The authors have no acknowledgements.

This study was approved by the local institutional review boards at all participating centers. All subjects in the multicenter trial provided written informed consent to participate in eTRIS at their respective institutions.
1. Image Acquisition
<ol>
	<li>Perform the MR imaging on a 1.5 T or 3 T whole body scanner using specialized phased-array coils for MRV such as a peripheral vascular coil, body matrix coils or run-off coils. Use these coils in conjunction with other body matrix coils or spine coils. If no suitable specialized coils are available, use the body coil instead. 
	NOTE: Use commercially available scanners such as Siemens Symphony, Sonata, etc.
	<ol>
		<li>Screen subject, and review MRI safety questionnaire prior to scan. Have subject change into a gown.</li>
	</ol>
	</li>
	<li>Place an intravenous line in the antecubital vein of subject for the injection of the contrast agent. Follow standard safety procedures for injecting a gadolinium-based contrast agent.</li>
	<li>Place subject in supine, feet first position in the MRI machine and position appropriate coils on regions to be scanned. Secure coils using Velcro straps as needed.
	<ol>
		<li>Fasten subject&#8217;s legs/feet to prevent motion artifacts during the MRI scan.</li>
		<li>Turn on the centering laser and move the table until the laser crossbeams are located just below the subject&#8217;s knees (patella). Accept this position for the iso-center of the scan and move the patient table to the center position of the scanner bore.</li>
	</ol>
	</li>
	<li>Measure the creatinine clearance (CrCL) and determine the dose of the contrast agent to be used for the subject based on body weight. If CrCL &#60;30 ml/min, subject is excluded from the study. For individuals with CrCL &#62;30 ml/min but less than 45 ml/min, 0.01 mmol/kg of contrast agent is used. For individuals with CrCL &#62;45 ml/min but &#60;60 ml/min, 0.02 mmol/kg of gadofosveset is injected. For individuals with normal renal function (creatinine clearance &#62;60 ml/min), a dose of 0.03 mmol/kg (0.12 ml/kg) gadofosveset trisodium is used. An MRI compatible power injector is used to inject the contrast agent.</li>
	<li>Perform bilateral imaging of both legs and lower pelvis in a single examination session lasting approximately 60 min as shown in Table 1 and described in MRI protocol section. 
	NOTE: Anonymize patient data prior to transfer of images to a central core laboratory for analysis.</li>
</ol>
2. MRI Protocol
<ol>
	<li>Execute the imaging protocol from the scanner console by selecting each protocol step from the protocol window and dragging it to the execution list. Once ready, run the sequence by pressing Scan/Execute or equivalent button.</li>
	<li>Acquire 2D gradient echo based sequences using sequence parameters in Table 1, in the three orthogonal axes to image from mid-calf to above the iliac crest and use as localizers/scouts.</li>
	<li>Perform time of flight (TOF) angiography scans in each segment as specified in Table 1 
	NOTE: These scans are also used as localizers to help differentiate arteries from veins as a long circulating contrast agent is used in steady state in this protocol. This view of the arterial tree is quite limited and only serves to help the image analyst differentiate the arteries from the venous vasculature and does not serve as a full-fledged angiogram.</li>
	<li>To avoid acquiring poor quality images at the inferior and superior extent of the imaging volume, acquire three coronal 40 cm segments in the foot to head direction overlapping by 10 cm during the 3D T1-weighted Gradient Echo scans. 
	NOTE: These three segments encompass: a) mid-calf to above knee b) above knee to thigh/lower pelvis, and c) thigh/pelvis to abdomen. Figure 1 shows sample images with the three acquisition locations illustrated.</li>
	<li>To distinguish between acute and chronic venous thrombosis, acquire T1W direct thrombus imaging (DTHI) and 3D gradient echo (GRE) sequences using parameters in Table 1. 
	NOTE: These 3 scans also serve as the pre-contrast scan for the MRV acquisition. The extent of the coverage of these acquisitions is the same as the initial localizers acquired in the three locations illustrated in Figure 1. GRE sequence is also easily implementable on various scanning platforms and across imaging field strengths.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52761/52761fig1.jpg" /> 
Figure 1: Sample images showing acquisition locations for the 3 stations used for imaging. The field of view in the coronal direction and axial directions was 40 cm for each bed position [acquisition: (mid-calf to above knee), (above knees to thigh/pelvis), (thigh/pelvis to abdomen)] with 10 cm overlap between each bed position. (a) axial image; (b) sagittal image and (c) coronal image. Red arrow indicates DVT. <a href="https://www.jove.com/files/ftp_upload/52761/52761fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="6">
	<li>Administer the contrast agent (gadofosveset trisodium) at a dose of 0.03 mmol/kg (0.12 ml/kg) intravenously into the subject at a rate of 2 ml/sec and flush with 20 ml of saline. Let the contrast agent circulate for 5 min to ensure a steady state in the blood pool.</li>
	<li>Acquire MRV images; post contrast 3D gradient echo sequences at 3 locations described previously. See the sequence parameters in Table 1 and Table 2. Use these images to determine the locations and size of the thrombi.</li>
</ol>
<img alt="Table 1" src="/files/ftp_upload/52761/52761table1.jpg" /> 
Table 1: Lower extremity MR venography protocol with the contrast agent, gadofosveset trisodium, including direct thrombus imaging.
<img alt="Table 2" src="/files/ftp_upload/52761/52761table2.jpg" /> 
Table 2: The specific imaging parameter for each sequence acquired in the protocol.
<ol start="8">
	<li>After scan is completed, remove subject from the MRI scanner and take out the intravenous line. Ask the subject to change out of gown and exit the facility.</li>
</ol>
3. Image Analysis
<ol>
	<li>Perform image analysis on a dedicated image analysis workstation running FDA-approved open source image processing software such as OsiriX MD20, by 2 trained image analysts. Ensure the analysts have a minimum of 3 years experience each.</li>
	<li>Temporarily blind all images before transfer to the image analyst workstation for analysis.</li>
	<li>Ensure that a radiologist assesses each MRI scan for the presence and location of thrombi. Provide the radiologist&#8217;s assessment to the image analyst to be used as a guideline throughout the analysis.</li>
	<li>Load all DICOM images from both MRI visits of a subject into the image processing software by selecting &#8220;import&#8221;, and compare the two imaging time-points of the MRV series for each subject to ensure adequate spatial coverage and registration across time points.
	<ol>
		<li>Select the &#8220;3D MPR&#8221; tool in viewer to provide simultaneous browsing of image data in 3 orthogonal views (axial, coronal, and sagittal). 
		NOTE: The image processing software&#8217;s 3D MPR mode enables the viewing of the vessels of interest.</li>
	</ol>
	</li>
	<li>Analyze the following vessels if DVT is present: external iliac, common femoral, superficial femoral, deep femoral, popliteal, anterior tibial, posterior tibial I &#38; II, gastrocnemius I &#38; II, and peroneal I &#38; II veins. All analysis is restricted to regions of the vessels that are present in images from both time-points.</li>
	<li>Assess the quality of the image for each vein with known DVT as indicated by the radiologist&#8217;s assessment on a 0-5 scale, with 0-2 being unanalyzable and 3-5 being analyzable for both the DTHI and MRV sequences. For the breakdown of scoring system see Figure 2.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52761/52761fig2.jpg" /> 
Figure 2: The breakdown of the scoring system used for assessing the quality of images for each vein of interest with known DVT.
<ol start="7">
	<li>For MRV analysis, analyze each vessel individually. After 3D curved multiplanar reconstruction, use contours following the centerline of each vein, traced by the analyst, to generate curved path for each vein and save to file.
	<ol>
		<li>Establish the position of the thrombus in the three dimensional space. From the Curved MPR plane, a 3D Bezier path will be displayed.</li>
		<li>Delineate the centerline of the vein by selecting of the &#8220;Contour Path&#8221; tool in &#8220;Creation Mode&#8221;.</li>
		<li>Place points repeatedly on any of the orthogonal MPR view to straighten the entire vessel of interest.</li>
		<li>Make adjustments when necessary to ensure that the vessel is entirely straightened in &#8220;Editing Mode&#8221;</li>
		<li>When the contour path is accurately delineated at centerline of vessel, save by selecting the &#8220;curved path&#8221; icon to export the file</li>
		<li>Generate 1 mm axial slices perpendicular to the curved path and save as DICOM files (Figure 3). Observe the the curved path, straightened vessel, and corresponding axial images for quantifying DVT from MRV images. 
		NOTE: The images rendered in the 3D Curved MPR viewer must then be exported in DICOM format and added to the database as a new series of images.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/52761/52761fig3.jpg" /> 
Figure 3: Sample DVT shown on MRV sequence. (i, left panel) Curved path (yellow line) illustrates the contour followed by the vein being analyzed. (ii, middle panel) Straightened vessel along centerline of vessel being analyzed (red dotted line) (iii, right panel) shows axial slices perpendicular to vein being analyzed at locations indicated by yellow lines (A, B, C) on longitudinal sections. <a href="https://www.jove.com/files/ftp_upload/52761/52761fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="7">
	<li>
	<ol start="7">
		<li>On these axial DICOM images, manually segment regions of interest encompassing the thrombus (see Figure 4) by using the &#8220;Closed Polygon ROI Tool&#8221;. Save regions of interests to file by selecting the &#8220;Save all ROIs of this Series&#8221; located in the &#8220;ROI&#8221; drop down menu and save ROI metrics by navigating to the the &#8220;Plugins&#8221; menu and select &#8220;ROI Tools&#8221; followed by &#8220;Export ROIs&#8221;. This should be saved in a CSV format.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/52761/52761fig4.jpg" /> 
Figure 4: Manually segmented regions of interest (green) are shown encompassing the thrombus on axial reformatted DICOM images. <a href="https://www.jove.com/files/ftp_upload/52761/52761fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="7">
	<li>
	<ol start="8">
		<li>Calculate thrombus volume by multiplying the area on each analyzed slice by the slice thickness (1 mm) using a custom built Matlab script. Calculate total thrombus volume in each subject by adding the thrombus volumes in each vessel.</li>
	</ol>
	</li>
	<li>For DTHI analysis, identify fresh thrombus as bright regions on pre-contrast T1W 3D gradient echo scans13 within the DVT regions segmented by the MRV.
	<ol>
		<li>On axial pre-contrast images, compute the volume of fresh thrombus by drawing regions of interest (ROI) manually as shown in Figure 5. In Figure 6, sample images indicating DVT measured by DTHI in conjunction with the MRV images are depicted.</li>
		<li>Open the pre-contrast and post-contrast sequences alongside each other. Select &#8220;axial&#8221; view and delineate bright regions along the vessel of interest using the &#8220;closed polygon ROI Tool&#8221;.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" src="/files/ftp_upload/52761/52761fig5.jpg" /> 
Figure 5: Sample DVT shown on DTHI imaging sequence. (a) coronal image, (b) axial image, and (c) axial image showing region of interest (green) traced around fresh thrombus (blue arrows) on pre-contrast images. The DTHI images are acquired prior to the injection of contrast agent and rely on met-hemoglobin content of thrombus to produce bright signal.
<img alt="Figure 6" src="/files/ftp_upload/52761/52761fig6.jpg" /> 
Figure 6: Sample DVT shown on DTHI imaging sequence. The left panels show MRV images with signal voids indicating total DVT (green arrows). The right panel show corresponding DTHI images with bright signal indicating presence of fresh thrombus (blue arrows). <a href="https://www.jove.com/files/ftp_upload/52761/52761fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Assessment of Reproducibility
<ol>
	<li>Evaluate a subset of ten subjects for the reproducibility of the analysis.</li>
	<li>Perform analysis, as described above, by two separate image analysts (primary and secondary readers) to assess inter-observer variability</li>
	<li>Ensure that primary reader also performs a second image analysis three months after the first analysis to assess intra-observer variability of the results.</li>
	<li>Compute intra-class-correlation coefficients (ICC) and perform a Bland-Altman analysis to assess inter and intra-observer reproducibility. Carry out a one sample t-test to look for bias on Bland-Altman analysis. ICC &#62;0.9 and no bias on Bland-Altman analysis are considered acceptable.</li>
</ol>

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Venkatesh Mani has received research grants from Novartis. Nadia Alie, Sarayu Ramachandran, Cecilia Besa, Philip Robson and Bachir Taouli have nothing to disclose. Michele Mercuri and Michael Grosso are employees of Daiichi Sankyo. Greg Piazza has received research grants from Daiichi Sankyo, BMS, EKOS, and the Thrombosis Research Institute. Samuel Goldhaber has received grants from BMS, Daiichi Sankyo, EKOS, NHLBI and the Thrombosis Research Institute. He is a consultant for Ariad, Bayer, Boehringer Ingelheim, Bristol Myers Squibb, Daiichi Sankyo, Genentech, Janssen, Merck, Pfizer and Portola. Zahi Fayad has received research grants from Daiichi Sankyo, Roche, GlaxoSmithKline, Merck, VBL Therapeutics, Novartis, Bristol-Myers Squibb, and Via Pharmaceuticals.

This study demonstrated the feasibility of quantification of deep vein thrombosis on MR venography using gadofosveset trisodium as a contrast agent, with excellent reproducibility of analysis for quantifying thrombus volume in a multicenter setting. To compute total thrombus volume, the primary method utilized the post-contrast MRV scan to measure thrombus volume. The secondary method used was the direct thrombus imaging approach (DTHI), which leverages the presence of met-hemoglobin within a fresh thrombus to produce a ...

Venous thromboembolism (VTE) affects 300,000-600,000 individuals in the United States every year1. Deep vein thrombosis (DVT) is the most common presentation of VTE, and most commonly affects the calf, thigh or pelvic veins. The diagnosis, management, and follow-up of subjects with DVT cannot be based solely on clinical examinations, since the signs and symptoms of this disease are non-specific2,3. While blood tests (such as D-dimer) can help rule out the diagnosis of DVT, imaging is required to establish the presence of DVT4. Compression ultrasound (CUS) is currently the most commonly used imaging test in the diagnosis of suspected acute DVT. CUS is inexpensive and has high sensitivity and specificity to detect acute DVT5. However, CUS cannot reliably assess the deep veins in the pelvis6. Additionally, CUS cannot directly quantify thrombus volume and composition, which are important when distinguishing between acute DVT (a potential source of pulmonary embolism (PE)) and chronic DVT (less likely to embolize) and for evaluation of therapeutic efficacy7.
Unlike computed tomography (CT), magnetic resonance imaging (MRI) does not deliver ionizing radiation, and is therefore suitable for serial examinations to evaluate thrombus evolution or regression. Compared with CUS, MRI can detect pelvic DVT and can more precisely define proximal (popliteal vein and above) and distal leg (below popliteal vein) DVT8, to better assess the risk of PE. MRI can characterize thrombus age and organization, and may help differentiate acute from chronic DVT9-11 (refs updated). Quantification of thrombus volume, an important metric to assess the disease evolution and response to treatment, is feasible with MR imaging. Current magnetic resonance venography protocols are performed after injection of gadolinium (Gd) based contrast agents12. These are small molecular weight molecules that extravasate quickly after injection, and require careful timing to capture the venous enhancement phase needed to correctly visualize the thrombus13,14.
A proof-of-concept study, edoxaban Thrombus Reduction Imaging Study (eTRIS), utilizing an open label design, investigated the efficacy and safety of edoxaban 90 mg once a day for 10 days, followed by edoxaban 60 mg once a day in the treatment of acute, symptomatic DVT (ClinicalTrials.gov Identifier: NCT01662908). eTRIS addresses whether edoxaban monotherapy, without concomitant low molecular weight heparin (LMW heparin) at the time of treatment initiation, is more effective than standard treatment with LMW heparin/warfarin therapy in subjects with DVT, as assessed by the percent (%) change from baseline in thrombus volume/size (measured by MRI) at Day 14-21.
Another goal of eTRIS was to develop and validate a straightforward MR venography (MRV) image acquisition and analysis protocol for the quantification of thrombus volume in DVT. To overcome some challenges faced by current MRV protocols in multicenter settings, we utilized a recently FDA-approved, long-circulating, gadolinium-based blood pool contrast agent (gadofosveset trisodium). Compared to the use of extracellular Gd-based chelates (e.g., Gd-DTPA) for MRV, gadofosveset has a significantly longer circulation time, which allows use of a simpler MR acquisition scheme, without any timing of acquisitions. Gadofosveset trisodium is a blood pool MRI contrast agent that circulates for 2-3 hr after intravenous injection15,16. Its safety profile is similar to those of traditional extravascular extracellular MRI contrast agents17. It allows steady-state imaging of the vasculature over a period of 1 hr. Therefore, no operator dependent timing of image acquisition is required after contrast agent injection. The additional advantage of using this contrast agent is that it is a small molecule (molecular weight 857 Da)18 and can permeate the sides of even a fully occluded thrombus, thereby providing excellent contrast of the DVT from surrounding areas on the MRV and enabling quantitative computation of DVT volumes. Previous studies have established the inter-rater reliability of visualizing veins using the MR Volume Interpolated Breath-hold Examination (VIBE) venography using gadofosveset trisodium19. Here, we use a similar approach in a multicenter clinical trial setting to evaluate deep vein thrombosis and use the volume of DVT measured by MRI as an endpoint. eTRIS provides an ideal platform to evaluate the feasibility and reproducibility of analysis of the MRV imaging approach proposed here, using a long-circulating Gd-based blood pool contrast agent for evaluating DVT volumes. We also evaluate the use of a direct thrombus imaging (DTHI) approach to quantify the extent of fresh DVT prior to the injection of contrast agents.
Two MRI examinations were performed during the course of the study: the first within 36 hr after randomization into the edoxaban monotherapy group or heparin/warfarin group, and the second between 14 to 21 days after randomization. The analyses of all the images were performed by a centralized core laboratory. Volume of fresh thrombus is calculated from a Direct Thrombus Imaging (DTHI) in the legs and lower pelvis before the injection of any contrast agent. The total thrombus volume (fresh and old) is computed from a post contrast magnetic resonance venography (MRV) images of the veins in the legs and lower pelvis.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ablavar (gadofosveset trisodium)</td>
			<td>Lantheus</td>
			<td></td>
			<td>Contrast Agent</td>
		</tr>
		<tr>
			<td>1.5T or 3T Scanners</td>
			<td>GE, Siemens, or Phillips</td>
			<td></td>
			<td>GE(Horizon, Signa, Hdx, 750), Siemens (Symphony, Avanto, Sonata, Trio, Aera) or Philips (Intera, Achieva)</td>
		</tr>
	</tbody>
</table>

a multicenter mri protocol for the evaluation and quantification of deep vein thrombosis

We evaluated a magnetic resonance venography (MRV) approach with gadofosveset to quantify total thrombus volume changes as the principal criterion for treatment efficacy&#160;in a multicenter randomized study comparing edoxaban monotherapy with a heparin/warfarin regimen for acute, symptomatic lower extremities deep vein thrombosis (DVT) treatment. We also used a direct thrombus imaging approach (DTHI, without the use of a contrast agent) to quantify fresh thrombus. We then sought to evaluate the reproducibility of the analysis methodology and applicability of using 3D magnetic resonance venography and direct thrombus imaging for the quantification of DVT in a multicenter trial setting. From 10 randomly selected subjects participating in the edoxaban Thrombus Reduction Imaging Study (eTRIS), total thrombus volume in the entire lower extremity deep venous system was quantified bilaterally. Subjects were imaged using 3D-T1W gradient echo sequences before (direct thrombus imaging, DTHI) and 5 min after injection of 0.03 mmol/kg of gadofosveset trisodium (magnetic resonance venography, MRV). The margins of the DVT on corresponding axial, curved multi-planar reformatted images were manually delineated by two observers to obtain volumetric measurements of the venous thrombi. MRV was used to compute total DVT volume, whereas DTHI was used to compute volume of fresh thrombus. Intra-class correlation (ICC) and Bland Altman analysis were performed to compare inter and intra-observer variability of the analysis. The ICC for inter and intra-observer variability was excellent (0.99 and 0.98, p &#60;0.001, respectively) with no bias on Bland-Altman analysis for MRV images. For DTHI images, the results were slightly lower (ICC = 0.88 and 0.95 respectively, p &#60;0.001), with bias for inter-observer results on Bland-Altman plots. This study showed feasibility of thrombus volume estimation in DVT using MRV with gadofosveset trisodium, with good intra- and inter-observer reproducibility in a multicenter setting.

For the purpose of the reproducibility assessments, the baseline and follow up scans were pooled and analyzed as separate cases. From the 10 randomly selected subjects (2 visits each), there were 59 vessels with DVT identified using the MRV approach and 29 vessels with fresh thrombus identified by the DTHI. In the subset of these 10 randomly selected subjects analyzed for the reproducibility metrics, no vessels with DVT were deemed to be of un-analyzable quality (defined as subjective scoring 0-2 for both MRV and DTHI im...

Watch this Scientific Journal Video about A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis at JoVE.com

A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis

The goal of this study is to use magnetic resonance venography with long-circulating gadolinium-based contrast agent and direct thrombus imaging for quantitative evaluation of DVT volume in a multicenter, clinical trial setting. Inter- and intra-observer variability assessments were conducted, and reproducibility of the protocol was determined.

We evaluated a magnetic resonance venography (MRV) approach with gadofosveset to quantify total thrombus volume changes as the principal criterion for ...

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17.0K Views.  Icahn School of Medicine at Mount Sinai. The goal of this study is to use magnetic resonance venography with long-circulating gadolinium-based contrast agent and direct thrombus imaging for quantitative evaluation of DVT volume in a multicenter, clinical trial setting. Inter- and intra-observer variability assessments were conducted, and reproducibility of the protocol was determined.

Video: A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Quantification of Optic Nerve Cross Sectional Area on MRI: A Novel Protocol using Fiji Software

Optic nerve assessment is an important aspect of glaucoma diagnosis and follow-up. This project describes a protocol for a unified methodology of optic nerve cross-sectional assessment and quantification using 3 T MRI for image acquisition and ImageJ's Fiji software for image processing quantification. Image acquisition was performed using 3 T MRI, with proper instructions for the patient to ensure straight fixation during imaging. A T2-weighted fat suppressed sequence was used. A coronal cut taken 3 mm behind the globe and perpendicular to the optic nerve axis should be uploaded to the software. Using the threshold function, the white matter area of the optic nerve is selected and quantified, thus, eliminating inter-individual measurement bias. We also described the normal limits for the optic nerve cross-sectional area according to age, based on previously published literature. We used the described protocol to assess optic nerve of a suspected glaucoma patient. The optic nerve cross-sectional area was found to be within the normal limits, a finding further confirmed via optical coherence tomography of the optic nerve.

We provided a detailed protocol for a standardized method of optic nerve assessment and quantification using MRI, utilizing a widely available imaging sequence, and open access software for image analysis. Following this standardized protocol would provide meaningful data for comparison between different patients and different studies.

Optic nerve assessment is an important aspect of glaucoma diagnosis and follow-up. This project describes a protocol for a unified methodology of optic ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...

A Real-World High-Intensity Interval Training Protocol for Cardiorespiratory Fitness Improvement

High-Intensity Interval Training (HIIT) has emerged as an interesting time-efficient approach to increase exercise adherence and improve health. However, few studies have tested the efficiency of HIIT protocols in a &#34;real world&#34; setting, e.g., HIIT protocols designed for outdoor spaces without specialized equipment. This study presents a &#34;real world&#34; training protocol, named &#34;beep training&#34;, and compares the efficiency of a HIIT regiment versus a traditional long-duration Moderate-Intensity Continuous Training (MICT) regiment using this beep training protocol on VO2 max of overweight untrained men. Twenty-two subjects performed outdoor running with MICT (n = 11) or HIIT (n = 11). Cardiorespiratory fitness was assessed before and after training protocols using a metabolic analyzer. Both training protocols were performed 3 days a week for 8 weeks using the Beep Test results. The MICT group performed the exercise program at 60%-75% of the maximum speed of the 20 m shuttle test (Vmax) and with a progression of the distance of 3,500-5,000 m. The HIIT group performed the interval exercise with 7-10 bouts of 200 m at 85%-100% of the maximum speed of the 20 m shuttle test (Vmax), interspersed with 1 min of passive recovery. Although the HIIT group presented a significantly lower training volume than the MICT group (p &#60; 0.05) after 8 weeks of beep training, HIIT was superior to MICT in improving VO2 max (MICT: ~4.1%; HIIT: ~7.3%; p &#60; 0.05). The &#34;real world&#34; HIIT regiment based on beep training protocol is a time-efficient, low-cost, and easy-to-implement protocol for overweight untrained men.

This study presents a low-cost and easy-to-implement "real world" high-intensity interval training (HIIT) protocol for scientific research and discusses its efficiency for cardiorespiratory fitness.

High-Intensity Interval Training (HIIT) has emerged as an interesting time-efficient approach to increase exercise adherence and improve health. However, ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

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 ...