A subscription to JoVE is required to view this content. Sign in or start your free trial.
High-speed video microscopy analysis is a relatively easy-to-perform, fast, cost-effective, and, in experienced hands, a considerably reliable tool for first-line diagnostics of primary ciliary dyskinesia, which should be available in every center involved in diagnostics and the treatment of severe lung diseases.
Primary ciliary dyskinesia (PCD) is a congenital disorder predominantly inherited in an autosomal recessive trait. The disorder causes disturbance in the motion of cilia, leading to severe impairment of mucociliary clearance (MCC). If undiagnosed or diagnosed too late, the condition leads to the development of bronchiectasis and serious damage to the lungs in later life. Most of the methods for diagnosing PCD are time-consuming and demand extensive economic resources to establish them. High-speed video microscopy analysis (HSVMA) is the only diagnostic tool to visualize and analyze living respiratory cells with beating cilia in vitro. It is fast, cost-effective, and, in experienced hands, very reliable as a diagnostic tool for PCD. In addition, classical diagnostic measures such as transmission electron microscopy (TEM) are not applicable for some mutations as morphological changes are absent.
This paper describes the process of collecting respiratory epithelial cells, the further preparation of the specimen, and the process of HSVMA. We also describe how brushed cells can be successfully kept unharmed and beating by keeping them in a nourishing medium for storage and transport to the investigation site in cases where a clinic does not possess the equipment to perform HSVMA. Also shown are videos with pathologic beating patterns from patients with a mutation in the dynein arm heavy chain 11 gene (DNAH11), which cannot be diagnosed with TEM; the result of an inconclusive HSVMA due to infection of the upper airways, as well as an unsuccessful brushing with superimposition of red blood cells. With this article, we would like to encourage every unit dealing with pulmonology patients and rare lung diseases to perform HSVMA as part of their daily routine diagnostics for PCD or send the specimens over to a center specializing in performing HSVMA.
Primary ciliary dyskinesia (PCD) is a rare, hereditary genetic disorder, which causes disturbances in the movement of beating cilia. If undiagnosed, it leads to severe lung damage in later life due to severe impairment of MCC. In the past, its prevalence has been estimated to be in the range of 1:4,000 to 50,000. Due to steadily improving diagnostics and a growing awareness of the condition, updates on the prevalence of PCD suggest that it might be much more common and probably in the range of 1:4,000 to 20,000 instead1,2. However, patients with PCD are still underdiagnosed or diagnosed too late
Ethics Statement:
This study was approved by the local ethics committee (69/2017) and was conducted in compliance with the declaration of Helsinki.
1. Collection and transport of respiratory epithelial cells
Video 1 and Video 2 show a normal control where CBF and CBP are in the normal range (see Figure 1). Video 3, Video 4, Video 5, and Video 6 represent two cases of PCD patients with a homozygous mutation in the DNAH11 gene (c.2341G > A; p. Glu781Lys)3. These representative videos were chosen because phenotypes of mutations in the DNAH11.......
Here, the diagnostic process for PCD using HSVMA is described and discussed in the light of first-line diagnostics. Despite being relatively easy to establish, cost-effective11, and a reliable method in experienced hands12, HSVMA is not a diagnostic measure without pitfalls. Abnormal CBF and CBP may be due to secondary infection, leading to inflammation of the broncho-respiratory epithelia15, and for the same reason, smoking individuals may have abno.......
We wish to express special thanks to the pediatric nurse Mrs. Johanna Juvankoski for her excellent help with the brushings. We would also like to express special gratitude to Professor Heymut Omran (University Clinic Münster, UKM) for granting permission to use the schematic figure of normal ciliary motion from their website. Finally, we would like to thank Mr. Alan Brown BA (Hons), PGCE, for proofreading the manuscript.
....Name | Company | Catalog Number | Comments |
Amoxiciline-clavulanic acid | Orion Oyj | 40 mg/kg divided in 2 doses/day, for adults 875/125 mg 1 tablet x2/day | |
Camera Software | Hamamatsu | HCI Image | |
Cold pack | any | for preservation and transport | |
Differential interference microscope | Carl Zeiss | Inverted, cell observer microscope | |
Digitial High Speed Video Camera | Hamamatsu | Orca Flash 4.0, digital camera type C11440 | |
Dulbecco´s Modified Eagle Medium | Thermo Fisher | 10565018 | basal cell culture medium |
Eppendorf tube | Eppendorf | 30120086 | 1.5 mL tube |
Glass-bottom microwell dish | MatTek | P35G-1.5-14-C | cuvette for microscopy |
Heating Unit | Carl Zeiss/PeCon | 810-450001 | Carl Zeiss incubation elements with PeCon TempModule S1 temperature control |
Interdental brush 0.6 mm | Doft | 872267 | Interdental brush on a long wire with a reusable handle and cap in zipbag |
Objective | Carl Zeiss | 100x/1.46, α Plan-Apochromat DIC objective | |
Small polystyrene box with lid | any | for transport |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved