Medicine
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High-speed Video Microscopy Analysis for First-line Diagnosis of Primary Ciliary Dyskinesia
Chapters
Summary January 19th, 2022
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.
Transcript
High speed Video microscopy analysis is a reliable tool for first line diagnostic of primary ciliary dyskinesia. This technique is relative easy to perform, fast, cost effective, and in experienced hands else a very reliable tool. And it is applicable for patients of all age groups.
Video microscopy also reveals secondary ciliary function. Like, for instance, due to bacterial or viral infections. But it has been mainly used to diagnose primary ciliary dyskinesia.
Scientists use this method also to visualize intracellular processes or biological functions in living cells. Assisting me in this procedure will be Mrs. Tina Peromaa, our laboratory technician.
Before collecting the respiratory epithelial cells, ask the patient to blow their nose thoroughly. When the patient is ready, keep the head of the patient fixed with one hand and use an interdental brush of 0.6 millimeter size in the other hand to brush the inferior turbinate of the nostril to collect sufficient epithelial cell strips. Drop the harvested cell strips into a 1.5 milliliter microcentrifuge tube containing the cell nourished DMEM.
After closing the lid of the microcentrifuge tube hold the tube against the light source and shake the tube to discover harvested cell strips and conglomerates. Later, place the microcentrifuge tubes with the lids properly closed in a polystyrene box. Before closing the box tightly ensure the tubes are fixed inside the box.
Then add a cold pack to the box while avoiding freezing the specimen. After removing the micro centrifuge tubes containing the samples from the polystyrene box, place one tube under a hood of the microscope heating unit to warm up to 37 degrees celsius for the high speed video microscopy analysis or HSVMA. Next, start the camera and the software of the video unit on the computer.
Use a pipette to take a small sample and place two drops of the sample into a cuvette. Cover the cuvette with a lid before placing it under the microscope. Use an oil immersion lens with a 100x magnification and put a drop of immersion oil onto the surface of the optic.
While approaching the bottom of the dish with the microscope lens, search for the cell clusters without red blood cells and with low mucus content. After finding a representative region of interest, or ROI, focus on one group of the beating cilia with the largest cilia movements and record a video sequence. Record the cells with beating cilia side wise and from above.
The representative analysis shows normal cilia motions seen side wise and from above. The sample of a primary ciliary dyskinesia, or PCD patient, with a mutation in the DNAH11 gene demonstrated the classic stiff minimally moving pattern of ciliary beat. When viewed from the side and above.
In another PCD patient with the same mutation, a different hyperkinetic but inefficient pattern of the beating cilia was observed. A pathological ciliary beat pattern and ciliary beat frequency were also seen in a patient having recurrent upper airway infections. If the brushing procedure is carried out rigorously HSVMA analysis could not be performed as ciliated epithelial cells were covered with a coating of red blood cells.
The most important step in the procedure is to get sufficient respiratory cells from the brushings and to keep this material well preserved for the video analysis. Remaining material can be used to perform electron microscopy or immunoflourescense microscopy to detect possible structural defects in cilia or to reveal defects and the synthesis of ciliary proteins.
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