Not applicable

All methods described here were approved by the Human Research Ethics Committee Institutional Review Board of Kanto Central Hospital.
NOTE: We used a commercially available portable thermal camera and dedicated software (see the Table of Materials), and have constructed our protocols based on the use of these specific instruments.
1. Preparation for Measurements
<ol>
	<li>Charge the thermal camera before use.</li>
	<li>Turn on the camera.</li>
	<li>Push the center button on the camera to display the menu. Select Color from the menu and select Iron or Rainbow color.</li>
	<li>Push the center button on the camera to display the menu. Select Measurement and then Center spot measurement to measure the BST in real time.</li>
</ol>
2. Patient Selection
<ol>
	<li>Test all patients with suspected WS. Patients with dizziness, vertigo, ptosis, hoarseness, dysphagia, anisocoria, or dissociated sensory disturbance may have WS.</li>
	<li>Subjectively determine the laterality of BST by palpating the patient with suspected WS.</li>
</ol>
3. Acquisition of Thermographic Images
<ol>
	<li>Instruct the patient to take off their socks and shoes. Have the patient remove clothing (if consent is granted) to enable examination of the abdominal region.</li>
	<li>Acquire images in an examination room or in the patient&#39;s bedroom, as soon as WS is suspected. Ask the patient to assume a supine posture or sitting position during image acquisition. Ask the photographer to stand 50-100 cm from the patient. 
	NOTE: Lighting conditions in the room do not influence the results of the thermographic measurements.</li>
	<li>Acquire one image at each of four areas for each patient: 1) frontal face, 2) bilateral palm, 3) abdomen, and 4) bilateral dorsum of the foot (Figure 1). These four areas are uncomplicated locations for image acquisition, even in bedridden patients who cannot move because of dizziness, vertigo, nausea and vomiting. 
	NOTE: Blankets or drafts caused by air conditioning may influence the BST. If such influence is suspected, cover all extremities and the trunk with blankets, then acquire additional images after an interval of &#62;10 min. Drip infusion therapy at the extremities also may influence the BST. If possible, slow down or stop the infusion, then acquire images after an interval of &#62;10 min.</li>
</ol>
4. Assessing the Laterality of BST
<ol>
	<li>Consider BST to exhibit laterality when the BST between the right and left sides is markedly different macroscopically and the degree of laterality is &#62;0.5 &#176;C18. If the laterality of BST is not obvious or appears to be &#8804;0.5 &#176;C, perform advanced analysis with dedicated software as described below. 
	NOTE: When laterality of BST is observed in multiple locations on the same side of the patient, there is a strong possibility that the patient has WS in the ipsilateral side of the medulla on the warmer side, as determined by BST.</li>
</ol>
5. Advanced Assessment of the Laterality of BST
<ol>
	<li>Initialize the thermal imaging and analysis software.</li>
	<li>Select and open the acquired image to be analyzed.</li>
	<li>Select the Ellipse measurement button on the left side tab, and indicate ellipses with diameters of &#62;5 mm at four locations: 1) the nasolabial fold on the face; 2) the palm of the hand; 3) thoracic spine levels 8-10, approximately 5 cm from the umbilicus of the torso; and 4) the center of the dorsum of the foot.</li>
	<li>Check the average BST from each location and compare it to the BST on the contralateral side of the body.</li>
</ol>
6. Confirming WS
<ol>
	<li>Perform a careful neurological examination if the patient exhibits laterality of BST to confirm a diagnosis of WS. In addition to screening by neurological examination, check for the presence of Horner&#39;s syndrome including ptosis and constricted pupil, as well as the presence of dissociated sensory disturbance, to determine whether the patient exhibits acute WS.</li>
	<li>Consider hospital admission when WS cannot be ruled out based on the results of thermographic measurement and neurological examination.</li>
	<li>Consider repeated thin-slice brainstem MRI after an interval of a few days to diagnose WS.</li>
</ol>

<ol>
	<li>Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pure+lateral+medullary+infarction:+Clinical-radiological+correlation+of+130+acute,+consecutive+patients.">Pure lateral medullary infarction: Clinical-radiological correlation of 130 acute, consecutive patients.</a> Brain. 126 (8), 1864-1872 (2003).</li><li>Kameda, W., 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=Lateral+and+medial+medullary+infarction:+a+comparative+analysis+of+214+patients.">Lateral and medial medullary infarction: a comparative analysis of 214 patients.</a> Stroke. 35 (3), 694-699 (2004).</li><li>Nowak, D. A., Topka, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+variability+of+Wallenberg's+syndrome:+the+anatomical+correlate+of+ipsilateral+axial+lateropulsion.">The clinical variability of Wallenberg's syndrome: the anatomical correlate of ipsilateral axial lateropulsion.</a> Journal of Neurology. 253 (4), 507-511 (2006).</li><li>Ogawa, K., Suzuki, Y., Oishi, M., Kamei, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+study+of+46+patients+with+lateral+medullary+infarction.">Clinical study of 46 patients with lateral medullary infarction.</a> Journal of Stroke and Cerebrovascular Diseases. 24 (5), 1065-1074 (2015).</li><li>Parathan, K. K., Kannan, R., Chitrambalam, P., Aiyappan, S. K., Deepthi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rare+variant+of+Wallenberg's+syndrome:+Opalski+syndrome.">A rare variant of Wallenberg's syndrome: Opalski syndrome.</a> Journal of Clinical &amp; Diagnostic Research. 8 (7), 8-9 (2014).</li><li>Korpelainen, J. T., Sotaniemi, K. A., Myllyl&#228;, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetrical+skin+temperature+in+ischemic+stroke.">Asymmetrical skin temperature in ischemic stroke.</a> Stroke. 26 (9), 1543-1547 (1995).</li><li>Takahashi, 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=Utility+of+thermographic+measurements+of+laterality+of+body+surface+temperature+to+prevent+misdiagnosis+of+acute+Wallenberg's+syndrome.">Utility of thermographic measurements of laterality of body surface temperature to prevent misdiagnosis of acute Wallenberg's syndrome.</a> Brain and Behavior. 8, (2018).</li><li>Kim, J. S., Lee, J. H., Lee, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+sensory+dysfunction+in+lateral+medullary+infarction.+Clinical-MRI+correlation.">Patterns of sensory dysfunction in lateral medullary infarction. Clinical-MRI correlation.</a> Neurology. 49 (6), 1557-1563 (1997).</li><li>Glass, T. A., 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=Outcome+at+30+days+in+the+New+England+Medical+Center+Posterior+Circulation+Registry.">Outcome at 30 days in the New England Medical Center Posterior Circulation Registry.</a> Archives of Neurology. 59 (3), 369-376 (2002).</li><li>Akhtar, N., 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=Ischaemic+posterior+circulation+stroke+in+State+of+Qatar.">Ischaemic posterior circulation stroke in State of Qatar.</a> European Journal of Neurology. 16 (9), 1004-1009 (2009).</li><li>Fitzek, S., 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=Time+course+of+lesion+development+in+patients+with+acute+brain+stem+infarction+and+correlation+with+NIHSS+score.">Time course of lesion development in patients with acute brain stem infarction and correlation with NIHSS score.</a> European Journal of Radiology. 39 (3), 180-185 (2001).</li><li>Choi, H. S., Park, S. C., Lee, Y. J., Kang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+medullary+infarction+presenting+with+vertigo+without+other+neurological+signs.">Lateral medullary infarction presenting with vertigo without other neurological signs.</a> Otolaryngology-Head and Neck Surgery. 147 (6), 1162-1163 (2012).</li><li>Oppenheim, C., 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=False-negative+diffusion-weighted+MR+findings+in+acute+ischemic+stroke.">False-negative diffusion-weighted MR findings in acute ischemic stroke.</a> American Journal of Neuroradiology. 21 (8), 1434-1440 (2000).</li><li>Tsuyusaki, Y., 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=&#34;Invisible&#34;+brain+stem+infarction+at+the+first+day.">&#34;Invisible&#34; brain stem infarction at the first day.</a> Journal of Stroke and Cerebrovascular Diseases. 23 (7), 1903-1907 (2014).</li><li>Tiu, C., 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=Vertebral+artery+dissection:+a+contemporary+perspective.">Vertebral artery dissection: a contemporary perspective.</a> Maedica. 11 (2), 144-149 (2016).</li><li>Low, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clinical Autonomic Disorders: Evaluation and Management. , (1997).</li><li>Takahashi, 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=Teaching+NeuroImages:+the+half-split+man.">Teaching NeuroImages: the half-split man.</a> Neurology. 87 (11), (2016).</li><li>Vardasco, R., Ring, F., Plassmann, P., Jones, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+symmetry+of+the+upper+and+lower+extremities+in+healthy+subjects.">Thermal symmetry of the upper and lower extremities in healthy subjects.</a> Thermology International. 22 (2), 53-60 (2012).</li><li>Nakazato, Y., Shimazu, K., Tamura, N., Hamaguchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+skin+surface+temperature+in+patients+with+unilateral+cerebral+infarction--with+special+reference+to+central+autonomic+regulation+of+skin+vasomotor+response.">A study of skin surface temperature in patients with unilateral cerebral infarction--with special reference to central autonomic regulation of skin vasomotor response.</a> Clinical Neurology. 35 (7), 758-763 (1995).</li><li>Motobe, K., 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=Cut-off+value+of+the+ankle-brachial+pressure+index+at+which+the+accuracy+of+brachial-ankle+pulse+wave+velocity+measurement+is+diminished.">Cut-off value of the ankle-brachial pressure index at which the accuracy of brachial-ankle pulse wave velocity measurement is diminished.</a> Circulation Journal. 69 (1), 55-60 (2005).</li><li>International Academy of Clinical Thermography. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Thermography Guidelines: Standards and protocols in clinical thermographic imaging. , (2002).</li></ol>

This work was supported in part by Ministry of Health, Labour and Welfare, Japan (UMIN000009958).

Critical steps of this protocol are the establishment the initial suspicion of WS and the decision to acquire thermographic images of the patient. Acquiring thermographic images and assessing the laterality of BST is a simple approach, even for non-neurologists who may examine patients upon initial presentation to the emergency department or a general clinic. If a patient exhibits laterality of BST, especially at multiple locations on the same side, the physician should consider the possibility of WS. Because most WS pat...

Wallenberg&#39;s syndrome (WS) is a type of brainstem infarction. Acute WS patients are sometimes initially misdiagnosed with non-stroke diseases because of the symptomatic and magnetic resonance imaging (MRI) characteristics of WS. To accurately diagnose acute WS, careful neurological examination is necessary, which may be difficult for non-neurologists to whom affected patients initially present. Here, we present a simple, rapid, noninvasive, and cost-effective method for the detection of acute WS using portable thermography.
WS is caused by the infarction of a wedge of the dorsal lateral medulla oblongata, due to occlusion of the vertebral artery or posterior inferior cerebellar artery1,2. WS may be misdiagnosed as a non-stroke disease because of a combination of unique symptomatic and MRI characteristics that contrast with those typically observed in cerebral infarction. Hemiparesis and tactile sensory disturbance, which tend to be observed in patients with other types of cerebral infarction, are rare in WS patients; however, they exhibit various combinations of clinical symptoms, including hoarseness and dysphagia, dissociated sensory disturbance, vertigo, gaze-induced nystagmus, ataxia, and Horner&#39;s syndrome1,2,3,4,5,6,7,. Another unique characteristic of WS patients is the limited severity of symptoms, which is similar to that in other types of brainstem infarctions7,8,9,10,11. Some patients with brainstem infarctions have arrived at the outpatient clinic on foot and reported only minor complaints7. In some patients with WS, vertigo is the only presenting symptom, and it can therefore be difficult to differentiate between WS and auditory vertigo12. Furthermore, WS can affect young patients, due to its potential etiology of artery dissection2. MRI analysis of brainstem infarction, including WS, is unique in that the high-intensity diffusion-weighted imaging signal may be delayed in some patients7,13,14.
The above characteristics are thought to cause misdiagnosis of WS. Dysphagia may cause aspiration pneumonia or asphyxia, and artery dissection may cause subarachnoid hemorrhage15; therefore, overlooking WS may result in the development of life-threatening conditions for the patient. Although careful neurological examination is necessary to prevent the misdiagnosis of WS, it is likely that a patient will first present to a non-neurologist. Therefore, a rapid and simple method for screening of acute WS may be clinically useful.
Previously, we reported that 89% of acute WS patients exhibit laterality of BST, which is presumed to result from disturbance of the central autonomic nervous tract due to infarction at the lateral medulla7. Because this autonomic nervous tract descends from the lateral brainstem (including the ventro-lateral medulla) and contains the connective pathway of sweating and skin blood flow16, disturbance of sweating and vasoconstriction lead to increased BST on the ipsilateral side of WS. In the prior report, we also showed that the laterality of BST can be easily detected within 2 min using thermographic measurement in most patients with WS7,17. Here, we report a method for the detection of laterality of BST using thermography, which may be useful in preventing misdiagnosis of acute WS.

<table><tbody><tr><td>FLIR E5</td><td>FLIR Systems</td><td>P/N: 63905-0501</td><td></td></tr><tr><td>FLIR Tools</td><td>FLIR Systems</td><td></td><td>RRID:SCR_016330</td></tr></tbody></table>

portable thermographic screening for detection of acute wallenberg's syndrome

Wallenberg&#39;s syndrome (WS) is a type of brainstem infarction. WS patients often show Horner&#39;s syndrome, dissociated sensory disturbance, truncal ataxia, and hoarseness. However, they rarely show tactile sensory disturbance and paralysis of the extremities. Additionally, acute brainstem infarction is often not apparent in magnetic resonance images. These symptomatic and imaging characteristics sometimes lead to misdiagnosis of WS as a non-stroke disease, including auditory vertigo. Although careful neurological examination is necessary to prevent misdiagnosis of WS, this type of examination may be difficult for non-neurologists to whom affected patients initially present. Lateral differences in body surface temperature (BST) constitute a recognized and widespread symptom of WS. We previously reported that most acute WS patients exhibit lateral differences in BST at multiple locations and that these lateral differences in BST could easily be detected by thermographic measurement. Here, we present the method for use of portable thermography to detect acute WS, using a simple, rapid, noninvasive, and cost-effective approach. To assess lateral differences in BST among patients with suspected WS, BST was measured as soon as possible in the examination room or in the patient&#39;s bedroom. Measurements were performed bilaterally at four locations where images could easily be acquired (face, palm of the hand, abdomen, and dorsum of the foot) using a portable thermal camera. When lateral differences in BST are observed macroscopically, especially in multiple locations on the same side, a diagnosis of WS should be suspected. Macroscopic assessment of BST laterality can be made within 2 min of the acquisition of thermographic images. This method may be useful in preventing misdiagnosis of acute WS as a non-stroke disease, especially when such patients initially present to non-neurologists.

Acquisition of thermographic images and macroscopic assessment to determine whether BST exhibits laterality may be performed within 2 min in most patients. Most acute WS patients exhibit laterality of BST at multiple locations. Some patients exhibit laterality of BST throughout the body (Figure 2A), whereas some exhibit laterality only in a few locations (Figure 2B). The warmer side, as determine...

Watch this Scientific Journal Video about Portable Thermographic Screening for Detection of Acute Wallenberg's Syndrome at JoVE.com

Portable Thermographic Screening for Detection of Acute Wallenberg's Syndrome

Acute Wallenberg&#39;s syndrome can be misdiagnosed as a non-stroke disease, such as auditory vertigo. Thus, careful neurological examination, which is sometimes difficult for non-neurologists, is necessary for precise diagnosis. Here, we present a simple, rapid, noninvasive, and cost-effective method for detection of acute Wallenberg&#39;s syndrome using portable thermography.

Wallenberg&#39;s syndrome (WS) is a type of brainstem infarction. WS patients often show Horner&#39;s syndrome, dissociated sensory disturbance, truncal ...

portable-thermographic-screening-for-detection-acute-wallenberg-s

Research

JoVE Journal

Neuroscience

6.5K Views.  Kanto Central Hospital. Acute Wallenberg's syndrome can be misdiagnosed as a non-stroke disease, such as auditory vertigo. Thus, careful neurological examination, which is sometimes difficult for non-neurologists, is necessary for precise diagnosis. Here, we present a simple, rapid, noninvasive, and cost-effective method for detection of acute Wallenberg's syndrome using portable thermography.

Video: Portable Thermographic Screening for Detection of Acute Wallenberg's Syndrome

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

Medicine

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Immunology and Infection

A Research Method For Detecting Transient Myocardial Ischemia In Patients With Suspected Acute Coronary Syndrome Using Continuous ST-segment Analysis

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture of an atherosclerotic plaque; hence, treatment is aimed at plaque stabilization in order to prevent cellular death. However, there is considerable debate among clinicians, about which treatment pathway is best: early invasive using percutaneous coronary intervention (PCI/stent) when indicated or a conservative approach (i.e., medication only with PCI/stent if recurrent symptoms occur).
There are three types of ACS: ST elevation myocardial infarction (STEMI), non-ST elevation MI (NSTEMI), and unstable angina (UA). Among the three types, NSTEMI/UA is nearly four times as common as STEMI. Treatment decisions for NSTEMI/UA are based largely on symptoms and resting or exercise electrocardiograms (ECG). However, because of the dynamic and unpredictable nature of the atherosclerotic plaque, these methods often under detect myocardial ischemia because symptoms are unreliable, and/or continuous ECG monitoring was not utilized.
Continuous 12-lead ECG monitoring, which is both inexpensive and non-invasive, can identify transient episodes of myocardial ischemia, a precursor to MI, even when asymptomatic. However, continuous 12-lead ECG monitoring is not usual hospital practice; rather, only two leads are typically monitored. Information obtained with 12-lead ECG monitoring might provide useful information for deciding the best ACS treatment.
Purpose. Therefore, using 12-lead ECG monitoring, the COMPARE Study (electroCardiographic evaluatiOn of ischeMia comParing invAsive to phaRmacological trEatment) was designed to assess the frequency and clinical consequences of transient myocardial ischemia, in patients with NSTEMI/UA treated with either early invasive PCI/stent or those managed conservatively (medications or PCI/stent following recurrent symptoms). The purpose of this manuscript is to describe the methodology used in the COMPARE Study.
Method. Permission to proceed with this study was obtained from the Institutional Review Board of the hospital and the university. Research nurses identify hospitalized patients from the emergency department and telemetry unit with suspected ACS. Once consented, a 12-lead ECG Holter monitor is applied, and remains in place during the patient's entire hospital stay. Patients are also maintained on the routine bedside ECG monitoring system per hospital protocol. Off-line ECG analysis is done using sophisticated software and careful human oversight.

Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.

Each year, an estimated 785,000 Americans will have a new coronary attack, or acute coronary syndrome (ACS). The pathophysiology of ACS involves rupture ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood flow that results in a net influx of blood to key organs and an increase in core temperature. This stress-induced hyperthermia is used to assess stress. However, measuring core temperature is invasive. As blood flow is redirected to the core, the periphery of the body can cool. This paper describes a protocol where peripheral body temperature is measured non-invasively in wild blue tits (Cyanistes caeruleus) using infrared thermography. In the field we created a set-up bringing the birds to an ideal position in front of the camera by using a baited box. The camera takes a short thermal video recording of the undisturbed bird before applying a mild stressor (closing the box and therefore capturing the bird), and the bird&#8217;s response to being trapped is recorded. The bare skin of the eye-region is the warmest area in the image. This allows an automated extraction of the maximum eye-region temperature from each image frame, followed by further steps of manual data filtering removing the most common sources of errors (motion blur, blinking). This protocol provides a time series of eye-region temperature with a fine temporal resolution that allows us to study the dynamics of the stress response non-invasively. Further work needs to demonstrate the usefulness of the method to assess stress, for instance to investigate whether eye-region temperature response is proportional to the strength of the stressor. If this can be confirmed, it will provide a valuable alternative method of stress assessment in animals and will be useful to a wide range of researchers from ecologists, conservation biologists, physiologists to animal welfare researchers.

There is a need for a non-invasive assessment of stress. This paper describes a simple protocol using thermal imaging to detect a significant response in eye-region temperature in wild blue tits to a mild acute stressor.

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

Infrared Thermography for the Detection of Changes in Brown Adipose Tissue Activity

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose (FDG) after a meal or in obese or diabetic patients fails as the method of choice. The main reason is that 18F-FDG competes with the postprandial high glucose plasma concentration for the same glucose transporter on the membrane of BAT cells. In addition, BAT uses fatty acids as a source of energy as well, which is not visible with PET-CT and could be changed along with glucose concentration in obese and diabetic patients. Therefore, to estimate the physiological importance of BAT in animals and humans, a new infrared thermography method used in recent publications is applied.
After overnight fasting, BAT activity was measured by infrared thermography before and after a meal in human volunteers and&#160;female wild-type mice. The camera software calculates the object&#39;s temperature using distance from the object, skin emissivity, reflected room temperature, air temperature, and relative humidity. In mice, the shaved area above the BAT was a region of interest for which average and maximal temperatures were measured. The phase of the estrous cycle in female mice was determined after an experiment by vaginal smears stained with cresyl violet (0.1%) stain solution. In healthy volunteers, two skin areas of the neck were selected: the supraclavicular area (above the collarbone, where BAT cells are present) and the interclavicular area (between the collarbones, where there is no BAT tissue detected). BAT activity is determined by the subtraction of those two values. Also, the average and maximal temperatures of skin areas could be determined in animals and human participants.
Changes in BAT activity after a meal measured by infrared thermography, a non-invasive and more sensitive method, were shown to be sex, age, and phase of the estrous cycle dependent in laboratory animals. As part of diet-induced thermogenesis, BAT activation in humans was also proven to be sex, age, and body mass index dependent. Further determining the pathophysiological changes in BAT activity after a meal will be of great importance for participants with high glucose plasma concentrations (obesity and diabetes mellitus type 2), as well as in different laboratory animals (knock-out mice). This method is also a variable tool for determining possible activating drugs that could rejuvenate BAT activity.

Here, we present a protocol for measuring brown adipose tissue activity after a meal in humans and laboratory animals.

Measuring brown adipose tissue (BAT) activity by positron emission tomography computed tomography (PET-CT) via the accumulation of 18F-fluorodeoxyglucose ...

Image Acquisition using Portable Sonography for Emergency Airway Management

With its increasing popularity and accessibility, portable ultrasonography has been rapidly adapted not only to improve the perioperative care of patients, but also to address the potential benefits of employing ultrasound in airway management. The benefits of point of care ultrasound (POCUS) include its portability, the speed at which it can be utilized, and its lack of invasiveness or exposure of the patient to radiation of other imaging modalities.
Two primary indications for airway POCUS include confirmation of endotracheal intubation and identification of the cricothyroid membrane in the event a surgical airway is required. In this article, the technique of using ultrasound to confirm endotracheal intubation and the relevant anatomy is described, along with the associated ultrasonographic images. In addition, identification of the anatomy of the cricothyroid membrane and the ultrasonographic acquisition of appropriate images to perform this procedure are reviewed.
Future advances include utilizing airway POCUS to identify patient characteristics that might indicate difficult airway management. Traditional bedside clinical exams have, at best, fair predictive values. The addition of ultrasonographic airway assessment has the potential to improve this predictive accuracy. This article describes the use of POCUS for airway management, and initial evidence suggests that this has improved the diagnostic accuracy of predicting a difficult airway. Given that one of the limitations of airway POCUS is that it requires a skilled sonographer, and image analysis can be operator dependent, this paper will provide recommendations to standardize the technical aspects of airway ultrasonography and promote further research utilizing sonography in airway management. The goal of this protocol is to educate researchers and medical health professionals and to advance the research in the field of airway POCUS.

Point of care ultrasound (POCUS) is increasingly being utilized in airway management. Presented here are some clinical utilities of POCUS, including differentiating endotracheal and esophageal intubation, identification of the cricothyroid membrane in the event a surgical airway is required, and measuring anterior neck soft tissue to predict difficult airway management.

With its increasing popularity and accessibility, portable ultrasonography has been rapidly adapted not only to improve the perioperative care of ...

Point-Of-Care Ultrasound Screening for Proximal Lower Extremity Deep Venous Thrombosis

Acute lower extremity deep venous thrombosis (DVT) is a serious vascular disorder that requires accurate and early diagnosis to prevent life-threatening sequelae. While whole leg compression ultrasound with color and spectral Doppler is commonly performed in radiology and vascular labs, point-of-care ultrasound (POCUS) is becoming more common in the acute care setting. Providers appropriately trained in focused POCUS can perform a rapid bedside examination with high sensitivity and specificity in critically ill patients. This paper describes a simplified yet validated approach to POCUS by describing a three-zone protocol for lower extremity DVT POCUS image acquisition. The protocol explains the steps in obtaining vascular images at six compression points in the lower extremity. Beginning at the level of the proximal thigh and moving distally to the popliteal space, the protocol guides the user through each of the compression points in a stepwise manner: from the common femoral vein to the femoral and deep femoral vein bifurcation, and, finally, to the popliteal vein. Further, a visual aid is provided that may assist providers during real-time image acquisition. The goal in presenting this protocol is to help make proximal lower extremity DVT exams more accessible and efficient for POCUS users at the patient&#39;s bedside.

Traditionally, lower extremity deep venous thrombosis (DVT) is diagnosed by radiology-performed venous duplex ultrasound. Providers appropriately trained in focused point-of-care ultrasound (POCUS) can perform a rapid bedside examination with high sensitivity and specificity in critically ill patients. We describe the scanning technique for focused POCUS DVT lower extremity examination.

Acute lower extremity deep venous thrombosis (DVT) is a serious vascular disorder that requires accurate and early diagnosis to prevent life-threatening ...