Name: nasal brushing sampling and processing using digital high speed ciliary videomicroscopy &ndash; adaptation for the covid-19 pandemic
Uploaded: 2020-11-07T14:00:00
Description: Watch this Scientific Journal Video about Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy Ã¢â‚¬â€œ Adaptation for the COVID-19 Pandemic at JoVE.com

We would like to thank Jean-Fran&#231;ois Papon, Bruno Louis, Estelle Escudier and all team members of PCD diagnostic center of Paris-Est for their availability and hearty welcome during the visit to their PCD diagnostic center, and the numerous exchanges. We also thank Robert Hirst and all team members at the PCD center of Leicester for their welcome and time, advice, and expertise.

Approval was obtained from the Liege hospital-faculty ethics committee and the University Department for Hygiene and Health Protection at Work.
1. Sampling respiratory ciliated epithelium
<ol>
	<li>Ensure that subjects are free of infection for at least 4-6 weeks, and free of nasal and inhaled medication, before sampling.</li>
	<li>Prepare supplemented M199 preparation: Supplement Cell Culture Medium 199 (M199) (500 mL) with antibiotic solution (5 mL of streptomycin/penicillin (50 &#956;g/mL...

This paper aims to provide a standard operating procedure for CFA using nasal brushing samples, with adjustments made for appropriate infection control considerations during the COVID-19 pandemic. PCD diagnosis is challenging, and currently requires a panel of different diagnostic tests, according to international recommendation, including nasal nitric oxide measurement, CFA using DHSV, ciliary ultrastructural analysis using transmission electron microscopy (TEM), labelling of ciliary proteins using immunofluorescence, a...

Primary ciliary dyskinesia (PCD) is an inherited heterogeneous motile ciliopathy, in which respiratory cilia are stationary, slow or dyskinetic, leading to impaired mucociliary clearance and chronic oto-sino-pulmonary disease1,2,3,4. The clinical manifestations of PCD are chronic wet cough and chronic nasal congestion starting in early infancy, recurrent or chronic upper and lower respiratory tract infections leading to bronchiectasis, and recurrent or chronic otitis media and sinusitis5,6,7. Approximately half of PCD patients present with organ laterality defects such as situs inversus or situs ambiguus. Some patients also present with infertility issues due to immotile sperm in men and immotile cilia in the Fallopian tubes in women1,2,8. PCD is rare, but the prevalence is difficult to define, and ranges from 1:10,000 to 1:20,0009,10. However, the real prevalence of PCD is thought to be higher due to difficulties in diagnosis and a lack of clinical suspicion. Symptoms of PCD mimic common respiratory manifestations of other acute or chronic respiratory conditions, and the diagnostic challenges of confirming the diagnosis are well known, leading to inadequate treatment and follow-up2,5,9,11.
Ciliary videomicroscopy, using Digital High-Speed Videomicroscopy (DHSV), is one of the diagnostic tools for PCD4,8,12,13. DHSV is considered the optimal method to perform ciliary functional analysis (CFA), comprising of ciliary beat frequency (CBF) and beat pattern (CBP) analysis2,14,15,16. DHSV uses living respiratory epithelium, usually obtained from nasal brushing13.
In view of the current COVID-19 outbreak, confirmation of a PCD diagnosis is now even more important as evidence suggests that underlying respiratory disease may lead to worse outcomes following COVID-19 infection17,18. A safe and efficient PCD diagnostic service during the current pandemic will also allow confirmed PCD patients to benefit from additional protective measures, compared with the general population19.
Transmission of COVID-19 occurs primarily through droplet spread20. High potential of transmission from asymptomatic (or minimally symptomatic) patients is suggested by the high viral load in nose sample20. Additionally, if viral particles become aerosolized, they stay in the air for at least 3 hours21. Therefore, respiratory healthcare workers are exposed to a high reservoir of viral load while performing clinical care and sample collection for diagnostic techniques22. Furthermore, manipulation of living respiratory samples exposes the technician to COVID-19 contamination. While best-practice recommendations for respiratory physicians and ENT surgeons caring for COVID-19 patients are being implemented23, there is a lack of recommendations for performing DHSV during the COVID-19 pandemic.
In order to continue providing a PCD diagnostic service, while ensuring the safety of the healthcare worker (performing sample collection) and technician (performing sample processing), the ciliary videomicroscopy protocol had to be adapted during the COVID-19 pandemic. The technique of ciliary videomicroscopy is currently limited to research service and specialized diagnostic centers, as CFA requires extensive training and experience. Furthermore, currently, there is a lack of standardization and precise operating procedure for processing and analyzing samples using DHSV4,13.
The aim of this paper is to describe standard operating procedures for DHSV, with particular reference to infection control measures and safety when sampling and processing living nasal epithelium. This will allow for high-quality PCD diagnosis and care to continue, despite the current COVID-19 outbreak.

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

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.

To illustrate the efficiency of the technique, we present the results of the CFA in a series of 16 healthy adult volunteers (5 males, age range 22-54 years).
Nasal brushing samples from 14 (4 males, age range 24-54 years) out of the total of 16 volunteers provided enough appropriate epithelial edges that satisfied the selection criteria needed to perform CFA. From these 14 nasal brushing samples, a total of 242 ciliated edges were recorded, and 212 edges met the defined inclusion criteria and ...

Watch this Scientific Journal Video about Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy Ã¢â‚¬â€œ Adaptation for the COVID-19 Pandemic at JoVE.com

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“ 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.

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 ...

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