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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

  1. Ensure that subjects are free of infection for at least 4-6 weeks, and free of nasal and inhaled medication, before sampling.
  2. Prepare supplemented M199 preparation: Supplement Cell Culture Medium 199 (M199) (500 mL) with antibiotic solution (5 mL of streptomycin/penicillin (50 μg/mL.......

Representative Results

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 .......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
15 mL conical tubesFisherScientific35209615 ml High-Clarity Polypropylene Conical Tube with lid
Amphotericin BLONZA17-836EAntifungal solution
Blakesley-weil nasal forcepsNOVO SURGICALE7739-12Used to hold the brush to perform the nasal brushing
Bronchial cytology brushCONMED129Used for nasal brushing
Cotton swabNUOVA APTACA2150/SGUsed for COVID-19 testing
Digitial high-speed videomicroscopy cameraIDTeu Innovation in motionCrashCam Mini 1510
Glass slideThermoScientific12372098Microscope slides used to create the visualization chamber
Heated BoxIBIDI cells in focus10918Used to heat the sample
Inverted Light microscopeZeissAXIO Vert.A1
Lens HeaterTOKAI HITTPiE-LHUsed to heat the oil immersion lens
Medium 199 (M199), HEPESTermoFisher Scientific12340030Cell Culture Medium
Motion Studio X64IDT Motionversion 2.14.01Software
OilFischerScientific, Carl Zeiss11825153
Rectangular cover slipVWR631-0145Used to cover the visualization chamber
Spacer (Ispacer) 0.25 mmSunjinlabIS203Used for the creation of the hermetic closed visualization chamber
Square cover slipVWR631-0122Used for the creation of lab-built open visualization chamber
Streptomycin/PenicillinFisherScientific, Gibco11548876Antiobiotics solution

References

  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.

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Nasal BrushingDigital High speed Ciliary VideomicroscopyPrimary Ciliary DyskinesiaCOVID 19Diagnostic TestSamplingProcessingInfection ControlSensitivitySpecificityElectron MicroscopyGenetic TestingWeil Blakesley Nasal ForcepsEpithelial StripsGlass SlideSpacer

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