Name: diagnosis of neoplasia in barrett’s esophagus using vital-dye enhanced fluorescence imaging
Uploaded: 2014-05-11T16:00:00.000+00:00
Duration: 6 min 55 s
Description: Watch this Scientific Journal Video about Diagnosis of Neoplasia in BarrettÃ¢â‚¬â„¢s Esophagus using Vital-dye Enhanced Fluorescence Imaging at JoVE.com

This work is supported by the National Cancer Institute at the National Institute of Health grant R01 CA140257-01.

NOTE: Informed consent was obtained from the patients. Also, this research has been performed in compliance with all institutional, national, and international guidelines for human welfare.
1. Prepare Computer
<ol>
	<li>Turn laptop on and connect USB from the DVI2USB Capture Card.</li>
</ol>
2. Prepare Monitor
<ol>
	<li>Connect DVI cable to monitor and PinP to allow the endoscopist to view videos from these screens rather than the computer.</li>
	<li>Make sure the standing monitor is on and is set to DVI.</li>
	<li>Connect PinP to the back of the Olympus system then press the input button on the Olympus system to view the image on the large monitor mounted on the wall.</li>
</ol>
3. Laser Diode Driver Setting
<ol>
	<li>Check to make sure the laser diode driver is OFF. It should only be turned on a couple minutes before imaging is performed.</li>
</ol>
4. Power Strip
<ol>
	<li>Plug in laptop, processor, laser diode driver, DVI splitter, and epiphan capture card into the power strip.</li>
</ol>
5. Run MDE Widefield on the Laptop Desktop
<ol>
	<li>Hit &#8216;Current Folder&#8217;.</li>
	<li>Type patient number and initials and press &#8216;Create Patient Folder&#8217;.</li>
</ol>
6. Prepare Cap and Filter
<ol>
	<li>When handling filter, always use gloves and Kimwipes to minimize the transfer of oil and debris to the filter. Gauze or alcohol swabs may leave fibers that can interfere with imaging.</li>
	<li>Place a few Kimwipes onto a table to create a platform to lay down the filter and cap. This disinfected area should be easily accessible to the gastroenterologist during the procedure.</li>
	<li>Lay down the filter and cap and keep Kimwipes close by for use during the procedure.</li>
</ol>
7. Patient Preparation
<ol>
	<li>Consent patient on use of VFI and proflavine dye before arriving to endo suite.&#160;</li>
	<li>Position patient for upper endoscopy procedure.</li>
	<li>Proceed with standard white-light imaging using a multispectral digital microscope (MDM)7.</li>
</ol>
8. Insert and Spray Proflavine
<ol>
	<li>After finishing with white-light imaging, insert and spray proflavine dye over tissue of interest. 1-5 mm should be sufficient depending on the area of the Barrett&#8217;s tissue. By spraying the proflavine dye at this step, ample time (at least 1 min) is given for sufficient tissue absorption before VFI. Proflavine, which is covered under an investigational new drug application from the FDA (IND 102,217), is a fluorescent contrast agent that concentrates within cell nuclei shortly after application. Although VFI cannot resolve individual cellular morphology, when the laser diode pans over tissue, the reflected light enables the endoscopist to appreciate overall glandular morphology.</li>
</ol>
9. Turn on Laser Diode
<ol>
	<li>Turn on the laser diode. Do this at least 2 min&#160;before imaging begins.</li>
</ol>
10. Prepare Endoscope for VFI
<ol>
	<li>Withdraw the endoscope completely.</li>
	<li>Prior to handling the endoscope, ensure that the assistant has two pairs of gloves on.</li>
	<li>Have the endoscopist hold the endoscope in front of the assistant who is next to the Kimwipe platform detailed above.</li>
	<li>With Kimwipes, have the assistant cleans the tip of the endoscope. Make sure to clean the front surface gently and also clean a few centimeters along the side of the endoscope for easier handling.</li>
	<li>After cleaning, have the assistant dispose of the outer pair of gloves.</li>
	<li>Have the assistant put on the filter. Insert the short cylindrical protuberance on the filter into its complementary hole in the endoscope. Keep the endoscope vertical to aid the process.</li>
	<li>Holding the filter in place, slide the cap over the filter and push it down over the tip of the endoscope. Make sure the cap is secure and pushed completely on with the edges flush with the endoscope. Make sure the lip of the cap slightly extends over the tip of the endoscope. Lastly, make sure the filter is still in place and flush with the tip of the endoscope.</li>
</ol>
11. Insert the Endoscope Back into the Esophagus and Image
12. Remove Endoscope from Esophagus
<ol>
	<li>Using Kimwipes carefully pull the cap and filter off the endoscope. Throw away the cap and clean the filter for the next case.&#160;</li>
</ol>
13. Clean Filter
<ol>
	<li>Gently clean the filter with Kimwipes.</li>
	<li>Fill a small cup with Cydex and submerge the filter for 12 min. Every few min&#160;turn the filter over.</li>
	<li>Gently clean the filter with Kimwipes.</li>
	<li>Submerge filter, this time in sterile water. After 5 min, use a squirt bottle and lightly spray sterile water over the filter.</li>
	<li>Place filter on a few Kimwipes and let it dry.</li>
	<li>Once dry, place in storage container in between Kimwipes.</li>
</ol>

<ol>
	<li>Modiano, N., Gerson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrett's+Esophagus:+Incidence,+etiology,+pathophysiology,+prevention+and+treatment.">Barrett's Esophagus: Incidence, etiology, pathophysiology, prevention and treatment.</a> Therapeutics and Clinical Risk Management. 3, 1035-1145 (2007).</li><li>Brown, L. M., Devesa, S. S., Chow, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+Adenocarcinoma+of+the+esophagus+among+white+Americans+by+sex,+stage,+and+age.">Incidence of Adenocarcinoma of the esophagus among white Americans by sex, stage, and age.</a> Journal of the National Cancer Institute. 100, 1184-1187 (2008).</li><li>Siegel, R., Naishadham, D., Ahmedin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Statistics,+2012.">Cancer Statistics, 2012.</a> A Cancer Journal for Clinicians. 62, 10-29 (2012).</li><li>Vieth, M., Ell, C., Gossner, L., May, A., Stolte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+analysis+of+endoscopic+resection+specimens+from+326+patients+with+Barrett's+esophagus+and+early+neoplasia.">Histological analysis of endoscopic resection specimens from 326 patients with Barrett's esophagus and early neoplasia.</a> Endoscopy. 36, 776-781 (2004).</li><li>Pohl, H., Rosch, T., Vieth, 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=Miniprobe+confocal+laser+microscopy+for+the+detection+of+invisible+neoplasia+in+patients+with+Barrett's+oesophagus.">Miniprobe confocal laser microscopy for the detection of invisible neoplasia in patients with Barrett's oesophagus.</a> Gut. 57, 1648-1653 (2008).</li><li>Thekkek, 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=Modular+video+endoscopy+for+in+vivo+cross-polarized+and+vital-dye+fluorescence+imaging+of+Barrett's-associated+neoplasia.">Modular video endoscopy for in vivo cross-polarized and vital-dye fluorescence imaging of Barrett's-associated neoplasia.</a> Journal of Biomedical Optics. 18, 026007 (2013).</li><li>Roblyer, D., 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=Multispectral+optical+imaging+device+for+in+vivo+detection+of+oral+neoplasia.">Multispectral optical imaging device for in vivo detection of oral neoplasia.</a> Journal of Biomedical Optics. 13, 024019 (2008).</li><li>Egger, 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=Biopsy+surveillance+is+still+necessary+in+patients+with+Barrett's+oesophagus+despite+new+endoscopic+imaging+techniques.">Biopsy surveillance is still necessary in patients with Barrett's oesophagus despite new endoscopic imaging techniques.</a> Gut. 52, 18-23 (2003).</li><li>Thekkek, 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=Vital-dye+enhanced+fluorescence+imaging+of+GI+mucosa:+metaplasia,+neoplasia,+inflammation.">Vital-dye enhanced fluorescence imaging of GI mucosa: metaplasia, neoplasia, inflammation.</a> Gastrointestinal Endoscopy. 75, 877-887 (2012).</li><li>Muldoon, T. J., Anandasabapathy, S., Maru, D., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+in+Barrett's+esophagus:+a+novel,+low-cost+endoscopic+microscope.">High-resolution imaging in Barrett's esophagus: a novel, low-cost endoscopic microscope.</a> Gastrointestinal Endoscopy. 68, 737-744 (2008).</li></ol>

There is nothing to disclose.

With standard endoscopic surveillance, neoplasia in BE is often missed8 because benign metaplasia can be indistinguishable from high-grade dysplasia and adenocarcinoma. As a tool that would better enable gastroenterologists to remedy this currently unavoidable error, vital-dye enhanced fluorescence imaging highlights a tissue&#8217;s glandular morphology thereby providing a distinct feature to differentiate the tissue types. Moreover, by providing a field of view up to 2.5 cm, VFI enables endoscopists to pan over the entire distal esophagus efficiently and systematically, making suspicious lesions more prominent.
To utilize this novel technique, first spray a topical contrast agent, proflavine, over the tissue of interest. Then, after removing the endoscope a 495-nm long-pass filter needs to be securely capped to the MDM tip. &#160;Finally, after reinserting the endoscope near the tissue of interest and then switching from the white light to the laser diode, a green fluorescence provides in vivo imagery of the tissue&#8217;s glandular framework.
In cases of continuous suboptimal fluorescence, using fresh proflavine can improve results as well as turning on the laser diode a few minutes before actual usage and adjusting its voltage. Separately, for patient safety, make certain the cap has been pushed completely over the filter, such that the filter is flush with the tip of the endoscope. This, in turn, will enhance image quality by preventing mucus buildup behind the filter itself.
While VFI is able to characterize glandular morphology, a tangible improvement to white-light endoscopy, it cannot resolve cellular dysplasia. That is, the spatial resolution of 50-100 &#956;m&#160;makes VFI incapable of differentiating benign metaplasia from low-grade dysplasia. To distinguish these diagnoses, higher resolution devices that visualize cellular traits, such as nuclear polarity and cellular crowding, are needed 9,10. As an example, high-resolution microendoscopy is a more novel technique that can differentiate neoplastic tissue on a cellular level as nuclei are illuminated bright white and nuclear to cytoplasmic ratio can be appreciated. Narrow band imaging is another option, but it requires use of two different scopes and processors and follow-up administration of either proflavine or fluorescein for microendoscopy; in contrast VFI allows immediate confocal imaging with one scope and one processor, lengthening anesthesia time by approximately 6-8 min. However, because these latter devices focus on minute tissue areas and consequently miss neoplasias, VFI, in addition to diagnosing neoplasia, may also act as a widefield, red-flagging device for the placement of higher-resolution probes. Thus, the future of endoscopic screening for Barrett&#8217;s dysplasia will likely involve a combination approach of widefield surveillance technology, like VFI, along with a cellular classification technology like microendoscopy.

Over the last forty years, the incidence of esophageal adenocarcinoma (EAC) has increased significantly1,2; yet due to late diagnosis, the five-year&#160;survival rate is less than 20%3. The current standard of endoscopic surveillance in BE, the precursor to EAC, is white light endoscopy with random four-quandrant forceps&#8217; biopsies of the segment. Unfortunately, this technique often misses neoplasia, which can be flat, subtle and difficult to differentiate on standard white light imaging4. While there has been success in using confocal laser microscopy to highlight cellular features in vivo, lesions can still be missed due to the decreased field of view5. Having a &#8216;bridge&#8217; technology that can highlight areas for further confocal microendoscopic imaging would be markedly valuable.
Consequently, an enhanced red-flag imaging modality that improves the ability to target and biopsy early neoplasia in BE would be instrumental in detecting EAC at an early, curable stage and could lead to more effective treatment and subsequently improved survival rates. VFI is a novel technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast, proflavine, to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal esophagus in hopes of improving the in vivo diagnosis6. Upon excitation of the proflavine, which concentrates within cell nuclei shortly after application, the fluorescent images provide spatial resolution of 50 to 100 &#956;m&#160;and a field of view up to 2.5 cm,&#160;allowing endoscopists to visualize glandular morphology. As a result, this approach enables gastroenterologists to distinguish classic Barrett&#8217;s metaplasia, which has continuous, evenly-spaced glands and an overall homogenous morphology, from BE with neoplasia, which has obliteration of the glandular architecture. Here we describe the protocol of this new technique with a multispectral endoscope, and provide representative results to demonstrate the utility of this device in depicting the morphological transformation from benign metaplasia to high-grade dysplasia and cancer.

<table><tbody><tr><td>Filter*</td><td>Schott North America, Inc., Duryea, Pennsylvania</td><td>Not Applicable</td><td>495-nm long-pass filter</td></tr><tr><td>Halo Cap &amp;ndash; Medium*</td><td>Barrx Medical</td><td>CP-002A</td><td></td></tr><tr><td>Processor*</td><td>Pentax</td><td>EPK-i</td><td></td></tr><tr><td>Multispectral Digital Microscope**</td><td>Not Applicable</td><td>Not Applicable</td><td></td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark</td><td>KimTech Science</td><td>S-8115</td></tr><tr><td>Cidex</td><td>Advanced Sterilization Products&amp;nbsp;</td><td>CIDEX OPA Solution</td><td></td></tr><tr><td>Proflavine hemisulfate (0.01% w/v)</td><td></td><td></td><td>FDA (IND 102,217)</td></tr><tr><td>Laser Diode*</td><td>Nichia Corporation</td><td>Not Applicable</td><td>455-nm</td></tr><tr><td>Image Capture*</td><td>Labview</td><td>Not Applicable</td><td></td></tr><tr><td>Spray Catheter</td><td>Olympus</td><td>Not Applicable</td><td></td></tr><tr><td colspan="4">*Equipment specifics within Reference 6. **Equipment specifics within Reference 7</td></tr></tbody></table>

diagnosis of neoplasia in barrett&#8217;s esophagus using vital-dye enhanced fluorescence imaging

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat and indistinguishable with white light imaging alone. As a result, a modality that highlights glandular architecture would be useful to discriminate neoplasia from benign epithelium in the distal esophagus. VFI is a novel technique that uses an exogenous topical fluorescent contrast agent to delineate high grade dysplasia and cancer from benign epithelium. Specifically, the fluorescent images provide spatial resolution of 50 to 100 &#956;m&#160;and a field of view up to 2.5 cm,&#160;allowing endoscopists to visualize glandular morphology. Upon excitation, classic Barrett&#8217;s metaplasia appears as continuous, evenly-spaced glands and an overall homogenous morphology; in contrast, neoplastic tissue appears crowded with complete obliteration of the glandular framework. Here we provide an overview of the instrumentation and enumerate the protocol of this new technique. While VFI affords a gastroenterologist with the glandular architecture of suspicious tissue, cellular dysplasia cannot be resolved with this modality. As such, one cannot morphologically distinguish Barrett&#8217;s metaplasia from BE with Low-Grade Dysplasia via this imaging modality. By trading off a decrease in resolution with a greater field of view, this imaging system can be used at the very least as a red-flag imaging device to target and biopsy suspicious lesions; yet, if the accuracy measures are promising, VFI may become the standard imaging technique for the diagnosis of neoplasia (defined as either high grade dysplasia or cancer) in the distal esophagus.

Figure 1B depicts classic Barrett's Esophagus with no dysplasia surrounded on the borders by normal squamous epithelium. Beginning with the flatter squamous tissue, which is peripherally located and indicated by the blue arrows, a homogenous area of dull fluorescence is present with no glandular architecture. The green arrows indicate a circular green line surrounding the squamous tissue. This outline is artifact resulting from the cap of the endoscope. Moving to the centrally located Barrett's tissue, glandular structures can be defined as green fluorescence surrounding a darker lumen. Although some glands are elongated, there is little distortion between adjacent glands, as the width of the glands is similar and the edges are clearly defined. Lastly, the glands and the lumens are evenly spaced with no clumping or crowding present.
Figure 2B depicts Barrett's Esophagus with Low-Grade Dysplasia. It is important to note that although there is Low-Grade Dysplasia present, this cannot be visualized by morphological criteria via this imaging modality. Thus, based on morphological patterns, this tissue is still classified as benign. While the homogenous glands and lumens in the yellow oval are suggestive of mere metaplasia, the blue oval indicates an area of largely coalesced glands. That is, the thickness of the glands have increased, while the darker luminal cavity has become thin and nearly absent. These crowded and slightly distorted glands, however, have discrete, borders and tend to be homogenous. Moreover, there is no effacement present as the edges of the glands are smooth, thus the tissue is still benign. The red arrow indicates tissue that is out of focus and in video can be readily distinguished. Lastly, the black arrow shows bubbles that are artifact.
In Figure 3B, the red oval indicates the most prominent area with high grade dysplasia. These glands are crowded as they have thin irregular borders along with areas of tissue with near effacement of the glandular architecture. That is, the glands are no longer distinct, but rather fusing together, with their lumens being small and irregular. Although small, the continued presence of some lumens likely indicates high-grade dysplasia as it is more often in invasive cancer where the lumens are completely lost. In contrast to the high-grade dysplasia, the yellow oval highlights malignant tissue. Here, the glandular architecture is obliterated and lumens are largely absent.
Figure 4B depicts a centrally located adenocarcinoma. Notice first the cancer within the red oval and the complete obliteration of glandular architecture with luminal absence. This obliteration can be further appreciated when comparing the cancer to the tissue indicated by the blue arrow, which possesses some glandular framework. On the left, the gray rectangle highlights squamous epithelium, which can be better appreciated in video when the endoscope pans over it in its entirety. This squamous tissue is a flat homogenous area of dull fluorescence with no glandular architecture.
<img alt="Comparison of Barrett's esophagus endoscopy; white-light vs. vital-dye fluorescence imaging." src="/files/ftp_upload/50992/50992fig1highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Neoplasia in Barrett's Esophagus: White-light endoscopy vs. vital dye fluorescence imaging. (Left) Images taken with white-light endoscopy. (Right) Corresponding images taken with vital-dye enhanced fluorescence imaging. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50992/50992fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Diagnosis of Neoplasia in BarrettÃ¢â‚¬â„¢s Esophagus using Vital-dye Enhanced Fluorescence Imaging at JoVE.com

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

Vital-dye enhanced fluorescence imaging (VFI) is a novel in vivo technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal esophagus.

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

diagnosis-neoplasia-barretts-esophagus-using-vital-dye-enhanced

Nanyang Technological University

Research

Education

JoVE Core

JoVE Journal

Medical-Surgical Nursing

Bioengineering

Unit 1

Disorders of the Esophagus

SCIENTISTS IN ACTION

12.0K Views.  Icahn School of Medicine at Mount Sinai.  Vital-dye verbeterde fluorescentie beeldvorming (VFI) is een roman in vivo techniek die hoge-resolutie epitheliale-beelden worden gecombineerd met exogeen actueel fluorescerende tegenstelling tot klier morfologie markeren en af te bakenen neoplasie (hooggradige dysplasie en kanker) in de distale slokdarm. 

Video: Diagnosis of Neoplasia in Barrett’s Esophagus using Vital-dye Enhanced Fluorescence Imaging

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Medicine

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Cancer Research

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Risk Prediction Model of Gastric Low-Grade Intraepithelial Neoplasia

Establishment and Evaluation of a Risk Prediction Model for Pathological Escalation of Gastric Low-Grade Intraepithelial Neoplasia

The study aims to explore the risk factors for pathological escalation after endoscopic surgery for gastric low-grade intraepithelial neoplasia (LGIN) and to establish and evaluate a risk prediction model for LGIN. A total of 120 patients diagnosed with gastric LGIN by biopsy and endoscopic submucosal dissection (ESD) between November 2020 and June 2022 were retrospectively analyzed. Gender, age, Helicobacter pylori (HP) infection, lesion size, lesion location, morphology, gastric mucosal congestion, nodules status, surface ulceration and erosion, and ME-observation of all patients were collected and divided into upgraded and non-upgraded groups according to the biopsy and ESD postoperative pathological diagnosis results. Independent risk factors for pathological escalation after ESD surgical treatment were screened by logistic regression analysis, and a risk prediction model was established. Among the 120 patients with gastric LGIN, 49 patients developed postoperative pathological upgrading; the rate of pathological upgrading was 40.83%. Among them, 42 patients were upgraded to high-grade intraepithelial neoplasia (HGIN), 1 case was upgraded to advanced gastric cancer, and 6 cases were upgraded to early gastric carcinoma (EGC). Univariate analysis showed that age, lesion size, gastric mucosal congestion, surface ulcers, and erosion were significantly different between the groups (p &lt; 0.05). Multivariate Logistic regression analysis revealed that age &#8805;60 years, focal length &#8805;2 cm, gastric mucosal congestion, and surface ulceration and erosion were independent risk factors for postoperative pathological escalation in patients with gastric LGIN. Final joint probability forecasting model for P = 1/[1 + e(26.515-0.161 x &#946;1-0.357 x &#946;2+0.039 x &#946;3-0.269 x &#946;4)]. Age, lesion size &#8805;2 cm, gastric mucosal congestion, and lesion surface ulceration and erosion are risk factors for postoperative pathological upgrading in patients with gastric LGIN. The risk prediction model established in this study based on risk factors has predictive value and can provide a scientific reference for the clinical treatment of patients with gastric LGIN.

Author Spotlight: Advancing Early Detection and Treatment of Gastrointestinal Tumors

Here, we performed a systematic evaluation of patients diagnosed with gastric low-grade intraepithelial neoplasia by previous endoscopic biopsy and obtained a pathological diagnosis by complete resection of the lesion by endoscopic submucosal dissection (ESD), analyzing factors that potentially increase the risk of pathological escalation.

The study aims to explore the risk factors for pathological escalation after endoscopic surgery for gastric low-grade intraepithelial neoplasia (LGIN) and ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett&#8217;s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

There is a discernible need for improved information on molecular drivers of Barrett&#8217;s Esophagus. Immunofluorescent staining is a useful technique for understanding the effects of cell signaling on cell morphology. We present a simple, effective protocol for the use of immunofluorescent staining to assess therapeutic treatment in Barrett&#8217;s Esophagus cells.

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...

Biology

Elastic Fiber Staining of Slide Sections: A Technique to Observe Gastric Cancer Metastasis

Source: Lei, G. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/58278" target="_blank">Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the elastic staining of sections obtained from gastrointestinal tumor tissue. This staining procedure enables visualization of fine elastic fibers and helps determine tumor cell invasion beyond the elastic lamina.

Source: Lei, G. et al. Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue. J. Vis. Exp. (2019)
In this video, we demonstrate ...

Diagnosis of Neoplasia in Barrett’s Esophagus using Vital-dye Enhanced Fluorescence Imaging

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video