Epithelial brushing storage conditions and the reliability of IF staining - implications for primary ciliary dyskinesia diagnostics

Immunofluorescence (IF) microscopy is gaining increased popularity as a pre-genetic diagnostic method in primary ciliary dyskinesia (PCD). Efficient IF-based diagnostics in PCD requires standardization of staining methods and antibodies. Obviously, specificity of the analysis highly depends on the quality of the analyzed material. The nasal epithelium brushing quality depends on the patient health status, but conditions of the slides storage are also very important. We applied automated image analysis to systematically examine the influence of samples storage conditions on the specificity of IF staining with eight polyclonal antibodies routinely used for PCD diagnostics, against epitopes of proteins representing various axoneme structural complexes. Seven various combinations of temperatures and storage times were tested to mimic the procedures of handling epithelial brushing on glass slides: at the clinic, during transport, or after reception at the diagnostic laboratory. Our study revealed that proper slide storage conditions are essential for the reliable PCD diagnosis via IF staining. If microscopic slides are prepared at the diagnostic laboratory, we suggest continuous storage at -80{degrees}C or -20{degrees}C. If the slides are prepared at a collaborating clinic and shipped, we suggest storage at -20{degrees}C or 4{degrees}C. The IF sensitivity to slide storage conditions differs among antibodies targeting various ciliary elements; for example, molecular ruler proteins appear the most sensitive to prolonged slide storage at room temperature. Moreover, to improve the reliability of the IF staining, additional control slides should be used to account for inter-individual differences. Finally, IF results indicative of PCD should be carefully confronted with patients clinical history.