Chapter 8 Endoscopic Biopsy Specimen Collection and Histopathologic Considerations

Albert E. Jergens, Michael D. Willard, Michael J. Day

Advances in endoscopic techniques have revolutionized the visualization and biopsy of parenchymal organs. Endoscopy facilitates direct, minimally invasive examination of mucosal and serosal surfaces and permits tissues to be obtained for histopathologic and cytologic examination or fluids to be procured for laboratory evaluation. The evaluation of endoscopically obtained tissue samples can be helpful in establishing a diagnosis, prognosis, and therapeutic approach. Sequential biopsy results might be useful in monitoring the response to therapy or progression of the disease process in specific cases, although results to date have not shown consistent benefit from follow-up biopsies. Endoscopic biopsy of the gastrointestinal (GI) tract has both advantages and disadvantages. Being able to directly visualize mucosal lesions that cannot be detected from the serosal surface allows targeted biopsy of abnormal tissue to be performed. Also, the ability to obtain six to ten or more endoscopic samples over a 5- to 15-cm length of the intestinal mucosa might be more likely to reveal the presence of lesions that are focal in distribution or severity, such as lymphoplasmacytic enteritis.

However, endoscopic biopsy specimens are small and delicate pieces of tissue. Pathologists often must examine multiple, minute, poorly oriented tissue specimens that are prone to a variety of handling and processing artifacts. This is particularly true of small intestinal biopsy specimens, which, if taken with improper technique, often contain only villus fragments or lack subvillus lamina propria. In addition, pathologists must be able to recognize the wide range of normal mucosal cellularity in intestinal mucosal biopsy specimens. Historically, interpretation of these specimens has been subjective (even for commonly diagnosed disorders such as inflammatory bowel disease [IBD]) and relied heavily on the experience and bias of the pathologist performing the histopathologic evaluation. These considerations are important if a meaningful correlation of abnormal histopathology and intestinal function is to be made.

This chapter will provide commentary on the following topics:

1. An overview of practical matters relating to endoscopic biopsy

2. A summary of endoscopic accessories available to the clinician

3. Standards for performing proper endoscopic examination of the alimentary tract including reporting of findings

4. Technical concerns regarding the handling and processing of biopsy specimens

5. Succinct morphologic descriptions of disease entities diagnosed most often by endoscopic biopsy

6. Introduction to recently published World Small Animal Veterinary Association (WSAVA) guidelines for defining the histopathologic changes that occur in GI inflammation

Standardized terminology for describing endoscopic abnormalities and their correlation to histopathologic findings are also reviewed. Emphasis is placed on the evaluation of biopsy specimens from the alimentary tract of dogs and cats.

Practical Considerations for Endoscopic Biopsy

Endoscopic examination and biopsy of the GI tract can be diagnostic of many mucosal disorders. However, endoscopy is an adjunctive procedure that complements information gained from a history, physical examination, selected laboratory and imaging studies, and well-designed therapeutic trials. Endoscopic biopsy is useful in detecting morphologic disease but typically fails to detect functional disorders, including abnormal GI motility, GI secretory disorders, and biochemical injury such as brush border enzyme deficiencies. A variety of clinical disorders characterized by primary or secondary GI signs but no significant changes in tissue morphologic features are commonly encountered (Box 8-1).

The decision to perform endoscopic biopsy should be made after salient clinicopathologic variables have been considered and the procedure has been discussed with the client. Patient status, procedural time, costs, and inherent risks should all be considered. Abdominal ultrasound is typically performed before endoscopy to ensure that there are not obvious lesions extending beyond the reach of the endoscope or that there are not lesions that can be diagnosed by fine-needle aspiration. Once endoscopic biopsy is deemed appropriate, the instrumentation needed for optimal specimen collection must be considered. Different alimentary organs require the use of different diagnostic sampling instruments and techniques to yield maximum results. Esophageal biopsy is uncommonly required. If examination of the esophagus is desirable and there is not a mass lesion, then exfoliative (brush) cytology is sometimes useful. Mucosal biopsy of the stomach, small intestine, and colon is a much more common procedure and is best performed with the use of pinch biopsy forceps. Whether the mucosal disease is focal or diffuse is a major consideration. Localized lesions (e.g., ulcers, solitary masses, and malignant strictures) require precise, directed biopsy (often at the periphery of normal- and abnormal-appearing tissue) to be diagnostic. In contrast, more generalized mucosal disorders (e.g., IBD, diffuse gastritis, and diffuse neoplasia) can usually be diagnosed with random biopsy of the affected organs. Lastly, the nature of the suspected lesion (e.g., superficial versus deep mucosal disease) will influence instrument selection and the biopsy technique. Nonlymphomatus neoplastic lesions are often best sampled deeply (i.e., repeated samples from the same site) because superficial biopsy specimens typically contain necrotic surface debris and superficial inflammatory cells that obscure the correct diagnosis.

Examination and Description of the Mucosa

A thorough, systematic examination of mucosal structures should precede all biopsy procedures because postbiopsy hemorrhage may obscure lesions. Excellent descriptive summaries of normal endoscopic mucosal examinations are provided elsewhere in this book. Recently, an attempt to standardize the procedure for upper and lower GI endoscopic examinations, as well as the descriptive terminology used to record the findings of the process, was produced by the WSAVA Gastrointestinal Standardization Group. The goal of this group was to ensure that a standardized protocol for performing thorough and complete endoscopic examinations was available for clinicians to use in practice. These protocols should also prove valuable for investigators undertaking multicenter clinical trials in which consistent endoscopic examination and biopsy specimen procurement are essential. Clinicians using these forms simply check off endoscopic observations, and these forms can then become a quick and convenient part of the patient’s medical record. The forms are available online and have been endorsed by the Comparative Gastroenterology Society and the European Society of Comparative Gastroenterology (http://www.wsava.org/StandardizationGroup.htm). These forms were not intended to be the final answer; rather, they incorporate features that are deemed valuable such as tick boxes, the ability to comment on the presence/absence of all possible lesions, and an area for identification of the extent of the examination, the equipment used, and any complications associated with the procedure. Videotape or photographic records serve to complement the endoscopic report on each animal. The important features of each endoscopic procedure should be documented (Box 8-2).

Consistent endoscopic terminology has been proposed to aid in lesion description and the formulation of a definitive diagnosis (Box 8-3). Erythema denotes mucosal redness, which may be a pathologic condition or a normal physiologic response to excessive insufflation or blood flow changes associated with anesthesia. Friability describes the ease with which the mucosa is damaged by contact with the endoscope or biopsy instrument. Alterations in surface texture are described as increased granularity. Granularity of the small intestinal mucosa may be influenced by villus height and crypt depth as the light of the endoscope reflects off these structures. Mass lesions are commonly seen with infiltrative mucosal diseases (e.g., inflammatory disorders, malignant neoplasia, and benign polyps) and may be pedunculated or sessile. As previously noted, masses should be sampled deeply to avoid misdiagnosis.

GI ulceration or erosion is defined as an endoscopically visible breach in mucosal integrity, which may or may not be associated with active hemorrhage. Ulcers are typically solitary, crateriform, well-circumscribed lesions that extend deeply into the mucosa and contain central fibrinous exudate. Erosions are discrete, superficial mucosal defects without raised margins or necrotic centers. Ulcers and erosions are characteristic of inflammatory and neoplastic lesions. Erosive lesions should be sampled directly. Ulcerative lesions should usually be sampled by obtaining specimens from the ulcer rim, where it merges with adjacent tissue. Alternatively, where ulcers are associated with lesions such as scirrhous gastric carcinoma in dogs, a diagnosis may sometimes only be achieved by sampling tissue as deeply as possible within the ulcer itself. Mucosal specimens from tissues surrounding an ulcer should also be procured to characterize benign from malignant ulcerative disease. Mucosal biopsy specimens obtained from focal lesions (e.g., ulcer, masses) should be placed in a separate biopsy cassette to distinguish the histopathologic features of these lesions from the histopathologic changes in biopsy specimens obtained from normal-appearing or less involved areas of mucosa.

Miscellaneous endoscopic observations should also be noted. Inadequate distension after air insufflation may be seen with extraluminal compression, strictures, infiltrative diseases, and severe fibrosis secondary to chronic inflammation. Normal submucosal blood vessels may not be visualized because of mucosal edema, the accumulation of exudate (e.g., blood, mucus, or necrotic debris), and the infiltration of inflammatory or neoplastic cells. Gross pitting of the duodenal mucosa has been associated with acute Giardia infection in the dog. Characteristic milky-white nodules (i.e., dilated lacteals) in the mucosa or a milky exudate within the intestinal lumen may be seen in dogs with lymphangiectasia. Mucosal abnormalities should always be observed as the endoscope is advanced forward to avoid misinterpretation as a result of operator-induced trauma. Linear lesions always suggest the possibility of an iatrogenic origin as opposed to a pathologic lesion.

Instrumentation for Specimen Collection

Selection of appropriate biopsy instruments is influenced by the type of endoscope (rigid versus flexible) and the range of equipment available for specimen collection. Practical endoscopic accessories include pinch biopsy forceps, cytology brushes, and aspiration catheters for acquiring intraluminal fluids. In most clinical situations, mucosal biopsy specimens obtained with flexible endoscopic techniques are required for definitive diagnosis. The relative merits of endoscopic accessories are discussed in the following sections.

Pinch Biopsy Forceps

Flexible, pinch biopsy forceps are most commonly used to obtain mucosal specimens from the GI tract. These small, flexible forceps with opposing 2- to 3-mm cups on their distal end are passed through the operating channel of the endoscope. There are numerous configurations (i.e., cups may be smooth or serrated, standard or fenestrated, with or without a central needle; forceps may be multiple use or disposable, and some are designed to allow multiple samples to be taken before withdrawing the instrument) (Figure 8-1). Fenestrated forceps are reported to cause fewer crush artifacts and yield larger biopsy specimens than nonfenestrated models. There is a lack of uniformity of opinion between endoscopists as to the best configuration for endoscopic biopsy forceps. Biopsy forceps with central needles can help stabilize the forceps and are useful for some endoscopists but in the hands of other clinicians yield inferior tissue samples. Endoscopists will need to decide which type works best in their own hands.

Figure 8-1 Biopsy cups with pinch forceps. A, Standard forceps with a central needle. B, Serrated-jaw forceps with a central needle. C, Serrated-jaw forceps without a central needle.

(Courtesy of Jergens AE: Gastrointestinal endoscopic biopsy techniques. In August JR, editor: Consultations in feline internal medicine, ed 3, Philadelphia, 1997, Saunders.)

Recent work has shown that the diagnostic quality of the endoscopic sample influences the likelihood that a pathologist will find a specific mucosal lesion. In general, six marginal or adequate samples should be collected to detect abnormalities in the feline gastric and duodenal mucosa, whereas six adequate or 10 to 15 marginal samples should be collected from the canine stomach and duodenum, depending on the lesion being sought. To be considered adequate, a biopsy sample should contain the full thickness of the mucosa and be wide enough to have at least three to four intact and preferably contiguous villi. Specimens containing submucosa are preferred, but it is not always possible to obtain tissue at this level, especially when the mucosa is relatively thick (e.g., large versus small dog; duodenum versus ileum) (Figure 8-2).

Figure 8-2 Small intestinal biopsy specimen procured with pinch forceps from a healthy dog. Note the excellent quality of this specimen, as evidenced by numerous intact villi, perpendicular orientation of crypts to surface epithelium, and inclusion of deeper lamina propria tissue (hematoxylin and eosin stain).

There are various techniques for sampling GI mucosa with flexible endoscopic forceps. We each have our preferences, but any technique that consistently works for an individual is acceptable. The procedure may be performed solely by the endoscopist; however, having an assistant to operate the biopsy forceps is advantageous. It is generally desirable to position the endoscope tip directly in front of and perpendicular to the area to be sampled (Figure 8-3). This may be difficult or impossible in smaller patients (e.g., cats) in which the intestinal lumen is relatively narrow. The pinch biopsy instrument is passed through the operating channel and extended beyond the tip of the endoscope, the jaws are opened, and the instrument is advanced into the mucosa with the use of forward pressure. In the case of animals with a small diameter intestinal lumen (e.g., small dogs and cats), one may first advance the forceps, open them, retract the tip of the forceps against the tip of the endoscope, and then maximally deflect the tip of the endoscope obliquely into the mucosa in an attempt to achieve as perpendicular an orientation as possible. This typically makes it impossible to visualize the mucosa (i.e., a “red out”), but it may allow one to advance the insertion tube tip into the mucosa at a more nearly vertical angle. Larger, deeper, and more diagnostically significant specimens are usually obtained by application of pressure at the time of biopsy, but if so much pressure is applied that the tip of the forceps turns and does not approach the tissue directly, then inferior samples may be obtained. Larger forceps (e.g., those that can pass through an accessory channel diameter of at least 2.8 mm) usually yield better quality specimens, although areas with relatively thin mucosa (e.g., ileum and feline duodenum) may be adequately sampled with smaller forceps (e.g., those that can pass through an accessory channel diameter of 2.2 mm or less). Adequate forward pressure is evidenced by displacement of the tissue away from the endoscope and gentle “bowing” of the biopsy instrument shaft (but not the tip of the forceps). The jaws of the forceps are then closed firmly, the instrument is retracted to the endoscope tip, and a firm steady tug is used to tear a tissue sample free. Except for the gastric antrum near the pylorus, it is not necessary to forcefully jerk the forceps back into the endoscope. If a forceful jerk is deemed necessary, it is important to straighten the tip of the endoscope to help avoid damage to the biopsy channel. The forceps instrument is withdrawn from the operating channel, and the tissue specimen is carefully removed from the open jaws of the forceps for processing. Specimens may also be used to make touch imprints for cytologic evaluation.

Figure 8-3 Gastric mucosal biopsy technique using pinch forceps. Note the perpendicular placement of the biopsy forceps along the angulus. Good biopsy technique is essential in assuring a high diagnostic yield from histopathologic examination by the pathologist.

Cytology Brushes and Catheters

Disposable guarded cytology brushes are useful for obtaining cell specimens. These brushes are passed through the operating channel of the endoscope under direct guidance and are protected from contamination by an outer sheath (Figure 8-4). Once the area to be sampled is identified, the brush is extended from its sheath, rubbed vigorously against the mucosa or surface of the lesion without causing excessive hemorrhage, and then retracted into the operating channel. The brush cytology instrument is then removed from the endoscope. Superficial cells and cellular debris are then rolled or rubbed onto microscopic slides, air dried, stained with a Romanowsky-type stain (e.g., Diff-Quik), and examined microscopically for evidence of inflammation, neoplasia, or infectious agents. Alternatively, touch cytology may be performed by transferring a mucosal specimen from the biopsy forceps to a glass slide with a needle and then gently touching the specimen onto the slide so that a cytologic imprint is obtained.