METHODS

Fifty three healthy children (31 male, age range 6 months to 17 years) were recruited from subjects undergoing elective surgery and 23 adult volunteers (16 male, age range 18–43 years) were also recruited. Subjects were excluded if they had a history of chronic respiratory or nasal disease or a symptomatic upper respiratory tract infection during the previous 6 weeks, were taking regular medication, or were known smokers.

Paediatric samples were obtained immediately after induction of anaesthesia with propofol. This agent has been shown to have no effect on ciliary beat frequency.²³ No premedication had been given to any subject before surgery. In all subjects ciliated samples were obtained by brushing the inferior nasal turbinate with a 2 mm cytology brush. Nasal brushings were placed in medium 199 (pH 7.3) which contained antibiotic solution (streptomycin 50 μg/ml, penicillin 50 μg/ml; Gibco, UK).

The study was approved by the Leicestershire ethical review committee and written consent was obtained before sampling.

Evaluation of ciliary structure and function

Tissue obtained by nasal brushing was fixed in 2.5% glutaraldehyde and processed through to resin by standard techniques as previously described.²⁴ Ultrathin sections were cut at 70 nm. These were collected on 200 mesh thin bar copper grids, stained in 1% uranyl acetate, counterstained in Reynold's lead phosphate, and examined by transmission electron microscopy.

The ciliated epithelium was assessed blindly for both epithelial and ciliary ultrastructural changes. Epithelial integrity was assessed by firstly examining cell type. The number of ciliated cells, mucous cells, and dead cells were totalled and expressed as a percentage of all cells examined. Disruption and damage to the tissue was quantified using the scoring system previously described.^24,25 Briefly, the tissue was scored for the following parameters: loss of cilia from ciliated cells: 0 (fully ciliated), 1, 2, 3 (a few cilia visible); projection of cells from the epithelial edge: 0 (normal alignment), 1, 2, 3 (cell projected from edge but some contact with other epithelial cells); cytoplasmic blebbing: 0 (absent), 1 (minor), 2 (major); mitochondrial damage: 0 (absent), 1 (present) (fig 1).

To give an overall evaluation of epithelial damage an epithelial integrity score was given to the epithelium which incorporated ciliary loss, cellular projection, cytoplasmic blebbing and mitochondrial damage. A healthy intact epithelial edge was scored 0 and a severely disrupted edge was scored 5 (0=no damage, 1=minor, 2=mild, 3=moderate, 4=major, 5=severe damage; fig 2). The scoring system was evaluated by comparing it against all measurements used to measure epithelial damage.

Damage to individual cilia was evaluated by examining the ciliary ultrastructure for microtubular and dynein arm defects. Alignment of individual cilia within a cell was assessed by measuring ciliary orientation as previously described.²⁶

The percentage of cells with loss of cilia, cellular projections, cytoplasmic blebbing, mitochondrial damage and with microtubular or dynein arm defects was calculated.

Ciliary beat frequency and beat pattern

Ciliary beat frequency and beat pattern were evaluated as previously described.¹ Briefly, ciliated strips of epithelium were suspended in a chamber created by the separation of a cover slip and glass slide by two adjacent cover slips. The slide was placed on a heated stage (37°C) of a Leitz Diaplan microscope mounted on an anti-vibration table (Wentworth Laboratories Ltd, UK). Specimens were examined using a ×100 interference contrast lens. Only undisrupted ciliated strips longer than 50 μm devoid of mucus were studied. Beating ciliated edges were recorded using a digital high speed video camera (Kodak Motioncorder Analyser, Model 1000) at a rate of 400 frames per second. The camera allows video sequences to be recorded and played back at reduced frame rates or frame by frame. The ciliated edge, projected onto a high resolution monitor, was divided into five adjacent areas measuring 10 μm. Two measurements of ciliary beat frequency were made in each area, resulting in a total of 10 measurements of beat frequency along each ciliated strip. A maximum of 10 edges were analysed per subject. Ciliary beat frequency was determined directly. Groups of beating cilia were identified and the number of frames required to complete 10 cycles recorded. This was converted to ciliary beat frequency (CBF) using the calculation (CBF= 400/(number frames for 10 beats) × 10).¹

As the digital high speed video system was to be used to establish reference ranges for the measurement of ciliary beat frequency, the reproducibility of the method was evaluated. A single point on the ciliated edge was identified on a grid placed on the monitor. Ciliary beat frequency at that point was measured independently by two observers (O1, O2), and this was repeated for each of the five areas displayed on the monitor. A total of five readings were obtained for each edge and the analysis was performed in 10 subjects. One observer repeated the series of measurements two days later (M1, M2). From this the inter-observer and intra-observer coefficient of variation (CV) could be calculated.

To assess the ciliary beat pattern each edge was analysed. Coordinated ciliary beating in a forward backward motion along the whole epithelial edge was defined as normal. Edges which appeared to have dyskinetically beating cilia were noted and the percentage of edges exhibiting areas of dyskinetically beating cilia was then calculated.

Analysis of data

As ciliary beat frequency may change with age,^17,18 we wanted to see if other parameters showed such variation. As suggested by Roth et al,¹⁸ a cut off was made at 18 years of age. To allow sufficient subjects in each age group, three age ranges were used: 0–6, 7–12, and 13–18 years of age. Adults were classified as ≥19 years.

To form reference ranges the mean ciliary beat frequency, standard deviation, 5th and 95th percentiles were calculated for individual age groups. A one way analysis of variance was performed between groups. Individual groups were compared using a Student's t test. Similarly, the mean percentage, 5th and 95th percentiles of edges exhibiting areas of dyskinetically beating cilia were calculated. For all ultrastructural parameters the mean and the 5th and 95th percentiles were calculated. A one way analysis of variance was performed between groups.

RESULTS

Ciliary beat frequency and beat pattern were measured in all subjects; 56 had sufficient tissue for epithelial integrity measurements and 60 for ciliary ultrastructure. Ciliary beat frequency and beat pattern were the initial measurements to be made after which samples were then processed for electron microscopy. During this procedure tissue may be lost. Consequently, some subjects had an inadequate sample for full ultrastructural analysis.

Table 1 shows the percentage of different cell types seen in the epithelial strips obtained. Analysis showed no difference between the percentage of different cells identified and the age of the subject. Ciliated cells formed 65% of the cell population.

Analysis of the factors involved in epithelial integrity are summarised in table 2. Even within the healthy population there is evidence of loss of cilia, cellular extrusion, cytoplasmic blebbing, and mitochondrial damage. Analysis of variation found no difference between groups for all measurements analysed. The epithelial integrity score, which reflects a combination of all the measurements used to assess epithelial damage, also showed no significant difference between the age groups.

A summary of the results of the ultrastructural analysis of individual cilia is shown in table 3. Dynein arm defects were found in less than 3% of cilia observed. It was possible to visualise, on average, seven of the expected nine dynein arms when counting both inner and outer dynein arms. Again no differences were found in the ultrastructural analysis of individual cilia between the various age groups. Microtubular abnormalities were uncommon in all age groups; ciliary orientation did not vary with age (table 3).

Ciliary beat frequency and the percentage of cells with ciliary dyskinesia are shown in table 4. No significant difference in mean ciliary beat frequency between individual age groups was found (ANOVA, p=0.10). However, there was a significant difference in ciliary beat frequency between patients under the age of 18 (12.8 Hz (95% CI 12.3 to 13.3)) and those over the age of 18 (11.4 Hz (95% CI 10.2 to 12.6 Hz), p<0.01, t test). Approximately 10% of all edges analysed exhibited areas of dyskinetically beating cilia. This was found to be higher in the 13–18 year group.

To establish a reference range the mean ciliary beat frequency was plotted against age (fig 3A). A weak negative correlation was found between mean ciliary beat frequency and increasing age (r=–0.30, p=0.008). This was modelled and a linear relationship was found. Quadratic and other relationships were modelled and found not to be significant.

Within each sample cilia were found to beat at different frequencies. To evaluate sample variation in ciliary beat frequency the edges with the highest and lowest ciliary beat frequency were plotted against age for each subject. The maximal ciliary beat frequency (fig 3C) of edges ranged from 7.1 to 22.4 Hz with 95% of subjects having a maximal ciliary beat frequency of >10 Hz. The lowest mean ciliary beat frequency (fig 3B) of edges ranged from 6.0 to 17.1 Hz with 85% of subjects having a minimum beat frequency of >8 Hz.

No significant difference was observed for the inter-observer (O1, O2) and intra-observer (M1, M2) measurements of CV. The mean (SD) CV for O1 and O2 was 11.6 (3.8)% (95% CI 8.3 to 14.9) and 10.7 (4.4)% (95% CI 6.3 to 14.6), respectively, and for M1 and M2 was 10.7 (4.4)% (95% CI 6.3 to 14.6) and 10.9 (5.1)% (95% CI 5.8 to 16.0), respectively. The mean (SD) difference in inter-observer CV was 0.9 (2.3)% (95% CI –1.1 to 2.9; range –1.4 to 4.9) and in intra-observer CV was 0.7 (2.0)% (95% CI –1.0 to 2.4; range –3.5 to 8.5).

DISCUSSION

Examination of the nasal ciliated epithelium from a large group of healthy children and a smaller group of adults has enabled us to establish normal age related reference ranges for both ciliary structure and function.

There are few data quantifying the ciliary epithelial ultrastructure following brush biopsy. We found evidence of minor epithelial damage in the tissue from healthy subjects. Our results show a greater degree of epithelial damage than previously described.²⁵ However, these data were from organ culture models and it is possible that, in the process of brushing and tissue preparation, minor damage may have occurred. Although two previous studies have evaluated the use of nasal brushing to sample cilia for ultrastructural measurement, they did not assess epithelial damage.^27,28

A scoring system for evaluation of epithelial integrity has been developed. This has been validated against the measurements used to assess epithelial ultrastructural damage and found to be representative of the minor epithelial damage observed in healthy subjects.

The percentage of dynein arm and microtubular abnormalities were both found to be less than 5%, which agrees with other published data.⁷ The mean orientation of cilia in the paediatric population has only been described in eight children under the age of 2 years and was reported to be 14.9°.²⁹ This is higher than the values we obtained (10.7–10.9°) in 60 subjects of differing ages.

The quantification of inner and outer dynein arms is important in the diagnosis of primary ciliary dyskinesia as dynein arm defects are the most common abnormality found in these patients.²⁸ The majority of inner and outer dynein arms were visualised in all subjects. Our results are consistent with other published data on the number of outer dynein arms visible, but we were able to identify a greater proportion of inner dynein arms than has previously been reported.³⁰ This may be because of the healthy nature of the tissue.

As suggested by Veale and colleagues, we examined ciliary beat frequencies from several edges and from different sites along an edge.³¹ The ciliary beat frequency was found to vary between edges within a sample, with a mean beat frequency of <11 Hz in some subjects. This is in keeping with other reports which have found cilia in healthy adults to beat maximally at a frequency of >10 Hz (range 10.2–14.6) and minimally at a frequency of >7 Hz (range 7.5–11.2).³¹ This was limited to 20 volunteers and no children were included. Adults were found to have slower beating cilia³¹ with frequencies as low as 6–9 Hz.^19,21 Two healthy children were also reported to have cilia beating as slowly as 6 Hz.²¹

The CV for measurement of ciliary beat frequency along an epithelial edge has been shown to vary from 9% to 58%³¹ compared with 10% in our study. We found no significant difference in inter-observer or intra-observer CV using the digital high speed video technique.

The ciliary beat frequency of the children was found to be significantly greater than the adult population. This is supported by studies which have shown ciliary beat frequency of neonates¹⁷ and teenagers¹⁸ to be greater than adult subjects. Our data suggest a slight fall in ciliary beat frequency with increasing age, which is in agreement with other studies,¹⁹ although Jorissen et al²⁰ found ciliary beat frequency to be independent of age. However, their readings were conducted at 22°C rather than at body temperature which makes the comparison difficult. At this temperature cilia beat at a much slower frequency and the association may therefore have been lost.³²

Digital high speed video imaging allowed us to visualise precisely the normal ciliary beat pattern in healthy subjects; 10% of edges had evidence of dyskinetically beating cilia. The remainder of the cilia were found to beat forward and backwards within the same plane without a classical sideways recovery sweep. This is consistent with our earlier description.¹ Analysis of ciliary beat pattern may improve our understanding of the actions of various respiratory pathogens—for example, cilia following infection have been found to have a dyskinetic beat pattern despite beating at a normal ciliary beat frequency.²⁴

In summary, we have established an extensive age related normal reference range for both ciliary structure and function. We have also examined the epithelial integrity in a healthy population. Such data will help with our evaluation of patients suspected of having primary ciliary dyskinesia and in research studies looking at the effects of various pathogens on nasal ciliary ultrastructure, function, and epithelial integrity.