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Abstract

Rationale: In North America, primary ciliary dyskinesia (PCD) is currently diagnosed through molecular genetic studies by identification of biallelic pathogenic mutations and/or demonstration of cilia ultrastructure defects by electron microscopy (EM). However, the equipment and expertise for EM testing is restricted to a few specialized centres across the country. These tests are also costly, take a prolonged amount of time to produce results, and miss approximately 30% of PCD cases. Objective: To determine the usability of a panel of 5 fluorescently labelled antibodies as a novel diagnostic tool for PCD, assess inter-rater reliability and ability of immunofluorescence (IF) to validate genetically inconclusive cases or variants of unknown significance (VUS). Methods: Immunofluorescence technique was used to label 24 nasal brushings collected from a discovery cohort of patients with confirmed PCD. Samples were incubated with antibodies to DNAH5, DNAH11, RSPH9, SPEF2, and GAS8. Specificity and sensitivity were calculated and compared against EM and genetic testing. All images were analyzed by three observers to validate the results and determine agreement amongst the raters. Once unblinded, the VUSs were re-evaluated and reclassified based on IF results, as well as supporting clinical and EM data. Results: This immunofluorescence antibody panel was able to accurately distinguish between healthy and PCD samples, detecting PCD cases with a 100% sensitivity and healthy controls with 100% specificity. The three raters agreed on a classification for 115 of 120 images, thus an excellent inter-rater reliability was observed (𝝹=0.85). Immunofluorescence results were available within 3 days and the cost was approximately $230 CAD per sample. Immunofluorescence was also able to provide functional validation for 26 of 34 VUSs, thus aiding in a more accurate diagnosis when genetic testing was inconclusive. Conclusions: This immunofluorescence antibody panel is a quick and sensitive test which holds promise to improve diagnostic turnaround time and cost effectiveness of PCD diagnosis in North America. The high agreement amongst raters highlights the feasibility of implementing and interpreting this test in a clinical setting. With improved diagnostic speed and accuracy comes earlier detection of PCD, and thus, improved patient outcomes and quality of life.