| Abstract|| |
Background: Celiac Disease involves the small intestine patchily affecting more frequently the proximal small bowel but the histological changes have been observed till terminal ileum. Of late in addition to D2, the duodenal bulb (D1 region) biopsies have been found helpful in identifying a small group of patients with CD. Therefore, multiple site biopsies are recommended as histological changes are not uniform throughout small intestine. Methods: During this present 1.5 years prospective study, we evaluated 84 cases of suspected celiac disease with respect to the light microscopy (D1, D2, and D3 biopsy) and serology (anti tTg and or EMA). Histological examination was done according to Modified Marsh grading system. Results: Out of 84 cases with raised anti tTg, the segmental biopsies significantly increased the diagnostic accuracy from 39/44 cases (88.6%) to 43/44 cases (97.7%) and 44/44 cases (100%) when D2 alone, D1 + D2 and D1 + D2 + D3 biopsies were evaluated, respectively. Of the suspected cases of celiac disease patients (tTg > 10 ULN and associated weight loss, diarrhea), additional D3 biopsy increased the diagnostic yield by 2.1%, compared to D1, D2 region biopsy and 6.38% compared to standard D2 biopsy alone. Of the 28 cases (tTg > 10 times ULN + EMA positive and associated weight loss, diarrhea), the potential celiac disease (histologically Type 1/Normal) cases reduced from 28.5% (standard D2 region alone) to 21.4% and 17.8% when additional biopsies were taken from D1 region and D3 region, respectively, and additional D3 biopsy increased the diagnostic yield by 10.8% (compared to standard D2 biopsy alone) and 3.7% (compared to D1 and D2 biopsy). Conclusion: We believe multiple sites duodenal biopsies including D3 region biopsies might increase the diagnostic accuracy of adult celiac disease in addition to sensitive and specific serologic tests.
Keywords: Celiac disease, diagnosis, duodenum, multiple biopsies, tissue transglutaminase
|How to cite this article:|
Narang V, Jindal A, Singh A, Varun Mehta BG, Sood N, Sood A. Diagnostic utility of multiple site duodenal biopsies in celiac disease. Indian J Pathol Microbiol 2021;64, Suppl S1:73-7
|How to cite this URL:|
Narang V, Jindal A, Singh A, Varun Mehta BG, Sood N, Sood A. Diagnostic utility of multiple site duodenal biopsies in celiac disease. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Jun 23];64, Suppl S1:73-7. Available from: https://www.ijpmonline.org/text.asp?2021/64/5/73/317925
| Introduction|| |
Celiac disease (CD) is a global disease, occurring in ~1% of the general population worldwide. Antibodies to tissue transglutaminase (tTg) and assays focused on endomysial antibodies.
(EMA) have been demonstrated to have high sensitivity but duodenal biopsies are considered the gold standard for diagnosis of celiac disease. The sensitivities of serological tests often vary and are difficult to reproduce in different laboratories, or even in the same laboratory using different sources of antigen. CD involves the small intestine patchily affecting more frequently the proximal small bowel but the histological changes have been observed even till terminal ileum. Traditionally, endoscopic postbulbar (D2) biopsies have been a standard protocol for studying the histological changes in celiac disease. Of late, the biopsies from the duodenal bulb (D1 region), previously disfavored because of the risk of artifacts (mainly from prominent Brunner glands), have been found helpful in identifying a small group of patients who would be missed by postbulbar biopsies (D2 region) alone., Multiple site biopsies are recommended as histological changes are not uniform throughout small intestine. The present study was planned to evaluate the significance of multiple site biopsies including D1, D2, and D3 in clinically suspected CD patients.
| Methods|| |
Source of data
This present 18 months longitudinal follow-up study (from January 2017 to June 2019) was conducted in the Department of Pathology and Department of Gastroenterology, Dayanand Medical College and Hospital, Ludhiana, Punjab, India.
All patients with clinical features like diarrhea, anemia, short stature, failure to grow, and increased anti tTg were considered as suspected cases.
Tissue transglutaminase (tTg)
The tTg assay was performed by enzyme-linked immunosorbent assay (ELISA) using fully automated immunoassay analyzer thermo/phadia 250 HSN code 3822. The kit (Euroimmune, Germany) was used and results >20 (IU/mL) were considered as positive (manufacture's cut-off value >20 IU/mL as positive).
Anti endomyseal antibodies (EMA)
The EMA Ig A was tested with glass microscope slides coated with middle third esophagus tissue sections. The patient serum was checked for the presence of antibodies by Fluorescein conjugated anti human immunoglobulins. A specific green fluorescent staining of antigen antibody complex was visualized with the aid of fluorescent microscope. The endomysium visualized as “fish net appearance” around the smooth muscle bundles. The intensity of 3+ and 4+ was considered positive.
All the enrolled patients underwent gastroduodenoscopy, performed by a single expert gastroenterologist (RM) using Olympus gastroduodenoscope (GIF-H170, Tokyo, Japan). A video was recorded maintaining the anonymity of the patient and a blinded second expert (AS) validated the findings after reviewing the video. Six biopsies (four from each quadrant in the second part of duodenum and two from the duodenal bulb) were collected in 10% formalin. The biopsy specimens were stained with hematoxylin and eosin and examined under 100× magnification by a single expert pathologist (VN) and the observations were recorded. These findings were then reviewed by a second expert (NS) and confirmed.
The histopathological features were noted according to the Modified Marsh criteria which included intraepithelial (IEL) lymphocyte counts at the tips of villi (>30 IEL/100 enterocytes were considered significant), villi to crypt ratio (>3:1 was considered normal), lymphoplasmacytic inflammation of lamina propria, villus atrophy, granulocytic infiltrates (neutrophils and eosinophils), increased crypt apoptotic activity, and reactive epithelial changes.
Other investigations like routine hemograms and albumin levels were also noted.
All the patients with borderline serology and poorly oriented biopsy were excluded.
Potential celiac disease
Patient with clinical symptoms like diarrhea, anemia, and weight loss with raised tTg > 10 times the normal and positive EMA with normal histology.
Confirmed celiac disease
Patient with clinical symptoms like diarrhea, anemia, and weight loss with raised tTg > 10 times the normal and positive EMA with type 2/type 3 changes on histopathology [Figure 1].
All patients were followed up for 3 months for response to gluten-free diet.
The study was approved by institutional ethical committee with letter BFUHS/2K18p-TH/14233.
| Results|| |
Of the 84 patients enrolled, the age ranged widely, with the youngest patient being 6 years old and the oldest being 69 years old. Female predominance was seen with M: F ratio of 1:1.8. Out of 84 cases with raised anti tTg, the histopathological changes of celiac disease, i.e., Modified Marsh Type II and III (considering the highest change among the D1/D2/D3 biopsy) were noted in 44 cases (40 cases with Type III changes and 4 cases with Type II changes) and the remaining 40 cases, despite clinical presentation and raised tTg, had either normal histopathology (14 cases) or Type I changes (26 cases). The segmental biopsies significantly increased the diagnostic accuracy from 39 cases (88.6%) to 43 cases (97.7%) and 44 cases (100%) when D2 alone, D1 + D2 and D1 + D2 + D3 biopsies were evaluated, respectively [Table 1], [Table 2] and [Figure 2]a,[Figure 2]b,[Figure 2]c.
|Figure 2: (a) Microphotograph showing Type 3 changes in D1 biopsy while D2 biopsy is normal (b) and D3 biopsy also showing Type 3 changes (c) in a single patient (H and E 200×)|
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|Table 1: Comparative histological changes in multiple site biopsies from patients with raised tTg|
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|Table 2: Diagnostic utility of multiple site biopsies in suspected celiac disease|
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The sensitivity and specificity of anti tTg was 87.88% and 32.43%, respectively, with P value 0.043. The positive predictive value (PPV) was 53.70% and negative predictive value (NPV) was 75%.
Of the 40 cases showing normal/Type I changes, anti tTg was markedly increased (>100 U/mL) in 14 cases while the remaining 26 cases had mild increase in anti tTg levels.
Of the 84 cases, 47 had raised tTg >10 times the ULN. On analyzing the D1, D2, and D3 region biopsies, it was observed that 36/47 (76.4%) were labeled as confirmed celiac disease and remaining 11 either had normal histology (02/47, 4.2%) of Type 1 changes (09/47,19.1%).
Compared with a standard D2 biopsy, an additional D1 biopsy increased the diagnostic yield by 4.7%. Among the 35 cases diagnosed as celiac disease (D1, D2), 01 had normal D2 biopsy specimens while another had Type 1 changes. Thus, a total of 2 patients (5.7%) of celiac disease would have been missed in the absence of additional D1 biopsy.
Similarly, additional D3 biopsy increased the diagnostic yield by 2.1%, compared to D1, D2 region biopsy) and 6.38% compared to standard D2 biopsy alone. Among the 36 cases diagnosed on D1, D2, and D3 region biopsies, two cases had Type 1 changes in D1 biopsies (Type 3 changes in D3 region) and one case had normal D2 biopsy. Thus, a total of one case (2.7%) and 3 cases (8.3%) of celiac disease would have been missed in the absence of additional D3 biopsy as compared to D1 D2 biopsy and standard D2 biopsy alone, respectively [Table 3].
|Table 3: Comparative histological changes in multiple site biopsies from patients with raised tTg >10 times the upper limit of normal|
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For further analysis, a subset of patients was selected who had EMA positivity in addition to raised (>10 times) tTg. Of the 47 cases, EMA was positive (3+ and 4+) in 28 patients. Remaining 19 cases EMA was not available.
In this group, compared to standard D2 biopsy, the additional D1 increased the diagnostic yield by 6.9% (78.5% vs. 71.4%). Similarly, additional D3 biopsy increased the diagnostic yield by 10.8% (compared to standard D2 biopsy alone) and 3.7% (compared to D1 and D2 biopsy).
In this subgroup, of the total 23 cases of diagnosed celiac disease on D1, D2, and D3 region biopsies, D2 biopsy was normal in one case and showed Type 1 changes in two cases in addition to one case with D1 biopsy showing Type 1 changes [Table 4].
|Table 4: Significance of multiple site biopsy in patients with raised tTg and EMA|
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The cases with raised tTg (>10 times) and, EMA positivity and normal or Type 1 histology were classified as potential celiac disease. The potential celiac disease cases reduced from 28.5% (standard D2 region alone) to 21.4% and 17.8% when additional biopsies were taken from D1 region and D3 region, respectively.
| Discussion|| |
In 2012, the European Society of Pediatric Gastroenterology and Nutrition (ESPGHAN) published guidelines for diagnosing CeD in children without a small intestinal biopsy, if the serum anti-tTg titer is >10 times upper normal limit, a positive anti-endomysial antibody test in a second blood sample, presence of suggestive HLA-DQ2/DQ8 alleles, with the consent of the first-degree-relatives of patients for this no biopsy approach. However, the American Gastroenterology Association (AGA) recommended intestinal biopsy in patients with strong clinical suspicion of CeD even if the serology is negative, while the United States National Institute of Health (NIH) recommends a biopsy only when the serological tests are doubtful. The Indian Council of Medical Research (ICMR) also favored the evaluation of intestinal biopsies for evaluation of mucosal pathologies along with other tests for establishing a diagnosis in adults. Variability of the outcome of commercially available anti-tTg ELISA kits and lack of the HLA haplotyping facility in all centers in India justify this recommendation.[1.2] In the present study, the sensitivity and specificity of anti tTg were 87.88% and 32.43%, respectively, with P value 0.043. The positive predictive value (PPV) was 53.70% and negative predictive value (NPV) was 75%. The sensitivity and specificity of anti EMA were 84.85% and 67.57%, respectively, with P value 0.001. The positive predictive value (PPV) was 70.00% and negative predictive value (NPV) was 83.33%, therefore reemphasizing the role of biopsy in the diagnosis of celiac disease.
Moreover, in the adult celiac disease, the biopsies in addition to serologic tests might help to rule out underlying IBS or Crohn's disease as patients with IBD also might get symptomatic relief with GFD. Baseline histological tests might enable the assessment of severity (degree of villous atrophy) and give the patient confidence about histological improvement if future biopsy samples are taken and patients might have a temporary celiac or gluten autoimmunity along with a negative biopsy.,,, Beyond the “classical” histologic features of active CD (ACD) mentioned in Modified Marsh grading system, there are additional findings that are common, although often overlooked. The enterocytes (absorptive intestinal epithelial cells) commonly have a reactive appearance, with mucin depletion, intracytoplasmic vacuolization, and a cuboidal rather than the typical columnar cell shape. Neutrophilic inflammation is common in the lamina propria, occurring in most newly diagnosed cases of ACD. This phenomenon has been shown to correlate with the degree of villus atrophy. Activated eosinophils are increased in ACD. Crypt apoptosis is often a subtle histologic finding.
Small-bowel biopsy remains the gold standard for diagnosing CD. It is also now well recognized that CD is a patchy disease. Historically, biopsies were taken from the jejunum to diagnose CD. Studies since the mid-1990s have shown that biopsies from the second part of the duodenum are sufficient for diagnosis without loss of sensitivity or specificity. However, there are no uniform, agreed-upon recommendations or guidelines for the number or site of biopsies required for diagnosis. In general, both the duodenal bulb and distal duodenum, first and second parts of the duodenum are exposed to high gluten load; hence, sampling the duodenum, especially the duodenal bulb along the second of duodenum is necessary. However, biopsy interpretation of the duodenal bulb has its challenges due to the presence of abundant submucosal Brunner's glands causing artifactual flattening of the overlying mucosa, and due to common inflammatory changes inflicted by the gastric acid or pancreatic juice reflux. Hence, if only the duodenal bulb is relied on for the pathological changes in CeD, the false positive rate will be high. Many investigators have demonstrated that CD-related histologic lesions are present at the bulb, and importantly, isolated mucosal abnormalities at this site can be seen in up to 10% of CD cases, in both adults and children., The duodenal bulb is also the most sensitive site to detect mucosal injury induced by gluten, as the severity of mucosal damage is thought to follow a proximal to distal gradient. Inadequate sampling may lead to false-negative diagnosis, and poorly oriented biopsy specimens can cause both under interpretation and over interpretation of the histologic abnormalities associated with CD.,,
Though the role of D1 and D2 biopsy has been studied in few studies, the present is first in the evaluation role of D3 biopsy in diagnosing celiac disease. We believe additional, deeper duodenum region (D3) biopsies might have a role in diagnosis and follow-up of patients of gluten-sensitive enteropathy because first deeper duodenum is less affected by peptic ulcer disease, etc., as compared to D1/D2 region which might be normal or have Modified Marsh Type 1 like changes, thus leading to the diagnosis of potential celiac disease in symptomatic seropositive, early celiac disease patients [in the present study, potential celiac disease cases reduced from 28.5% (standard D2 region alone) to 21.4% and 17.8% when additional biopsies were taken from D1 region and D3 region, respectively]. Second, D3 region can have more severe histological changes as compared to D1 region [in the present study 5.5% cases had more severe changes (Marsh Type 3) as compared to D1 region]. Third, gluten-free diet might cause reduction in the severity of duodenal mucosa in D1/D2 region, whereas in D3 can have persistent severe changes (in the present study, one case had Type 1 changes in D1D2 region but Type 2 changes in D3 region).
The present analysis not only emphasized the role of biopsy in addition to serological but also highlighted the significance of additional D3 biopsy in diagnosing celiac disease and reducing the number of potential celiac disease with additional multiple site biopsies, thus avoiding unnecessary gluten-free diet in clinically and serologically suspected celiac disease patients.
| Conclusion|| |
Precise diagnosis of adult celiac disease is essential. We believe multiple sites duodenal biopsies including D3 region biopsies might increase the diagnostic accuracy of celiac disease in addition to sensitive and specific serologic tests and help in avoiding gluten-free diet in all suspected celiac disease patients.
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Conflicts of interest
There are no conflicts of interest.
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Department of Pathology, Dayanand Medical College and Hospital, Ludhiana, Punjab
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]