Endoscopic, Biopsy, and Histopathologic Guidelines for the Evaluation of Gastrointestinal Inflammation in Companion Animals

Diagnosis and treatment of companion animal gastrointestinal tract disorders have long been complicated by the absence of clinical, diagnostic, histopathologic, and therapeutic standards. Accordingly, the World Small Animal Veterinary Association (WSAVA) International Gastrointestinal (GI) Standardization Group was convened in 2004 for the purpose of developing standards for history taking, physical examination, laboratory diagnostic tests, imaging procedures and reports, endoscopic procedures and reports, biopsy procedures and reports, histopathologic interpretation, immunohistochemistry (IHC), treatment trials, and patient response and outcome in dogs and cats with gastrointestinal disease. The Standardization Group first met at the American College of Veterinary Internal Medicine (ACVIM) Forum in Minneapolis in 2004, and several abstracts of its work were presented at national and international meetings (WSAVA Congress, European College of Veterinary Internal Medicine [ECVIM] Congress, and ACVIM Forum). A final summary of Phase I studies was presented at the WSAVA Congress in Dublin in 2008. During Phase I (2004–2008), the GI Standardization Group published proposed standards for endoscopy,1 biopsy,2 and histopathological evaluation of inflammation3 in endoscopic biopsies of the gastrointestinal tract of dogs and cats. In 2008, the GI Standardization Group was invited to develop an ACVIM Consensus Statement on "Endoscopic, Biopsy, and Histopathologic Guidelines for the Evaluation of Gastrointestinal Inflammation in Companion Animals" for presentation at the 26th Annual ACVIM Forum in San Antonio, TX. After presentation at the ACVIM Forum, a written draft of the Consensus Statement was prepared by the Group and posted to the ACVIM Website for additional commentary from the membership of the ACVIM Internal Medicine Specialty. The manuscript was further independently reviewed by a series of experts in the field. A revised manuscript was submitted to the ACVIM Board of Regents and editors of the Journal of Veterinary Internal Medicine for final review and approval. An evidence-based medicine approach was used by the Group to develop the Consensus Statement. Where evidence was conflicting, ambiguous, or lacking, the Group adopted interpretive recommendations on the basis of its collective expertise. IBD broadly refers to a group of idiopathic, chronic gastrointestinal disorders characterized by mucosal inflammation.4, 5 Although the prevalence of IBD is unknown, it is arguably the most common histopathologic diagnosis obtained in dogs and cats with chronic vomiting or diarrhea. Despite perceived importance, the accuracy of these diagnoses has been the subject of some contention. IBD may represent one or more forms of chronic enteropathy that are distinguished from food-responsive and antibiotic-associated causes by their therapeutic responsiveness to immunosuppressive agents but not to dietary or antibiotic therapy alone.5 Although the underlying cause of IBD remains unknown, accumulating evidence in animal models suggests that intestinal inflammation results from altered interaction between gut microbes and the mucosal immune system in a susceptible host.6-8 Aggressive host immune responses directed against bacteria or their products are believed to play a central role in the pathogenesis of chronic mucosal inflammation.6 The concept of impaired immunoregulation in IBD is supported by observations of increased numbers of immunoglobulin-secreting plasma cells and T cells in inflamed tissues,9-13 upregulated mucosal or luminal expression of nitric oxide metabolites,5, 14 and altered serum concentrations of selected acute phase proteins, such as C-reactive protein (CRP), in diseased dogs.15, 16 Genetic predispositions are recognized in several breeds, including Siamese cats, and German Shepherd Dogs, Basenjis, soft-coated Wheaten Terriers, and Shar Peis.4, 5, 17 Diagnosis of IBD currently is defined by (1) chronic (ie, > 3 weeks) persistent or recurrent gastrointestinal signs; (2) histopathologic evidence of mucosal inflammation; (3) inability to document other causes of gastrointestinal inflammation; (4) inadequate response to dietary, antibiotic, and anthelmintic therapies alone; and (5) clinical response to anti-inflammatory or immunosuppressive agents. Clinical signs (eg, vomiting, small bowel diarrhea, large bowel diarrhea, weight loss, alterations in appetite) are attributed to mucosal cellular infiltrates, inflammatory mediators, and inflammation-associated enterocyte dysfunction and intestinal dysmotility.4, 5, 18, 19 Histopathologic evaluation of biopsy specimens is required for definitive diagnosis, but no standard microscopic grading system of IBD lesions has been universally accepted. Histopathologic examination is performed to distinguish normal from diseased tissue, characterize the nature and severity of tissue changes, and provide an accurate morphological or etiological diagnosis, thus facilitating formulation of prognosis and appropriate therapy. Some histopathological diagnoses (eg, adenocarcinoma) can be made relatively simply. By contrast, interpretation of mucosal inflammatory changes, and distinguishing them from alimentary lymphoma has proved to be far more complex. Characterization of gastrointestinal inflammation has been hampered by lack of accepted, standard criteria for measuring the histopathological changes within a sample of mucosal tissue. Over the past 2 decades, several independent groups have developed and applied classification systems for characterizing the nature and severity of gastrointestinal inflammatory changes.4, 9-13, 17, 20-33 In most of these studies, the nature of gastrointestinal inflammation is portrayed primarily by the dominant population of inflammatory cells (eg, lymphoplasmacytic, eosinophilic, pyogranulomatous) within the lamina propria. Such populations, however, may overlap and occur in various combinations and patterns. In many instances, the morphologic or cytoarchitectural changes of the epithelium and mucosa have been inappropriately underemphasized. The severity of gastrointestinal inflammation usually has been graded by a simple 4-point scale (ie, normal, mild, moderate, marked or severe). Although this approach appears logical, the specific criteria defined by various groups have differed so that it is impossible to conclusively compare the histopathological changes described in different studies. Even when specific criteria are applied, substantial variation may occur among pathologists' interpretations of changes in gastrointestinal tissue samples. Willard et al,34 for example, reported lack of uniformity in the assessment of 50% of biopsy samples examined by 5 veterinary pathologists. This interpretive variation poses problems for the routine diagnosis of gastrointestinal disease as well as for monitoring the progress of patients undergoing posttherapeutic endoscopy. Moreover, multicenter diagnostic and therapeutic clinical trials are not possible with such variation. With this background, a GI Standardization Group was convened with the support of the WSAVA, with the purpose of developing standards for the diagnosis and treatment of gastrointestinal diseases in the dog and cat. One of the 1st tasks of this group was to develop a consensus on the normal histology of the gastrointestinal tract with the subsequent aim of developing a set of histopathological standards for the nature and severity of mucosal inflammatory and associated morphological changes. The normal histology of the canine and feline gastrointestinal tract is affected by variables such as developmental stage (eg, age of the animal,35, 36 dietary history, medication history) and therefore remains the subject of considerable controversy. Lack of agreement on normal histology has been one reason for erroneous diagnosis of gastrointestinal inflammation in many veterinary patients. Lack of agreement on standards for normal histology also has limited universal acceptance of grading systems in the evaluation of IBD. The GI Standardization Group used an evidence-based medicine approach37 to establish a reference range for normal histologic findings in the gastrointestinal tract of dogs and cats. Several examples of Class II and III evidence-based data were found in the Group's review of the scientific literature. Most of the studies employed microscopic evaluation of hematoxylin and eosin (HE) stained tissues,21-23, 35-39 whereas others used IHC to label and count leukocyte populations.11-13, 40-48 Representative examples from the GI Standardization Group's archives have been published already,3 but summaries of studies in each anatomic area follow. Two studies have characterized the leukocyte subpopulations within the superficial region of the normal canine gastric fundic mucosa.41, 48 In one of these studies,48 a "mucosal unit" was defined as a 250 μm length of mucosa, in which CD3+ intraepithelial lymphocytes (IEL) (mean, 0.9; range, 0–2), CD3+ lamina propria lymphocytes (mean, 4.2; range, 0.5–13), lamina propria eosinophils (mean, 0.5; range, 0–2), and lamina propria plasma cells (mean, 1.6; range, 0–5.8) were enumerated. Biopsy samples were derived from 8 dogs, in which considerable interanimal variation in cell counts was noted. The leukocyte subpopulations within the superficial region of the normal canine antral mucosa have been characterized in 2 studies.41, 48 In one of the studies,48 a "mucosal unit" was defined as a 250-μm length of mucosa, in which CD3+ IEL (mean, 4.4; range, 1.5–8), CD3+ lamina propria lymphocytes (mean, 10.7; range, 2.5–16.5), lamina propria eosinophils (mean, 2.7; range, 0–6) and lamina propria plasma cells (mean, 6.8; range, 0.5–15.5) were enumerated. Biopsy samples were derived from 8 dogs in that study, in which considerable interanimal variation in cell counts was noted. Several studies have evaluated the normal canine and feline duodenal mucosa with HE and immunohistochemical staining.43, 47, 49, 50 The normal villus length for an adult dog is 722 ± 170 μm, the normal crypt depth is 1,279 ± 203 μm, and the normal villus to crypt ratio is 0.7 ± 0.3.39, 49, 50 Normal dogs have a mean number of 3.6 ± 3.6 goblet cells per stretch of 100 villous enterocytes, and 9.3 ± 3.1 goblet cells per stretch of 100 cryptal enterocytes.43 Villous IEL are less numerous in the dog (20.6 ± 9.5 per 100 enterocytes) than in the cat (47.8 ± 11.7 per 100 enterocytes), but the number of cryptal IEL in the dog (5.2 ± 2.3 per 100 enterocytes) is similar to that in the cat (4.6 ± 1.7 per 100 enterocytes).43, 47, 50 In the dog, the total leukocyte count is greater in the cryptal lamina propria (156.3 ± 24.9 per 10,000 μm2) than in the lamina propria of the base (128.3 ± 26.6 per 10,000 μm2) or tip (100.7 ± 43.9 per 10,000 μm2) of the villus.43 Similarly, there are more eosinophils in the canine cryptal lamina propria (9.8 ± 7.5 per 10,000 μm2) than in the lamina propria of the villus base (3.7 ± 3.5 per 10,000 μm2) or tip (3.8 ± 6.1 per 10,000 μm2).43 In cats, a population of globular leukocytes sometimes is recognized within the intestinal epithelium. These cells have distinctive eosinophilic granules within the cytoplasm and express the molecule perforin as shown by IHC labeling with crossreactive antisera.51 This observation suggests that the cells are granular lymphocytes with cytotoxic function. In general, these cells do not appear to increase in number in feline inflammatory enteropathy, but neoplasia of this lineage is documented.46 In the colonic mucosa, there are, on average, 7.7 ± 3.7 IEL per stretch of 100 colonocytes in the normal canine basal crypt epithelium.43 In the lamina propria between the basal crypts of the canine colon there are approximately 5.5 ± 4.3 plasma cells and 3.8 ± 3.7 eosinophils per 10,000 μm2.20, 43, 44 Some studies have assessed the number of goblet cells in normal canine colonic cryptal epithelium (25.6 ± 7.3 per 100 colonocytes).43, 44, 52 The GI Standardization Group recognized that measurement of goblet cells in colonic epithelium is not straightforward and that the number of such cells may be artifactually decreased by discharge of mucus during the biopsy process. For that reason, assessment of alteration in goblet cell number (specifically goblet cell hyperplasia) was not incorporated into the final version of the standard template. The recognition and interpretation of inflammatory change in endoscopically derived biopsies of gastrointestinal tract mucosa historically has posed great challenges for veterinary pathologists. Fundamental questions in this diagnostic process include the following: (1) Are the biopsies of sufficient size and quality for accurate diagnosis? (2) What is the nature of the inflammatory response (eg, neutrophilic, eosinophilic, granulomatous, pyogranulomatous or lymphoplasmacytic)? (3) How severe is the inflammatory response? and (4) When may an inflammatory response be a precursor to lymphoid neoplasia? Given these limitations in the microscopic examination of HE-stained sections, it has been suggested that IHC evaluation of mucosal biopsies might provide a more accurate means of assessing inflammation. When coupled with computer-aided morphometry (ie, counting numbers of cells of specific phenotypes per unit area of lamina propria or epithelium), subtle changes in cellular content may be identified in tissue that may not be regarded as abnormal on evaluation of HE-stained sections.10 It is unlikely, however, that this time-consuming and costly procedure will become standard for routine clinical diagnosis. In response to these limitations, the GI Standardization Group developed guidelines for the standardized interpretation of inflammatory change in the gastrointestinal mucosa of the dog and cat.3 These recently published guidelines provide a simple visual and textual description of the major inflammatory changes in the gastric body and antrum, duodenum and colon, and define what constitutes mild, moderate, and severe pathological change. The guidelines are applicable to tissues from both dogs and cats; the only distinction between the species being with respect to the numbers of duodenal IEL, which are greater in cats compared with dogs.43, 47 The guidelines are designed to be used "microscope-side" by veterinary pathologists and define changes at the level of the 40 × microscope objective, which is considered to be the magnification at which most pathologists will refine their morphological diagnosis. Morphologic and inflammatory changes typical at each of the 4 anatomic sites are outlined in Tables 1–4. The guidelines adopted by the GI Standardization Group are accompanied by a set of standard reporting forms, which encourage pathologists to evaluate biopsies and record findings in a consistent fashion (Appendices 1 and 2). The forms could serve as the basis for numerical scoring of inflammatory changes as would be undertaken in research investigations. Pathologists are encouraged to report the total number of tissue samples present on the microscope slide and document the quality of these samples using descriptions of "adequate,""marginal," and "inadequate" as defined by the GI Standardization Group.2 If such information is not included in a biopsy report, the group recommends that clinicians specifically request that it be included in the final report. The importance of this request will be seen below when the effect of biopsy quality upon diagnosis is discussed ("Guidelines for Endoscopic Examination and Biopsy"). These histopathology guidelines have been presented to the clinical and research community for evaluation and the GI Standardization Group anticipates that they will be continually refined. The GI Standardization Group pathologists have used the guidelines to evaluate a large slide set, derived from both dogs and cats from 9 referral institutions in 6 different countries. The Group currently is using these guidelines to identify factors affecting interpathologist variation and histologic lesions associated with hypoalbuminemia. Although the interpretation of endoscopically obtained biopsies of gastrointestinal mucosa will remain a diagnostic challenge, acceptance and refinement of the GI Standardization Group's guidelines should help address current problems related to lack of standardization. Additional studies will be needed to evaluate the relative importance of each criterion and whether a weighted or nonweighted cumulative score is appropriate. The ultimate value of any grading system will be determined by its ability to accurately diagnose disease, direct therapy, and predict outcome. Representative tissue samples containing lesions of interest are crucial for the diagnosis of most gastrointestinal tract diseases. There are 3 means of obtaining such biopsies: flexible endoscopy, laparoscopy, and surgery. Flexible endoscopy has 5 advantages. (1) Endoscopy permits the operator to see mucosal changes that cannot be visualized by the serosal approach of the surgeon. This in turn permits directed biopsy at these sites. (2) Endoscopy also permits the collection of multiple tissue biopsies (eg, 10 or more, if necessary) from each site, which is potentially important because some diseases may have a multifocal distribution, even within 1 section of the intestine. (3) In some instances, endoscopy permits diagnosis of selected lesions without the need for tissue biopsy (eg, ulceration, erosion, lymphangiectasia). (4) Endoscopic procedures have minimal risk of perforation and septic peritonitis, compared to surgical biopsy. (5) The procedure is quicker, less stressful, and less invasive to the patient, and may be less expensive than surgery. Flexible endoscopy has some disadvantages. Standard duodenoscopy cannot access the entire gastrointestinal tract (although enteroscopy could), and duodenoscopy alone in animals with severe gastrointestinal tract disease might not permit detection of the most important lesions. It is very easy to obtain inadequate tissue samples (eg, mostly tips of villi) that do not readily permit diagnosis. Finally, even well-trained endoscopists cannot reliably sample duodenal muscularis mucosa or dense, submucosal infiltrative lesions with flexible endoscopic forceps. Endoscopy is not necessarily appropriate for every animal with chronic gastrointestinal disease. It is impossible to make an all-encompassing list of when to do and when not to do gastrointestinal endoscopy. Substantial latitude must be given to the clinician who continually must weigh the specifics of the case, client expectations, monetary concerns, risk to the patient, and other factors. Nonetheless, certain general principles can be stated. First, endoscopy seldom benefits patients with acute diarrhea (ie, <3 weeks in duration) unless the disease is particularly severe or a specific disease needs to be quickly diagnosed or eliminated (eg, histiocytic ulcerative colitis, histoplasmosis, neoplasia). Second, clinical assessment usually seems more useful and appropriate than endoscopic biopsy in determining response of inflammatory diseases to therapy. Third, endoscopy is primarily of value in diagnosing infiltrative, erosive, or other anatomic (eg, lacteal dilatation, foreign body) problems. It seldom allows diagnosis of dietary-responsive enteropathy, antibiotic-responsive diarrhea, or gastrointestinal motility disorders. The the patient (ie, to no weight loss, relatively body normal serum not not no evidence of infiltrative the more should be given to therapeutic trials (eg, dietary, antibiotic, or of endoscopic at the more the patient (eg, severe weight loss, very body evidence of infiltrative the more it usually is to endoscopic biopsy therapeutic if the it is to the endoscopy in an to that infiltrative lesions of of the (eg, are not When endoscopy is and examination of the small and large is the 1st endoscopic report forms have been developed that a and examination of the gastrointestinal endoscopy forms have several include patient and reason for specific used (ie, biopsy foreign body of examination (ie, far the was of specific and final are in such report forms so as to document whether specific lesions were or were not seen and whether specific problems or not also are useful in to that are Such forms, developed by the GI Standardization have been by the and the European for and are at the WSAVA Website are included in 3 and 4 of this Consensus Statement. biopsy is being recognized as potentially information not found in duodenal or colonic The usually can obtain biopsies by the into the or biopsy the in dogs and cats. should be given to obtaining biopsies in animals or seems the Group has to for tissue. tissue quality is as important as endoscopic because tissue samples may not be by There are many studies and recommendations and for endoscopic biopsy of the gastrointestinal but similar data are not as readily for the dog and cat. Some may be It seems that (eg, biopsy and tissue samples than (eg, forceps. tissue quality upon mucosal Willard et reported that duodenal biopsy quality was between dogs and cats, the that feline intestinal biopsies the of and biopsy forceps. The feline duodenal mucosa may be more readily even into the muscularis mucosa, than the canine duodenal This observation also may the mucosa is more readily than the duodenal in tissue quality may from in sample as well as tissue in the diagnostic tissue samples should be from the biopsy and submitted in such a as to and to permit tissue in the The clinician must and work with the laboratory to tissue on because the approach may from laboratory to for and tissue samples have been described If samples are to be submitted in the only to they will be is for the histopathology to each of tissue with a microscope as they are biopsy samples are needed to establish a diagnosis as the quality of the tissue from inadequate to to there are some important between the dog and the cat. 6 or tissue samples from the feline or duodenum are sufficient to diagnose villus and to cellular In the dog, however, approximately or gastric or duodenal tissue samples are required to reliably diagnose villus and or cellular duodenal crypt lesions are more to diagnose and approximately or samples may be The numbers will change as more studies evaluate this but samples the diagnostic of the the total number of tissue samples that should be during a procedure will upon the of the In general, endoscopists must samples than endoscopists to the number of samples on the histology The of diagnosis on the quality of the tissue samples the that clinicians should upon including both the total number of tissue samples submitted and the quality of these samples (ie, to the level of in the reported diagnosis If most of the samples are inadequate or the clinician should or for If the clinician could request a on the to their of a biopsy sample of canine villus tips are This is considered an "inadequate" tissue and eosin with of a biopsy sample of canine This is an of a tissue and eosin with of a biopsy sample of canine This is an of an tissue sample that has at 3 and the entire depth of the intestinal mucosa as seen by lamina which to the mucosa Even muscularis mucosa is not the of the tissue sample that it to the muscularis and eosin with The clinical of IBD is characterized by and or Gastrointestinal signs are and may upon and anatomic of the disease. Several clinical have been developed to IBD in dogs including clinical 5 histopathologic of mucosal of immune and measurement of inflammatory such as of nitric acute phase such as serum and altered expression of 17, for in the cat have been described only Clinical remain the most used in assessing disease A clinical scoring (ie, disease has been used to disease to histopathologic findings and serum In that study, clinical with a of histopathologic severity and at the of histopathologic assessment was not In clinical signs and endoscopic lesions in dogs in dogs with and but treatment not in changes in the severity of gastric or duodenal histopathologic lesions. The between histopathologic change and clinical findings has been or in other studies. et that total numbers in the duodenal mucosa of dogs with IBD not change treatment with et to a between of therapy by and severity of histologic lesions. a dogs with chronic enteropathy to an between severity of histologic changes and outcome 3 in in any of these studies may to the of histologic scoring systems or in The WSAVA GI Standardization Group has reported that one specific histologic change (ie, lacteal was associated with In a review of the evidence currently has not identified a between clinical findings and histopathologic lesions in dogs with when changes in disease are compared to histopathologic There is some evidence that dogs with IBD accompanied by increased concentrations are more to have histologic lesions than dogs only clinical and dogs with are more to have certain histologic These findings the that endoscopic biopsy is important to document inflammation (ie, 1 of the 4 criteria needed to diagnose but cannot be used by to diagnose or establish a prognosis in these patients. In cats with a