Most endoscopic procedures involve tissue acquisition with diagnostic and monitoring intent. Endoscopic mucosal (submucosal) biopsies are routinely procured for varied gastrointestinal diseases. Other settings where tissue acquisition is commonly required are biliopancreatic pathologies, usually strictures with or without a visible mass. These situations usually require intra-ductal brushings and microforceps biopsies (Table 1). Endoscopic ultrasound (EUS) has enabled extra-luminal tissue sampling from adjacent structures. EUS-guided tissue acquisition for mural and peri-luminal pathologies requires familiarity with the sampling needle devices.

Biopsy forceps are used to acquire pinch mucosal biopsy specimens, which sometimes includepart of the submucosa. Standard biopsy forceps can generally obtain a full-thickness mucosal specimen except for the greater curve of the gastric body, where the folds usually are thicker.

Most pinch biopsy forceps fit through a 2.8 mm biopsy channel with a closed cup diameter of 2.2-2.3 mm. Infants and young children often have biopsy specimens taken with smaller forceps that fit through a 2.2 mm biopsy channel. These smaller forceps are indicated for children weighing less than 10 kg. The large-cup (‘jumbo’) forceps require a larger biopsy channel of 3.6 mm or more. These larger capacity forceps biopsies yield two to three times the surface area than standard forceps but are not generally much deeper.^3,4

1. Rotating the endoscope to display the target lesion close to the 6 O clock position on the screen makes obtaining biopsies easier. 

2. Increasing tension or stretch of the biopsy site by pushing the biopsy forceps reduces the quality of the specimen obtained. Partial lumen deflation usually helps capture better specimens by bunching up the mucosa. Folds or valvulae may also be selectively targeted for biopsy specimens. 

3. Slow pulling back the closed forceps to tent the mucosa may provoke a crush artifact. The closed forceps should be snapped back quickly.

4. The ‘turn-in technique’ may be used when biopsies must be taken at an oblique angle, as in the esophagus. The opened forceps is first drawn back flush with the endoscope tip. The endoscope tip is then deviated towards the area to be biopsied with the open forceps; the forceps may be advanced a bit and some suction may be used to improve tissue capture. There is usually a visual red-out at the moment of obtaining the biopsy with the turn-in technique. 

5. The biopsy specimen should be removed from the forceps cup using a blunt probe to push the specimen out from the base of the opened cups. Pushing the specimen out from the top side of the forceps cup, or picking at it, may lead to squashing of the specimen. 

6. Shaking biopsy specimens off the forceps into a fixative bottle should be avoided. Extracting the captured specimen by shaking the forceps may traumatize the tissue and cause epithelial denudation, especially in gastric body specimens. Detached epithelium in gastrointestinal mucosal biopsies is a common feature encountered by pathologists. 

7. Shaking off biopsy specimens with surface exudate into the fixative may peel off the exudate that often contains the evidence for the infecting organisms, especially Candida and Herpes simplex. These organisms reside in the surface exudate or epithelial slough.

Biopsies that include the muscularis mucosa can contract, making the mucosal surface convex. This can impact the interpretation of small bowel biopsies where poor orientation will lead to crypts cut tangentially during sectioning (Figure 2a). This may falsely increase the villous-to-crypt ratio, leading to an underestimation of histological abnormalities. Inaccurate orientation can also result in crushing or partial fusion of adjacent villi, wrongly suggesting partial villous atrophy or causing difficulties in intra-epithelial lymphocyte (IEL) count.⁵ A well-oriented small bowel biopsy specimen is de?ned by four consecutive, parallel, crypt-villous units that are visualized along their entire lengths (Figure 2b). In contrast, round cross-sectional crypts indicate a non oriented biopsy. In practice, duodenal biopsies are not oriented in most endoscopy suites or pathology departments before processing and embedding biopsy specimens in paraf?n tissue blocks. It has been reported that over 30% of the duodenal biopsy specimens may be poorly oriented.⁶

There have been many modifications to the biopsy forceps, some of which have been detailed above. This section will discuss two specialized versions of the biopsy forceps for use through a cholangioscope (SpyBite^TM forceps) and a 19G EUS-FNA needle (Moray^TM microforceps).

Indeterminate biliary strictures are those in which cross-sectional imaging (computed tomography or magnetic resonance imaging) and endoscopic retrograde cholangiopancreatography (ERCP) with cytologic brushing or biopsy are non-diagnostic.⁹ Although most of these biliary strictures have a malignant etiology, up to one-fourth of these suspected malignant biliary strictures ultimately turn out benign.¹⁰ Cholangioscopic visual impression has higher sensitivity than directed biopsies for malignant etiology of a biliary stricture; however, the specificity of visual impression is lower than cholangioscopic guided biopsies.^11-13 Hence it may not be appropriate to conclude the diagnosis based on visual impression alone and directed intraductal biopsies should be taken whenever feasible.¹⁰

There is limited evidence from a retrospective study that balloon dilatation before intraductal biopsies of malignant bile duct stenosis may increase the diagnostic yield.¹⁷ However, the role of routine stricture dilatation before acquiring intraductal biliary biopsies is unsettled. Several intraductal biopsy sampling technique modifications have been used to improve diagnostic accuracy. These include triple sampling (bile cytology and brush cytology and intraductal biopsies), fluorescent in-situ hybridization (FISH), ROSE of touch imprint cytology, and genetic mutational analysis with next-generation sequencing (NGS). Discussion of these ancillary techniques is beyond the scope of this review.

MorayTM through the needle (micro) biopsy (TTNB) forceps These microforceps have been used to obtain histologic samples from pancreatic cyst walls and subepithelial wall tissue (Figure 4). An extraction pick is included to facilitate specimen removal from the microforceps. In addition to the cyst wall biopsy, FNA cytology of the cyst wall or any solid component can also be performed with the 19G needle.

Various cytology brushes are available for tissue sampling in the luminal gastrointestinal tractand the pancreatic and biliary ducts. Design modifications include brushes of variable sizes and stiffness, wire-guided or non-wire-guided, single or multi-lumen, and with or without a ?exible guide tip. Outer sheaths for brushes used in ERCP are 6Fr to 8Fr.^25,26 Brush cytology remains an established diagnostic technique for biliary strictures, but its diagnostic yield remains low, with sensitivity ranging between 40-56% and a specificity between 90-97% for detecting cholangiocarcinoma.^27-30 In a systematic review and meta-analysis, the pooled sensitivity of brush cytology for cholangiocarcinoma was 45% (95% confidence interval [CI], 40-50%).³¹ The accuracy of brush cytology is negatively influenced by flat-type bile duct cancer, non-bile duct cancer, short length (<30 mm) of the stricture, and underlying inflammatory cholangiopathies like primary sclerosing cholangitis (PSC).³² The effect of prior balloon dilatation of the stricture on the yield of biliary brushings is inconclusive.^33-35

Newer cytology acquisition devices are being developed for a wide-field sampling of Barrett’s epithelium (BE) for screening and surveillance. 

All EUS-FNA needles have the same basic design and are single-use devices. The hollow FNA needles are made entirely of metal to permit effective longitudinal force delivery, unlike injector needles which usually have a metal tip affixed to a long hollow plastic cylinder. The FNA needles have a rounded or beveled stylet pre-loaded inside. These needles can be extended up to 8 - 8.5 cm from the distal end of the sheath. There are markings at 1 cm intervals on the handle and a ‘needle stopper’ or ‘safety screw’ on the handle to limit needle advancement beyond the intended depth. Approximately 1cm of distal FNA needle is modified (by laser etching, mechanical dimpling, or sandblasting) to enhance its echogenicity and visibility in the ultrasound field. 

Two meta-analyses have shown the superiority of the 25G over the 22G FNA needles for solid pancreatic lesions. This is likely due to ease of passage of the thinner 25G needle in angulated scope positions into often firm pancreatic cancer tissue.^43,44 The diagnostic yields of 22G and 25G needles are similar for non-pancreatic lesions and lymph nodes.^45,46 In addition, 25G needles may provide superior cytological smears with less blood contamination compared with the larger 22G needles.There is no incremental diagnostic yield of cytology with the 19G needle compared with 22G or 25G needles.^47,48

The rationale of using a stylet inside the FNA needle is to unclog the needle of any plugging gastrointestinal tract contaminants after needle advancement the target lesion. However, no advantage of using a stylet regarding specimen quality and diagnostic yield has been demonstrated.^49-53 FNA needles may be used with or without stylet as per operator preference.

Poor handling of the FNA aspirates negates the utility of the entire procedure. Using an air-filled syringe to expel the tissue from the FNA needle often leads to an uncontrolled spray of the aspirate and should be avoided. We prefer slowly reintroducing the stylet to expel the aspirated material from the needle in a controlled droplet-by-droplet manner on the slide. With a proper oval-shaped smear, the larger tissue fragments are seen in the center and single cells are dispersed at the periphery.  

Alcohol-fixation, air-drying, or both methods can be used to fix the prepared slides and should be discussed with the pathology department. The smears must still be wet when immersed in a 95% ethanol fixative jar. These alcohol-fixed slides are stained with Papanicolaou (Pap) or Hematoxylin & Eosin stains. Pap smear highlights the nuclear details, chromatin quality, and 3-dimensional cellular clusters. Air-dried slides are stained with Romanowsky-type stains (Diff-Quick, May-Gruenwald-Giemsa, or Hemacolor).⁵⁸ Romanowsky stains highlight the intra-cytoplasmic material and extra-cellular substances. Air drying of slides also allows rapid on-site evaluation (ROSE). Hence, alcohol-fixed and air-dried smears provide complementary information. 

The presence of an on-site cytopathologist leads to a 10-15% increase in the diagnostic yield.^59,60 There is an approximate 20% rate of non-diagnostic aspirates without ROSE.⁶¹ In our experience, on-site reporting with EUS-FNA is more complex than with percutaneous FNA because of frequently encountered esophageal, duodenal, and gastric contaminants. ROSE also helps to limit the number of passes and directs additional sample collection for ancillary tests such as microbiological cultures or flow cytometry.

Core biopsies should be obtained when tissue architectural details are required to establish a specific diagnosis. These situations include certain well-differentiated tumors, tumors with extensive desmoplasia, and tumors like GIST when adequate cellular specimens are difficult to obtain. Lymphomas, mainly low-grade varieties, also need histological (architectural) evaluation for a conclusive diagnosis. Unlike malignancies, many benign pathologies like autoimmune pancreatitis (AIP) are less cellular and need tissue architectural details for a diagnosis. Like cell blocks, core biopsies allow extensive IHC panels and molecular analysis.

Standard bevel 19G, and even 22G FNA needles can be used to procure core tissues for histology.⁶⁴ Studies have demonstrated yields of tissue cores or cell blocks ranging from 75-80% when using standard bevel 22G FNA needles and 59-100% when using standard bevel 19G FNA needles.^65,66

Needles with reverse bevel are commercially available in three sizes: 19G, 22G, and 25G (EchoTip ProCore™ needles, Cook Medical). A new 20G ProCore needle (ECHO-HD-3-20C™, Cook Medical) with an automatic recoiling stylet, forward bevel, and coil spring sheath for increased flexibility is now available. The side bevel on these needles hooks and cut the tissue like a cheese grater and traps it in the needle during its motion. In addition, the side hole is also postulated to function as a vent for the air column, facilitating the filling of the needle with tissue cores. Because of the side bevel size, these needles are unsuited for small (<10 mm) lesions or vascular lesions. 

The Franseen FNB needle(Acquire™, Boston Scientific)has a crown-tip with three sharp cutting edges 120 degrees apart and three symmetric cutting surfaces instead of the usual single bevel. The Franseen needle geometry incorporates a larger inclination angle and a smaller included angle.⁷⁷ A high inclination angle translates into more force to penetrate tissue, while a smaller included angle translates into maximal sharpness at cutting.

The fork-tip needle (SharkCore™, Medtronic) has a needle tip designed with six distal cutting surfaces. It has a longer sharp ‘access tip’, and an opposing ‘catch bevel’ to improve tissue capture as the needle movement shears it off.

There are two related ways to handle the specimens obtained from core biopsy needles:

1. Expel the entire material in 10% formalin and process it as tissue cores.

2. Expel the material on a glass slide or petri-dish, and micro-dissect out the tissue cores with a small tweezer or a needle.

Commonly used ancillary tests in EUS-guided aspirates include polymerase chain reaction (PCR) and culture for tuberculosis, flow cytometry (FC), and mutational analysis by reverse transcriptase polymerase chain reaction (PCR) with or without direct sequencing methods, like Sanger sequencing or next-generation sequencing (NGS). The discussion of these techniques is beyond the scope of this review.

Collecting appropriate gastrointestinal luminal and extra-luminal samples during endoscopic procedures not only requires a thorough knowledge of the endoscopic tools, but also close understanding of the pathologic and laboratory processing of the collected samples. A close liaison with the concerned laboratory personnel is essential for obtaining optimal results of endoscopic tissue acquisition.