Variation of Ciliary Beat Pattern in Three Different Beating Planes in Healthy Subjects

Accepted Manuscript Variation of ciliary beat pattern in 3 different beating planes in healthy subjects Celine Kempeneers, MD, Claire Seaton, BM BCh, Mark A. Chilvers, MB ChB MD PII:

S0012-3692(16)59262-9

DOI:

10.1016/j.chest.2016.09.015

Reference:

CHEST 698

To appear in:

CHEST

Received Date: 27 May 2016 Revised Date:

24 July 2016

Accepted Date: 9 September 2016

Please cite this article as: Kempeneers C, Seaton C, Chilvers MA, Variation of ciliary beat pattern in 3 different beating planes in healthy subjects, CHEST (2016), doi: 10.1016/j.chest.2016.09.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Word count: abstract: 248; text: 2499

Variation of ciliary beat pattern in 3 different beating planes in healthy subjects

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Running Head: Variation of beat pattern in healthy subjects

Author list : Celine Kempeneers, MD; Claire Seaton, BM BCh; Mark A. Chilvers ; MB ChB

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MD

Institutional affiliations: Division of Respirology, Department of Pediatrics, University of

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British Columbia and British Columbia Children’s Hospital, Vancouver, BC, Canada Corresponding author information : M. Chilvers, Division of Respirology, BC Children’s Hospital, 4480 Oak Street, Vancouver, BC, V6H 3V4, Canada; e-mail: [email protected]

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Conflict of interests: No potential conflicts of interest with any companies/organizations whose products or services may be discussed in this article exist for the specified authors. Funding information: This study was supported by the BCCH Telethon Grant. Dr Celine

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Kempeneers has received the following grants: “Citadelle Recherche et Formation” Grant

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from CHR, Liege, Belgium, Horlait-Daspens Foundation Grant, Belgium Notation of prior abstract publication/presentation: -

Kempeneers C, Seaton C, Chilvers MA - Ciliary Videomicroscopy: Defining a Standard Methodology. 2014 International Conference of the American Thoracic Society, San Diego, CA, USA, May 16th-21st, 2014

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Kempeneers C, Seaton C, Chilvers MA - Primary Ciliary Dyskinesia Diagnosis: variation of the Ciliary Beat Pattern in Healthy Subjects - 44th Annual Congress of the Belgian Society of Pediatrics (BVK-SBP), Brussels, Belgium, March 10th-11th, 2016

ACCEPTED MANUSCRIPT Abbreviations list CBF = ciliary beat frequency; CBP = ciliary beat pattern; DHSV = digital high speed videomicroscopy; DKS = dyskinesia score; %DK = percentage of dyskinetic edges; IMI =

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immotility index; PCD = primary ciliary dyskinesia; ROI = region of interest Abstract

Background: Digital high speed videomicroscopy(DHSV) allows analysis of ciliary beat

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frequency(CBF) and pattern(CBP) of respiratory cilia in 3 planes. Normal reference data use a sideways edge to evaluate ciliary dyskinesia, and calculate CBF using the time for a cilium

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to complete 10 beat cycles. Variability in CBF within respiratory epithelium has been described, but data concerning variation of CBP is limited in healthy epithelium. This study aims to document variability of CBP within normal samples, to compare ciliary function in 3 profiles, and CBF calculated over 5 or 10 beat cycles.

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Methods: Nasal brushing samples from 13 healthy subjects were recorded using DHSV in 3 profiles. CBP and CBF over 10 beat cycle were evaluated in all profiles and CBF re-

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evaluated over 5 beat cycles in the sideways edges. Results: 82.1% of edges exhibited a uniform CBP. In the sideways profile, uniformity within

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the edge was lower(uniform normal CBP: 69.1%(sideways), 97.1%(towards), 92.0%(above)), and dyskinesia was higher. Inter-observer agreement for dyskinesia was poor. CBF was not different between profiles(p=0.8097), or between 10 and 5 beat cycles(p=0.1126). Conclusions: Our study demonstrates a lack of uniformity and consistency in manual CBP analysis of healthy samples, emphasizing the risk of automated CBP analysis in limited regions of interest(ROIs), and of single and limited manual CBP analysis. The towards and above profiles may be used to calculate CBF, but may be less sensitive for evaluation of

ACCEPTED MANUSCRIPT ciliary dyskinesia and CBP. CBF can be measured reliably by evaluation of only 5 ciliary beat cycles. Text

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Mucociliary clearance relies on interactions between cilia, periciliary fluid, and overlying mucus. Regular, coordinated beating of cilia allows mucus clearance from the

airways and provides the primary defence mechanism in the lungs1–3. In patients with primary

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ciliary dyskinesia(PCD), an inherited disorder in which cilia are either stationary, slow, or dyskinetic, mucociliary clearance is impaired or absent, leading to significant sinopulmonary

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disease3–5. Digital High Speed Videomicroscopy(DHSV) allows real time evaluation of ciliary function, including ciliary beat frequency(CBF) and beat pattern(CBP)6,7. This technique allows beating cilia to be viewed in three distinct planes: a sideway profile, beating directly towards the observer and from directly above 7(Fig 1, Video 1).

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CBF is determined by the time required for a group of cilia to complete a given number of ciliary beat cycles7–9. Current normal reference data calculate CBF using the time to complete 10 beat cycles on a sideways edge10. The development of automated analysis of

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CBF has improved the ease of CBF calculation11–14. One study has compared automated

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analysis with direct measurement of DHSV recordings of cilia and found it to be comparable14. However, the automated analysis did not use nasal respiratory epithelium but cell cultures samples11,13,14, with differences in CBF reported between the two types of cells15. In addition, CBF assessment by «Sisson-Ammons Video Analysis11» has been reported to be inaccurate in mutations associated with specific CBP and required the additional manual evaluation of CBF by DHSV16. CBP analysis has been demonstrated to be more sensitive and specific than CBF measurement alone in the diagnosis of PCD17. CBP is measured qualitatively by comparing

ACCEPTED MANUSCRIPT the observed ciliary beat cycle with the normal beat pattern16,18, and normal data for semiquantitative CBP analysis in the sideways profile have been published10,17. Papon et al8 proposed quantitative parameters, such as beat amplitude or distance travelled by the cilium per second, to objectively characterize CBP viewing cilia only in a sideway profile, and

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reported better sensitivity for diagnosing PCD than semi-quantitative analysis.

Despite the advancement in automation of CBF measurement, CBP evaluation still requires manual analysis with DHSV16. A ciliary beat waveform extraction system has been

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developed, involving comparison between a standard and studied waveform19, but is

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unsuitable for analysis of non planar or irregular CBP, and is not used in clinical practice. Quinn et al20 developed an automated method for analysis of CBP, involving the evaluation of a digital signature of ciliary motion within a region of interest(ROI), and reported a good agreement between the automated analysis and observer. One drawback is that the method only evaluates CBP in limited ROIs and not within the whole sample, and fails to provide a

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description of variation in ciliary motion.

However, despite the advancement of ciliary functional analysis using DHSV, issues

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still persist. Firstly, there is variability in ciliary beating within individual respiratory ciliated epithelial edges. Published reference ranges show that cilia do not all beat synchronously

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within a sample, and CBF will vary between edges or ROIs10,16,21. Data evaluating the variation of CBP within edges of healthy individuals is limited to either a qualitative ciliary dyskinesia score(DKS) or immotility index(IMI)8,10,17. Only one study quantified different types of CBP within healthy samples8, but precise evaluation within a normal ciliated epithelial edge has not been reported. This is of relevance, as it has been recently shown that in patients with PCD, cilia may have varying beat patterns with specific CBPs not seen uniformely within epithelial edges16.

ACCEPTED MANUSCRIPT Secondly, Thomas et al9 reported that ciliary function will vary depending on the quality of the edges. This may significantly reduce the number of acceptable edges available for analysis within a sample. Often ciliated edges may be viewed beating towards the observer or from above, but limited data is available to evaluate ciliary function in these

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directions.

Finally, low CBF is often seen in patients with PCD. Manual evaluation can be

challenging using DHSV, as 10 beat cylces may not always be obtainable. The evaluation of

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only 5 beat cycles in CBF analysis has been reported22, but has not been compared with 10

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beat cycles.

The primary aim of this study was to document the variable beat patterns observed in normal samples and to compare measurements of CBF and CBP in the three different profiles.

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A secondary aim was to compare CBF when measured by evaluation of 5 and 10 beat cycles.

MATERIALS AND METHODS

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Subjects

Samples of ciliated epithelium were obtained by brushing the inferior nasal turbinate

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of 13 healthy subjects(age range:19 days to 62 years). Exclusion criteria included: chronic respiratory disease, a family history of PCD, respiratory tract infection during the previous 4 weeks, regular nasal or inhaled medication, or active smoking. The study was approved by the UBC Children’s and Women’s Research Ethics Board(H10-02168), and written consent was obtained from all subjects. Ciliary function was evaluated at a controlled temperature of 37 degree, using the experimental DHSV system previously reported7(e-Appendix 1). Sample Selection

ACCEPTED MANUSCRIPT Using the quality scoring system by Thomas et al9, only normal edges or edges with minor projections were included. Subject samples and ciliated edges selection criteria are described in e-Appendix1.

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Ciliary Beat Frequency The mean CBF was calculated(method described in e-Appendix 1) from 10 beat cycles using the 3 profiles, and from 5 beat cycles using only the sideways profile.

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Ciliary Beat Pattern

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The CBP was evaluated using the 3 different profiles(Fig 1). Three markers were used to assess for ciliary dyskinesia: the IMI18,the DKS, scored from 1-49, and the percentage of dyskinetic edges(%DK). The beat pattern was categorized into 5 distinct CBPs by modification of previous reported descriptions(normal, immotile, stiff, circular and asynchronous)8,18,23,24, and the percentage of each type of CBP was recorded for each edge.

Data analysis

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Details are described in e-Appendix 1.

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All images and corresponding clinical details were recorded in a database for

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secondary verification and audit purposes. All videos were re-analysed by a second observer(C. S.) and on a second occasion by the original observer(C. K.) to calculate interand intra-observer agreement. Statistical analysis was performed using GraphPad Prism 5(Graph Pad, San Diego,

CA, USA). CBF, IMI, %DK and % of each CBP were described as mean, standard deviation, 5th and 95th percentiles. DKS was described as median, range. To study the evaluation of CBF and CBP between the 3 profiles, ANOVA was performed using the one way analysis of variance for parametric data, and Kruskal-Wallis test for nonparametric data. The evaluation

ACCEPTED MANUSCRIPT of CBF using 10 or 5 beat cycles was compared using a paired t test. The inter-observer and intra-observer correlation were calculated with the Pearson correlation coefficient for parametric data, and the Spearman correlation coefficient for the nonparametric data. A p-

RESULTS

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value of < 0.05 was taken as the threshold for statistical significance.

A total of 256 ciliated edges were recorded, and 179 edges met the defined inclusion

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criteria and were analyzed(Table 1). A total of 1081 CBF measurements were obtained(Table 1). The mean CBF for all subjects calculated from 10 beat cycles using the sideways profile

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was 13.5 Hz(12.0-14.9, 95%CI)(n=94 edges). This agrees with published reference data10. Only 2/13 subjects exhibited a completely normal CBP in the 3 different profiles. The variability of CBP in the 3 profiles is reported in Table 2. In our healthy subjects, uniformity of CBP was lower in the sideways profile(normal uniform CBP in 15.4% of subjects),

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compared to the other profiles. In the 3 profiles, 82.1% of edges analyzed exhibited a completely uniform CBP. Of edges beating uniformly, the beat pattern observed was normal (sideways:69.1%; towards: 97.1%; above: 92.0%), with only one edge(1.1%) exhibiting a

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stiff CBP, and one edge(1.1%) exhibiting an asynchronous CBP, in the sideways profile.

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The percentage of each type of CBP was different between profiles(Fig 2). In the sideways profile, the percentage of normal CBP was significantly lower(p=0.050), and the percentage of stiff beating cilia was higher(p=0.0030), when compared to the other profiles. The percentage of immotile CBP was also higher in the sideways profile when compared to the above profile only(p = 0.0067). No significant difference was observed for cilia beating asynchronously between profiles(p=0.2318). As reported previously8, no cilia were found to beat in a circular pattern for any of the 3 profiles.

ACCEPTED MANUSCRIPT The results of CBF and ciliary dyskinesia analysis for the three different profiles are reported in Table 3. There was no significant difference in mean CBF between the three profiles(p=0.8097). However, differences were observed when evaluating ciliary dyskinesia. The %DK and the IMI were significantly higher(p=0.0012, p=0.0283, respectively) in the

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sideways profile comparative to the other profiles. A difference was observed for the median DKS between the sideways and the other profiles(p=0.0667), this was non-significant as median DKS was 0 in each profile in the healthy subjects.

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A subset of the edges were re-analyzed, and the CBF calculated by evaluation of 10 and 5 complete beat cycles(75 edges). No significant difference in mean CBF(95%CI) was

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found between 10 and 5 beat cycles, 12.6Hz(11.3-14.0) and 12.5 Hz(11.2-13.9), respectively(p=0.1126).

The intra- and inter-observer agreement(Table 4a) for measurement of CBF was excellent for each profile. The agreement varied for measures of ciliary dyskinesia(Table 4a)

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and the percentage of different CBPs(Table 4b), with the correlation generally higher for the sideways profile, and the inter-observer agreement generally lower than the intra-observer agreement. We were unable to calculate the correlation for every marker of

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dyskinesia(calculation not feasible if median or mean value is zero), as few dyskinetic edges

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were observed in our healthy subjects due to the high quality of the edges selected for analysis.

DISCUSSION

PCD diagnosis is difficult and different diagnostic tests have been developed, each with its own advantages and limitations5,25,26. No “gold standard” diagnostic test exists, and a panel of different tests are required to confirm a PCD diagnosis, with diagnostic algorithms differing by patient location and age25. The use of CBP analysis is important in making a

ACCEPTED MANUSCRIPT diagnosis of PCD as some specific genetic mutations are associated with a normal CBF and ciliary ultrastructure, with abnormal CBP being the only finding16,27. Ciliary motility pattern abnormalities may be very subtle, and experienced investigators using DHSV will detect almost all defects in CBP28.

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Recently, lack of uniformity of CBP in respiratory ciliated samples of patients with PCD has been described16. DNAH11 mutation is associated with a normal ultrastructure, but an abnormal CBP, with normal or high CBF29–31. Genes encoding radial spoke head protein,

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such as RSPH4A16,32,33, RSPH916,32,33, RSPH134, or central microtubules, such as HYDIN35, are associated with central microtubular pair abnormalities, and with a circular16,32,34 or a stiff

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CBP16,35, with normal or slightly decreased CBF16.

Our study demonstrates the variability of CBP in healthy nasal ciliated epithelium, and a lack of uniformity of CBP within high quality epithelial edges(Table 2, Fig 2). This variability of CBP in healthy and PCD patients suggests that automated CBP analysis within

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limited ROIs may be inaccurate, and that manual CBP evaluation requires extensive sample analysis and rigorous edges selection. Furthermore, the data emphasizes the need for quantification of different types of CBP. This will also allow evaluation of phenotypic

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variation of ciliary structure and function within distinct genetic variants of PCD16. Previous studies have emphasized the need for standardization in the methodology for

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the assessment of ciliary function using DHSV23,28,29, including environmental factors such as pH and temperature36–38, and the quality of the edges analyzed9. There is limited data to assess the value of using different beating profiles. Studies have only reported normal data for CBF, CBP, and markers of ciliary dyskinesia for cilia viewed from a sideway profile10,17. The above and towards profiles have only been used to subjectively characterize the type of CBP18.

ACCEPTED MANUSCRIPT Our results indicate that in healthy controls, CBF may be calculated in the above and towards profiles, as shown by the similar CBF and good agreement between the 3 profiles. Utilizing these additional planes allowed a 50% increase in the number of edges available for analysis. Poor sample quality can lead to insufficient sideways edges for CBF analysis. Cell

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culture may increase the quality of the sample, but is technically challenging, not readily available and prolongs time to diagnosis39. Analysis of different profiles may reduce the need for culturing of samples, ultimately improving productivity of DHSV. However, the 3

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markers of ciliary dyskinesia in the towards or above profiles may be less sensitive than in the sideways profile in detecting dyskinesia. This was highlighted in the proportion of each

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CBP.

Caution is required when quantifying ciliary function from different profiles in samples from patients with PCD. In this patient population, ciliated edges may have a higher proportion of immotile cilia17. A recording of 0Hz for immotile cilia decreases the mean CBF

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of a sample. Compared to the sideways profile, less immotile cilia were observed in the towards profile, and no immotile cilia were observed in the above profile, as stationary points

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couldn’t be reliably discriminated as ciliary tips(Video 1). Consequently, these profiles may have a higher CBF and a lower IMI than the sideways profile in PCD samples. Further work

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is needed to clarify the use of CBF and dyskinesia measurements in profiles beating towards and above in PCD patients. CBF can be measured reliably and efficiently by the evaluation of only 5 complete

ciliary beat cycles without compromising accuracy. The longer video duration is thought to increase accuracy as CBF may change and a variation of less than 10% is considered to be non-significant40,41. However, by comparing the mean CBF in the sideways profile over both 5 and 10 beat cycles, we have shown a difference of only 0.7%.

ACCEPTED MANUSCRIPT Despite the mean CBF closely matching previous publications9,10, less correlation was observed when evaluating measures of ciliary dyskinesia(Table 4). There are few reports evaluating consistency or observer agreement for CBP assessment. Only one study has evaluated the agreement for DKS and reported a strong correlation9. Further work is needed

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to study consistency for CBP assessment in healthy subjects and PCD patients in both

automated and manual CBP evaluation, and raises the question of whether 2 independent

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analysis are needed.

CONCLUSIONS

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The data suggest a lack of uniformity of CBP within healthy ciliated respiratory epithelium, and a lack of consistency in manual CBP analysis. Our results suggest that manual CBF assessment using DHSV is reliable, but that a single manual CBP analysis may be innacurate. It also highlights the risk of automated CBP analysis, especially if relying on

sample.

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binary output in limited ROIs without a precise report of variation of CBP throughout the

In addition, the results demonstrate that, in healthy subjects, CBF can be evaluated

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analyzing cilia beating in planes towards the observer and from above, increasing the number

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of edges available for analysis. However, these planes were less sensitive in detecting ciliary dyskinesia. The use of the above and towards profiles may improve the efficiency of DHSV, but requires further evaluation in patients with PCD, who have a higher degree of abnormally beating cilia.

Finally, our results suggest that in healthy subjects, CBF can be measured reliably by evaluation of only 5 ciliary beat cycles on a sideways profile.

ACCEPTED MANUSCRIPT ACKNOWLEDGEMENTS Author contributions: Dr Kempeneers had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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Dr Kempeneers: contributed to study design, acquisition and analysis of the data, and assembly of the manuscript.

Dr Seaton: contributed to acquisition and analysis of the data, and to review and final

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approval of the manuscript.

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Dr Chilvers: contributed to the conception of the study, analysis of the data, review and final approval of the manuscript, and is the clinical leader of the PCD service in Vancouver. Financial/nonfinancial disclosures: No potential conflicts of interest with any companies/organizations whose products or services may be discussed in this article exist for

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the specified authors.

Role of sponsors: The sponsors had no role in the design of the study, the collection and

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analysis of the data, or in the preparation of the manuscript. Other contributions: We are grateful for the statistical advice of Dr Ruth Milner and Boris

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ACCEPTED MANUSCRIPT Figure legends: FIGURE 1. Representative images of cilia beating in the three different profiles recorded through Digital High Speed Videomicroscopy (DHSV)

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a) Sideway profile: cilia viewed beating on a sideway direction b) Towards profile: cilia viewed beating directly towards the observer

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c) Above profile: cilia viewed beating from above the observer

above profiles. Data are expressed as mean, 95% CI.

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FIGURE 2. Analysis of each type of ciliary beat pattern (CBP) in the sideway, towards and

a) Percentage of normal CBP within the sample (ANOVA one way analysis of variance,

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p = 0.0050): sideway profile: 89.0%(81.6-96.5), towards profile: 99.3%(97.7-100.0), above profile: 98.3%(96.6-100.0).

b) Percentage of immotile CBP within the sample (ANOVA one way analysis of

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variance, p = 0.0067): sideway profile: 2.8%(0.9-4.6), towards profile: 0.7%(0.0-2.3), above profile: 0.0%(0.0-0.0).

AC C

c) Percentage of stiff CBP within the sample (ANOVA one way analysis of variance, p = 0.0030): sideway profile: 3.5%(0.9-6.1), towards profile: 0.0%(0.0-0.0), above profile:(0.0-0.0).

d) Percentage of asynchronous CBP within the sample (ANOVA one way analysis of variance, p = 0.2318): sideway profile: 4.7%(0.0-10.8), towards profile: 0.0%(0.00.0), above profile: 1.7%(0.1-3.4).

ACCEPTED MANUSCRIPT Tables: Table 1 - Description of the ciliary beat frequency (CBF) measurements , and number of edges recorded and analyzed in every subject in each profile, and according to the quality of the edge (THOMAS et al description9) NUMBER OF EDGES RECORDED

NUMBER OF EDGES ANALYZED

Total sideway profile

510

153

94

Normal edge

224

65

Minor projection

286

77

Major projection

0

8

Isolated ciliated cell

0

3

Single cell

0

RI PT

NUMBER OF CBF MEASUREMENTS DONE

40

M AN U

SC

54 0

0

0

0

45

35

18

15

26

20

218

Normal edge

110

Minor projection

108

Major projection

0

1

0

0

0

0

0

0

0

353

58

50

TE D

Total towards profile

Isolated ciliated cell

Total above profile

EP

Single cell

AC C

CBF: ciliary beat frequency.

ACCEPTED MANUSCRIPT Table 2 – Percentage of subjects and edges exhibiting a uniform distinct ciliary beat pattern (CBP) in the 3 different profiles

TOWARDS PROFILE

Percentage of edges exhibiting a uniform distinct CBP in each profile

N

15.4

69.1

I

0.0

0.0

S

0.0

C

0.0

A

0.0

N

88.9

I

0.0

S

0.0

C A N I S C A

1.1

0.0

1.1

97.1

EP

0.0 0.0

0.0

0.0

69.2

92.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

CBP: ciliary beat pattern; N: Normal; I: Immotile; S: Stiff; C: Circular; A: Asyncronous. Data are expressed as % of the total number of subjects or edges.

AC C

0.0

0.0

TE D

ABOVE PROFILE

RI PT

Percentage of subjects exhibiting a uniform distinct CBP in each profile

SC

SIDEWAY PROFILE

CBP

M AN U

Profile

ACCEPTED MANUSCRIPT Table 3 - Analysis of ciliary beat frequency (CBF) and ciliary dyskinesia in the sideway, towards and above profiles Sideway

Towards

Above

p-value

5th, 95th centile

Mean(SD)

5th, 95th centile

Mean(SD)

5th, 95th centile

CBF (Hz)

13.5 (2.4)

12.0-14.9

14.0 (3.0)

11.7-16.3

13.3 (2.8)

11.6-15.0

0.8097

IMI (%)

5.3 (7.6)

0.7-10.0

1.5 (3.1)

0.0-4.0

0.0 (0-0)

0.0-0.0

0.0283

% DK (%)

25.2 (20.7)

12.7-37.8

2.8 (8.3)

0.0-9.2

5.8 (9.4)

0.2-11.5

0.0012

Median

Range

Median

Range

Median

Range

0

0-1.5

0

0-0

0

0-0

DKS

RI PT

Mean(SD)

0.0667

SC

CBF: ciliary beat frequency; IMI: immotility index; % DK: percentage of dyskinetic edges; DKS: dyskinesia score. Statistical tests used: mean CBF, IMI, %DK: ANOVA one way analysis of variance; Median DKS: ANOVA Kruskal-Wallis.

M AN U

Table 4a - Intra and inter observer correlation for ciliary beat frequency (CBF) and markers of ciliary dyskinesia for each of the three profiles Interobserver

CBF sidewaya

0.9316c

0.8278c

CBF towardsa

0.9796c

0.9772c

CBF abovea

0.9840c

0.9688c

TE D

Intraobserver

% DK sidewaya

0.4689

0.4031

0.8488c

0.2380

0.7283c

0.6864c

0.8277c

0.8707c

0.6482

0.5649

IMI abovea

d

d

Median DKS sidewayb

0.6528c

0.2552

Median DKS towardsb

d

d

Median DKS aboveb

d

d

% DK towardsa

IMI sidewaya

AC C

IMI towardsa

EP

% DK abovea

CBF: ciliary beat frequency, %DK: Percentage of dyskinetic edges, IMI: Immotility index, DKS: Dyskinesia score. Results are expressed as Pearson correlation coefficient (a) or Spearman correlation coefficient (b). c

P<0.05

d

correlation calculation not feasible

ACCEPTED MANUSCRIPT

Table 4b - Intra and inter observer correlation for percentage of cilia with normal, immotile, stiff or asynchronous ciliary beat pattern (CBP) for each of the three profiles for each subject Interobserver

Normal CBP sideway

0.8851a

0.5898

Normal CBP towards

0.8490a

0.2380

Normal CBP above

0.8581a

0.4851

Immotile CBP sideway

0.6015

0.9398a

Immotile CBP towards

0.8490a

0.7151

Immotile CBP above

b

b

Stiff CBP sideway

0.7492a

Stiff CBP towards

b

Stiff CBP above

b

b

Circular CBP sideway

b

b

Circular CBP towards

b

b

Circular CBP above

b

b

Dyskinetic CBP sideway

0.9418a

0.4741

b

b

0.6366a

0.4851

Dyskinetic CBP above

SC 0.5159 b

M AN U

TE D

Dyskinetic CBP towards

RI PT

Intraobserver

P<0.05.

b

correlation calculation not feasible

AC C

a

EP

CBP: ciliary beat pattern. Results are expressed as Pearson correlation coefficient.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT