Tissue sampling is the most basic but important technique for gastrointestinal endoscopy. However, tissue sampling is often taken for granted, and guidelines for the various biopsy methods are surprisingly sparse. The purpose of this program is to demonstrate and provide practical recommendations regarding endoscopic tissue sampling techniques and indications for the GI tract. Indications for endoscopic tissue sampling are broad. Indications include suspicious mucosal tissue due to malignant, inflammatory, and infectious processes, the assessment of tissue response to therapy, and confirmation of normal tissue in any part of the GI tract. Although there is no universal consensus, the risks and benefits of tissue sampling should be weighed in patients at high risk for bleeding. The same consideration should be taken for patients on antithrombotic agents. The AASG has classified endoscopic procedures according to bleeding risk. The AASG has classified endoscopic procedures according to bleeding risk. Routine endoscopic biopsies are considered low-risk procedures, while polypectomy and EUS-guided FNA are higher-risk procedures. The risk of thromboembolic events in patients discontinuing medication should also be considered. Current guidelines recommend continuing antithrombotic agents, including aspirin, clopidogrel, and warfarin, for routine endoscopic biopsies. However, for more advanced tissue sampling procedures, such as polypectomy and EUS-guided FNA, clopidogrel and warfarin should be discontinued. For high-risk patients, discontinuation with other bridge therapy can also be considered. Despite published guidelines that suggest it is safe to perform an endoscopic biopsy during warfarin therapy or polypectomy while on aspirin medication, application in the clinical field varies according to personal experience and population. In a recent study, eastern endoscopists did not typically perform an endoscopic biopsy while their patients were on warfarin therapy and did not perform a polypectomy while their patients were taking aspirin compared to western endoscopists. Adequate biopsy samples are essential for accurate diagnosis. In general, large biopsies, including at least the muscular smucosal layer and with minimal crush artifacts, are adequate for histopathologic examination. Various commercially available biopsy forceps are used for endoscopic biopsy. Most endoscopic accessory manufacturers have a lineup according to the size, the presence of a needle, and the shape of the forceps head. The ASGE update on endoscopic tissue sampling devices stated that needle forceps enhanced directed lesion sampling and provided deeper biopsies. However, there are few studies that prospectively compared needle to non-needle forceps, and results according to histology scores showed no significant difference for specimen adequacy between needle and non-needle versions. Results comparing jumbo, large, and standard capacity forceps showed that although the diameter of the specimen was greatest for jumbo forceps, depth, tissue orientation, and overall adequacy were the poorest. Other variations of forceps have been developed to improve the efficiency and adequacy of tissue sampling. Hot biopsy forceps are not recommended for routine tissue sampling. They are currently widely used for hemostasis during EMR and ESD. Complication rates of tissue sampling devices used in patients without coagulopathies are exceedingly rare. Hot biopsy forceps are generally associated with a higher complication rate than cold biopsy forceps. Bleeding or perforation after hot biopsy may occur as late as two weeks after the procedure. Data regarding tissue adequacy according to forceps type are conflicting, and current studies do not show a significant advantage for a specific type of forceps. One practical note is that the specimen size and depth are also dependent on the skill of the endoscopist and on the pressure applied to the forceps. Tissue sampling can be difficult in this regard. Tissue sampling can be difficult in the esophagus, especially if active peristalsis is present. Due to the long and narrow nature of the esophagus, biopsy forceps are oriented parallel to the esophageal wall, causing easy slippage. Forceps with needles may help in this situation. Biopsies of lesions located at the 12 o'clock position may be difficult due to the angle created with the forceps. It is easier to rotate the scope and reposition the target lesion at the 6 o'clock position. Malignant tumors of the esophagus can be diagnosed by biopsy alone in 95% of cases, except when obstruction prevents adequate visualization. Approximately 8 to 10 biopsy specimens should be obtained. However, if endoscopic treatment is planned, it may be necessary to perform a biopsy of the entire esophagus. Chromoscopy using Lugol's solution can help detect suspicious lesions and guide target biopsies. Dysplasia and early esophageal cancer can be treated with endoscopic resection. Lugol's solution reveals an unstained area at the mid-esophagus. It can be treated with endoscopic resection. Lugol's solution can be treated with endoscopic resection. Dysplasia and early esophageal cancer can be treated with endoscopic resection. Lugol's solution reveals an unstained area at the mid-esophagus. If endoscopic resection is not planned, multiple biopsies should be performed at the base and the margins, as well as any other suspicious lesions below and above the segment. This patient was treated with ESD. Today, most cytology brushes are used for pancreatic and biliary ducts. Today, most cytology brushes are used for pancreatic and biliary ducts. Brush cytology may increase the diagnostic yield of esophageal biopsies. However, recent literature regarding brush cytology of the esophagus is limited. Cytology may still be useful in esophageal cancer when obstruction prevents adequate visualization and biopsy of the lesion. There are numerous direct and over-the-guidewire cytology brushes for the GI tract. Cytology brushes may be useful for obstructive esophageal cancer. In this patient, biopsies cannot be taken due to incomplete visualization. A cytology brush is passed to and fro through the stricture for tissue sampling. Barrett's esophagus is specialized intestinal metaplasia of the esophagus, irrespective of length. Although Barrett's esophagus is a pre-religion condition, screening for Barrett's esophagus remains controversial because of lack of documented impact on mortality. from esophageal adenocarcinoma. Endoscopic screening should be considered in high-risk patients with chronic long-standing GERD. After a negative screening examination, further screening endoscopy is not indicated. Methylene blue selectively stains specialized columnar epithelium in Barrett's esophagus with high accuracy. Methylene blue is inexpensive and can be used for target biopsies of Barrett's esophagus. Previous reports have shown that highly dysplastic or malignant Barrett's esophagus stains differently with methylene blue than non-dysplastic Barrett's. Increased heterogeneity and decreased methylene blue stain intensity have been reported to be significant independent predictors of high-grade dysplasia or cancer. Surveillance endoscopy remains controversial because of the lack of randomized trials supporting its value. Critical analysis of the literature does suggest a survival advantage of endoscopic surveillance. Multiple retrospective studies have been published, all of which indicate that survival is significantly enhanced if the cancers are detected by endoscopic surveillance rather than presenting with symptoms. The grade of dysplasia determines the appropriate surveillance interval. Any grade of dysplasia by histology should be confirmed by an expert pathologist. Patients with no dysplasia can be followed up every three years. The finding of low-grade dysplasia warrants follow-up within six months. If high-grade dysplasia is found, the patient should be given the option of intervention or close follow-up. In this patient with short-segment Barrett's esophagus, methylene blue is used to direct biopsies. After spraying and washing of the dye, stained columnar epithelium is observed. The surrounding gastric metaplasia remains unstained. For long-segment Barrett's, a systematic biopsy protocol has been used. This method uses a turn-in-suction technique to perform four-quadrant biopsies every two centimeters from the EG junction. In one report, the prediction of cancer using the Seattle Protocol prior to surgery was as high as 93%. The Seattle Biopsy Protocol uses a turn-in-suction technique for four-quadrant biopsies. Biopsies are taken every two centimeters or one centimeter in the case of high-grade dysplasia from the EG junction to the squamo-columnar junction. Biopsies from a given segment should be submitted in a separate container. However, the Seattle Protocol has several limitations. First, it is a time-consuming procedure and requires compliance from both patients and physicians. Second, only a very small percentage of the mucosa is sampled, and as such there is always a risk for sampling error. Third, there are problems with inter-observer agreement among pathologists in distinguishing high-grade dysplasia from intramucosal cancer. Gastric neoplasia can present as flat, ulcerative, polypoid, or as thick gastric folds. Lesions located at the posterior wall of the lower body, or the cardia, may be difficult to approach. In high-risk populations for gastric cancer, it is prudent to biopsy even subtle mucosal changes. For lesions near or on the cardia, the scope should be J-turned and withdrawn slowly to approach the target lesion. The updated Sydney system recommends that biopsy specimens be taken at five different sites for optimal assessment of both gastritis and H. pylori status. There have been conflicting reports about the most appropriate biopsy site for detecting H. pylori. Initially, it was reported that the antral, or angular, specimens were highly sensitive for detecting H. pylori infection. More recently, however, the gastric body was suggested as the best biopsy site to get the most reliable results using the rapid urease test in gastric ulcer patients. A gastric ulcer is a defect in the gastric wall that extends through the muscularis mucosa into the submucosa, or proper muscle layer. Typically, four to six biopsies should be taken from the ulcer margin and base if possible. In the past, biopsies of all ulcers was recommended for differential diagnosis with malignant ulcers. However, recent data is insufficient to support this. The decision to take biopsies should be individualized, and patients with low risk of gastric cancer with typical NSAID-induced ulcer findings on endoscopy can be excluded from biopsy. On diagnosis of a peptic ulcer, testing for H. pylori should be performed. Although endoscopic appearance is a good predictor of the absence of malignancy, some malignant ulcers may initially appear benign. Repetitive endoscopic and histologic examination should be performed for such ulcers even if biopsy is negative. When performing biopsies, the direction of blood flow according to body position should always be kept in mind. The order of each biopsy should be planned ahead to prevent blood from obscuring the lesion. With the patient in the left lateral decubitus position, the direction of gravity is toward the 12 o'clock position. The biopsy order is planned accordingly. For malignant ulcers, multiple biopsies should be performed from the ulcer margins and islets in the center of the ulcer. A flat depressed lesion is seen on the antrum. For such lesions, biopsies should be performed from the center of the depression. However, if endoscopic resection is planned, excessive biopsies can cause submucosal fibrosis, and resection may be difficult in such cases. Biopsies can also be used to determine the resection margin during or before endoscopic resection. In one study comparing chromoendoscopy and frozen margin biopsies for prediction of the lateral border in early gastric cancers with obscure margins, frozen biopsies resulted in a higher complete resection rate. Before ESD in this patient, the lateral margin was unclear. One biopsy prior to ESD with standby frozen histopathologic assessment showed that the suspicious margin was free from tumor involvement, and ESD was performed. Diffuse infiltrating gastric tumors are usually associated with marked submucosal fibrosis and gastric wall thickening, with or without mucosal ulceration. The endoscopic appearance is usually nonspecific, however, poor distensibility has been a frequent finding. The endoscopic differential diagnosis includes in addition to linitis plastica, Menetrier's disease, and lymphoma, all of which are difficult to diagnose without an adequate tissue biopsy. It has been reported that in up to 30% of linitis plastica cases, forceps tissue biopsy and brushings can be negative. Tissue sampling of subepithelial tumors is often challenging. Forceps biopsy using the bite-on-bite method can be applied to these tumors. Although this method is simple, reported diagnostic yield is relatively low. A prospective head-to-head comparison of jumbo forceps biopsy and endoscopic resection for subepithelial tumors showed that the diagnostic yield was significantly higher for endoscopic resection than jumbo forceps biopsy. Advanced tissue sampling techniques have evolved from big particle biopsy to strip biopsy and EMR. Recently, more treatment-oriented procedures, such as ESD and notes, have been used for combined treatment and diagnosis. Strip biopsy uses standard snare polypectomy techniques, with or without the addition of submucosal injection, to separate the proper muscle layer from the more superficial layers. A two-channel endoscope, grasping forceps, are used for the lift-and-cut technique. Strip biopsy was used for tissue diagnosis in this patient with linitis plastica after failure of forceps biopsy. Following submucosal injection, grasping forceps and a snare are used to cut and resect the submucosal layer. Strip biopsy was successful, and signet ring cell carcinoma was diagnosed. Initial evaluation of subepithelial lesions with EUS helps identify those lesions where tissue diagnosis may be necessary. If a subepithelial lesion is found to be a hypoechoic mass in the 3rd or 4th echoic layer on EUS examination, then tissue sampling should be strongly considered to establish the diagnosis, because malignant or potentially malignant lesions are included in the differential diagnosis, and EUS findings have been shown to be insufficient to correctly establish the diagnosis. A preoperative tissue diagnosis may not be necessary for large and symptomatic lesions that require surgery, irrespective of histology. The need to obtain tissue from small hypoechoic masses of the gastric wall is controversial and has not been adequately studied, but should be considered given the potential for malignant behavior of such lesions. Endoscopic resection of SCTs are usually reserved for lesions up to the submucosal layer. Although the complication rate is higher than forceps biopsy, the tissue yield is superior. Endoscopic resection requires various accessories, such as snares, hemostatic and grasping forceps, various electrosurgical knives, injection needles, and distal attachments. EMR involves lifting of a lesion from the GI tract either by injection of saline or suction of the lesion into the cap attached to the distal end of the endoscope, followed by electrosurgical snare resection. In this case, a submucosal tumor is observed on endoscopy at the mid-esophagus. Saline solution was injected beneath the SCT to elevate it from the proper muscle. A snare is placed around the base of the tumor and tightened for successful resection. Reports of EMR for tissue sampling of SCTs have shown excellent rates of pathological diagnosis. However, higher rates of bleeding were also reported. The unroofing method utilizes a snare over the middle of the SCT, creating an opening in the overlying mucosa. Indications of the unroofing method are diagnosis and treatment of SCTs from the third layer, and partial resection and tissue diagnosis of SCTs from the fourth proper muscle layer. This case shows the unroofing technique for diagnosis of a hypoechoic tumor in the fourth layer. A snare is used to resect the overlying mucosa, and then the snare is placed over the tumor and slowly tightened for partial resection and tissue sampling. Final diagnosis was a GIST positive for C-kit. The unroofing technique for SCTs of the fourth layer showed a high diagnostic rate. Immunohistochemical staining and mitotic index was possible for all GIST tissue samples. However, careful assessment of the need for partial resection with disruption of the tumor capsule of potentially malignant tumors should be addressed. Recently, endoscopic submucosal dissection and natural orifice transluminal endoscopic surgery have been used for the complete resection of SCTs. With the lack of current data, and given the therapeutic nature of these techniques, it is difficult to recommend them for routine diagnosis of SCTs. Possible indications could be resection and diagnosis of small GISTs. US guided needle biopsy is indicated for cytopathological diagnosis of mass lesions of and adjacent to the gastrointestinal tract when these lesions cannot be sampled by less invasive methods or if sampling by alternative methods have failed. USFNA is the method of choice for tissue diagnosis in hypoechoic SCTs and for diffuse infiltrating gastric cancer after failure of other biopsy methods. USFNA is contraindicated in all conditions in which the risks of the procedure outweigh the expected benefits of its diagnostic information. Severe impairment of coagulation, lack of patient cooperation or informed consent, failure of ultrasonic control of needle position, and high infectious risk lesions are contraindications to USFNA. USFNA is only performed using echoendoscopes with electronic curved linear transducers. Some of these echoendoscopes have an Alboran elevator to facilitate needle positioning. The diameter of the accessory channel varies from 2 mm to 3.8 mm. A wide range of disposable and reusable needle kits are produced by various manufacturers. Most USFNA needles have a stiff needle stylet inside a steel or 19-0 needle with an outer diameter of 25, 22, or 19 gauge. The tip of the stylet is blunted or pointed and may have a side port near the distal tip for additional tissue entry. The needle tip is usually dimpled for optimized ultrasonic needle visualization. Until only recently, only one 19-gauge needle device was available for obtaining core biopsies by means of a spring-loaded true-cut mechanism. The inner stylet of this QuickCore needle has a specimen notch of 20 mm and contains a spring-loaded mechanism built into the handle that permits automated procedurement of biopsy specimens. Recently, a new needle developed specifically for core biopsies with a built-in core trap has been developed. This ProCore needle is available in 19 and 22 gauges and dimensions allow sampling of 10 mm lesions. USFNA was used for tissue diagnosis in this patient with diffuse gastric wall thickening. A 22-gauge needle was pushed into the gastric folds. An enlarged lymph node was also aspirated. Pathologic analysis diagnosed undifferentiated adenocarcinoma for both specimens. A 73-year-old female with a past history of breast cancer was admitted for dysphagia. Endoscopy revealed a stricture just distal to the upper esophageal sphincter. CT scans showed extrinsic compression of the esophagus by enlarged lymph nodes. EUS-guided fine needle aspiration was performed at the stricture and pathologic findings were consistent with metastatic adenocarcinoma. The diagnostic yield of USFNA varies depending on the nature of the target lesion, the skill and experience of the endoscopist, as well as the cytopathologist. The reported diagnostic yield of USFNA for SCTs are conflicting and range from 40 to 97%. Limitations of USFNA are hampered cytologic interpretation by the presence of blood and benign epithelial cells. Additionally, USFNA typically yields a small biopsy sample and destroys the tissue architecture, thereby limiting the diagnostic sensitivity for lesions such as GI stromal tumors. Finally, USFNA cannot assess mitotic index, a parameter essential to assessing the malignant potential of gastrointestinal stromal tumors. In this 72-year-old female, a submucosal elevation with bridging folds was observed at the antrum. Radial EUS showed a hypoechoic mass originating from the fourth layer. For diagnosis, EUS-TRUCA biopsy was performed. On immunohistochemistry, spindle cells with diffuse and intense positivity for S100 protein supported the diagnosis of schwannoma. A submucosal elevation is observed at the upper body of the stomach wall in a 42-year-old female. EUS showed extrinsic compression from an enlarged lymph node. We performed EUS-guided needle biopsy for tissue sampling. We used a 19-gauge Procore needle for EUS needle biopsy. Locking the needle and adjusting the sheath adjuster and safety ring are identical to other EUS needles. The needle is inserted into the enlarged lymph node, and four passes were performed. Tissue samples from the Procore needle biopsy showed an elongated core of tissue. Histopathology revealed casus necrosis, and Ziehl-Nielsen stain confirmed the diagnosis of tuberculosis. Previous reports have shown that TRUCUT needle biopsy more accurately diagnosed SCTs compared to EUS-FNA. In one report, the accuracy of TRUCUT for diagnosis of GISTs was twice as high as that of FNA. Current evidence shows that EUS TRUCUT biopsy should be favored for diagnosis of GISTs and other hypoechoic SCTs. Optical biopsies use advanced endoscopic imaging techniques, such as narrowband imaging and confocal endomicroscopy, to predict the histopathology of lesions without actual tissue sampling. Optical biopsies are noninvasive and have no sampling limitation. Normal mucosa can be immediately ruled out, and if needed, precise targeted biopsies can be performed. MBI is an optical filter technology that radically improves the visibility of capillaries, veins, and other subtle tissue structures by optimizing the absorbance and scattering characteristics of light. MBI uses two discrete bands of light, one blue at 415 nm and one green at 540 nm. Narrowband blue light displays superficial capillary networks, while green light displays subepithelial vessels. When these two lights are combined, they offer an extremely high contrast image of the tissue surface. Capillaries on the surface are displayed in brown, and veins in the subsurface are displayed in blue. MBI combined with magnifying endoscopy further enhances the diagnostic capabilities of MBI. Visualization of the fine vascular network and surface texture is possible, and tissue characterization and differentiation is more evident. Identifying the lateral extensions of superficial neoplasia by changes in microvascular pattern is also possible. The mucosal and vascular patterns seen on MBI in Barrett's esophagus have been reported to be associated to the level of dysplasia. Irregular and distorted mucosal and vascular patterns were more frequently found in high-grade dysplasia. MBI with magnification is used to detect dysplasia in this patient with long Barrett's esophagus. Circular patterns with normal vasculature indicate normal mucosa or non-dysplastic metaplasia. Irregular vascular or mucosal patterns are more indicative of dysplastic mucosa. A circular mucosal pattern is seen on MBI with magnification. Target biopsies of this area show columnar epithelium without goblet cells on alshin blue stain. This figure shows the normal vascular pattern observed on MBI with magnification. Biopsies of this area showed columnar epithelium devoid of goblet cells. This image shows the ridge and villus mucosal pattern observed on MBI with magnification. Biopsy results showed intestinal metaplasia with dysplastic change. In a study comparing white light endoscopy with MBI, 18% of patients had more advanced dysplasia detected using MBI compared to standard white light endoscopy. There are no patients in whom a finding of dysplasia was found on white light endoscopy with random biopsy that was not found on MBI. The total number of biopsies was also significantly less in the MBI group. MBI with magnification can distinguish intricate IPCL patterns of the surface mucosa. The IPCL patterns are known to change with the degree of inflammation and dysplasia. IPCL pattern changes in severe dysplasia or carcinoma include meandering, caliber change, and irregular form. The IPCL pattern may also be able to predict the infiltration depth of cancerous lesions. More prominent and elongated IPCL patterns correlate with deeper invasion. In tumors with some mucosal invasion, total destruction of the IPCL pattern can be observed. Endoscopy shows a superficial erosion on the mitosophagus. MBI with magnification shows multiple dilated and torturous IPCLs. The shapes of the IPCLs vary and caliber change can be seen. The patient was eventually diagnosed with early esophageal cancer and underwent ESD. Studies of MBI with magnification for detection of early esophageal cancer showed that MBI with magnification was more sensitive and accurate than white light endoscopy. When compared to iodine stain, although sensitivity was not different between both groups, MBI was more specific for detection of early esophageal cancer. A large flat depressed lesion is observed on the greater curvature. Previous forceps biopsy results showed differentiated adenocarcinoma. On MBI with magnification, a corkscrew vascular pattern can be observed. This patient underwent surgery and was diagnosed with cignate ring-celled carcinoma. Studies have shown that the microvascular pattern in depressed-type early gastric cancer can predict the differentiation of the tumor. A corkscrew vascular pattern was significantly associated with undifferentiated cancer. One of the most anticipated new imaging modalities for endoscopy is the endoscopic endoscopy. One of the most anticipated new imaging modalities for endoscopy is confocal endomicroscopy. The confocal microscope has the ability to select images in depth by substantially rejecting out-of-focus light through the use of a confocal pinhole. This optical sectioning capability enables high-resolution imaging of thick biological samples, such as tissue in vivo, without the need for tissue excision, fixation, and sectioning, as is required for conventional histological processing. A point of illumination source is focused into the sample, illuminating a single point at a time. The detection pinhole aperture passes light from the focus, but significantly rejects light from depths in the sample above and below the focal plane. The growing body of evidence for confocal endomicroscopy can be seen by the increasing number of publications every year. There are two major systems for confocal endomicroscopy. The first is the probe-based type confocal endomicroscopy. The probe type is compatible with all flexible endoscopes and enables acquisition of dynamic microscopic videos anywhere in the GI tract. The second type is an integrated system with the confocal microscope located inside the endoscope. The integrated type confocal endomicroscopy can provide images at 1000 magnification with a penetration depth of 250 micrometers. On confocal endomicroscopy, gastric, intestinal, and neoplastic epithelium can be differentiated according to capillary and epithelial cell pattern. In contrast to normal squamous epithelium, Barrett's esophagus without dysplasia shows columnar epithelium and black goblet cells. As the degree in dysplasia increases, glandular structure is lost, and irregular glands and black cells with irregular borders can be seen. In a recent study comparing CLE with standard biopsy protocols for detection of Barrett's esophagus, CLE-guided target biopsy had double the diagnostic yield compared to standard 4-quadrant biopsies. Confocal images of barrett's esophagus can be seen on the right side of the slide. Confocal findings of gastric, intestinal, and neoplasia show columnar epithelium with numerous goblet cells. Differentiated adenocarcinoma, seen on CLE, shows a disorganized configuration of glands. However, overall glandular structure is still present. Confocal findings of undifferentiated adenocarcinoma show destruction and absence of all ductal structures. In a recent study, the overall accuracy of CLE diagnosis of gastric adenomas and gastric cancer was significantly higher than endoscopic biopsy. Although not significant, the overall accuracy of CLE diagnosis of differentiated and undifferentiated adenocarcinomas was also higher. To conclude, during routine tissue sampling, adequate biopsy samples are of the utmost importance. Biopsies should be planned ahead and targeted. Advanced tissue sampling techniques can be used as an alternative method for tissue sampling after failure of other methods. Current trends of application of endoscopic resection for advanced tissue sampling are focused on both treatment and diagnosis at the same time. US-guided tissue sampling is the method of choice for hypoechoic fourth-layer SCTs. US-guided tissue sampling can be used for diagnosis of diffuse infiltrating gastric lesions and extrinsic compressing lesions of the GI tract. Optical biopsies can achieve non-invasive real-time in vivo histological tissue analysis. There is no limited sample size. In concept, confocal endomicroscopy could be the ideal endoscope.

tissue sampling

gastrointestinal endoscopy

endoscopic procedures

biopsy samples

Barrett's esophagus

gastric neoplasia

endoscopic ultrasound-guided needle biopsy

optical biopsies