithelium

Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified airway epithelium that mimics the apical side of the lung airway. While the differentiation of primary lung airway cells is expected, the assessment of biophysical properties like ciliary function and membrane impermeability provides a quality assessment of the airway epithelium and ensures the reliability of the experiment. Here, we describe a straightforward protocol for the development of multi-cellular pseudostratified airway epithelium in ALI culture and assess two important biophysical properties: ciliary function and membrane impermeability. To determine ciliary function, we captured the ciliary movement of a 4-week differentiated airway epithelium using a high-speed camera attached to an inverted microscope, followed by quantifying ciliary beat frequency (CBF) using the Simon Amon Video Analysis (SAVA) system. We also measured transepithelial electrical resistance (TEER) using a volt-ohm meter to determine the epithelial barrier integrity of the airway epithelium.

Air-liquid interface culture is commonly used to develop pseudostratified airway epithelium by differentiating primary normal human bronchial epithelial cells that mimic the apical side of the lung airway. Here, we describe an easy protocol for determining its quality by monitoring its biophysical properties, such as ciliary function and membrane integrity.

Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified ...

Volumetric Imaging and Analysis of Primary Cilia in Musculoskeletal Tissue using the ARL13B-CENTRIN-2 Mouse Model

The primary cilium is a non-motile, solitary organelle assembled by most cell types. It is essential to the development and homeostasis of the skeleton and in coordinating cell responses to biochemical and mechanical cues from their surrounding environment. Due to its micrometer-scale size, the primary cilium is challenging to image in situ to characterize in tissues, in a high throughput manner and without optical biases. An understanding of its organization in its 3D environment in vivo will be key to understanding ciliary roles in physiology and disease. The ARL13B-CENTRIN-2 mouse line presents an mCherry tag on the ciliary axoneme protein ARL13B and a green fluorescent protein (GFP) tag on the centriole protein CENTRIN-2, localized to the base of the cilium. This mouse line allows for imaging of the primary cilium without the need for staining, which reduces the number of steps in image collection and circumnavigates issues with signal-to-noise. This publication describes a step-by-step protocol to image primary cilia in musculoskeletal tissues whilst preserving the fluorescent signal. It also describes a universally applicable image analysis pipeline, enabling unbiased quantification of cilia features, such as length and 3D orientation.

This method describes the exploitation of the endogenous signal in an ARL13B-CENTRIN-2 transgenic mouse model to image primary cilia in situ, in three dimensions across large areas of mineralized tissue, from tissue collection to analysis of cilia organization. This protocol may also be applied to other types of tissues.

The primary cilium is a non-motile, solitary organelle assembled by most cell types. It is essential to the development and homeostasis of the skeleton ...

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

Samenvatting

Meer video's verkennen