Culture of Primary Ciliary Dyskinesia Epithelial Cells at Air-Liquid Interface Can Alter Ciliary Phenotype but Remains a Robust and Informative Diagnostic Aid

Abstract

BackgroundThe diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns.MethodsWe retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n = 111) was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture.ResultsCiliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced.ConclusionsThe change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia.

Diagnosis of primary ciliary dyskinesia: searching for a gold standard

“…Moreover, abnormal ciliary function can be secondary to infectious or inflammatory damage to the native tissue before biopsy or to damage during biopsy or mishandling of the sample prior to recording of HVMA. Repeat testing or reanalysis following culture at the air-liquid interface (ALI) [18] or under submerged conditions [19] has, therefore, been recommended [4] to confirm primary rather than secondary defects. Most defects of ciliary ultrastructure defined in adequate numbers of cilia with good preparation and sampling of images have 100% specificity; however, analysis of cilia by TEM will miss the 30% of patients who do not have ''hallmark'' ultrastructural defects [20].…”

Diagnostic Methods in Primary Ciliary Dyskinesia

“…This removes the need to repeat nasal brushing to obtain a sample for reanalysis which is required if diagnosis depends on HVMA. The ciliary phenotype changes following cell culture helping to differentiate primary from secondary dyskinesia and this change is therefore advantageous [99]. Jorissen et al [57] first reported the use of cell culture to aid PCD diagnosis using a submerged method (monolayer-suspension cell culture).…”

“…Jorissen et al [57] first reported the use of cell culture to aid PCD diagnosis using a submerged method (monolayer-suspension cell culture). Culture followed by ciliation at air-liquid interface (ALI) [99][100][101] has the advantage of yielding more cells and cilia than the submerged method. Both submerged and ALI-culture techniques allow reanalysis by HVMA [57,99,101] and TEM [99,101,102] aiding the diagnosis of PCD.…”

“…Culture followed by ciliation at air-liquid interface (ALI) [99][100][101] has the advantage of yielding more cells and cilia than the submerged method. Both submerged and ALI-culture techniques allow reanalysis by HVMA [57,99,101] and TEM [99,101,102] aiding the diagnosis of PCD. ALI-culture of respiratory cells from PCD patients additionally provides an excellent ex vivo model for research [103,104].…”

Diagnostic testing in primary ciliary dyskinesia

“…The UK PCD reference centres regularly audit each other's analyses, and discuss difficult cases [6]. We routinely analyse de novo cilia following culture of the original sample at air liquid interface allowing us to differentiate primary and secondary functional and structural defects [7]. These methods are technically demanding and not available in many centres.…”