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  Table of Contents    
GUIDELINE PAPER  
Year : 2021  |  Volume : 64  |  Issue : 5  |  Page : 8-31
Best practices of handling, processing, and interpretation of small intestinal biopsies for the diagnosis and management of celiac disease: A joint consensus of Indian association of pathologists and microbiologists and Indian society of gastroenterology


1 Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Pathology, Post Graduate Institute of Medical Sciences and Research, Chandigarh, India
3 Department of Pathology, Lokmanya Tilak Municipal General Hospital Sion Hospital, Mumbai, Maharashtra, India
4 Department of Pathology, Govind Ballabh Pant Institute of Postgraduate Medical Education and Research, New Delhi, India
5 Department of Pathology, School of Medicine, Amrita Vishwa Vidyapeetham, Kochi, Kerala, India
6 Department of Pathology, King Edward Memorial Hospital and Seth Gordhandas Sunderdas Medical College, Mumbai, Maharashtra, India
7 Department of Pathology, Asian Institute of Gastroenterology and AIG Hospitals, Hyderabad, Telangana, India
8 Department of Pathology, St. John's Medical College, Bangalore, Karnataka, India
9 Department of Pathology, Institute of Liver and Biliary Sciences, New Delhi, India
10 Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
11 Department of Pathology, Christian Medical College, Vellore, Tamil Nadu, India
12 Department of Pathology, KPC Medical College and Hospital and Peerless Hospital, Kolkata, West Bengal, India
13 Department of Pathology, Armed Forces Medical College, Pune, Maharashtra, India
14 Department of Pathology, Chacha Nehru Bal Chikitsalya, New Delhi, India
15 Department of Gastroenterology, Govind Ballabh Pant Institute of Postgraduate Medical Education and Research, New Delhi, India
16 Department of Gastroenterology, Kuwait Hospital, Sharjah, UAE
17 Department of Gastroenterology, Post Graduate Institute of Medical Sciences and Research, Chandigarh, India
18 Department of Gastroenterology, Institute of Post Graduate Medical Education and Research, Kolkata, West Bengal, India
19 Department of Gastroenterology, SRM Institute of Medical Sciences, Chennai, Tamil Nadu, India
20 Department of Gastroenterology, Dayanand Medical College and Hospital, Ludhiana, Punjab, India
21 Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
22 Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India
23 Department of Pathology, MLN Medical College, Allahabad, Uttar Pradesh, India

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Date of Submission06-Dec-2020
Date of Decision11-Dec-2020
Date of Acceptance07-Jan-2021
Date of Web Publication7-Jun-2021
 

   Abstract 


The Indian Association of Pathologists and Microbiologists (IAPM) and Indian Society of Gastroenterology (ISG) decided to make a joint consensus recommendation for handling, processing, and interpretation of SI biopsies for the diagnosis and management of celiac disease (CD) recognizing the inhomogeneous practice of biopsy sampling, orientation, processing, and interpretation. A modified Delphi process was used to develop this consensus document containing a total of 42 statements and recommendations, which were generated by sharing the document draft, incorporating expert's opinion, followed by three cycles of electronic voting as well as a full-day face-to-face virtual ZOOM meeting and review of supporting literature. Of the 42 statements, 7 statements are on small intestinal (SI) biopsy in suspected patients of CD, site and the number of biopsies; 7 on handling, fixative, orientation, processing, and sectioning in pathology laboratories; 2 on histological orientation; 13 statements on histological interpretation and histological grading; 3 on the assessment of follow-up biopsies; 2 statements on gluten-free diet (GFD)-nonresponsive CD; 4 on challenges in the diagnosis of CD; 2 statements each on pathology reporting protocol and training and infrastructure in this area. The goal of this guideline document is to formulate a uniform protocol agreed upon both by the experienced pathologists and gastroenterologists to standardize the practice, improve the yield of small bowel biopsy interpretation, patients' compliance, overall management in CD, and generate unified data for patient care and research in the related field.

Keywords: Celiac disease, guideline, mimickers, mucosal biopsies, orientation, pathology, processing, recommendations, reporting format, small intestine, training

How to cite this article:
Das P, Vaiphei K, Amarapurkar AD, Sakhuja P, Nada R, Paulose RR, Chaturvedi R, Sekaran A, Kini U, Rastogi A, Kumari N, Pulimood A, Banerjee M, Kinra P, Singh L, Puri A, Pai G, Kochhar R, Dhali GK, Ramakrishna B S, Sood A, Ghoshal UC, Ahuja V, DattaGupta S, Makharia GK, Misra V. Best practices of handling, processing, and interpretation of small intestinal biopsies for the diagnosis and management of celiac disease: A joint consensus of Indian association of pathologists and microbiologists and Indian society of gastroenterology. Indian J Pathol Microbiol 2021;64, Suppl S1:8-31

How to cite this URL:
Das P, Vaiphei K, Amarapurkar AD, Sakhuja P, Nada R, Paulose RR, Chaturvedi R, Sekaran A, Kini U, Rastogi A, Kumari N, Pulimood A, Banerjee M, Kinra P, Singh L, Puri A, Pai G, Kochhar R, Dhali GK, Ramakrishna B S, Sood A, Ghoshal UC, Ahuja V, DattaGupta S, Makharia GK, Misra V. Best practices of handling, processing, and interpretation of small intestinal biopsies for the diagnosis and management of celiac disease: A joint consensus of Indian association of pathologists and microbiologists and Indian society of gastroenterology. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Jun 17];64, Suppl S1:8-31. Available from: https://www.ijpmonline.org/text.asp?2021/64/5/8/317903





   Introduction Top


Celiac disease (CD) is a major public health problem with a pooled global seroprevalence of 1.4%, including 0.7% of biopsy-confirmed CD worldwide.[1] Its prevalence in Asia and India are 0.5% and 0.67%, respectively.[2] In India, its seroprevalence is highest in northern (1.23%) and north-Eastern states (0.87%) than in southern parts (0.10%).[3] The Indian Council of Medical Research (ICMR) initiated and supported Indian Guidelines on CD, which recommends diagnosing CD based on a combination of positive celiac-specific serology and demonstration of villous abnormalities of modified Marsh grade 2 or more on small intestinal (SI) biopsies.[4] Therefore, obtaining a duodenal mucosal biopsy is an integral part of the workup for patients with CD. However, the protocol of biopsy processing, orientation, and methods of interpretation varies widely leading to heterogeneity in reports. Due to the recognition of the high CD burden and its expanding extra-intestinal manifestations, pathologists from both Institutional and stand-alone laboratories are getting SI biopsies from suspected patients. Improper handling of biopsies will impart artifacts, impact interpretation, generate heterogeneous patient information, and make management difficult especially when a patient relocates to another city. Additionally, the high interobserver disagreement (up to 50%) reported in the histological interpretation of SI biopsies and grading is worrisome.[5],[6] A need was perceived for the standardization of the processing and interpretation protocols of proximal SI mucosal biopsy samples. The Indian Association of Pathologists and Microbiologists (IAPM) and the Indian Society of Gastroenterology (ISG) decided to make a joint consensus recommendation for handling, processing, and interpretation of SI biopsies for the diagnosis and management of CD. The authors believe that this joint consensus will not only refine and standardize the laboratory practices of the collection and processing of SI and further interpretation and bring uniformity.


   Methods Top


The areas covered in this guideline are based on the data collected from a Nationwide survey under the aegis of the IAPM and ISG. A set of 40 questionnaires were divided into 3 sections covering different aspects of biopsy handling, processing, and interpretation and circulated among 2000 registered members of the IAPM using the Survey Monkey platform. For most questions, the participants were asked if they strongly agree, agree, neither agree nor disagree, disagree or strongly disagree with the options provided and a total of 261 IAPM members completed the survey, whereas the others mentioned that they do not practice GI Pathology and others did not respond or given reasons for not participating. In summary, it was observed that only 40% of the biopsy fragments are filter paper mounted and separately labeled as per the site of biopsy sampling and about 50% of routine SI biopsy load is either not oriented or unfit for interpretation as stated by the participants. There were heterogeneity of site of proximal SI biopsies, number of biopsies sampled, clinical and endoscopic details received with samples, method of orientation, processing, sectioning, number of step sections examined, method of histopathological assessment of IELs, crypt depths (Cd), and Villous heights (Vh), histological grading of biopsies, and assessment of follow-up biopsies post-GFD. Unanimously 99.5% of the participants said that a uniform protocol agreed upon both by the pathologists and gastroenterologists is the need of the hour to standardize pathology practice, which will impact the patient compliance and management, as well as generate unified data for patient care and research in related filed.

Methods of formulating this guideline: A modified Delphi process was followed and this guideline document was made on the approval of the Executive Council and General Body of the IAPM and written approval from the Secretary-General of the ISG.[7] Thereafter, under the chairmanship of the current President and Past-President of the IAPM and the current Secretary-General ISG, a core committee was formed comprising of senior gastrointestinal pathologists and gastroenterologists with proven expertise in this field. A project coordinator was selected who, with the approval of the committee, formulated the questions and coordinated all activities related to the survey, formulated guideline draft, made revisions as per the members' suggestions, conducted online and final virtual voting for each recommendation mentioned in this document. A systematic review was performed on the relevant areas based on literature searched in the MEDLINE, EMBASE, and regional and International consensus statements and guidelines given on CD. The issues identified and discussed with the core committee members were detailed and uniform and acceptable terminologies to be used in this guideline document were formulated and finalized with the consent of all committee members [Table 1]. Voting was done anonymously.
Table 1: Uniform Terminologies used in this document

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The categorization of evidence, classification of recommendation, and voting scheme were according to the Canadian Task Force on the Periodic Health Examination [Table 2].[8] A consensus statement was 'completely' accepted or 'accepted with some reservation' when voted favorably by ≥80% of members, and a statement was rejected when ≥80% of the members rejected a statement 'completely' or 'with some reservation'. Each statement was graded to indicate the level of evidence available and the strength of recommendation. There were three rounds of online voting by the core-group members and an online final face-to-face meeting to discuss each statement followed by real-time voting to finalize the document.
Table 2: Criteria for defining the quality of evidence, type of recommendations, and voting on recommendations

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Target group to follow this guideline: This guideline is recommended to be followed and implemented by all centers and laboratories irrespective of their affiliations and size who deal with the SI biopsies from patients suspected of CD. In this guideline, a few recommendations have also been suggested for the Gastroenterologists/Endoscopist colleagues and it is expected that they will also follow these recommendations to help the pathologists to achieve a meaningful outcome of SI biopsy assessment. After the final approval of the draft guideline by the core-committee, the final guideline was published in Society Journals, displayed on the respective society's websites, and mailed to the participating members of the preceding Survey.

The Consensus Statements and Recommendations on handling, processing, and interpretation of SI biopsies for the diagnosis and management of celiac disease

1. Historical perspectives and SI biopsies in CD



Over the past 70 years, the diagnostic method of CD has changed substantially. Being an enteropathy, demonstration of mucosal histological changes post-gluten intake was considered the mainstay of diagnosis in CD. Before the 1970s, whereas the 3-biopsy approach was the standard before and after the gluten intake, with the availability of celiac-specific serological tests as anti-gliadin antibody (AGA) and anti-reticulin antibody enzyme-linked immunosorbent assay (ELISA) kits, followed by anti-endomysium (anti-EMA) and anti-tissue transglutaminase (tTG) antibody kits, gradually the focus shifted towards less invasive and a combined clinical-serological-histology based diagnostic approach. Gradually, the European Society of Pediatric Gastroenterology and Nutrition (ESPGHAN) criteria published in 1990 with one biopsy approach became the accepted norm for diagnosis both in pediatrics and adults. In early 2000, anti-tTG screening became the standard of practice, although histological confirmation was required. In 2012, the revised ESPGHAN criteria recommended a non-biopsy approach of diagnosis, especially in symptomatic children based on the serum anti-tTG titer of >10 times upper normal limit, positive anti-EMA antibody test performed on a second blood sample in patients having HLA-DQ2/DQ8 alleles, with the consent of patient's first-degree-relatives to use the 'no-biopsy approach' for diagnosis in children consuming gluten before the biopsy. This approach was found to have a 99% positive predictive value (PPV) as assessed by an International prospective study group,[9],[10] and was later adopted by the British Society for Pediatric Gastroenterology, Hepatology, and Nutrition, with the modification of replacing the anti-EMA test with a second line anti-tTG test using an ELISA kit of different principle than that was used initially.[11] The no-biopsy approach in children was favored as the endoscopic biopsies needed multiple passes for collecting >2 mucosal fragments under anesthesia, with inherited risks of invasive procedure and added high-cost of diagnosis.[12] The American Gastroenterology Association (AGA) recommended intestinal biopsy in patients with strong clinical suspicion of CD even when the serology is negative, whereas the United States National Institute of Health (NIH) recommended a biopsy only when the serological tests are doubtful.[13] The ICMR also favored the evaluation of intestinal biopsies for mucosal changes along with other tests for establishing a diagnosis of CD in adults.[4] Variability of the outcome of commercially available anti-tTG ELISA kits, lack of facilities for doing HLA haplotyping in India, and the question of affordability were the justifications for this recommendation. Besides, HLA-typing has a low PPV in the diagnosis of CD, and the HLA-DQ2/DQ8 haplotype/s is/are present in 13–30% of the Indians of which only <1% develop CD.[4] In a study from the Central European region on the utility of intestinal biopsies in children, 20.6% of children with CD were diagnosed with 'no biopsy approach', and 51.9% of symptomatic children who underwent intestinal biopsy were also later assessed to be eligible for no-biopsy approach; however, the interval to diagnosis was significantly longer in children with 'no-biopsy approach' than in whom biopsy was done.[14] HLA haplotyping in the 'no-biopsy approach' was responsible for this delayed diagnosis, and when it was excluded, the interval to diagnosis was similar for both the 'no-biopsy' and 'with-biopsy' approaches.[15] However, intestinal biopsies in these patients can rule out a false positive anti-tTG ELISA test (in about 10%) and co-existing SI pathologies, not uncommon in South Asian countries.

Symptoms of malabsorption, diarrhea, weight loss, or anemia, are not specific to CD and may be seen in other disorders also. Although anti-tTG serology titer >5-times upper normal limit (UNL) is mostly pathological, anti-tTG titers between 2 and 5 times UNL, and <2 times the UNL are dubious, and in such scenarios a SI biopsy is necessary.[16] Kori M et al. identified mucosal pathological findings of CD in 17.4% of anemic children who underwent routine endoscopic biopsies for evaluation of anemia.[17] Although it is possibly right that the no-biopsy approach works well in children aged <3 years old, in older children experts differ in their approach to diagnosis. Especially in India and Asia Pacific region, gastroenterologists prefer taking SI biopsies in suspected patients of CD when the age of children is >10 years in addition to serological assessment. Gluten challenge is not an easy option in all centers and needs patients and their parents' cooperation.[18] The problem using only serological tests for diagnosis is the development of transient immunity to gluten peptide or false- positivity in patients with other chronic autoimmune diseases having high serum proteins and an endoscopic biopsy can identify the etiology or rule out CD.[19] Studies have shown that combining serological tests based on different principles can help in diagnosis in up to 78% of patients without a biopsy; however, an intestinal biopsy is indispensable in cases showing discordance among various serological tests and in patients with IgA deficiency.[20] A SI biopsy can also help to assess mucosal response to GFD on follow-up intestinal biopsies. Although the symptomatic improvement is achieved within a few weeks of starting GFD, the mucosal healing can take months to a few years to become normal.[21] SI biopsies also would be instrumental when the patients primarily present with dermatitis herpetiformis (DH), non-celiac gluten sensitivity (NCGS), extra-intestinal manifestations as in ataxia to rule out the possibility of CD and in children with IgA deficiency or patients with borderline high serology titer. However, the availability of IgG anti-deamidated gliadin peptide (anti-DGP) ELISA kit has high sensitivity and specificity for diagnosing CD especially in children who are IgA deficient. Overall, with expanding spectrum and recognition of extra-intestinal manifestations of CD even in the absence of primary GI symptoms, the number of patients undergoing diagnostic workup including SI biopsies is likely to increase in near future; hence, pathologists are expected to encounter more proximal SI biopsies for assessments.

2. Site of SI biopsies in patients suspected of CD



In patients with CD, the SI mucosa is said to be involved in a craniocaudal direction and the resolution of pathological changes occurs in the opposite direction.[22],[23] Most changes of CD have been documented in post ampullary parts of the duodenum (D2) and at duodenojejunal flexure. Duodenum is also a convenient site for taking endoscopic biopsies.[24] But, in about 10% of cases, the duodenal bulb is the primary site of the disease, either isolated (often named as ultra-short segment CD) or in combination with the involvement of the distal duodenum.[25] Duodenal bulb biopsies particularly those from 9 o'clock and 12 o'clock regions, with 4 mucosal fragments from the distal duodenum (D2) show histological changes of CD in almost all cases.[26],[27],[28] Mooney PD et al. found that the addition of a D1 biopsy fragment from any site was superior to a distal duodenal biopsy alone, increasing the diagnostic yield by 9.3%–10.8% (P < 0.0001).[27] However, interpretation of duodenal bulb biopsies is challenging because of mucosal stretching by submucosal Brunner's glands [Figure 1]a, or inflammatory changes inflicted by the gastric or pancreatic juice reflux.
Figure 1: A duodenal bulb biopsy shows artifactual effacement of mucosa over Brunner's gland lobules [a x40]. Typical leafy villi in distal duodenum (arrows) [b x100], and villi with branching or fused tips (arrows) may lead to erroneous impression [c x100]. Figure D shows patchy pathological changes in patients with celiac disease, whereas the left circle shows changes of advanced celiac disease, the circle in the right shows relatively unremarkable villi [d x 40]

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Like duodenum, jejunal biopsies also show similar histological changes in CD. However, there are differences in the shape of villi and pathological cut-off of IELs between the duodenum and jejunum. Although the jejunal villi are finger-shaped, the duodenal villi are usually leaf-shaped. The proximal jejunal villi are taller and the distal jejunal villi are shorter than the duodenum [Figure 1]b and [Figure 1]c.[29],[30] Very rarely, histological changes of CD canbe restricted to jejunum only (2.4%), whereas in the rest pathologies can be identified both in the duodenum and jejunum.[31] The SI pathologies in CD can be patchy and evolving, hence, in 5–13.6% of patients with newly diagnosed CD, the diagnosis may be missed in the first endoscopic biopsy [Figure 1]d.[32] Ravelli et al. compared the mucosal pathological changes in the duodenal bulb, D1, D2, and D3 biopsies and found that the histological changes are more prominent in an aboral direction.[24] Hence, in clinically suspected cases where the initial D2/bulb biopsies are noncontributory, repeat biopsies from the same sites, and additional biopsy fragments from the D3 or jejunum may reveal pathological changes.

3. The ideal number of SI biopsy fragments



The estimated probability of identifying pathological changes in CD in SI mucosal biopsies is 100% with 4-biopsy fragment protocol, 95% with 3-biopsy fragment protocol, and 90% with a 2-biopsy protocol.[33],[34] However, in a large pathology database-based study from the United States on 1, 32, 352 patients with malabsorption or suspected CD undergoing endoscopic SI biopsies adherence to 4-biopsy fragment protocol was observed only in 39% of patients, which improved the documentation of pathological changes in 1.8% cases versus 0.7% cases in those in whom <4 mucosal biopsy fragments were sampled (P < 0.0001).[35] Bonamico, et al., in a study on 102 children with suspected CD, found duodenal bulb biopsy changes in 16 (15.6%) patients, whereas in 4.2% of biopsies the pathological changes were restricted to the duodenal bulb.[22] Even with the 8-biopsy fragment protocol, the sensitivity of identifying pathological change reached 100% only when at least one biopsy fragment was obtained from the duodenal bulb, and the pathological findings were missed in 40–45% of cases when at least a biopsy fragment was not included from the duodenal bulb.[24],[27],[36] Based on the above mentioned published data, it seems that obtaining least 5-6 duodenal biopsy fragments, including at least four fragments from the distal post-ampullary parts of the duodenum, measuring 3 mm each in length,[3] as well as at one to two fragments from the 9 o'clock and 12 o'clock positions of the duodenal bulb will yield the pathological changes of CD in suspected patients most efficiently.

4. Ideal fixative and duration of fixation for SI mucosal biopsies



10% neutral buffered formalin (pH 6.8–7.4) is an ideal fixative for optimum tissue sections and staining.[37] The buffer used in 10% formalin preserves the efficacy of formalin for a longer time and prevents its degradation.[38] For fixation of tissue and to produce minimal artifacts though alcohol-based or acid-alcohol-formalin-based fixatives are often preferred; for routine H and E staining and histological analysis neutral buffered formalin suffices. The use of picric acid-based fixatives has fallen short of routine use as it leads to tissue pigmentation and creates challenges in histological identification of the Paneth cell metaplasia. Formalin can also produce tissue pigmentation if the pH of the solution is 5.5 and hence a buffered formalin is recommended. In situations, where there is a clinical possibility of diseases such as Whipple's disease or microvillus inclusion disease (MVID), additional intestinal mucosal biopsy fragments should also be obtained and fixed in 2.5% glutaraldehyde solution for electron microscopy. In suspected abetalipoproteinemia, additional biopsy fragments should be sent fresh on foil paper on ice for frozen sectioning and fat staining. In all such special situations, pathology laboratories must be communicated in advance. Depending upon the clinical indications, special staining such as Periodic acid Schiff (PAS) with and without diastase digestion, Zeihl Nielsen stain, Auramine Rhodamine stain, Giemsa stain, Warthin starry silver stains can easily be performed on formalin-fixed tissues. Although not commonly used in SI biopsies, immunohistochemical (IHC) stains may be required to highlight organisms as cytomegalovirus inclusions, H pylori, or to establish the phenotypes of IELs in patients suspected to have a refractory CD or lymphoreticular lesions. The immunophenotyping can also be performed on FFPE tissue. One should avoid using undiluted 40% formalin as it produces a charring artifact, making subsequent interpretation difficult, or unnecessary heat to avoid producing smudgy or washed out nuclei appearance with inappreciable chromatin details.[39]

An optimum fixation time for SI mucosal biopsies is about 5-6 hours. If biopsies are left in formalin for >36 hours (especially on weekends), the tissues become hard, brittle, and difficult to cut. Fixing the biopsies in formalin for a prolonged time also (>16 hours) leads to loss of immunoreactivity.[38] However, not entirely the biopsies fixed for more than 6 hours become uninterpretable histologically, as can be unavoidable in centers that do not have in-house pathology laboratories. The ideal volume of formalin solution to the tissue volume should be between 25:1 and 10:1 to generate optimum osmotic driven forces and capillary driven forces for tissue penetration and should be adhered to.[39]

5. Biopsy handling & transport to Pathology laboratory



6. Processing of biopsy samples in Pathology Laboratory



All individual biopsy fragments should be separately mounted on a one cm2 size rectangular piece of Whatman type I filter paper, white or colored, as per the preference of the pathologist. Cellulose acetate paper, gel foam, thick black paper, thin cards, or vegetable matrices such as thin slices of cucumber or potato can also be used for mounting the biopsy fragments. However, there are many centers, where pathologists do not prefer to mount biopsies on filter papers. The gastroenterologists/endoscopists play an important role in the biopsy orientation and mounting them on mediums mentioned above after gently separating the biopsy fragments from the endoscopic forceps cup by a needle tip without causing crushing artifact, keeping the shiny mucosal surface up. After separating, they may be gently flattened using clean sterile needles/pins, not over-stretched, so that the mucosal epithelium does not fall apart or edema like artifact due to overstretching is not produced [Figure 2].[40] Biopsy fragments mounted on tissue paper face-down can cause artifactual mucosal flattening and best be avoided.[41] Waiting for 10-20 seconds thereafter will result in adhesion of the biopsy fragments to mounting media by coagulation of plasma proteins. Then the biopsy fragments adhered to a filter paper should be dropped in 10% neutral buffered formalin keeping the tissue face-down so that the fragments do not float away [Figure 2]. It is essential to note that the biopsy fragments should not be pressed with forceps, fingers, or glass slides as they cause irretrievable damage. The use of a dissection microscope or the handheld lens may aid in proper tissue mounting on filter paper. The biopsy fragments obtained from different sites, as the duodenal bulb, D1, D2, D3, or jejunum should be put in separate labeled containers to enable pathologists to recognize the site of biopsies, apply appropriate criteria of interpretation, and generate site-wise biopsy reports. Within the containers, the biopsy fragments should be submerged with adequate fixative with a desirable fixative solution to biopsy volume ratio of at least 10:1 (ideal 25:1 to 10:1).[38],[42] It is best to transport the tightly capped sample containers as soon as possible to the pathology laboratory keeping them at room temperature. Inadvertent storage in the freezing chamber of a refrigerator may result in the formation of ice-crystal artifacts in tissue sections. Heating in a microwave may produce smudgy nuclear artifacts. The fixation of biopsy fragments in fixative solution follows Fick's law. The diffusion coefficient depends on the concentration of the fixative solution, size of fragments, temperature, and potency of the fixative solution. Tissue fixated for a long time becomes brittle and difficult to cut, however, with experienced hands tissue fixed for a long time can be satisfactorily processed and interpreted under a light microscope. Tissues fixed for more than 24 hours lose antigenicity for further IHC based workup and become unsuitable for nuclei acid-based works.[43]
Figure 2: The diagram shows step by step method of handling, orientation, and processing of duodenal biopsy fragments. The three rows summarize the critical contribution of the endoscopy team, pathologist and pathology technicians

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The biopsies should always be accompanied by a properly filled requisition form. As the histological changes in most of the causes of malabsorption syndrome are similar, additional information will allow the pathologists to look for specific findings [Table 3]. Biopsy forms should at least include the following information[40],[44]:
Table 3: Protocol of collection of samples from patients having malabsorption syndrome

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  • Patients name, age, sex, address, name of guardian/relative, hospital identification number
  • Number of containers sent, number of biopsy fragments in each container, and site of biopsies
  • Brief clinical history mentioning the clinical features, endoscopic indications, relevant investigation findings as celiac serology titer, stool examination findings, any relevant medical or surgical details, and clinical differential diagnoses. Mandatorily the HIV/Covid-19 or similar contiguous infective disease status should be mentioned to alters pathologists for taking extra precaution for processing.
  • Mandatorily, a history of medications, especially non-steroidal anti-inflammatory drugs, angiotensin II receptor blockers, proton pump inhibitors, chemotherapeutic medications, anti-PD-L1 medications, etc., should be mentioned.
  • If the patient is already on a gluten-free diet (GFD), the status of follow-up nutritional assessment, clinical symptoms, and serology titer should be mentioned.
  • The contact number of the treating physician or resident must be made available to the pathologist so that issues can be easily discussed when needed. Changing the requisition form and converting it to a synoptic format including all these points will help to follow it on routine practice.


The biopsies can be stained with 0.01% Eosin solution during handling at the laboratory for easy identification of darkly stained already fixed mucosal pole. The filter paper mounted biopsy fragments should not be separated, and individually mounted biopsy fragments should be wrapped into a bigger piece of filter paper during grossing like an envelope, keeping the mucosal fragments up and kept in the cassette [Figure 2]. Only ≤4 biopsy tissue fragments should be put in one cassette to prevent overcrowding at a time depending on the size of the biopsy fragments. Just before embedding the biopsy fragments in paraffin blocks, the individual biopsy fragments should be carefully separated from filter paper mounts one by one by the technicians and rotated 90° to bring all layers of the biopsy fragment in the cutting plane of the FFPE tissue blocks [Figure 2].

Post-trimming, at least 5-step sections should be cut from each FFPE-tissue block keeping the level-1-cut towards the slide label for identifying the sequence of step sections during light microscopic interpretation [Figure 2].[16] Examining each serial section helps one to choose the most appropriate section for interpretation. If needed, pathologists can ask for a second set of 5-step sections on another glass slide, and so on till an optimally oriented mucosal biopsy fragment is identified. Of all the step-sections, the most oriented step section should be marked for future reference. During review meetings or inter-departmental meetings, such marked sections should be used to make a presentation, rather than going over all the sections. All sections should be 4 μm thick, as thick sections with cutting artifacts and nicks cause problems in microscopic evaluation, especially during IEL counting. Routinely, no special stain is needed for interpretation.

Related issues (collection of biopsies in a special situation)

Unmounted floating biopsy fragments

Some endoscopists do not prefer to mount the biopsy fragments on a mounting media and directly shake the biopsy forceps cups in the fixative solution and make their biopsy fragments freely float in the container. In an interesting study, Ruiz GC et al., compared the occurrence of post-fixation mucosal contraction, crushing artifacts, and interpretability of the biopsies amongst those free-floating gastric and duodenal biopsies, biopsy fragments mounted on the cucumber slice, and also those mounted on the moistened synthetic foam-sponge. They observed that the contraction of the biopsy fragments was most observed most in free-floating samples. The orientation, absence of artifacts, and interpretability of the biopsies were appropriate on synthetic sponge mounted biopsies, followed by those mounted on the cucumber slices.[42]

Post-fixation, identification of mucosal pole is difficult due to change of color of mucosa to tan-brown owing to the formation of acid formalin hematin and loss of mucosal shine. But, in most situations, the muscularis mucosae contract, leaving the mucosal surface on the concavity of the contracted biopsy fragments. Pediatric mucosal biopsies tend to be superficial and often the fibers of muscularis mucosae are not sampled, leading to further difficulty in orientation if they are not mounted. In an interesting study by Garg N et al. the authors compared orienting free-floating biopsies sent in containers over biopsies mounted on Whatman filter paper and lens papers, with and without application of albumin as an adhesive substance during grossing and found that with the use of lens paper and albumin, the % length orientation of the biopsy fragments was best, whereas those mounted on Whatman filter paper % orientation was the worst. The use of albumin to adhere to the biopsy fragments according to them also improved the orientation of biopsy fragments in Whatman filter paper.[45] Such techniques should be standardized in laboratories when filter paper mounted biopsy fragments are not sent to the pathology laboratory.

When mounting media other than Whatman filter paper is used

When instead of laboratory-grade 1 Whatman filter paper cellulose acetate paper, gel foam, or vegetable matrices are used for mounting, the biopsy fragments can be embedded with the mount in-situ and cut through with microtome. In such cases, care should be taken during embedding so that the tissue is perpendicular to the cutting surface of the tissue block.[16],[46],[47] If the biopsy fragments are not mounted, post-fixation, the contracted tissue fragments should be embedded on the edge. With experience, some technicians can straighten the curved tissue fragments in such a way that the sections provide a relatively straight orientation of the villi perpendicular to the muscularis mucosae. Many pathologists use a hand-held lens to determine the post-fixation orientation, but its utility is not beyond doubt.

7. Section thickness for interpretation



Cutting the processed tissue on glass slides of optimum thickness is one of the important steps for optimum microscopic interpretation. Sections cut at 4 μm thickness are most optimum for counting the IELs and sections thicker than this may pose difficulty due to overlapping of lymphocytes and epithelial cell nuclei. This may result in erroneous reporting of otherwise normal biopsies as Marsh 1 lesion. Some pieces of evidence tend to suggest that a section with higher thickness has a higher cut-off for IELs. Ferguson and Murray reported the upper normal limit of IELs as 40/100 ECs with 7 μm thick sections,[48] Batman, et al., reported 33/100 ECs as cut off with 5 μm thick sections,[49] Hayat et al., 25/100 ECs with 4 μm thick sections,[50] and Mahadeva et al. 22 IELs/100 ECs with 3 μm thick sections.[51] Later, 4 μm thick sections of duodenal biopsies were reported to uniformly define a pathological cut-off of IELs ≥25/100 ECs across countries including North India.[5],[52] Hence, 4 μm thick sections of duodenal biopsies for counting the IELs seems optimum. Looking at the prevailing rate of gastrointestinal infection in India, setting a cut-off value of IELs to 30 IELs/100 epithelial cells at the tip of the villi appears ideal to avoid overdiagnosis of CD.[52],[53]

8. The orientation of mucosal biopsy fragments



One of the keys for appropriate interpretation and reporting of the intestinal mucosal biopsies is to have properly oriented mucosal biopsy fragments, characterized by 3-4 consecutive crypts perpendicularly arranged over the muscularis mucosae with properly aligned overlying villi visible from base to tips (as shown schematically in [Figure 3]a).[40] The oriented crypts should reach the muscularis mucosae. The presence of only perpendicularly oriented crypts may not be adequate to assess orientation as overlying villi may show slanting, fusion, denudation, or they may be artifactually flattened when biopsies are taken over Brunner's glands or lymphoid follicles. The presence of even a single tall villus in a flattened mucosa should raise doubt on the quality of orientation and such biopsies should be interpreted with caution [Figure 3]b and [Figure 4]a,[Figure 4]b,[Figure 4]c. Similarly, despite having aligned villi, the underlying crypts may be transversely cut round crypts [Figure 3]c. Interpretation in such areas should be avoided [Figure 3]d and [Figure 4]d. Even when the biopsy fragments do not fulfill the criteria of orientation and the villi are cut transversely round, some pathologists still prefer to count IELs in them. Though this is not an ideal scenario the same should be internally validated and correlated with serological and clinical findings.
Figure 3: Schematic diagrams showing different situations a pathologist can encounter. (a) depicts an oriented mucosa; in (b) the crypts are oriented, but the villi are not aligned; in (c) the villi are aligned but the crypts are not oriented and in (d), neither the villi nor the crypts are properly aligned. The biopsy fragments mimicking (a) is most appropriate for interpretation

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Figure 4: (a) shows a mucosal fragment, with crypts oriented perpendicularly over the muscularis mucosae (black lines) and villi with intact surface epithelium perfectly aligned [x100]. (b) shows villous abnormalities of modified Marsh grade 3c. The horizontal black line indicates the shoulder of the biopsy [x100]. (c) shows perpendicularly oriented crypts, with an aligned villous in the left (arrows), whereas the circled villous is inclined backward and uninterpretable[x 100]. (d) shows all transversely cut, round crypts (arrows), not suitable for the interpretation [x100]

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The orientation of each biopsy fragment should be assessed individually and there may be instances where only one of 5-6 biopsy fragments or none would be found oriented. Mounting the mucosal fragments on filter papers improves the orientation as discussed earlier. Identification of fibers of muscularis mucosae at the bottom of the mucosal fragments not only helps in the assessment of orientation but also prevents false flattening of the biopsy fragments.

Interpretation of histological parameters

9. IEL counting



10. Assessment of mucosal Cd and Vh



In the normal SI, each villous is supported by 3–5 crypts, which are instrumental to produce new enterocytes and other differentiated mucosal cells when there is cell loss. The pathological changes in CD start with mucosal epithelial infiltration of IELS resulting in programmed cell loss. Initially (Marsh grades 1 & 2) the crypts can replenish the surface cell loss, however, when the disease is severe the crypt cannot keep up the supply of new enterocytes and the villi become flattened, which further is sub-graded into modified Marsh grades 3a, 3b, and 3c. These changes are sequential and follow the disease course. Not only does the surface mucosa lose enterocytes when there is increased IEL infiltration in patients with CD, but this is followed by crypt hyperplasia or varying degrees of villous flattening, the common histological features identified on light microscopy in patients with CD. However, none of these histological changes are specific and most infective, autoimmune, allergic, and drug-induced SI injuries will produce similar changes. [Table 4] shows the causes of IELosis, villous flattening, and crypt hyperplasia in various diseases.
Table 4: Diseases where similar histological findings are in patients with celiac disease

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In some HLA DQ2/DQ2 positive symptomatic patients with increase serum anti-tTG titer, a duodenal biopsy may fail to show significant pathological changes (Marsh grades 0 & 1), called potential CD. On the other hand, patients having mucosal pathological changes may show serum titer within normal limits. In such condition's possibilities of false-negative and false-positive serum tests should be considered. Routinely, gastroenterologists advocate GFD to patients only fulfilling ≥Marsh grade 2 changes. However, it has been shown that symptomatic patients with early-enteropathy changes (Marsh 0 and 1) also respond to GFD with a reduction of epithelial programmed cell death.

Interpretation of IEL count

The orientation of the biopsy fragments and thickness of tissue sections can greatly impact the counting of IELs as discussed earlier. In duodenal biopsies from normal individuals, the IELs most often are concentrated towards the base of the villous. A villous cut transversely/obliquely through the base may appear flattened and IELs may be counted erroneously high [Figure 4]. The optimum site for IEL counting is the upper one-third of the villous,[53],[54] as the villous tips are commonly exposed to luminal gluten peptide, whereas the lower part of the villi and the crypts are not exposed to luminal contents unless the intestine is distended.[55] The preferential infiltration of IELs at the villous tip is also known as the 'crescendo pattern', characteristically seen in CD. In certain clinical conditions such as that in tropical sprue (TS), IELs should be counted both at the upper part and the base of the villous. Predominant infiltration of the epithelium by the IELs covering the villous base is called a 'decrescendo pattern' [Table 5].
Table 5: Histological differences of SI biopsies from patients with celiac disease, tropical sprue, Crohn's disease, and SI lymphoma/IPSID

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There are various methods for counting IELs. One of the methods is to start counting from the peak of the villus tip and continue both side slopes till 10 epithelial cells (nuclei) are counted on each side. Thus, one will get IELs per 20 epithelial cells at the villous tip [Figure 5]a and [Figure 5]b.[53] The same procedure should be followed in 5 oriented villous tips to get IEL counts per 100 epithelial cells.[53] The other method is to count at least 500 epithelial cells in a row and express the average IEL count per 100 epithelial cells.[52] However, while counting the enterocytes in a row the high IEL count at the villous base, may result in a falsely high IEL count. Therefore, counting 500 epithelial cells only at the upper one-third of the villi is a preferred method. Demonstration of the crescendo pattern of IEL infiltration is one of the important histological pre-requisites in patients with CD.[56] The pathological cutoff of IELs described in CD varied from time to time. A cut-off of ≥40/100 ECs was described in the original Marsh classification, Oberhuber modified grading system and modified grading system by Corazza and Villanacci.[6],[57] Marsh MN, in an article in 2017 described 27/100 ECs as the cut-off as per his ROC curve analysis.[58] In recent publications, a pathological cut-off of ≥25/100 ECs has been described from various countries including India. However, to prevent an overdiagnosis it is better to stick to the cut-off of ≥30 IELs/100 epithelial cells until further validation is done with other IEL cut-offs.
Figure 5: (a) shows the method of identifying the IELs (arrows), in contrast to the cigar-shaped elongated epithelial cell nuclei [a x 200]. (b) shows the method of counting of IELs in the upper 1/3rd of villi starting from the tip on both sides, covering at least 5 oriented villi [b x200]. Anti-CD3 stain highlighting the IELs in normal villi [c x100] and a biopsy showing modified Marsh 3c changes (arrows) [d x 100]. (e) shows elongated and tortuous hyperplastic crypts (arrows) [e 100]. Figures show Marsh 1 (f), Marsh 2 (g), modified Marsh 3a (h), 3b (i), and 3c changes (j). The shoulder of the biopsy fragments has been highlighted with green lines [f-j x 100]

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Although not recommended routinely, the IELs can be counted after immunohistochemical (IHC) staining with anti-CD3 (pan T cell marker) or CD8 antibodies.[59] However, this is not routinely necessary and will add extra time and cost [Figure 5]c and [Figure 5]d. IHC staining may be restricted to situations such as i) high serum anti-tTG antibody titer but microscopic Marsh 0 lesion or biopsies borderline high IEL count, ii) characterization of IEL phenotype in patients suspected to have either type I or type II refractory CD, or iii) when there is suspicion of the lymphoproliferative disease.[51] Cooper R et al. compared the IEL counting in H and E stained slides with IEL counting on anti-CD3 stained sections and found that the number of IELs was higher in CD3 stained slides than the count on H and E stained slides. Hence, in CD3 stained slides, cases may be over-diagnosed as CD though interobserver agreements do not significantly differ between IEL counting on H and E stained slides and CD3 stained slides.[60] To summarize, counting of IELs should ideally be done at the tips of at least 5 oriented villi on a 4 μm thick section-glass slide. The routine use of IHC for IEL counting is not necessary.[52]

Interpretation of crypt hyperplasia

Hyperplastic crypts appear visually as elongated and tortuous crypts with cytoplasmic basophilia [Figure 5]e. Crypt hyperplasia can be a result of the mucosal regenerative activity. Due to the nonspecific nature, it was not considered as a significant histological parameter in the grading systems proposed by Corazza & Villanacci or the Q-histological classification systems.[5],[6] But, in an appropriate clinical context with a positive celiac specific serological test, reporting of crypt hyperplasia along with IELs and with or without villous flattening is valuable [Table 4].

Both the crypt hyperplasia and the villous flattening can alter the crypt depth to the villous height (Cd:Vh) ratio. In comparison to Cd:Vh ratio of 1:5 to 1:3 in the European-Americans,[13] the Cd:Vh ratio in healthy Indian adults is lower (1:2) as identified in a study from the Northern part of India.[5] Similar findings have also been reported both in adults and children by other investigators.[50],[61],[62] It remains to be determined whether this morphological variation is due to the constitutively shorter villi in South-eastern Asian countries or because of mucosal damage following frequent gastrointestinal tract infections.

Villous height (Vh) interpretation

Flattening of the villi is the most characteristic and reliable histological feature among mucosal changes in CD [Figure 4]. Branching of villi is common in the duodenum and post-formalin fixation they can sway in any direction due to contraction of the mucosal fragments. It is important to identify the villous to crypt junction or the 'shoulder of the biopsy' correctly and in most cases, the shoulder is not a horizontal line. As the shape and Vh vary in the duodenum and the jejunum, labeling of mucosal biopsies sampled from different sites separately is essential. It is important to recognize that most available histological grading systems are based on qualitative criteria for sub-grading of the villous flattening, as 'mild', 'moderate' or 'severe' flattening which is subjective and thus results in high inter-observer disagreement.[56],[63] For better concordance, one can consider modified Marsh grade 3a change when the Vh is shorter than normal (Cd:Vh <1: 2) but still longer than the Cd, modified Marsh grade 3b changes when the Vh and the Cd are equal (Cd:Vh ratio 1:1), and modified Marsh 3c change when the Cd is longer than the Vh (Cd:Vh ratio ≥1: 1) [Figure 5]f,[Figure 5]g,[Figure 5]h,[Figure 5]i,[Figure 5]j.[64] It is still unclear whether the subclassification of villous abnormalities into modified Marsh grade 3a, 3b, or 3c has any clinical connotation. But it will be appropriate to use a histological classification system for uniform reporting and monitoring the mucosal changes post-GFD.[64],[65] Importantly, there can be regional variations of histological grades of severity of villous abnormalities in different fragments of the biopsies from the same patient, in such a situation, the highest-grade of severity should be reported, along with making a note about the heterogeneous nature of the histological changes.[56]

It is also a relevant question that how many oriented crypt-villous axes are necessary at minimum for the interpretation of the Cd: Vh ratio? Although there is no defined standard, the finding of at least four to five oriented crypt-villous axes should suffice. However, keeping in mind the regional variations, as many oriented crypt-villous axes available should be assessed. Many pathologists also use the Cd:Vh and Vh:Cd ratio interchangeably, which may create confusion and misinterpretation not only by pathologists but also by the gastroenterologists.

Other histological findings in patients with CD

Additional histological findings in patients with CD are the presence of eosinophils in the lamina propria, shortening of the height of columnar enterocytes infiltrated with IELs, thickening of the subepithelial basement membrane, and mucosal neutrophilic infiltration, mostly in those having clinically severe CD. Some of these features help in making differentiation from the mimickers, but when identified isolated are not diagnostic [Table 5].[64],[65] These additional findings may be mentioned in the histopathology report for better interpretation.

11. Interpretation of SI biopsies, fallacies, and histological mimickers



There can be the following fallacies that may impact histological interpretation:

  • The changes can be patchy and may not be sampled at all
  • The biopsy fragments are too few to represent the pathology
  • The possibility of a bulb or D1-limited CD cannot be ruled out when not sampled
  • Overdiagnosis in a case of misoriented biopsy fragments
  • Overdiagnosis when the biopsy fragments have been taken only from D1
  • Overdiagnosis of CD, when the pathologists do not know the serum antibody titer, clinical features, and clinical possibilities in case of malabsorption.
  • Biased diagnosis in a case of false-high or false-low serology titer
  • Overdiagnosis or under-diagnosis when the tissue is not properly fixed, cut with optimum thickness, optimally stained, and adequate efforts have been made to take step-sections to achieve an oriented fragment
  • Use of improper pathological criteria to interpret the histological changes
  • Overdiagnosis or underdiagnosis by pathologists who do not have enough experience to interpret the small bowel biopsies.


All have been discussed in detail. Not only the processing of SI biopsies needs expert technical hands, the pathologists interpreting the biopsies should make effort to gain experience to understand the nitty and gritty of their job. In discordant situations, always the case may be referred to an experienced pathologist for the second option and the issues may be discussed with the gastroenterology colleagues to effectively resolve them.

Histological mimickers of CD: The histological changes in patients with CD are not diagnostic and a diagnosis of CD is based on correlations of clinical symptoms, serology titer, and suggestive histological changes.[66] [Table 4] shows the diseases where similar histological changes.

The closest mimickers of CD in South-East Asia are the TS, parasitic infestations, and Crohn's disease, at least clinically and histologically. Though less commonly seen in North India, TS is quite prevalent in Southern India and some parts of Asia. In [Table 5], the histological similarities and differences among these common entities have been highlighted. However, it is difficult to differentiate these conditions on histology alone, and attention should be given to the severity of clinical and endoscopic changes, serological titers, travel history, findings of stool examinations, etc. Most of the bacterial, viral, and parasitic infections of the SI, Whipple's disease, food allergy, autoimmune enteropathy, drugs (NSAIDs, Olmesartan, immune checkpoint inhibitors), SI bacterial overgrowth, autoimmune enteropathy can result in mucosal pathology similar to that seen in CD.[64],[65] Hence, the availability of the relevant clinical details to the pathologists will help them to rule out the common mimickers and state if CD can be a possibility. If the villous abnormalities are of high grade and the anti-tTG antibodies are negative, one should repeat the serological tests from a different laboratory and an anti-EMA test should also be done. A short course of gluten challenge followed by a repeat biopsy, including those from distal SI and/jejunum may help to establish the pathological changes in such a scenario.[67] A negative HLA DQ2/DQ8 haplotype will rule out CD.[66],[68],[69],[70] Programmed cell death of the enterocytes not only seen in CD, but are prominent in autoimmune enteropathy, graft versus host disease, and chronic intake of NSAIDs and Olmesartan. Avoidance of NSAIDs at least 7 days before the biopsy procedure may help to exclude this change. However, Olmesartan induced changes are mediated by IL-15 take 4-6 months to appear or regress; hence, short time discontinuation before biopsy may not help.[71] Regardless of geographical locations, pathologists should not forget about parasitic infestations as giardiasis or H pylori infection especially in India and Southeast Asia, which may produce similar histology.[72] The histological changes of combined variable immune deficiency (CVID) may also mimic that of CD. The absence of mature mucosal plasma cells, prominent lymphoid aggregates, and a low level of serum immunoglobulins should help in the identification [Table 5].

A diagnosis of lymphoma or immune proliferative SI disease (IPSID) should be considered when sheets of lymphoid cells or plasma cells are observed in the lamina propria along with destruction and/or loss of crypt [Table 5]. Levels of serum IgA, immunoblotting, or immunostaining with cryptic IgA, kappa, and lambda light chains may help in differentiation. The presence of prominent mucosal histiocytes should raise the considerations of Whipple's disease, histoplasmosis, or Mycobacterium avium intracellulare infection, particularly in immune-compromised patients. Prominent fat vacuoles in the enterocyte cytoplasm should raise the possibility of abetalipoproteinemia. In such a diagnosis, the biopsies should be repeated, and they should be collected in a specific manner as described in [Table 5] for further workup. Although the presence of intracytoplasmic inclusions suggests a diagnosis of Cytomegalovirus infection, the presence of an intact glycocalyx layer on epithelial helps to rule out the possibility of MVID in infants with intractable diarrhea. Type of diarrhea, as histology of chronic protein-losing enteropathy, should raise the possibility of lymphangiectasia, or the presence of watery diarrhea should raise the possibility of microscopic enteritis. Also, sometimes pathological conditions may co-exist as giardiasis and CD, or CD and CVID, or IPSID in a background of CD, which needs multidisciplinary discussions and correlations.[73]

12. Histological grading of SI biopsies in CD



Though a histological grading system described by Rubin et al. was already available,[74] Marsh MN was the first to classify the histological changes as per the sequence of pathological changes,[54] followed by the introduction of Oberhuber's modified Marsh classification system.[57] The basic difference between the original Marsh and modified Marsh classification system was that the qualitative sub-grading of villous flattening was described in the latter, as mild, moderate, and severe villous flattening, leading to interobserver and intraobserver agreements in only about 50% of biopsies.[5],[56],[72] This problem was recognized, followed by the introduction of subsequent modified Corazza/Villanacci classification,[6],[75] Ensari classification,[53] which were also based on qualitative criteria, and the off-late described Q-histological methods incorporating quantifiable criteria [Table 6].[76],[77]
Table 6: Table showing a comparison of different histological classification systems available for SI biopsy interpretation from patients with celiac disease

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Further sub grading of the villous flattening as mild, moderate, and severe villus flattening was questioned based on the findings of 3D image reconstruction studies, inconsistent correlation of mucosa infiltrating lymphocyte density, and grades of villous flattening, mathematical regression equation analysis, questionable clinical relevance.[58] Clinically it is enough to know if seropositive patients have ≥Marsh 2 histological changes to advocate GFD.[76] Seropositive patients with Marsh 0 or Marsh 1 changes are called potential CD, and their progression to higher modified Marsh grades has been documented in 30% of these patients after 12-months of follow-up.[77]

Subjective criteria used for the sub grading results in low interobserver agreements. Werkstetter et al. highlighted the disagreement between the local and central pathologist in differentiating no CD (Marsh 0 and 1) and CD (Marsh 2 or 3) categories in up to 7% biopsies, and the disagreement rose to 58% when they were further asked for sub grading the villous atrophies into modified Marsh 3a-3c changes.[10] For better concordance one can consider modified Marsh grade 3a change when the Vh is shorter than normal but still longer than the Cd, modified Marsh grade 3b changes when the Vh and the Cd are equal, (Cd:Vh ratio 1:1), and modified Marsh 3c change when the Cd is longer than the Vh (Cd:Vh ratio ≥1:1) [Figure 5]f,[Figure 5]g,[Figure 5]h,[Figure 5]i,[Figure 5]j.[64] But the most important justification for following the extended modified Oberhuber classification system is to assess the mucosal healing post-GFD, as symptomatic or serological improvements do not go hand in hand with the mucosal histological normalization.[78] In a study by Lee SK, 12 months post-GFD, whereas 77% of patients had normal serology, only 21% of them showed histological reversal.[79] Lanzini A, et al., after 16 months post-GFD showed mucosal normalization only in 8% of patients. Interestingly, 83% of patients having baseline modified Marsh 3c changes showed a mismatch of histological and serological response after an average period of 16 months post-GFD.[80] Among all serological tests available only EMA was found to be a fair indicator of mucosal healing, though not routinely performed during the initial screening and diagnosis, especially in India.[81] In the presence of systemic autoimmune inflammation, both the anti-tTG titer and EMA can be falsely elevated/positive despite achieving mucosal healing.[82] In the United States, persistent villous flattening was identified in up to 66% of patients on GFD after two-years of follow-up.[83] In another study, after a year of post-GFD follow-up, only 50.5% of adult patients achieved mucosal recovery, whereas 81% of them showed mucosal healing after 8.1 years of follow-up.[84] As the compliance to GFD in children is relatively better than in adults, increasing weight or height in children may be used as reliable clinical indicators of mucosal healing, besides the availability of biomarkers such as I-FABP (intestinal fatty acid-binding protein) or anti-DGP serology test.[85],[86] However, less than half of the adult patients with CD, especially those over 40 years of age, and who had modified Marsh 3c changes at baseline, the serological or symptomatic changes are reliable indicators of mucosal healing, necessitating follow-up biopsies.[84],[85],[86],[87],[88],[89] Noninvasive biomarkers as serum I-FABP or Citrulline though have come up, large validation studies are needed before their clinical application in comparison to continuing with the gold standard SI biopsies.[87] Keeping in mind the limitations of the light-microscopic histological classification systems, the Q-histological assessment based on quantifiable parameters improved intra-observer agreement by up to 85% and interobserver agreements up to 71%. This assessment also brought objectivity, is labor-intensive, time-consuming, and increases reporting time. Another advantage of the Q-histological grading system is that minor changes in villous dimensions can be measured, valuable in clinical trials. The application of artificial intelligence-based automation may simplify its use during routine reporting, and till that is available, its use does not seem practical for routine evaluation. Two simple approaches which can be adopted are (1) to train the pathologists who deal with SI biopsies through online webinars, hands-on-workshops, and (2) to improve routine assessment of biopsy dimensions is using eye-piece reticles in reporting microscopes to help assisted near-quantitative eyeballing of Vh and Cd, rather than directly measuring them as per the experience of the authors. However, this system needs further validation [Figure 6].
Figure 6: The image shows the utility of using a microscope eye-piece reticle for quick quantitative eyeballing for assessment of Cd and Vh in a duodenal biopsy [x 40]

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13. Assessment of healing and follow-up biopsies in patients with CD



Follow-up SI biopsies help in identifying GFD-compliant patients. However, it needs to be recognized that there is no established guideline for follow-up intestinal biopsies in patients with CD and the protocol can vary depending on the patient's response and treating physician's choice. Literature review shows adult patients aged >40 years at diagnosis, males, and with baseline modified Marsh 3c changes would preferably need follow-up biopsies. Adult females on the other hand show relatively quick mucosal healing post-GFD.[78],[89] Unless sub grading of villous flattening into modified Marsh 3a, 3b, and 3c changes are done at baseline, triaging and follow-up assessment will be difficult.[73] Follow-up SI biopsies will help to work up for refractory celiac disease (RCD) in GFD-non compliant patients, or rule out a coexisting disease, in patients with high anti-tTG serology titer but normal histology; or in seropositive patients having histology corresponding to Marsh grade 0/1 changes prospectively after a period of 2-years while on a gluten-containing diet to establish the progression of mucosal pathology necessitating GFD, however, there is lack of established guideline regarding follow-up biopsies in potential CD patients.[89],[90],[91] Generally, follow-up biopsies should be taken 12-months post-GFD as the histological changes of mucosal pathologies take time to reverse. The AGA recommends a second follow-up biopsy after a gap of 12-months if the first follow-up biopsy had Marsh 2 changes.[86],[92],[93],[94],[95] There is also no established protocol on how to report the follow-up biopsies as the sequence of reversal of changes is exactly not known. Reporting of follow-up biopsies exactly in a similar manner as done for baseline biopsies with mentioning of current modified Marsh grade will help to compare existing mucosal changes with changes in baseline biopsies and assessing response to GFD. Applying Q-histological measurement in the follow-up biopsies also can provide accurate parameters for assessing response to GFD, but the use of this system seems is not yet easy to implement in routine practice and restricted to clinical trials.[10]

14. GFD nonresponsive CD and Refractory CD



GFD is the only recommended treatment for patients with CD. However, non-response to GFD has been reported in up to 10-50% of adults on follow-up.[96] Non-response can be due to non-adherence to GFD, or mistaken diagnoses followed by GFD treatment or an associated disease which complicates the course of CD including the RCD type 1 and 2, followed by the risk of systemic complications of chronic malabsorption, lymphomas, and carcinomas reported especially in RCD type 2.[3],[97] On 12-months post-GFD follow-up, only about half of the patients with CD show mucosal healing. On the other hand, even after about 8 years of post-GFD follow-up up to 81% of patients showed mucosal healing.[84] The diagnoses of GFD-nonresponse are not straight forward, need clinical and nutritional assessment, with a battery of investigations. Histologically all GFD-unresponsive patients of CD show the persistent villous flattening and the validated histological features to predict mucosal response are not known. The annual incidence of RCD is about 0.83/10.000 CD patients,[98] and immunophenotyping of the IELs by either IHC or flow cytometry are needed for diagnosis and typing, as follows: RCD type 1: CD3+CD103+CD8+IELs having polyclonal TCR γ chain gene, and RCD type 2: ≥50% by IHC or ≥25% of IELs by flowcytometry having CD3-CD103+/–CD8-/CD335+phenotype with clonal TCR γ chain genes.[99] Hence, pathologists need to play an important role in the workup of patients with GFD-nonresponsive CD and to identify patients with RCD. A multidisciplinary approach and the collection of formalin-fixed biopsy fragments for IHC, and fresh biopsy fragments for isolation of IELs, followed by flow cytometry and clonality assessment are required.

15. Challenges in pathology interpretation of SI biopsies



Scenario 1: Assessment of SI biopsies from those already on GFD (without a definitive diagnosis)

A perplexing clinical scenario is to confirm CD in patients who started GFD of their own without relevant investigations. In these cases, patients should be advised to undergo anti-tTG serology and a positive serology does not pose a difficulty in diagnosis. But seronegative patients should undergo HLA DQ2/DQ8 typing, which if comes negative rules out a CD. In seronegative patients having HLA DQ2/DQ8 haplotypes, a gluten challenge with 3–10 g of gluten (one to four slices of bread) every day for 2–8 weeks followed by duodenal biopsy would help to document gluten-induced mucosal pathologies. As the amount of gluten to use and duration needed to produce the mucosal changes vary according to different studies the protocol needs to be individualized.[67] However, gluten challenge should not be done in children <6 years old and during the pubertal spurt. It is strongly recommended to send such patients to tertiary care centers or refer them to experienced gastroenterologists before conducting gluten challenge because of the rare but fatal risk of developing celiac crisis.

Scenario 2: Negative serology in suspected patients

In patients having histological features like that in CD but negative IgA anti-tTG serology should be considered for IgA deficiency, unsolicited GFD, seronegative CD, non-celiac enteropathy as detailed in [Table 3]. In short, from an Indian perspective, possibilities of TS, Crohn's disease, parasitic infestation, food allergy, enzyme deficiencies, NASID induced changes, or CVID should not be forgotten and investigated accordingly.[100] Before releasing the report, a second opinion on the biopsy and ruling out all the mentioned conditions would be advisable. Careful nutritional assessment, stool gluten Immunogenic Peptide (GIP) test, serum Ig levels estimation, IgG based serology, or HLA typing would be advisable.[99]

Scenario 3: Positive serology and normal biopsy

Positive IgA anti-tTG serology can be seen in patients having normal duodenal histology. In up to 10% of cases, serology can be falsely positive, especially in patients having other autoimmune systemic diseases, chronic liver disease, congestive heart failure, or hypothyroidism. As discussed earlier, in such a scenario, the number of biopsy fragments included, their orientation should be checked, and a second opinion should be asked from an experienced GI-pathologist. Repeating the anti-tTG serology by an ELISA kit of a different make than the earlier used one, or a second-line anti-EMA test would also help. HLA DQ2/DQ8 typing would help to rule out CD in haplotype negative patients. A gluten challenge may be an option. If positive serology is confirmed or supported by other tests as detailed above in histologically normal patients, the patient should be categorized as a potential CD.[67],[101],[102]

Applicability of IgA anti-TG2 immune deposit in tissues in a difficult diagnostic situation

In the above-mentioned situations where the diagnosis of CD is difficult, identification of IgA anti-TG2 immune deposits in the SI biopsies by dual-color IHC or dual-color confocal IF methods have shown high sensitivity and specificity.[103],[104] IgA anti-tTG2 deposits have been identified in CD, potential CD, seronegative CD, and dermatitis herpetiformis.[104] Facilities for the same should be established in centers receiving a high load of celiac patients.



16. Reporting of SI biopsies

An ideal pathology report [Table 7] should describe the histological findings in a synoptic manner, followed by overall interpretation based on the clinical, serological, and histological findings and the modified Marsh grade based on the highest degree of pathological changes across all the biopsy fragments.[3],[16],[44],[105],[106]
Table 7: A model histology report should look like the following format

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When serology titer is not high or not known, but histological features mimic CD, the pathology interpretation should be descriptive. E.g., when the biopsy shows 34 IELs/100 ECs with a C:V of 1:2 and crypt hyperplasia in a patient with anti-tTG serology titer of 6 (say the lab cut off is 4 IU/mL), the pathology report should be as 'Overall features are of mild villous abnormality with increased IELs and crypt hyperplasia. Although features may be consistent with CD, the patient should be further investigated to rule out other mimics.' In such cases modified Marsh grading should not be mentioned.

Section 16: Communication between two disciplines for optimal interpretation of SI biopsies

There is some basic but essential information that pathologists expect from the gastroenterology colleagues and the gastroenterologists expect from pathologists. Adherence to this checklist will help both to get the maximum yield of a SI biopsy procedure.

What Pathologists expect from gastroenterologists?

  • Information on site of biopsies & biopsy fragment numbers
  • Diagnostic biopsies or follow-up biopsies
  • Biopsy fragments from different site labeled and sent in separate containers
  • Information regarding the time of biopsy (to calculate the time of fixation)
  • Biopsy fragments are oriented on mounting media or not
  • Appropriate and relevant clinical information including serotiter
  • Relevant investigation results
  • Clinical differential diagnoses
  • Any specific clinical question in mind


What is expected from the pathologists?

  • Site wise descriptive report
  • Reporting in a specified format
  • Mention regarding the number of step cuts examined & biopsy orientation
  • Final Interpretation
  • Comments on limitations
  • Suggestions
  • Additional tests needed


If the diagnostic issue is still not resolved, the following steps should be taken periodically:

  • Joint conferences (physical, virtual, phone, What's App)
  • Multidisciplinary review meetings.


17. Training and infrastructure



A joint and coordinated effort from all those involved in this process including the endoscopists, pathology technical staff, and experienced pathologists is essential for optimum yield of this invasive procedure. In medical schools, during post-graduate training of doctors as well as nursing staff and paramedics training on intestinal biopsy handling is ignored. Arguelles-Grande C et al. evaluated agreement for primary diagnosis between pathologists working in community hospitals, commercial laboratories, smaller institutions, and University hospitals. The agreement was very good between this institution and university hospitals (k = 0.888) but moderate in community hospitals (k = 0.465) or commercial laboratories (k = 0.419). Within different Marsh score categories, the agreement was poor (k < 0.03) for Marsh 1 and 2 categories, both missed at other centers, and fair or moderate for scores 3a and 3b. Information regarding the degree of villous flattening and IELs was lacking in 26% and 86% of reports and non-quantifiable terminologies as 'blunting' or 'marked atrophy' were commonly used.[107]

Trongwongsa T et al. evaluated the outcome of training of the pathology technicians handling SI biopsies and endoscopy nurses who mount the biopsied fragments on filter paper.[108] Without training, 46% of the biopsy fragments were interpretable, however, after training of technical staff, the interpretability rose to 60%. Furthermore, when endoscopy nurses and assistants were also trained regarding the handling and mounting techniques, interpretability rose to 74% of all biopsies (P 0.004). Interestingly, when only the pathology technicians or the endoscopy nurses were trained, the improvement was not statistically significant.[91] The authors also observed that the procedures followed by trained staff did not take extra time than the time taken by untrained staff.[108] A focus on practical aspects during staff training, periodical evaluation of the quality of biopsy and interpretability, holding inter-disciplinary periodical meetings and internal quality assessments would help to improve the outcome. For smaller centers and non-academic establishments periodical, hands-on-workshop, and training courses both on biopsy handling and interpretation would help to generate a sense of awareness and uniformity in reporting. This guideline should be followed by all, irrespective of their nature of the practice, and implement to achieve the goal of this guideline. Pathologists should also take the lead to implement these SOPs in their respective laboratories and train their staff as per the guidelines. Institutions and pathology laboratories should strive to create a database of SI biopsies following the uniform reporting format to create a National database on SI biopsies in patients suspected of CD.

Knowledge gap and areas of priorities

Though the awareness regarding CD, and access to specialist gastroenterologists, and endoscopes have increased, still there is a heterogeneity of understanding, practice, interpretability, and yield of the SI biopsy procedure. The following are the areas of priorities that were identified before this guideline was made. However, this is only the first step and efforts should be made to work systematically on each aspect to improve the overall quality of practice:

  1. Formulation of a detailed pathologist centered practice guideline on SI biopsy handling, processing, and interpretation, in consultation with the experienced gastroenterologists.
  2. Improve the quality of practice and to bring uniformity by formulating a guideline acceptable to all.
  3. Establishing a facility of centralized or regional training of pathologists and technical staff and endoscopy nurses with periodical assessment courses.
  4. Working on the improved histological grading system of SI biopsies in patients with CD, including Q-histological classification and deep learning algorithm
  5. Establishing facilities to workup nonresponsive CD and refractory CD
  6. Establishing facilities to identify IgA anti TG2 deposits in biopsy tissues
  7. Establishing laboratory diagnostic methods to confirm the extra-intestinal CD.



   Conclusions Top


Small bowel biopsy still holds the position of the gold standard for evaluating the mucosal pathologies in patients with CD, especially in adults, and for evaluation of mucosal healing in follow-up biopsies. As the histological changes in CD are not specific, access to clinical, endoscopic findings and serological titer will improve the pathology reporting. Pathologists should make effort to rule out all mimickers of CD and follow the laid down reporting format for uniformity. Gaining experience in the work by practice, undergoing training, and periodic assessment should be undertaken by pathologists, technical staff, and endoscopy nurses. A periodic quality assessment would give an insight into the nature of practice and difficult cases should be discussed in the multi-disciplinary meeting to understand the areas of deficiencies. Histological grading of the biopsies from patients with treatment naïve CD will help to identify patients who should be necessarily followed up and compare the findings of follow-up biopsies with baseline biopsies. To ensure the availability of clinical and serological detail, the requisition forms should be modified. Finally, pathologists should try to use digital image analysis software or a microscope eye-piece reticle for quick assisted semi-quantitative Cd and Vh assessment. This guideline intends to give a standard operating protocol formulated with the help of experienced pathologists and gastroenterologists and should be implemented by all for a uniform outcome.

Guideline Summary of Essential Recommendations



Acknowledgements

We acknowledge and deeply appreciate the office bearers of the IAPM and ISG to consider and approve this important guideline work under their aegis, all respected members of the IAPM who took part in the online National survey, and Ms. Yogyata Joshi for conducting and helping in technical aspects of face to face virtual meeting.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Govind K Makharia
Professor of Gastroenterology and Human Nutrition, All India Institute of Medical Sciences, New Delhi
India
Siddhartha DattaGupta
Professor of Pathology, All India Institute of Medical Sciences, New Delhi
India
Vatsala Misra
Department of Pathology, MLN Medical College, Allahabad - 211 001, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_1405_20

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