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%) were 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.
Highlights
Primary ciliary dyskinesia (PCD) is a rare disease usually inherited as an autosomal recessive condition autosomal dominant and X-linked cases exist [1]
Ex vivo nasal or bronchial samples obtained by brushing or curette biopsy are imaged by high-speed video microscopy analysis (HSVA) [10,11] and ciliary motility analysed as a frontline functional test [12]
Some genetic defects result in subtle ciliary beat pattern abnormalities, which are difficult to differentiate from secondary defects (e.g., GAS8 [21], DNAH9 [18], CCDC103 [22,23] mutations) by HSVA and appear normal by Transmission electron microscopy (TEM)
Summary
Primary ciliary dyskinesia (PCD) is a rare disease usually inherited as an autosomal recessive condition autosomal dominant and X-linked cases exist [1]. The incidence of PCD is approximately 1:10,000, higher in consanguineous populations [2], and it is associated with impaired function of motile cilia in the airways, embryonic node, and reproductive system [3]. This causes a spectrum of symptoms including unexplained neonatal respiratory distress, persistent wet cough from infancy, repeated respiratory infections, rhino-sinus disease, organ laterality abnormality and subfertility [4]. Genotyping can detect pathogenic bi-allelic or X-linked hemizygous mutations in 50 PCD-related genes to confirm the diagnosis in approximately 70% of well characterized cases [1,3,20]. MCIDAS [24], CCNO [25] and FOXJ1 [26] mutations cause a lack of cilia rather than dyskinesia, and this could be mistaken for severe secondary epithelial damage
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