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A revised protocol for culture of airway epithelial cells as a diagnostic tool for primary ciliary dyskinesia

A revised protocol for culture of airway epithelial cells as a diagnostic tool for primary ciliary dyskinesia
A revised protocol for culture of airway epithelial cells as a diagnostic tool for primary ciliary dyskinesia
Air-liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling, infection or inflammation confounding PCD diagnostic results. ALI-culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI-culture method adopted from April 2018 across three collaborating PCD diagnostic sites, including current University Hospital Southampton COVID-19 risk mitigation measures, and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI-culture and 199 (82.9%) ciliated. Fifty-four of 83 (63.9%) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated, scanning electron microscopy demonstrated excellent ciliation, and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary, our ALI-culture protocol provides high ciliation rates across three centres, minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful, facilitating PCD research
pcd, ALI-culture, bio-resource, primary nasal epithelium, diagnostics
Coles, Janice
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Thompson, James
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Horton, Katie Leanne
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Hirst, Robert A.
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Griffin, Paul
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Williams, Gwyneth
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Goggin, Patricia Mary
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Doherty, Regan Jean
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Lackie, Peter
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Harris, Amanda
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Walker, Woolf T.
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O'Callaghan, Christopher
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Hogg, Claire
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Lucas, Jane
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Blume, Cornelia
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Jackson, Claire
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Coles, Janice
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Thompson, James
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Horton, Katie Leanne
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Hirst, Robert A.
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Griffin, Paul
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Williams, Gwyneth
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Goggin, Patricia Mary
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Doherty, Regan Jean
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Lackie, Peter
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Harris, Amanda
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Walker, Woolf T.
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O'Callaghan, Christopher
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Hogg, Claire
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Lucas, Jane
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Blume, Cornelia
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Jackson, Claire
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Coles, Janice, Thompson, James, Horton, Katie Leanne, Hirst, Robert A., Griffin, Paul, Williams, Gwyneth, Goggin, Patricia Mary, Doherty, Regan Jean, Lackie, Peter, Harris, Amanda, Walker, Woolf T., O'Callaghan, Christopher, Hogg, Claire, Lucas, Jane, Blume, Cornelia and Jackson, Claire (2020) A revised protocol for culture of airway epithelial cells as a diagnostic tool for primary ciliary dyskinesia. Journal of Clinical Medicine, 9 (11), [jmc-988553]. (doi:10.3390/jcm9113753). (In Press)

Record type: Article

Abstract

Air-liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling, infection or inflammation confounding PCD diagnostic results. ALI-culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI-culture method adopted from April 2018 across three collaborating PCD diagnostic sites, including current University Hospital Southampton COVID-19 risk mitigation measures, and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI-culture and 199 (82.9%) ciliated. Fifty-four of 83 (63.9%) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated, scanning electron microscopy demonstrated excellent ciliation, and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary, our ALI-culture protocol provides high ciliation rates across three centres, minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful, facilitating PCD research

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Accepted/In Press date: 19 November 2020
Keywords: pcd, ALI-culture, bio-resource, primary nasal epithelium, diagnostics

Identifiers

Local EPrints ID: 445128
URI: http://eprints.soton.ac.uk/id/eprint/445128
PURE UUID: d7d849d6-dfe3-46b4-b044-ea155401cab3
ORCID for Patricia Mary Goggin: ORCID iD orcid.org/0000-0003-4730-0206
ORCID for Peter Lackie: ORCID iD orcid.org/0000-0001-7138-3764
ORCID for Cornelia Blume: ORCID iD orcid.org/0000-0001-6133-7318

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Date deposited: 20 Nov 2020 17:32
Last modified: 19 Nov 2021 02:49

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Contributors

Author: Janice Coles
Author: James Thompson
Author: Katie Leanne Horton
Author: Robert A. Hirst
Author: Paul Griffin
Author: Gwyneth Williams
Author: Patricia Mary Goggin ORCID iD
Author: Regan Jean Doherty
Author: Peter Lackie ORCID iD
Author: Amanda Harris
Author: Woolf T. Walker
Author: Christopher O'Callaghan
Author: Claire Hogg
Author: Jane Lucas
Author: Cornelia Blume ORCID iD
Author: Claire Jackson

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