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 M&#252;nster, 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.

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
<ol>
	<li>Brushing
	<ol>
		<li>Before brushing, administer a two-week course of oral amoxicillin-clavulanic acid to the patient to eradicate biofilms interfering with cilia function. Ensure the antibiotic is terminated 2 days before the procedure.</li>
		<li>To diminish an overlay of mucous material on the brushed epithelial cell strips, ask the patient to blow their nose thoroughly. Ask parents to help their small children blow their noses.</li>
		<li>For brushing, use an interdental brush of 0.6 mm size (see the Table of Materials). Keep the head of the patient fixed with one hand, and with the other hand, brush the inferior turbinate of both nostrils to collect sufficient epithelial cell strips. 
		NOTE: A slight resistance is usually experienced when inserting the interdental brush deeply under the inferior turbinate. Nasal brushing is carried out by quick turning and picking for approximately 2 s. Longer and intense brushing might otherwise cause severe epithelial injury and consecutive epistaxis.</li>
		<li>If bronchoscopy is planned for reasons other than suspected PCD, collect samples during bronchoscopy by brushing the carina or bronchial epithelium or by biopsy13.</li>
		<li>Drop the harvested cell strips into a 1.5 mL microcentrifuge tube containing the cell-nourishing Dulbecco&#180;s Modified Eagle Medium (DMEM). 
		NOTE: Epithelial cell strips usually drop off the brush more easily by tossing and turning the brush against the tube wall.</li>
		<li>Close the lid of the microcentrifuge tube and hold the tube against a light source. Shake the tube to discover harvested cell strips and conglomerates.</li>
	</ol>
	</li>
	<li>Transport of the specimen
	<ol>
		<li>Place the microcentrifuge tubes in a tightly closed polystyrene box and ensure that the lid of the tubes is properly closed and the tubes are fixed inside the box.</li>
		<li>Add a cold pack; however, avoid freezing the specimen. 
		NOTE: The optimal temperature for preserving the specimen before investigation is approximately 4-8 &#176;C (39.2-46.4 &#176;F).</li>
		<li>Ensure that the preserved samples are analyzed during the next 24 h. 
		NOTE: In these experiments, the mean time between brushings and HSVMA was 3 h.</li>
	</ol>
	</li>
</ol>
2. High-speed video microscopy analysis (HSVMA)
<ol>
	<li>Video microscopy of the samples
	<ol>
		<li>After receiving the microcentrifuge tubes containing the samples, warm them up to 37 &#176;C (98.6 &#176;F) to mimic an optimal, in vivo-like environment.</li>
		<li>If the microscope is equipped with a heating unit, place the tubes under the hood and warm them up there. Alternatively, use an incubator to warm up the samples.</li>
		<li>Start the camera and the software of the video unit on the PC.</li>
		<li>Using a pipette, take a small amount of the sample and place two drops into a cuvette or glass-bottom dish (see the Table of Materials). Cover the cuvette or dish with a lid and place it under the microscope.</li>
		<li>For evaluation of the samples, use a differential-interference microscope equipped with a cold-light source and a video camera able to record at high speed (at least 200 frames/s). Use an oil immersion lens with a 100x magnification and put a drop of immersion oil onto the surface of the optic. 
		NOTE: Microscopes with a lens approaching from below are recommended.</li>
		<li>Approach the bottom of the dish with the microscope lens and search for cell clusters without red blood cells and with low mucus content. After finding a representative region of interest (ROI), focus on one specific group of beating cilia with the largest cilia movements and record a video sequence. Record cells with beating cilia sidewise and from above. After that, search for another representative cluster of cells and repeat the recording. 
		NOTE: The cilia coated cells must be investigated under a 1,000x magnification, and beating cilia should be recorded with a digital high-speed video (DHSV) camera set on a frame rate of 200/s or more (256/s in this protocol). Because the data obtained from the recorded video clips requires a lot of space on the hard drive, an external SSD hard drive is recommended.</li>
	</ol>
	</li>
	<li>Analysis of video sequences
	<ol>
		<li>To determine CBF and CBP, play the video clips back frame by frame.</li>
		<li>To determine CBF, set the frame rate into slow motion with 15 frames/s and count 10 consecutive beats.</li>
		<li>Record the number of frames passing by during a single cycle of 10 beats and insert the result into Eq (1). Determine the frequency by calculating the mean of all recorded cilia beat cycles and compare the result with the reference values for age (see Table 1)14. 
		<img alt="Equation 1" src="/files/ftp_upload/63292/63292eq01.jpg" /> (1) 
		Where X is the number of frames passing by during a cycle of 10 beats.</li>
		<li>For evaluating CBP, watch if the movement of the beating cilia is in full range (see Figure 1) and synchronized. Have two independent operators evaluate the CBP to prevent selection bias.</li>
		<li>Report the results of the HSVMA analysis to be either compatible, unlikely, or inconclusive with PCD.</li>
	</ol>
	</li>
</ol>

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G., Omran, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+primary+ciliary+dyskinesia.">Diagnosis and management of primary ciliary dyskinesia.</a> Cilia. 4 (1), 2 (2015).</li><li>Mirra, V., Werner, C., Santamaria, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+ciliary+dyskinesia:+An+update+on+clinical+aspects,+genetics,+diagnosis+and+future+treatment+strategies.">Primary ciliary dyskinesia: An update on clinical aspects, genetics, diagnosis and future treatment strategies.</a> Frontiers in Pediatrics. 5, 135 (2017).</li><li>Schultz, R., Elenius, V., Lukkarinen, H., Saarela, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+novel+mutations+in+the+DNAH11+gene+in+primary+ciliary+dyskinesia+(CILD7)+with+considerable+variety+in+the+clinical+and+beating+cilia+phenotype.">Two novel mutations in the DNAH11 gene in primary ciliary dyskinesia (CILD7) with considerable variety in the clinical and beating cilia phenotype.</a> BMC Medical Genetics. 21 (1), 237 (2020).</li><li>Knowles, M. 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R., Pachigolla, R., Deskin, R. W., Hawkins, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+technique+to+diagnose+primary+ciliary+dyskinesia.">Optimal technique to diagnose primary ciliary dyskinesia.</a> The Laryngoscope. 110 (9), 1548-1551 (2000).</li><li>Chilvers, M. A., Rutman, A., O&#180;Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+cilia+and+ciliated+epithelial+ultrastructure+in+healthy+children+and+young+adults.">Functional analysis of cilia and ciliated epithelial ultrastructure in healthy children and young adults.</a> Thorax. 58 (4), 333-338 (2003).</li><li>Lucas, J. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cigarette+smoking+on+nasal+mucociliary+clearance+and+ciliary+beat+frequency.">Effect of cigarette smoking on nasal mucociliary clearance and ciliary beat frequency.</a> Thorax. 41 (7), 519-523 (1986).</li><li>Kobbernagel, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+azithromycin+maintenance+therapy+in+primary+cilia+dyskinesia+(BESTCILIA):+a+multicentre,+double-blind,+randomised,+placebo-controlled+phase+3+trial.">Efficacy and safety of azithromycin maintenance therapy in primary cilia dyskinesia (BESTCILIA): a multicentre, double-blind, randomised, placebo-controlled phase 3 trial.</a> Lancet Respiratory Medicine. 8 (5), 493-505 (2020).</li><li>Takeyama, K., Tamaoki, J., Chiyotani, A., Tagaya, E., Konno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+macrolide+antibiotics+on+ciliary+motility+in+rabbit+airway+epithelium+in-vitro.">Effect of macrolide antibiotics on ciliary motility in rabbit airway epithelium in-vitro.</a> Journal of Pharmacy and Pharmacology. 45 (8), 756-758 (1993).</li><li>Toskala, E., Haataja, J., Shirasaki, H., Rautliainen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+cells+harvested+with+nasal+brushing:+a+method+for+evaluating+ciliary+function.">Culture of cells harvested with nasal brushing: a method for evaluating ciliary function.</a> Rhinology. 43 (2), 121-124 (2005).</li><li>Pifferi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+cell+culture+method+for+the+diagnosis+of+atypical+primary+ciliary+dyskinesia.">Simplified cell culture method for the diagnosis of atypical primary ciliary dyskinesia.</a> Thorax. 64 (12), 1077-1081 (2009).</li><li>Hirst, R. 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The authors have nothing to disclose.

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...

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 late1,3. Therefore, infants with either congenital situs inversus and/or heterotaxy or perinatal rhinorrhea, neonatal respiratory distress, a blocked nose, and feeding difficulties should be suspects for PCD. In later life, chronic otitis, recurrent pneumonia, rhinosinusitis, and a chronic, typical wet cough due to impaired MCC are the hallmark symptoms of PCD, which in combination with bronchiectasis and impaired lung function, continue into adulthood2. 
Patients suspected of having PCD can be diagnosed by using different diagnostic tools. TEM has been considered the gold standard for first-line diagnostics in the past. However, up to 30% of PCD cases do not show abnormal ultrastructure1,3,4,5,6, demanding a different diagnostic approach. Therefore, a growing number of centers and the guidelines of the European Respiratory Society (ERS) suggest a combination of nasal nitric oxide (nNO) and HSVMA as first-line diagnostics1,7,9,10. HSVMA and nNO are also the most cost-effective options in identifying a patient with PCD11. However, even if genetic testing were included in the diagnostics, it must be kept in mind that there is currently no stand-alone test or combination of tests that can exclude PCD with 100% certainty8,9,10. 
Out of the available diagnostic options, HSVMA is the only test that focuses on living, cilia-coated respiratory cells and evaluates ciliary beat pattern (CBP) and ciliary beat frequency (CBF). In contrast to TEM, the results of HSVMA are available quickly, usually on the day of testing, whereas results of TEM might arrive months after the specimen has been taken. HSVMA can be applied for all age groups, whereas nNO demands a high degree of compliance; attempts to use it under the age of 5 years are usually unsuccessful10. In experienced hands, HSVMA has excellent sensitivity and specificity to diagnose PCD at 100% and 96%, respectively12.
This paper describes the step-by-step procedure to perform HSVMA, including the harvesting of cilia-coated respiratory cells from the inferior turbinate of the nose, the preservation of harvested cells in a cell-nourishing medium for transport to the site of investigation, and the process of microscopic video analysis to determine CBF and CBP. Additionally, some video clips from patients are shown, comparing normal CBPs and CBFs with abnormal cilia function (Video 3, Video 4, Video 5, Video 6, Video 7, and Video 8).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amoxiciline-clavulanic acid</td>
			<td>Orion Oyj</td>
			<td></td>
			<td>40 mg/kg divided in 2 doses/day, for adults 875/125 mg 1 tablet x2/day</td>
		</tr>
		<tr>
			<td>Camera Software</td>
			<td>Hamamatsu</td>
			<td></td>
			<td>HCI Image</td>
		</tr>
		<tr>
			<td>Cold pack</td>
			<td>any</td>
			<td></td>
			<td>for preservation and transport</td>
		</tr>
		<tr>
			<td>Differential interference microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Inverted, cell observer microscope</td>
		</tr>
		<tr>
			<td>Digitial High Speed Video Camera</td>
			<td>Hamamatsu</td>
			<td></td>
			<td>Orca Flash 4.0, digital camera type C11440</td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Modified Eagle Medium</td>
			<td>Thermo Fisher</td>
			<td>10565018</td>
			<td>basal cell culture medium</td>
		</tr>
		<tr>
			<td>Eppendorf tube</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td>1.5 mL tube</td>
		</tr>
		<tr>
			<td>Glass-bottom microwell dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td>cuvette for microscopy</td>
		</tr>
		<tr>
			<td>Heating Unit</td>
			<td>Carl Zeiss/PeCon</td>
			<td>810-450001</td>
			<td>Carl Zeiss incubation elements with PeCon TempModule S1 temperature control</td>
		</tr>
		<tr>
			<td>Interdental brush 0.6 mm</td>
			<td>Doft</td>
			<td>872267</td>
			<td>Interdental brush on a long wire with a reusable handle and cap in zipbag</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>100x/1.46, &#945; Plan-Apochromat DIC objective</td>
		</tr>
		<tr>
			<td>Small polystyrene box with lid</td>
			<td>any</td>
			<td></td>
			<td>for transport</td>
		</tr>
	</tbody>
</table>

high-speed video microscopy analysis for first-line diagnosis of primary ciliary dyskinesia

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.

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 &#62; A; p. Glu781Lys)3. These representative videos were chosen because phenotypes of mutations in the DNAH11...

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High-speed Video Microscopy Analysis for First-line Diagnosis of Primary Ciliary Dyskinesia

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 ...

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4.1K Views.  Tampere University Hospital. 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.

Video: High-speed Video Microscopy Analysis for First-line Diagnosis of Primary Ciliary Dyskinesia

FISH for Pre-implantation Genetic Diagnosis

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Video Movement Analysis Using Smartphones (ViMAS): A Pilot Study

The use of smartphones in clinical practice is steadily increasing with the availability of low cost/freely available &#34;apps&#34; that could be used to assess human gait. The primary aim of this manuscript is to test the concurrent validity of kinematic measures recorded by a smartphone application in comparison to a 3D motion capture system in the sagittal plane. The secondary aim was to develop a protocol for clinicians on the set up of the smartphone camera for video movement analysis.
The sagittal plane knee angle was measured during heel strike and toe off events using the smart phone app and a 3D motion-capture system in 32 healthy subjects. Three trials were performed at near (2-m) and far (4-m) smartphone camera distances. The order of the distances was randomized. Regression analysis was performed to estimate the height of the camera based on either the subject&#39;s height or leg length.
Absolute measurement errors were least during toe off (3.12 &#177; 5.44 degrees) compared to heel strike (5.81 &#177; 5.26 degrees). There were significant (p &#60; 0.05) but moderate agreements between the application and 3D motion capture measures of knee angles. There were also no significant (p &#62; 0.05) differences between the absolute measurement errors between the two camera positions. The measurement errors averaged between 3 - 5 degrees during toe off and heel strike events of the gait cycle.
The use of smartphone apps can be a useful tool in the clinic for performing gait or human movement analysis. Further studies are needed to establish the accuracy in measuring movements of the upper extremity and trunk.

Video Movement Analysis Using Smartphones ViMAS: A Pilot Study

This manuscript describes the method to test the concurrent validity of kinematic measures recorded by the smartphone application in comparison to a 3D motion capture system in the sagittal plane. This protocol will enable clinicians to set up smartphones for video capture of human movement.

The use of smartphones in clinical practice is steadily increasing with the availability of low cost/freely available &#34;apps&#34; that could be used to ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Hydra, a Computer-Based Platform for Aiding Clinicians in Cardiovascular Analysis and Diagnosis

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect of different cardiovascular risk factors (e.g., diabetes, raised blood pressure, unhealthy diet, tobacco use, stress, etc.) that commonly coexist and act multiplicatively. Most CVDs can be prevented by an early identification of the highest risk factors and an appropriate treatment. The stratification of cardiovascular risk factors involves a wide range of parameters and tests that specialists use in their clinical practice. In addition to cardiovascular (CV) risk stratification, ambulatory blood pressure monitoring (ABPM) also provides relevant information for diagnostic and treatment purposes. This work presents a list of protocols based on the Hydra platform, a web-based system for clinical decision support which incorporates a set of functionalities and services that are required for complete cardiovascular analysis, risk assessment, early diagnosis, treatment and monitoring of patients over time. The program includes tools for inputting and managing comprehensive patient data, organized into different checkups to track the evolution over time. It also has a risk stratification tool to compute a CV risk factor based upon several risk stratification tables of reference. Additionally, the program includes a tool that incorporates ABPM analysis and allows the extraction of valuable information by monitoring blood pressure over a specific period of time. Finally, the reporting service summarizes the most relevant information in a set of reports that aid clinicians in their clinical decision-making process.

This article presents a protocol based on Hydra &#8212; a web-based system for clinical decision support that integrates a full and detailed set of functionalities and services required by physicians for complete cardiovascular analysis, risk assessment, early diagnosis, treatment, and monitoring over time.

Cardiovascular diseases (CVDs) are the leading cause of death throughout the world. The total risk of developing CVD is determined by the combined effect ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

Measurement of Myocardial Lactate Production for Diagnosis of Coronary Microvascular Spasm

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina attack. Since the pressure-rate product is almost identical at rest and the onset of attack in those patients, the decrease in coronary blood flow rather than increased myocardial oxygen consumption is likely to explain myocardial ischemia, indicating a substantial involvement with coronary microvascular spasm (MVS). Myocardial lactate production, which could be defined as a negative myocardial lactate extraction ratio (ratio of the coronary arterial-venous difference in lactate concentration to arterial concentration), is considered indicative of objective evidence to support the emerging myocardial ischemia. Thus, monitoring of the myocardial lactate production and the emergence of chest pain and ischemic electrocardiographic changes during acetylcholine (ACh) provocation testing is of significant value for detecting the entity of MVS. Practically, 1 min after incremental doses of ACh (20, 50, and 100 &#956;g) are administered into the left coronary artery (LCA), paired samples of 1 mL of blood are collected from the LCA ostium and coronary sinus for measurement of lactate concentration by a calibrated automatic lactate analyzer. Then, the development of MVS could be confirmed by negative myocardial lactate extraction ratio despite the absence of angiographically demonstrable epicardial coronary spasm or before its occurrence throughout ACh provocation testing. In conclusion, assessment of myocardial lactate production is essential and valuable for the diagnosis of MVS.

Myocardial lactate production (coronary arterial-venous difference in serum lactate level) during coronary spasm provocation testing is considered as a highly sensitive marker that reflects acetylcholine-induced myocardial ischemia due to microvascular spasm. This article presents the procedures to assess myocardial lactate production for the diagnosis of coronary microvascular spasm.

In about a quarter of patients with angina and non-obstructive coronary arteries, no epicardial spasm is noted on coronary arteriography during an angina ...

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...

Objectification of Tongue Diagnosis in Traditional Medicine, Data Analysis, and Study Application

Tongue diagnosis is an essential technique of traditional Chinese medicine (TCM) diagnosis, and the need for objectifying tongue images through image processing technology is growing. The present study provides an overview of the progress made in tongue objectification over the past decade and compares segmentation models. Various deep learning models are constructed to verify and compare algorithms using real tongue image sets. The strengths and weaknesses of each model are analyzed. The findings indicate that the U-Net algorithm outperforms other models regarding precision accuracy (PA), recall, and mean intersection over union (MIoU) metrics. However, despite the significant progress in tongue image acquisition and processing, a uniform standard for objectifying tongue diagnosis has yet to be established. To facilitate the widespread application of tongue images captured using mobile devices in tongue diagnosis objectification, further research could address the challenges posed by tongue images captured in complex environments.

The present study employed U-Net and other deep learning algorithms to segment a tongue image and compared the segmentation results to investigate the objectification of tongue diagnosis.

Tongue diagnosis is an essential technique of traditional Chinese medicine (TCM) diagnosis, and the need for objectifying tongue images through image ...