Pediatric Allergy, Immunology, and Rheumatology
Who Should Be Evaluated?
The most serious manifestations of Primary Ciliary Dyskinesia are its adverse respiratory health effects. Inasmuch as the syndrome is genetically conferred, respiratory symptoms are present from birth. Individuals with PCD develop chronic sinusitis and bronchiectasis and may experience frequent episodes of recurrent otitis media and pneumonia. Because mucociliary clearance is ineffective, patients may present with a chronic cough, a collateral mechanism for clearance of secretions. Half of patients with PCD exhibit malrotation of organs (situs inversus), specifically the heart (dextrocardia). Individuals with polysplenia and retinitis pigmentosa also have been described with respiratory symptoms and ultrastructural level ciliary abnormalties consistent with PCD. There is an emerging understanding of effective management of PCD which includes early identification in pediatric populations. However, the natural history of early childhood infections in children makes evaluation of the very young child problematic. For this reason, children under three years of age are not good candidates for evaluation unless there are specific indicators such as situs inversus, chronic cough, radiographic findings of sinusitis and bronchiectasis, and/or a family history of PCD.
Acquisition of Epithelium for Examination
The lining of the conducting airways to the lungs is a superficial layer of cells comprising only a small percentage of the total airway. For this reason it is important to optimize the size and quality of the specimen for examination. Because the same specimen is used for light microscopic screening as well as electron microscopic examination, it is important to obtain a sample comprised primarily of ciliated epithelial cells. This may be accomplished conveniently and non-invasively by sampling the nasal epithelium using a cytological brush, a sterile stainless-steel ear currette, or a commercially available disposable plastic curette designed for this specific purpose. In our experience, the curettage procedure is preferable to cytologic brushing inasmuch as the anatomic organization and orientation of the cells is retained with less fragmentation. Respiratory epithelium may be easily obtained from the inferior surface of the inferior nasal turbinate by gentle brushing or curettage under direct vision with an illuminating otoscope. This brief procedure (10-15 seconds) causes a minimum of discomfort for patients and since local anesthetics may interfere with ciliary kinetics, they should not be used when obtaining the sample. Patients may exhibit transient rhinorrhea, a tickling sensation, minor acute focal pain in the naris, and watery eyes. Since the procedure acquires only the most superficial layer of cells, frank epistaxis is a rarity although a small amount of blood may be noted in secretions for a short period of time following the procedure. Samples of both nasal and lower airway epithelium also may be obtained for evaluation at times when flexible bronchoscopic procedures are being performed. Pinch biopsies and nasal polyps obtained during surgical procedures generally do not contain a large complement of epithelial cells relative to the size of the biopsy, therefore use of such tissues for evaluation is discouraged.
Light Microscopic Screening of Airway Epithelium
Immediately upon retrieval of the specimen, it may be transferred to 10-15 ml of any standard tissue culture medium for transport to the laboratory. There, the small fragments of tissue can be easily retrieved from the specimen container using a GLASS pipette and transferred to a microscope slide for viewing. Ciliary activity can be easily detected at relatively low magnifications however careful examination of ciliary kinetics and beat frequency generally require examination with a 100x oil immersion objective. The best imaging of the epithelium is obtained using either phase contrast optics or Nomarski Differential Interference Contrast (DIC) optics.
Viable epithelial cells exhibit a gray to faint straw yellow color. The epithelial cells may be organized into clumps having a “pillowy” appearance. Even among healthy individuals, some totally non-motile ciliated cells often will be evident in the sample. These are dead cells and may be identified by their granular, “contrasty” cytoplasm.
Among many patients with PCD, light microscopic examination of ciliary motility often will be overtly abnormal. Ciliary beat frequency is significantly reduced and individual cilia will appear out of synchrony with the neighboring cilia even to the point of exhibiting an opposing beat relative to one another. Also, cilia often will appear to be motile but in a stiffened “rigor” pattern.
See a video clip of characteristic ciliary dyskinesia in nasal respiratory epithelium of a patient with pcd.
Although abnormal ciliary beat frequency and kinetics may be obvious in respiratory epithelium of the patient with PCD, ciliary beat among some patients may appear avid and within normal limits. Evaluation of such specimens requires additional careful inspection of beat kinetics. In some of these cases, it is possible to discern a “jump rope” pattern of motility in which the two ends of the axoneme appear stationary thus imposing a twirling motility to the shaft. Still, some specimens from patients with a clinical presentation consistent in every way with PCD will exhibit a pattern of motility which appears normal even to the experienced eye. For this reason, light microscopic evaluation should be considered only as a screening procedure which may support the subsequent findings of ultrastructural studies. Because there are presently no molecular genetic reagents for probing, the standard for diagnosis in PCD is the identification of characteristic ultrastructural lesions of the ciliary axonemes of affected patients presenting with a characteristic pattern of clinical symptoms.
Ultrastructural Analyses of Cilia Lining the Conducting Airways in PCD
Specimens examined by light microscopy are suitable for subsequent fixation and examination by transmission electron microscopy to identify ultrastructural features consistent with PCD. Based on published reports, the characteristic ultrastructural features conferring a diagnosis of PCD are:
- Dysmorphology of either or both dynein arms.
- Absence of radial spokes.
- Transposition of an outer peripheral microtubular pair to the center of the axoneme.
- Dysorientation of the central microtubular pairs of adjacent cilia relative to one another.
Among patients with PCD, abnormalities of the dynein arms are the most prevalent of the ultrastructural lesions seen in PCD (See panels B & C below).
A. Cross-section of airway cilium in a healthy subject.
B. Cross-section of an airway cilium in a patient with PCD illustrating absence of both inner and outer axonemal dynein arms.
C. Cross-section of an airway cilium in a patient with PCD illustrating absence of only inner axonemal dynein arms.
Dysorientation of central microtubular pairs (Panel D below) also may be observed in many individuals although this may be a feature of acute respiratory epithelial injury also. In this micrograph of cilia in a nasal biopsy from a patient with PCD (note the absence of both dynein arms), the straight lines with arrows adjacent to two neighboring cilia depict the direction of ciliary beat of those two cilia (A parallel line would pass between the central microtubules.) Note that the direction of beat of these cilia is opposing and not synchronous and that similar orientations occur among other cilia in the same field.