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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 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.
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....
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
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 .......
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.......
We would like to thank Jean-François 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.
....Name | Company | Catalog Number | Comments |
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 |
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