High-Speed Video Microscopy for Primary Ciliary Dyskinesia Diagnosis: A Study of Ciliary Motility Variations with Time and Temperature

Abstract

Primary ciliary dyskinesia (PCD) is a rare disease resulting from a defect in ciliary function that generates, among other issues, chronic upper and lower respiratory tract infections. European guidelines recommend studying ciliary function (pattern (CBP) and frequency (CBF)), together with characteristic clinical symptoms, as one of the definitive tests. However, there is no “gold standard”. The present study aims to use high-speed video microscopy to describe how CBF and CBP alter over time and at different temperatures to reduce the error rate in the diagnosis of PCD. Samples of nasal epithelium from 27 healthy volunteers were studied to assess CBF and CBP at 0, 3, 24, 48, and 72 h, at room temperature and 4 °C. It was observed that CBF increased while CBP became dyskinetic, both at room temperature and at 4 °C, as time passed, especially after 3 h. In order to preserve all ciliary function parameters and to perform a reliable analysis to improve the diagnostic process of PCD, analysis should be performed within the first 3 h of sample collection, preferably in reference centers.

Quantifying cilia beat frequency using high‐speed video microscopy: Assessing frame rate requirements when imaging different ciliated tissues

“…Ns = not significantly different (p > 0.05). Mateos-Quiros et al, 2021;Mikhailik et al, 2021;Reula et al, 2021;Smith et al, 2012;Zahid et al, 2020). The lack of a standardized fps for imaging motile cilia is especially problematic because it hinders high-speed video microscopy from being clinically adapted as a diagnostic tool to help uncover motile cilia diseases, such as primary ciliary dyskinesia (Bricmont et al, 2021;Shapiro et al, 2018).…”

“…The easiest way to assess motile cilia function is by quantifying CBF via highspeed video microscopy (Francis & Lo, 2013 ; O'Callaghan et al, 2012 ; Sisson et al, 2003 ) and this forms a sizable chunk of the cilia literature. However, no standardized frame rate exists when imaging cilia motility for subsequent CBF calculation, this has resulted in a large range of frames rates used to image cilia motility within the published literature (Abdelhamed et al, 2018 ; Abdelhamed et al, 2020 ; Bustamante‐Marin et al, 2019 ; Chen et al, 2016 ; Hagiwara et al, 2020 ; Hennessy et al, 1986 ; Liu et al, 2021 ; Mateos‐Quiros et al, 2021 ; Mikhailik et al, 2021 ; Reula et al, 2021 ; Smith et al, 2012 ; Zahid et al, 2020 ). The lack of a standardized fps for imaging motile cilia is especially problematic because it hinders high‐speed video microscopy from being clinically adapted as a diagnostic tool to help uncover motile cilia diseases, such as primary ciliary dyskinesia (Bricmont et al, 2021 ; Shapiro et al, 2018 ).…”

“…The apparent higher accuracy of CBFs calculated manually versus calculated by FFT‐based algorithm from lower fps movies is not surprising as the human brain is well known for its superlative pattern recognition (Mattson, 2014 ) which would make it excellent for quantifying CBF from noisy cilia kymographs (Figure S1 ). While the MATLAB script utilized a common technique for analyzing CBF (i.e., fast Fourier transform) (Awatade et al, 2021 ; Lindberg et al, 1996 ; Reula et al, 2021 ; Sisson et al, 2003 ; Smith et al, 2012 ), it may be possible to refine the automated analysis to provide more accuracy at lower fps movies. A possible solution for automated analysis for data collected with low framerates, is to employ the spatio‐temporal pixels' cross‐correlation over varying field of view centred at ciliary region (Chioccioli et al, 2019 ) to compensate for the loss in temporal resolution.…”

“…The most used method to assess motile cilia function is to quantify cilia beat frequency (CBF) via high‐speed video microscopy (Reula et al, 2021 ; Rubbo et al, 2019 ; Shapiro et al, 2018 ). However, a large heterogeneity exists within the published literature as to what frame rate per second (fps) to use when imaging cilia motility for subsequent CBF calculation; with published frame rates varying between 28–500 fps (Abdelhamed et al, 2018 ; Abdelhamed et al, 2020 ; Bustamante‐Marin et al, 2019 ; Chen et al, 2016 ; Hagiwara et al, 2020 ; Liu et al, 2021 ; Mateos‐Quiros et al, 2021 ; Mikhailik et al, 2021 ; Reula et al, 2021 ; Smith et al, 2012 ; Zahid et al, 2020 ). The large variation in sampling rates suggests some studies may be under‐sampling or oversampling cilia motility, which could result in inaccurate quantification of CBF or generation of large volumes of oversampled data requiring analysis and storage.…”

Ciliary Videomicroscopy: A Long Beat from the European Respiratory Society Guidelines to the Recognition as a Confirmatory Test for Primary Ciliary Dyskinesia

“…Reula et al [49] studied ciliary motility variations with time and temperature. In this study, 27 nasal curettages from healthy volunteers were divided equally; half of the samples were kept at room temperature (22-24 • C), and the other half were kept in a refrigerator (4 • C).…”