Development and Validation of a Method of Cilia Motility Analysis for

Documento descargado de http://www.elsevier.es el 18/11/2016. Copia para uso personal, se prohíbe la transmisión de este documento por cualquier medio o formato.
Acta Otorrinolaringol Esp. 2012;63(1):1---8
www.elsevier.es/otorrino
ORIGINAL ARTICLE
Development and Validation of a Method of Cilia Motility Analysis
for the Early Diagnosis of Primary Ciliary Dyskinesia夽
Miguel Armengot,a,∗ Mireya Bonet,a Carmen Carda,b María José Gómez,a
Javier Milara,c Manuel Mata,c Julio Cortijoa
a
Sección de Rinología, Servicio de Otorrinolaringología, Hospital General Universitario, Universidad de Valencia, Valencia, Spain
Departamento de Patología, Universidad de Valencia, Valencia, Spain
c
Fundación para la Investigación, Hospital General Universitario, Universidad de Valencia, Valencia, Spain
b
Received 10 June 2011; accepted 10 July 2011
KEYWORDS
Sinusitis;
Bronchiectasis;
Dynein;
Ciliary beat;
Situs inversus;
Fertility;
Sinus hypoplasia
Abstract
Background: Primary ciliary dyskinesia (PCD) is a clinically uniform entity, but cilia motility
and structure can vary between patients, making the diagnosis difficult. The aim of this study
was to evaluate the sensitivity and specificity in diagnosing PCD of a system of high-resolution
digital high-speed video analysis with proprietary software that we developed for analysis of
ciliary motility (Desinsoft-Bio 200). The secondary aim was to correlate nasal ciliary activity
with clinical and structural abnormalities in PCD.
Material and methods: We analysed nasal mucociliary transport, cilia ultrastructure, nasal ciliary beat frequency and beat pattern studied by high-resolution digital high-speed video in
25 cases of PCD (11 Kartagener syndrome), 27 secondary ciliary dyskinesia and 34 healthy
volunteers.
Results: Nasal mucociliary transport was defective in both primary and secondary ciliary dyskinesia. Ciliary immotility was observed only in 6 patients with Kartagener syndrome and
correlated with the absence of dynein. We observed a correlation between partial dynein deficiencies and ciliary dyskinesia. Cilia activity and structure were normal in secondary ciliary
dyskinesia.
Conclusion: Nasal mucociliary transport showed high sensitivity for PCD diagnosis with a low
specificity. High-resolution digital high-speed video has a high sensitivity and specificity for
diagnosing PCD. This system of video analysis is more useful than ultrastructural study and
mucociliary transport for PCD screening. Dynein absence is correlated with cilia immotility and
is more common in patients with Kartagener syndrome.
© 2011 Elsevier España, S.L. All rights reserved.
夽 Please cite this article as: Armengot M, et al. Desarrollo y validación de un método de análisis de la movilidad ciliar para el
diagnóstico precoz de la discinesia ciliar primaria. Acta Otorrinolaringol Esp. 2012;63:1---8.
∗ Corresponding author.
E-mail address: miguel.armengot@gmail.com (M. Armengot).
2173-5735/$ – see front matter © 2011 Elsevier España, S.L. All rights reserved.
Documento descargado de http://www.elsevier.es el 18/11/2016. Copia para uso personal, se prohíbe la transmisión de este documento por cualquier medio o formato.
2
M. Armengot et al.
PALABRAS CLAVE
Sinusitis;
Bronquiectasias;
Dineína;
Batida ciliar;
Situs inversus;
Fertilidad;
Hipoplasia de senos
paranasales
Desarrollo y validación de un método de análisis de la movilidad ciliar para el
diagnóstico precoz de la discinesia ciliar primaria
Resumen
Introducción: La discinesia ciliar primaria (DCP) es una entidad clínica de difícil diagnóstico
debido a que la motilidad y la estructura ciliar pueden variar según los pacientes. El objetivo
principal ha sido evaluar la sensibilidad y especificidad para el diagnóstico de la DCP de un
sistema de videoanálisis de alta resolución digital junto con un software propio desarrollado
por nosotros para el análisis de la movilidad ciliar (Desinsoft-Bio 200). El objetivo secundario
ha sido relacionar la actividad nasal ciliar con la clínica y las anomalías estructurales del cilio
respiratorio.
Material y métodos: Se analizaron el transporte mucociliar mediante un método isotópico, la
ultraestructura con microscopia electrónica y la frecuencia y el patrón de batida ciliar mediante
el sistema Desinsoft-Bio 200. Veinticinco pacientes con DCP (11 de ellos con síndrome de
Kartagener), 27 con discinesia ciliar secundaria y 34 sanos.
Resultados: El transporte mucociliar fue defectuoso en los pacientes con DCP y discinesia ciliar
secundaria. La inmovilidad ciliar se observó en 6 de los pacientes con síndrome de Kartagener
correlacionándose con la ausencia de dineína. Un movimiento ciliar alterado (discinesia) aconteció en el resto de pacientes con DCP, correlacionándose con un déficit parcial de dineína o
ulraestructura normal. La movilidad ciliar y la estructura fueron normales en las discinesias
ciliares secundarias y en sanos.
Discusión y conclusiones: El transporte mucociliar nasal posee una gran sensibilidad cercana
al 100% para el diagnóstico de la DCP, pero muy baja especificidad. El sistema de análisis de
la movilidad ciliar mediante el sistema videodigital de alta velocidad y resolución tiene una
sensibilidad y especificidad elevadas para el diagnóstico de la DCP. Este sistema de videoanálisis
es más útil que el estudio ultraestructural y el transporte mucociliar como cribado de la DCP.
La ausencia de dineína se correlaciona con la inmovilidad ciliar y es más frecuente en pacientes
con síndrome de Kartagener.
© 2011 Elsevier España, S.L. Todos los derechos reservados.
Introduction
Primary ciliary dyskinesia (PCD) or ciliary immotility syndrome (CIS) is a rare, usually inherited, autosomal disease
with a recessive pattern. It affects between 1:20 000 and
1:60 000 individuals.1 It includes a group of diseases among
which there are a number of alterations in the motility of
respiratory cilia, which may present ciliary immotility (CIS),
dysmotility (PCD), or both. Both CIS and PCD are uniform
and similar clinical entities and therefore clinically synonymous and considered as such in this text. The coexistence
of PCD and situs inversus is called Kartagener syndrome (KS)
and has a frequency between 40% and 50% among patients
with PCD.2 Both PCD and CIS are characterised by the presence of simultaneous chronic infections of the upper and
lower respiratory tracts, including the middle ear, starting
from the time of birth. Early diagnosis is very important
because, once it has been established, it is possible to institute a number of respiratory measures that help to relieve
the irreversible lung damage.3
Although PCD is a uniform clinical entity, studies conducted with electron microscopy have shown that there are
different subgroups within it. Dynein deficit affects 70%---80%
of patients.4,5 However, there are cases of patients with PCD
and SK with a completely normal ciliary ultrastructure.4 On
the other hand, it has been observed that there may be
ciliary abnormalities in the context of infection and inflammation (secondary ciliary dyskinesia, SCD). For these two
reasons, the diagnostic validity of the ultrastructural study
is limited.6
The study of mucociliary transport by different methods,
such as saccharin or isotopic agents, does not distinguish
between a deficit in transport due to alterations in mucus
or in ciliary activity, thus not being able to differentiate between PCD and SCD. A recently described model
using high-speed and precision digital video imaging makes
it possible to study the pattern and frequency of ciliary
beat, which may be useful in the diagnosis of PCD.7,8
Although various genetic alterations have been implicated
in the aetiopathogenesis of PCD, there are currently no
genetic tests available that have been validated for its
diagnosis.9,10
The main objective of this study was to evaluate the sensitivity and specificity for PCD diagnosis of a high-resolution
digital system used in combination with proprietary software
developed by our group for the analysis of ciliary motility
(Desinsoft-Bio 200, Spain). The secondary objective was to
link nasal ciliary activity with clinical and structural abnormalities and to evaluate the usefulness of this system in the
diagnosis of PCD and SCD.
Methods
Collection and Preparation of Nasal Epithelial Cells.
Imaging Analysis of the Ciliary Beat Frequency
With a High-resolution Digital System
Samples of airway epithelial cells were obtained by curettage of middle turbinate and/or middle meatus mucosa
Documento descargado de http://www.elsevier.es el 18/11/2016. Copia para uso personal, se prohíbe la transmisión de este documento por cualquier medio o formato.
Development and Validation of a Method of Cilia Motility Analysis
without the use of local anaesthesia, to avoid any external influence, during a non-acute infectious period. The
experiments performed were approved by the Ethics Committee of the Hospital and informed consent was obtained
from patients prior to sampling. The topical treatment that
patients were following was interrupted 48 h before sampling. This manoeuvre does not produce pain or epistaxis
and can be performed in children of all ages as well as
adults.
The tissue sample was introduced into 1 ml of Dulbecco’s
modified Eagle’s medium (DMEM, Cambrex) supplemented
with 10% foetal bovine serum, 2 mM glutamine, penicillin
(100 U/ml) and streptomycin (100 ␮g/ml). The nasal biopsy
was partially disaggregated and dissolved in this medium.
Culture dishes treated with 0.5% gelatine (Corning Incorporated Costar 3513, 12-wells) to facilitate adherence of
ciliated cells were used to grow 150 ␮l samples of nasal
epithelium biopsy.
The rest of the sample was used to determine the pattern and frequency of ciliary beat in both healthy volunteers
and patients with PCD, SCD and KS in an immediate manner,
no more than 30 min after dosing. The study was conducted
in a room at a temperature between 23 and 27 ◦ C. To corroborate the results, the nasal biopsy was seeded in culture
dishes treated with human collagen (type IV, Vitrogen 100;
Cohesion Technologies) in a DMEM medium at 37 ◦ C in an
atmosphere of 5% CO2 for 24 h until a new determination of
ciliary beat frequency.
The samples were visualised using the high-speed digital
video imaging system with a Nikon Eclipse TS100 microscope
(Nikon, Tokyo, Japan), using a phase contrast objective
with a 400× increase. The method used for measuring the
ciliary beat frequency was based on methods previously
published by Salathe et al. in 1999,11,12 with appropriate
modifications. We also used a MULTIMETRIX® XA3051 device
(Dewsintec, SL., Valencia, Spain) for measuring ciliary beat
frequency online. Transillumination was performed with a
150 W green light filter. The beam was aimed directly at a
CCD camera (CV-A33 CL Digital Quad High Speed Progressive Scan Camera, Jai UK Ltd., Unbridge, United Kingdom),
with a resolution of 649(horizontal)×494(vertical) pixels,
and a maximum resolution speed of 128 frames per second.
Video signals were digitised and processed using software
developed by our group (Desinsoft-Bio 200, Spain). To do
so, video images were selected by monitoring the change
in light intensity of each pixel, frame by frame. The variation frequency of the light intensity of each pixel was
obtained using fast Fourier transform (FFT). The resolutions obtained for the frequency and time values were
0.5 Hz and 2 s, respectively. We analysed the intensity
variations in 6 different regions measuring 3×3 pixels in
each cilium, for a minimum of 10 cells per patient. To
view the ciliary beat pattern, we relied on the classification of Chilver et al.13 , with some modifications.
We considered 5 different wave or beat modes: (1) ciliary immotility; (2) a coordinated and rigid ciliary beat
pattern; (3) an uncoordinated vibratile and rigid ciliary
beat pattern; (4) uncoordinated; and (5) normal ciliary
beat pattern. We considered a pattern to be vibratile
when the amplitude of ciliary beat was decreased and
uncoordinated when the cilia beat in non-metachronous
phases.
3
Figure 1 Normally shaped ciliary axonemes. Dynein arms can
be observed in most peripheral tubules.
Nasal Mucociliary Transport
Nasal mucociliary transport was determined using the
radioisotope technique of albumin labelled with metastable
technetium 99 (Tc-99m) according to prior protocols.14
Mucociliary transport rates over 4 mm/min were considered
normal.
Ciliary Ultrastructure
Biopsy specimens obtained by curettage of middle turbinate
mucosa were processed in the usual manner.15 Ciliary ultrastructure was classified following the latest Afzelius ranking4
according to abnormalities in dynein and microtubules.
We studied 100 cross sections of cilia per patient. Dynein
arms (internal, external or both) were considered absent
(‘‘absence of external and internal arms’’) when the mean
number of dynein arms counted in all sections was less than
2 per cross section. The ‘‘absence of internal dynein arm’’
was considered when the mean recorded was less than 0.6
arms per cross section. The ‘‘absence of external dynein
arm’’ was considered when the mean recorded was less
than 1.6 arms per cross section. ‘‘Sparse number of internal
and external dynein arms’’ was considered when the mean
recorded was <7 and <3 per section, respectively. ‘‘Short
dynein arms’’ represented a scarce projection of dynein
from the microtubule compared with normal cilia. Ciliary
orientation was considered normal if the deviation from the
axis of the cilium was less than 28 ◦ C. Alterations from the
9+2 microtubule form were considered in cases where their
prevalence exceeded 30% of the cilia studied (Figs. 1---3).16,17
Patients
To differentiate the various subgroups in this study, the PCD
group included those patients without situs inversus and the
KS group included those patients with situs inversus. We
studied a total of 34 healthy volunteers to measure the
Documento descargado de http://www.elsevier.es el 18/11/2016. Copia para uso personal, se prohíbe la transmisión de este documento por cualquier medio o formato.
4
M. Armengot et al.
manifestations, ciliary ultrastructure, CT (for patients with
situs inversus, pneumatization of the sinuses, sinusitis or
bronchiectasis) and nasal mucociliary transport. A sweat
test was performed to rule out cystic fibrosis. In addition, 27
patients with a prior diagnosis of PCD established by primary
mucociliary transport (Table 2) were reviewed and diagnosed as suffering SCD, after a normal frequency and pattern
of ciliary beats were observed using the high-resolution
motility analysis system.
Statistics
Figure 2 Cross sections of ciliary axonemes corresponding to
a patient with PCD. Cilia with predominant deficit of internal
dynein arms (arrows) can be seen. This was correlated with
present but dyskinetic ciliary motion.
ciliary beat frequency and observe the normal pattern of
ciliary beats in our population. Patients were classified
according to clinical data: sinus pneumatization deficit,
classified as frontal sinus hypoplasia and frontal sinus aplasia
(not applicable in patients under 15 years); pansinusitis (partial or total occupation of all paranasal sinuses,
not applicable in patients under 15 years), bronchiectasis, secretory otitis media, polyposis, family history of
chronic respiratory disease, respiratory allergy or infertility
in patients older than 18 years (male infertility was determined by a spermiogram when patients gave their consent,
or else was considered ‘‘not applicable’’; female infertility
was considered after 3 years of failed pregnancy attempts,
or else was considered ‘‘not applicable’’).
We included 14 patients with PCD and 11 with KS
(Table 1). Patients were diagnosed by different clinical
Data are presented as mean±standard deviation in all experiments. Statistical analysis was performed using analysis of
variance (ANOVA), followed by a Bonferroni test (GraphPad
Software Inc., San Diego, CA, USA). We accepted a level of
statistical significance for values of P<.05.
Results
Determination of the Frequency and Pattern of
Ciliary Beats in Healthy Patients: Differences
Between Patients With PCD and KS
The mean ciliary beat frequency among the 34 healthy volunteers was 10.89±0.29 Hz with a CV of 2.66%. Optical
microscopy observation of ciliary beat pattern was normal,
characterised by planar motion in 2 distinct and coordinated
phases. There were no ultrastructural alterations.
Patients With Primary Ciliary Dyskinesia
In 14 patients with PCD, the mean ciliary beat frequency was
6.31±0.38 Hz, with a CV of 6.02%. A total of 6 patients had
a normal presence of dynein with low ciliary beat frequency
(42.8%). A further 8 patients were classified as suffering a
partial dynein deficiency. There were no patients with an
absence of dynein or immotile cilia.
The presence of stasis in mucociliary transport was
observed in all patients in this group. Their clinical characteristics are shown in Table 1. The pattern of ciliary
motility showed a 100% correlation between dynein alterations and the uncoordinated, vibratile and rigid ciliary
movement pattern. Four patients with presence of dynein
presented a normal ciliary beat pattern despite having a low
beat frequency. Two patients with normal dynein presented
an uncoordinated ciliary beat pattern (Table 3) (Fig. 1). The
ciliary beat frequency in patients with PCD did not change
after 24 h of primary cell culture.
Patients With Kartagener Syndrome
Figure 3 Cross sections of ciliary axonemes corresponding to
a patient with KS. Cilia with absence of dynein (arrows) can
be seen. This was correlated with ciliary immotility and the
presence of situs inversus.
In our cohort of 11 patients with KS, we observed 5 patients
with presence, albeit dyskinetic, of ciliary motility (Table 1).
Of these, 4 patients (patients 2, 3, 6 and 11) presented a partial dynein deficiency, with ciliary beat frequencies of 5, 2.5,
1.5, and 2 Hz, respectively. Patient number 7 with KS showed
the presence of dynein along with ciliary beat frequency of
7 Hz. The remaining 6 patients suffered an absence of dynein
and ciliary immotility. Only in 1 case of KS (9%) (patient 7,
Documento descargado de http://www.elsevier.es el 18/11/2016. Copia para uso personal, se prohíbe la transmisión de este documento por cualquier medio o formato.
Development and Validation of a Method of Cilia Motility Analysis
5
Table 1 Ciliary Beat Frequency, Clinical and Demographic Data and Ciliary Ultrastructure in Patients Diagnosed With Primary
Ciliary Dyskinesia and Kartagener Syndrome.
Patient
Age/Gender
1 (PCD)
2 (PCD)
3 (PCD)
4 (PCD)
5 (PCD)
6 (PCD)
7 (PCD)
8 (PCD)
9 (PCD)
10 (PCD)
11 (PCD)
12 (PCD)
13 (PCD)
14 (PCD)
1 (KS)
2 (KS)
3 (KS)
4 (KS)
5 (KS)
6 (KS)
7 (KS)
8 (KS)
9 (KS)
10 (KS)
11 (KS)
35/F
4/M
41/F
27/F
7/M
50/F
60/F
1/M
33/F
15/M
12/M
9/M
46/F
49/F
15/M
14/M
40/M
49/M
39/M
19/F
66/F
34/M
14/M
17/M
<1/M
CBF, Hz
5.0
6.0
6.0
6.3
5.5
6.1
4.6
3.5
5.7
7.7
6.8
4.2
6.1
6.5
Immobile
5.0
2.5
Immobile
Immobile
1.5
7.0
Immobile
Immobile
Immobile
2
Ciliary Ultrastructure
PPS
PS
B
SOM
P
Presence dynein
Partial dynein deficiency
Partial dynein deficiency
Partial dynein deficiency
Presence dynein
Presence dynein
Partial dynein deficiency
Partial dynein deficiency
Presence dynein
Presence dynein
Partial dynein deficiency
Partial dynein deficiency
Presence dynein
Partial dynein deficiency
Absence dynein
Partial dynein deficiency
Partial dynein deficiency
Absence dynein
Absence dynein
Partial dynein deficiency
Presence dynein
Absence dynein
Absence dynein
Absence dynein
Partial dynein deficiency
FSH
NA
FSA
FSA
NA
FSA
FSA
NA
FSH
FSH
NA
NA
FSA
FSH
FSH
NA
FSH
FSA
FSH
FSH
FSH
FSA
NA
FSH
NA
Yes
NA
Yes
Yes
NA
Yes
Yes
NA
Yes
Yes
NA
NA
Yes
Yes
Yes
NA
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
NA
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
FHCRD
Fertility
MNT
RA
Yes
No
Yes
No
No
No
No
No
No
Yes
No
No
No
Yes
No
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
No
Yes
NA
NA
NA
NA
No
Yes
NA
NA
NA
NA
NA
Yes
No
NA
NA
No
No
NA
NA
No
No
NA
No
NA
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
NA
B, bronchiectasis; CBF, ciliary beat frequency; F, female; FHCRD, family history of chronic respiratory disease; FSA, frontal sinus aplasia;
FSH, frontal sinus hypoplasia; KS, Kartagener syndrome; M, male; MNT, mucociliary nasal transport; NA, not applicable; P, polyposis;
PCD, primary ciliary dyskinesia; PPS, pneumatization of paranasal sinuses; PS, pansinusitis; RA, respiratory allergy; SOM, secretory otitis
media.
a Patients who presented microtubule abnormalities.
Table 3) was dynein normal, but with a coordinated, vibratile
and rigid movement pattern. The absence of dynein and ciliary immotility was more common in patients with KS than
in patients with PCD (Table 3).
The absence of mucociliary transport affected all
patients. The clinical characteristics are summarised in
Table 1. The pattern of ciliary beat in patients with KS
was immotility for 100% of patients with a total absence of
dynein (Fig. 3). However, patients with a partial deficiency
of dynein (Fig. 2) presented an uncoordinated, vibratile and
rigid movement pattern. An exception to this was the case
of patient 7, who presented a coordinated, vibratile and
rigid movement pattern (Table 3). The mean ciliary beat
frequency in KS patients was 1.6±0.79 Hz. Ciliary beat frequency was unchanged after 24 h of primary cell culture at
37 ◦ C in an atmosphere of 5% CO2.
Diagnosis of Secondary Ciliary Dyskinesia in 27
Patients Previously Diagnosed With Ciliary
Dyskinesia
We reviewed 27 patients with PCD diagnosed previously by
observation of stasis in nasal mucociliary transport. Their
clinical characteristics are summarised in Table 2. The mean
ciliary beat frequency was 10.17±0.18 Hz, almost the same
as for healthy patients. The ciliary beat pattern observed
was completely normal. All patients showed the presence
of dynein without microtubule alterations.
Table 4 shows a comparative summary of the results
obtained in the different clinical groups studied.
Discussion
In this study we compared the use of a high-resolution digital
system to correlate the ciliary beat frequency and pattern
with clinical and structural PCD changes. The first step in the
diagnosis of PCD was the study of nasal mucociliary transport. As some previous studies have demonstrated,18,19 our
negative results for this test (stasis) showed 100% sensitivity
for the diagnosis of PCD (Table 1) but low specificity because
all patients with SCD presented stasis.
As previously described,4 the clinical characteristics of
patients with PCD and KS were similar. However, situs inversus was more common in our group of patients with absence
of dynein. Therefore, this study suggests that patients
with PCD suffer dynein deficiencies that allow the cilia to
move, albeit in a flawed manner. However, the absence of
dynein and ciliary immotility was predominant in cases of
Documento descargado de http://www.elsevier.es el 18/11/2016. Copia para uso personal, se prohíbe la transmisión de este documento por cualquier medio o formato.
6
M. Armengot et al.
Table 2 Ciliary Beat Frequency, Demographic and Clinical Data and Ultrastructure of Patients Diagnosed With Secondary Ciliary
Dyskinesia.
Patient
Age/Gender
CBF, Hz
Ciliary Ultrastructure
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
7/F
3/M
47/M
50/M
31/M
6/M
3/M
5/M
4/F
7/F
4/F
11/M
20/M
6/F
4/M
32/F
35/M
37/F
15/F
12/M
15/M
3/M
1/M
2/M
1/M
64/M
13/F
10.5
10.6
11.5
10.0
9.5
9.0
9.1
10.0
9.0
10.0
12.0
8.0
10.0
9.5
9.0
12.0
10.5
10.0
10.5
11.0
11.3
10.8
9.5
9.8
10.5
11.2
10.0
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
Presence
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
dynein
PPS
PS
B
SOM
P
NA
NA
Normal
Normal
Normal
NA
NA
NA
NA
NA
NA
NA
Normal
NA
NA
Normal
Normal
Normal
Normal
Normal
Normal
NA
NA
NA
NA
FSA
NA
NA
NA
No
No
No
NA
NA
Na
NA
NA
NA
NA
No
NA
NA
Yes
No
No
No
No
No
NA
NA
NA
NA
Yes
NA
No
No
Yes
Yes
Yes
No
No
No
No
No
No
No
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
No
NA
NA
YES
Yes
No
Yes
No
Yes
No
No
No
No
Yes
Yes
No
No
No
No
Yes
Yes
Yes
Yes
No
Yes
Yes
No
Yes
NA
NA
NA
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
NA
NA
NA
No
No
FHCRD
Fertility
MNT
RA
No
No
No
No
Yes
No
No
No
No
No
No
Yes
No
No
Yes
No
Yes
Yes
Yes
Yes
No
No
No
No
So
No
No
NA
NA
No
Yes
Yes
NA
NA
NA
NA
NA
NA
NA
Yes
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Yes
NA
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Stasis
Yes
Yes
No
No
Yes
No
No
No
Yes
No
Yes
No
No
No
No
No
No
No
Yes
Yes
Yes
No
No
No
No
No
Yes
B, bronchiectasis; CBF, ciliary beat frequency; F, female; FHCRD, family history of chronic respiratory disease; FSA, frontal sinus aplasia;
M, male; MNT, mucociliary nasal transport; NA, not applicable; P, polyposis; PPS, pneumatization of paranasal sinuses; PS, pansinusitis;
RA, respiratory allergy; SOM: secretory otitis media.
a Patients presenting microtubule abnormalities.
situs inversus-KS.16 In accordance with other studies,4 we
observed that infertility was common among men with PCDKS. This infertility also affected women, although to a lesser
degree.
To improve the diagnosis of PCD, we used a highresolution digital model that enabled us to define the ciliary
beat pattern and frequency associated with dynein and
microtubule abnormalities. However, except for the 9+0
form, microtubule alterations are characteristic of SCD and
therefore not significant in PCD. Deficiencies in dynein arms
were observed in congenital cases only and were the most
frequent.6,16 Our results for ciliary beat frequency among
healthy volunteers were consistent with previous results,
ranging between 9 and 15 Hz.20,21 We correlated the absence
of dynein (absence of internal and external arms) with ciliary
immotility in 100% of the cilia from the 6 patients with KS.
Moreover, the combined defects of the internal and external arms (scarce or short) were found in 3 patients with PCD
and 3 with KS, all of them presenting a very low mean ciliary beat frequency (3.3 Hz). When we considered patients
with an isolated defect of the internal or external dynein
arms separately, we found 6 patients with a mean ciliary
beat frequency of 5.31 Hz, with no differences between the
defects of the internal or external arms. Nevertheless, more
patients would be required to corroborate these results,
since other studies correlate ciliary motility with specific
deficiencies in the internal and external dynein arms.13,22,23
As established in the Materials and Methods section,
we considered 2 groups of patients; PCD patients and KS
patients. In the first group we observed isolated or combined deficiencies of the internal and external dynein arms,
as well as the presence of normal dynein. There were no
cases of total absence of dynein (Table 3). However, 6 of
the 11 KS patients presented a complete lack of dynein.
This fact explains the low ciliary beat frequency observed
in patients with KS.
Among our patients we found 4 different types of ciliary
beat pattern24---26 : (1) immotile cilia, associated in 100% of
cases with the absence of internal and external dynein arms;
(2) an uncoordinated, vibratile and rigid movement pattern,
associated with partial defects of the internal or external
dynein arms; (3) uncoordinated movement pattern, found
in 2 patients with normal dynein; and (4) pattern of normal ciliary motility but with a low beat frequency, found in
patients with presence of dynein. Among our patients we did
not find isolated microtubule alterations. Consequently, neither the movement pattern nor the frequency of movement
can be attributed to these alterations (Table 4).
Documento descargado de http://www.elsevier.es el 18/11/2016. Copia para uso personal, se prohíbe la transmisión de este documento por cualquier medio o formato.
Development and Validation of a Method of Cilia Motility Analysis
7
Table 3 Evaluation of the Ciliary Beat Frequency, Ciliary Beat Pattern and Microtubule and Dynein Abnormalities Through the
High-speed Digital Video and Electron Microscopy System in Patients With Primary Ciliary Dyskinesia and Kartagener Syndrome.
Patient
CBF, Hz
Microtubule Abnormalities
Dynein Abnormalities
Ciliary Beat Pattern
Presence dynein
Short external dynein arm
Short external dynein arm
Absence internal dynein arm
Normal
Uncoordinated vibratile rigid
Uncoordinated vibratile rigid
Uncoordinated vibratile rigid
5.5
6.1
4.6
3.5
No abnormalities
Peripheral microtubules
Peripheral microtubules
Central and peripheral
microtubule
No abnormalities
No abnormalities
No abnormalities
No abnormalities
Normal
Uncoordinated
Uncoordinated vibratile rigid
Uncoordinated vibratile rigid
9 (PCD)
10 (PCD)
11 (PCD)
5.7
7.7
6.8
No abnormalities
No abnormalities
No abnormalities
12 (PCD)
4.2
No abnormalities
13 (PCD)
14 (PCD)
1 (KS)
6.1
6.5
Immobile
No abnormalities
No abnormalities
No abnormalities
2 (KS)
5.0
No abnormalities
3 (KS)
4 (KS)
2.5
Immobile
No abnormalities
No abnormalities
5 (KS)
Immobile
No abnormalities
6 (KS)
1.5
No abnormalities
7 (KS)
8 (KS)
7.0
Immobile
No abnormalities
No abnormalities
9 (KS)
Immobile
No abnormalities
10 (KS)
Immobile
No abnormalities
11 (KS)
Immobile
No abnormalities
Presence dynein
Presence dynein
Short external dynein arm
Scarce external and internal dynein
arms
Presence dynein
Presence dynein
Scarce external and internal dynein
arms
Scarce external and internal dynein
arms
Presence dynein
Absence of internal dynein arm
Absence of external and internal
dynein arm
Short external and internal dynein
arms
Absence of internal dynein arm
Absence of external and internal
dynein arm
Absence of external and internal
dynein arm
Scarce external and internal dynein
arms
Presence dynein
Absence of external and internal
dynein arm
Absence of external and internal
dynein arm
Absence of external and internal
dynein arm
Scarce external and internal dynein
arms
1
2
3
4
(PCD)
(PCD)
(PCD)
(PCD)
5.0
6.0
6.0
6.3
5
6
7
8
(PCD)
(PCD)
(PCD)
(PCD)
Normal
Normal
Uncoordinated vibratile rigid
Uncoordinated vibratile rigid
Uncoordinated
Uncoordinated vibratile rigid
Ciliary immotility
Uncoordinated vibratile rigid
Uncoordinated vibratile rigid
Ciliary immotility
Ciliary immotility
Uncoordinated vibratile rigid
Coordinated vibratile rigid
Ciliary immotility
Ciliary immotility
Ciliary immotility
Uncoordinated vibratile rigid
CBF, ciliary beat frequency; KS, Kartagener syndrome; PCD, primary ciliary dyskinesia.
Table 4
Summary of Results.
Subjects
Ciliary Motility
Mean CBF, Hz
Dynein
MNT
Volunteers (n=34)
PCD (n=14)
Present (100%)
Present (100%)
10.89±0.29
6.31±6.02
>4 mm/min
Stasis
KS (n=11)
Present (45.45%)
Absent (54.55%)
SCD (n=27)
Present (100%)
100% normal
42.8% normal
57.2% partial
9.10% normal
36.36% partial
55.54% absent
100% normal
3.6±2.08
0
10.17±0.18
Stasis
Stasis
CBF, ciliary beat frequency; KS, Kartagener syndrome; MNT, mucociliary nasal transport; PCD, primary ciliary dyskinesia; SCD, secondary
ciliary dyskinesia.
Documento descargado de http://www.elsevier.es el 18/11/2016. Copia para uso personal, se prohíbe la transmisión de este documento por cualquier medio o formato.
8
M. Armengot et al.
Conclusions
Our results show that the high-resolution and speed video
analysis system has an elevated sensitivity for detecting
abnormalities in the ciliary beat pattern and frequency, as
well as a high specificity for differentiating between PCD
and SCD. Furthermore, it is also a rapid technique that can
serve to guide the differential diagnosis between PCD and
SCD. The mucociliary transport test has a low specificity. The
absence of dynein is correlated with ciliary immotility and
is more common among patients with KS. The presence of
situs inversus could be related to ciliary immotility and the
absence of dynein.
11.
12.
13.
14.
Financing
15.
This project was funded by ERDF (European Regional Development Funds), CIBER CB06/06/0027 by Instituto de Salud
Carlos III of the Spanish Ministry of Health and by Research
Funds from the Regional Government of Valencia, Spain.
16.
17.
Conflict of Interests
The authors have no conflicts of interest to declare.
18.
References
19.
1. Afzelius BA, Mossberg B. Immotile-cilia syndrome (primary ciliary dyskinesia), including Kartagener syndrome. In: Scriver C,
Beaudet AL, Sly W, Valle D, editors. The metabolic and molecular bases of inherited diseases. 7th ed. New York: McGraw-Hill;
1995. p. 3943---54.
2. Afzelius B. Situs inversus and ciliary abnormalities. What is the
connection? Int J Dev Biol. 1995;39:839---44.
3. Ellerman A, Bisgaard H. Longitudinal study of lung function in a cohort of primary ciliary dyskinesia. Eur Resp J.
1997;10:2376---9.
4. Afzelius BA. Cilia-related diseases. J Pathol. 2004;204:470---7.
5. Jorissen M, Bertrand B, Eloy P. Ciliary dyskinesia in the nose and
paranasal sinuses. Acta Otorhinolaryngol Belg. 1997;51:353---66.
6. Bush A, Chodhari R, Collins N, Copeland F, Hall P, Harcourt J,
et al. Primary ciliary dyskinesia: current state of the art. Arch
Dis Child. 2007;92:1136---40.
7. Chilvers MA, O’Callaghan C. Analysis of ciliary beat pattern
and beat frequency using digital high speed imaging: comparison with the photomultiplier and photodiode methods. Thorax.
2000;55:314---7.
8. Santamaria F, de Santi MM, Grillo G, Sarnelli P, Caterino M,
Greco L. Ciliary motility at light microscopy: a screening technique for ciliary defects. Acta Paediatr. 1999;88:853---7.
9. Geremek M, Witt M. Primary ciliary dyskinesia: genes, candidate
genes and chromosomal regions. J Appl Genet. 2004;45:347---61.
10. Lonergan KM, Chari R, Deleeuw RJ, Shadeo A, Chi B, Tsao MS,
et al. Identification of novel lung genes in bronchial epithelium
20.
21.
22.
23.
24.
25.
26.
by serial analysis of gene expression. Am J Respir Cell Mol Biol.
2006;35:651---61.
Salathe M, Bookman RJ. Mode of Ca2+ action on ciliary beat
frequency in single ovine airway epithelial cells. J Physiol.
1999;520 Pt 3:851---65.
Nlend MC, Bookman RJ, Conner GE, Salathe M. Regulator of
G-protein signaling protein 2 modulates purinergic calcium and
ciliary beat frequency responses in airway epithelia. Am J Respir
Cell Mol Biol. 2002;27:436---45.
Chilvers MA, Rutman A, O’Callaghan C. Ciliary beat pattern is associated with specific ultrastructural defects in
primary ciliary dyskinesia. J Allergy Clin Immunol. 2003;112:
518---24.
Armengot M, Ruiz G, Romero de Avila C, Carda C, Basterra J.
Diagnostic usefulness of radioisotope study of nasal mucociliary
transport in patients with recurrent respiratory infections. Rev
Esp Med Nucl. 1998;17:21---6.
Armengot Carceller M, Carda Batalla C, Escribano A, Samper GJ.
Study of mucociliary transport and nasal ciliary ultrastructure
in patients with Kartagener’s syndrome. Arch Bronconeumol.
2005;41:11---5.
Carlen B, Stenram U. Primary ciliary dyskinesia: a review. Ultrastruct Pathol. 2005;29:217---20.
Rossman CM, Lee RM, Forrest JB, Newhouse MT. Nasal ciliary
structure and function in patients with primary ciliary dyskinesia compared with that in normal subjects with various
respiratory diseases. Am Rev Respir Dis. 1984;129:161---7.
Marthin JK, Mortensen J, Pressler T, Nielsen KG. Pulmonary
radioaerosol mucociliary clearance in diagnosis of primary ciliary dyskinesia. Chest. 2007;132:966---76.
De Boeck K, Proesmans M, Mortelmans L, Van Billoen B, Willems
T, Jorissen M. Mucociliary transport using 99mTc-albumin colloid: a reliable screening test for primary ciliary dyskinesia.
Thorax. 2005;60:414---7.
Chilvers MA, Rutman A, O’Callaghan C. Functional analysis of
cilia and ciliated epithelial ultrastructure in healthy children
and young adults. Thorax. 2003;58:333---8.
de Iongh RU, Rutland J. Ciliary defects in healthy subjects,
bronchiectasis, and primary ciliary dyskinesia. Am J Respir Crit
Care Med. 1995;151:1559---67.
Rossman CM, Forrest JB, Lee RM, Newhouse AF, Newhouse MT.
The dyskinetic cilia syndrome; abnormal ciliary motility in association with abnormal ciliary ultrastructure. Chest. 1981;80(6
Suppl):860---5.
Pedersen M. Specific types of abnormal ciliary motility in
Kartagener’s syndrome and analogous respiratory disorders.
A quantified microphoto-oscillographic investigation of 27
patients. Eur J Respir Dis Suppl. 1983;127:78---90.
Armengot M, Milara J, Mata M, Carda C, Cortijo J. Cilia motility
and structure in primary and secondary ciliary dyskinesia. Am J
Rhinol Allergy. 2010;24:175---80.
Milara J, Armengot M, Mata M, Morcillo EJ, Cortijo J. Role
of adenylate kinase type 7 expression on cilia motility: possible link in primary ciliary dyskinesia. Am J Rhinol Allergy.
2010;24:181---5.
Armengot Carceller M, Mata Roig M, Milara Payá X, Cortijo
Gimeno J. Primary ciliary dyskinesia. Ciliopathies. Acta Otorrinolaringol Esp. 2010;61:149---59.