Analysis of ciliary beat frequency and beat pattern measurements
We recruited 164 children (88 males, age range 2-17 years) and 50 adult volunteers from December 2015 to November 2016. We excluded 34 subjects because of known underlying diseases or inadequate samples and 1 adult subjects who was an active smoker. Samples from the remaining 135 children subjects (67 males, age range 3-17 years) and 44 adults (25 males, age range 18-60 years) were analysed. Forty-six children were exposed to SHS at home. Ten adults were exposed to SHS at home while 34 did not. The mean CBF and the percentage of dyskinetically beating edges for all subjects were summarized in Table 1. No significant difference was found in mean CBF between individual age groups (ANOVA, p=0.542) and dyskinetically beating edges between individual age groups (ANOVA, p=0.212).The normal ciliary beat pattern from a subject showed a coordinated ciliary beating in a forward backward motion along the whole epithelial edge (see Video 1) and the ciliated edges analysed that exhibited areas of dyskinetically beating cilia ranged from 18.0 to 24.2% (representative images of ciliated edge exhibited static cilia, seeVideo 2). There was also no significant difference in mean CBF between children with and without exposure to SHS for the 3 different age groups (ANOVA, p=0.89 for children aged 2-6, p=0.29 for children aged 7-12, p=0.58 for children aged 13-17) but there was slightly higher mean CBF in adults with SHS exposure compared to adults without (ANOVA, p=0.04) (Table 2). The mean CBF for children aged < 18 years was slightly higher than the adult group but it did not reach statistical significance [10.1Hz (95% CI 9.8 to 10.4) versus 9.5Hz (95% CI 8.9 to 10.0), ANOVA, p=0.05]. Yet, it was lower than that of the European study [12.8Hz (95% CI 12.3 to 13.3, ANOVA, p<0.05) [1].
Table 1
Analysis of ciliary beat frequency and beat pattern measurements
Age (years)
|
N
|
Mean*
|
SD
|
5th, 95th percentiles
|
Dyskinetically beating edges (%)¶
|
<18
|
135
|
10.1
|
2.0
|
6.3, 13.5
|
20.9 (0.0, 56.4)
|
2-6
|
51
|
10.3
|
2.0
|
6.9, 13.8
|
24.2 (0.0, 60.0)
|
7-12
|
43
|
10.1
|
2.1
|
5.8, 13.5
|
18.0 (0.0, 50.0)
|
13-17
|
41
|
9.9
|
1.9
|
6.1, 13.1
|
19.8 (0.0, 50.0)
|
>18
|
44
|
9.5
|
1.9
|
5.4, 11.9
|
14.9 (0.4, 44.7)
|
*Mean ciliary beat frequency(Hz), standard deviation (SD), and 5th, 95th percentiles ¶mean (5th, 95th percentiles) percentage of edges exhibiting areas of ciliary dyskinesia
UK reference CBF mean for <18 years 12.8 (95% CI 12.3 to 13.3)1 vs ours <18 mean CBF 10.1 (95% CI 9.8 to 10.4) (p<0.05, t-test)
|
Table 2
Effect of exposure to second-hand smoking on ciliary beat frequency
Age (years)
|
Family member(s) smoked
|
|
|
|
|
|
|
Yes
|
No
|
|
N
|
|
*Mean + SD
|
N
|
|
*Mean + SD
|
P value
|
<18
|
46
|
|
9.9 + 1.68
|
89
|
|
10.2 + 2.16
|
0.284
|
|
|
2-6
|
11
|
|
10.4 + 1.53
|
40
|
|
10.3 + 2.09
|
0.886
|
|
|
7-12
|
21
|
|
9.7 + 2.13
|
22
|
|
10.4 + 2.15
|
0.289
|
|
|
13-17
|
14
|
|
9.7 + 0.81
|
27
|
|
10.0 + 2.33
|
0.575
|
|
>18
|
10
|
|
10.5 + 1.65
|
34
|
|
9.2 + 1.82
|
0.044
|
|
*Mean ciliary beat frequency(Hz), standard deviation(SD)
|
|
|
|
|
For ciliary beat pattern, 1 subject in the 13-17 year group had a mixed ciliary beat pattern with dyskinetic cilia, cilia with a normal pattern and cilia with a circular beat pattern when viewed from above on occasional ciliated cells(see Video 3).The mean CBF and TEM was normal. Normal beat pattern was observed in the ciliated epithelium from all other subjects.
To establish a reference range, mean CBF was plotted against age of each subject (Figure 1A). A weak negative correlation was found between mean CBF and increasing age (r2 = 0.021). As cilia were found to beat at different frequencies within each sample, we also plotted sample variation in CBF, the ciliated edges with the highest and lowest CBF against age. The highest mean CBF of edges ranged from 7.3 to 24.3Hz (Figure 1B) with 93% of subjects having a maximal CBF of >10 Hz. The lowest mean CBF of edges ranged from 2.3 to10.4Hz(Figure 1C) with 45% of subjects having a minimum CBF of >6 Hz.
No significant difference was found for inter-observer and intra-observer measurements of CBF. The mean (SD) for inter-observer measurement was 0.57 (1.87) (95% CI -0.77 to 1.9; range -2 to 4) and intra-observer was 0.01 (1.4) (95% CI -1.02 to 1.03; range -3 to 3).
Transmission electron microscopy examination of cell types
Some subjects had an inadequate sample for ultrastructural analysis as tissue might have been lost during initial HSVM assessment performed before sample processed for TEM [1]. Among 179 subjects analysed with CBF and beat pattern, 121 subjects had sufficient tissue for epithelial integrity measurements and 159 subjects had tissue processed for ultrastructure analysis. The percentages of different cell types observed in the ciliated epithelial strips were summarized in Table 3. There was no significant difference between the percentages ofdifferent cells types across age groups (ANOVA, p>0.1). Ciliated cells formed about 50% of the cell population.
Table 3
Transmission electron microscopy examination of cell types
Age (years)
|
n
|
Ciliated cells (%)
|
Unciliated cells (%)
|
Mucous cells (%)
|
Dead cells (%)
|
<18
|
95
|
52.0 (0.0, 100.0)
|
35.8 (0.0, 100.0)
|
12.2 (0.0, 52.0)
|
0.0 (0.0, 0.0)
|
2-6
|
37
|
53.6 (0.0, 100.0)
|
37.1 (0.0, 100.0)
|
9.3 (0.0, 34.3)
|
0.0 (0.0, 0.0)
|
7-12
|
30
|
46.0 (0.0, 100.0)
|
38.3 (0.0, 100.0)
|
15.7 (0.0, 78.7)
|
0.0 (0.0, 0.0)
|
13-17
|
28
|
56.4 (0.0, 100.0)
|
31.3 (0.0, 77.8)
|
12.3 (0.0, 55.5)
|
0.0 (0.0, 0.0)
|
>18
|
26
|
51.0 (17.8, 93.0)
|
36.5 (3.6, 77.5)
|
12.3 (0.0, 43.0)
|
0.2 (0.0, 2.7)
|
Results are expressed as the mean percentage (5th, 95th percentiles) for each age group.
|
Assessment of ciliary epithelial integrity and ultrastructure by transmission electron microscopy
The integrity of ciliated epithelium was assessed by examining factors including loss of cilia, cellular extrusion, cytoplasmic blebbing, and mitochondrial damage in Figure 2. Normal with a normal healthy mitochondrion (arrow, bar=1µm) in Figure 2A. Loss of cilia, grade 3, and a cell with a damaged mitochondrion (arrow, bar=1µm) in Figure 2B. Cellular extrusion, grade 2, (bar=2µm) in Figure 2C. Cytoplasmic blebbing, grade 2 (arrow, bar=2µm) in Figure 2D. Evidence of minor epithelial damage was observed and the analysis was summarised in Table 4.There was no significant difference of epithelial integrity score across age groups (ANOVA, p=0.07). Normal nasal epithelium with an intact ciliated surface and minimal disruption (epithelial integrity score=0, bar=2µm) was shown in Figure 3Aand abnormal epithelium with severely disrupted cell surface and marked loss of cilia (epithelial integrity score=4, bar=2µm) in Figure 3B.
Table 4
Assessment of epithelial integrity by transmission electron microscopy
Age (years)
|
n
|
Cells with loss of cilia (%)
|
Cells extruding from epithelial edge (%)
|
Cells with cytoplasmic blebbing (%)
|
Cells with mitochondrial damage (%)
|
Epithelial integrity
|
<18
|
95
|
41.4 (0.0, 56.8)
|
31.5 (0.0, 67.0)
|
21.1 (0.0, 100.0)
|
16.3 (0.0, 100.0)
|
1.83 (0.8, 3.0)
|
2-6
|
37
|
55.1 (0.0, 100.0)
|
26.1 (0.0, 67.0)
|
10.8 (0.0, 50.0)
|
14.9 (0.0, 100.0)
|
1.8 (0.0, 3.1)
|
7-12
|
30
|
44.4 (0.0, 100.0)
|
41.0 (0.0, 90.9)
|
29.2 (0.0, 100.0)
|
14.4 (0.0, 100.0)
|
2.1 (0.0, 3.5)
|
13-17
|
28
|
20.3 (0.0, 56.8)
|
28.6 (0.0, 67.0)
|
25.9 (0.0, 100.0)
|
20.2 (0.0, 100.0)
|
1.6 (0.0, 3.0)
|
>18
|
26
|
49.3 (0.0, 100.0)
|
40.2 (6.0, 67.0)
|
24.7 (0.0, 61.1)
|
13.4 (0.0, 100.0)
|
2.0 (1.0, 3.0)
|
Results are expressed as the mean percentage (5th, 95th percentiles) for each age group.
|
Ultrastructural analysis was summarised in Table 5. Abnormal cilia were observed in some subjects. Microtubule defects including disarranged tubules (0.7%), extra-tubule including extra-single tubule and extra-microtubular pair (2.1%), 8+gap (1.0%) and single tubule (5.5%); central microtubules defects including extra-inter tubule (0.3%) and central pair damage (1.2%); compound cilia (0.5%) and combination of defects (0.9%) were identified in all of the cilia counted in children aged <18 years (Figure 4 B-I). The commonest defect identified was single micro-tubule (Figure 4E).
The mean microtubule defects in 13-17 year group (14.4%) were significantly higher than those in the other age groups (8.3% in 2-6 year group, 5.3% in 7-12 year group; ANOVA, p<0.01). Central microtubules defects, compound cilia and other microtubule defects referring to a combination of defects were found in less than 3% of all of the cilia counted in children aged <18 years (Table 5). There was no significant difference between groups for the central microtubules defects, compound cilia and other microtubule defects (ANOVA, p>0.05).
Table 5
Analysis of ciliary ultrastructure by transmission electron microscopy
Age (years)
|
n
|
Outers
|
Inners
|
Dynein arm defects (%)
|
Microtubules defects (%)
|
Central microtubules defects (%)
|
Compound cilia (%)
|
Other defects (%)
|
<18
|
121
|
8.5 (7.5, 9.0)
|
7.8 (6.5, 8.6)
|
0.0 (0.0, 0.0)
|
9.3 (0.0, 28.4)
|
1.5 (0.0, 9.1)
|
0.5 (0.0, 5.8)
|
0.9 (0.0, 6.6)
|
2-6
|
46
|
8.5 (7.8, 9.0)
|
7.8 (6.9, 8.5)
|
0.0 (0.0, 0.0)
|
8.3 (0.0, 27.9)
|
1.5 (0.0, 10.4)
|
0.2 (0.0, 0.0)
|
0.5 (0.0, 7.0)
|
7-12
|
36
|
8.3 (6.9, 9.0)
|
7.6 (4.2, 8.5)
|
0.0 (0.0, 0.0)
|
5.3 (0.0, 22.6)
|
2.7 (0.0, 19.7)
|
1.0 (0.0, 9.2)
|
0.3 (0.0, 3.1)
|
13-17
|
39
|
8.7 (8.3, 9.0)
|
8.1 (6.7, 8.8)
|
0.0 (0.0, 0.0)
|
14.4 (0.0, 37.8)
|
0.4 (0.0, 3.7)
|
0.4 (0.0, 6.3)
|
1.8 (0.0, 11.1)
|
>18
|
38
|
8.5 (7.0, 9.0)
|
7.3 (5.8, 8.5)
|
0.0 (0.0, 0.0)
|
10.1 (0.0, 31.5)
|
1.9 (0.0, 16.1)
|
0.7 (0.0, 6.3)
|
0.9 (0.0, 13.1)
|
Results are expressed as the mean percentage (5th, 95th percentiles) for each age group.
|