Quantifying intraepithelial lymphocytes and subepithelial collagen band in microscopic colitis,extracting insights into the interrelationship of lymphocytic and collagenous colitis

Microscopic colitis (MC) is the umbrella term for the conditions termed lymphocytic colitis (LC) and collagenous colitis (CC). LC with thickening of the subepithelial collagen band or CC with increased number of intraepithelial T- lymphocytes (IELs) is often seen in MC and may lead to difficulties in correct histological classification.We investigated the extent of overlapping features of CC and LC in 60 cases of MC by measuring the exact thickness of the subepithelial collagen band in Van Gieson stained slides and quantifying number of IELs in CD3 stained slides by digital image analysis. A thickened collagen band was observed in nine out of 29 cases with LC (31%) and an increased number of IELs in all 23 cases of CC (100%). There was no correlation between the thickness of the collagen band and number of IELs.Due to the increased number of IELs in all cases of CC we consider the lymphocytic inflammatory infiltration of the mucosa to be the essential histopathological feature of MC. However, although LC and CC are related due to the lymphocytic inflammation, the non-linear correlation of number of IELs and thickness of the collagenous band indicate differences in their pathogenesis.     

Changes in phenotypically distinct mucosal macrophage populations may be a prerequisite for the development of inflammatory bowel disease.

Previous studies have demonstrated the presence of much more marked macrophage heterogeneity in colonic mucosa affected by the idiopathic inflammatory bowel diseases (ulcerative colitis and Crohn's disease) than in normal mucosa. This study examines the morphology, distribution and phenotypic expression of mucosal macrophage-like cells in biopsies from patients with idiopathic inflammatory bowel disease in comparison with disease control samples from patients with colonic infection or ischaemia. Approximately 80% of macrophage-like cells in histologically normal mucosa co-express the antigens recognized by the monoclonal antibodies RFD1 (an interdigitating cell marker) and RFD7 (a marker for mature tissue macrophages). In idiopathic inflammatory bowel disease, the normal colonic macrophage population is partly replaced by cells staining positively with RFD7 alone, and, to a lesser extent, with RFD1+ dendritic cells. Sections from patients with infections and ischaemia exhibited epithelial HLA-DR positivity and infiltration of the lamina propria by a more heterogeneous population of macrophages than that seen in histologically normal mucosa. However, the displacement of the normal colonic macrophage phenotype by RFD7+ tissue macrophages occurred to a significantly greater extent in idiopathic inflammatory bowel disease than in disease control mucosa. A pathognomonic feature of the ulcerative colitis and Crohn's colitis sections was the clustering of RFD9+ epithelioid cells at the bases of disrupted crypts and adjacent to areas of mucosal damage. It is concluded that a degree of macrophage heterogeneity and macrophage infiltration can occur as a non-specific response to colonic mucosal damage. The distinctive feature of idiopathic inflammatory bowel disease mucosa is the almost complete replacement of the normal colonic mucosal macrophage population by tissue macrophages and epithelioid cells, and this phenomenon may be important in promoting the development of a chronic inflammatory state.     

Is Epstein-Barr virus infection associated with the pathogenesis of microscopic colitis?

BackgroundEpstein-Barr virus (EBV) has been associated with inflammation in the colon, particularly in patients with inflammatory bowel disease (IBD). Even if a relevant plasmocytosis, similar to IBD, is present in microscopic colitis (MC), the frequency of EBV infection in this setting is unknown.ObjectivesWe aimed to compare the frequency of colonic EBV infection in patients with MC, ulcerative colitis (UC), and irritable bowel syndrome (IBS).Study designThe frequency of colonic EBV infection in biopsies of 30 patients with MC, 30 patients with UC, and 30 controls with IBS was retrospectively assessed. PCR was performed to detect viral EBV DNA in colonic biopsies. In situ hybridization was also performed to identify and localize EBV-encoded small RNA1 and 2 (EBERs) within cells.ResultsThe presence of EBV DNA was detected in 27 out of 30 MC patients, in 20 out of 30 UC cases, and in none of IBS group. The frequency of EBV DNA in MC was significantly higher compared with that reported in UC (90.0% vs. 66.7%, p = 0.03). EBERs+ cells were observed in 18 out of 30 MC patients, in only 3 out of 30 UC patients (60.0% vs. 10.0%, p < 0.001), and in none of IBS group.ConclusionsEBV infection is almost always detectable in the colonic mucosa of patients with MC. Further studies are necessary to confirm this association and to clarify the role of EBV in MC and, more generally, in colonic inflammation.     

Cryptal lymphocytic coloproctitis: a new phenotype of lymphocytic colitis?

BACKGROUND/AIMS: Lymphocytic colitis is a clinicopathological entity characterised by protracted watery diarrhoea and an increased number of intraepithelial lymphocytes (IELs) in the surface epithelium of the colonic mucosa. This report describes two patients with symptoms similar to those of lymphocytic colitis and an increased number of IELs, but within the cryptal epithelium. METHODS: The numbers of IELs were assessed in colorectal biopsies from the two patients. Sections were stained immunohistochemically for CD3, CD8, CD20, and TIA1. RESULTS: The colorectal biopsies had an abnormally high number of IELs in the epithelium of the crypts but not in the surface epithelium. The IELs in the crypts were CD3+++, CD8+, TIA1+, and CD20-. CONCLUSIONS: The histological diagnosis in these two patients was cryptal lymphocytic coloproctitis. Patients with similar symptoms and an increased number of IELs in the surface epithelium are now filed at this department as having surface lymphocytic coloproctitis. Immunohistochemistry showed that the cryptal IELs were cytotoxic suppressor T cells. Interestingly, a case of cryptal lymphocytic colitis was recently recorded in a non-human primate dying after years of protracted chronic diarrhoea. It is possible that antigens present in the lumen of the crypts elicit a lymphocytic reaction within the cryptal cells.     

Digital image analysis: a review of reproducibility, stability and basic requirements for optimal results

Digital image analysis (DIA) is increasingly implemented in histopathological research to facilitate truly quantitative measurements, decrease inter-observer variation and reduce hands-on time. Originally, efforts were made to enable DIA to reproduce manually obtained results on histological slides optimized for light microscopy and the human eye. With improved technical methods and the acknowledgement that computerized readings are different from analysis by human eye, recognition has been achieved that to really empower DIA, histological slides must be optimized for the digital 'eye', with reproducible results correlating with clinical findings. In this review, we focus on the basic expectations and requirements for DIA to gain wider use in histopathological research and diagnostics. With a reference to studies that specifically compare DIA with conventional methods, this review discusses reproducibility, application of stereology-based quantitative measurements, time consumption, optimization of histological slides, regions of interest selection and recent developments in staining and imaging techniques.     

Colonic mucosal inflammatory cells in children and adolescents with lactase deficiency

Introduction and objectivesMany of the symptoms of patients with lactose intolerance are due to fermentation of undigested lactose in the colonic lumen, which may also lead to inflammatory cell changes in the colonic mucosa. The objective of our project was to understand the histopathological changes involving infiltration of eosinophils and mast cells in the colonic mucosa of children with lactase deficiency (LD).MethodsIn this retrospective study we studied colonic mucosa of children and adolescents with LD to determine if any pathological changes or inflammatory cell changes were present. Pathology reports and Hematoxylin and eosin stained slides were reviewed. Tryptase immunohistochemistry was performed for mast cell assessment.ResultThere were 30 subjects in the study who had a LD and 15 presented with diarrhea and 15 without diarrhea. The colonic mucosa of 35.5 % of the subjects revealed increased mucosal eosinophils. There was no increase of mast cells or lymphocytic colitis in any of the subjects. Excepting for the increased eosinophils in a subset of the subjects, all had a normal appearance of the colonic mucosa.ConclusionColonic mucosa of children and adolescents with LD has a normal histological appearance in majority of the patients. However, 35 % of the patients could demonstrate elevated eosinophils. In primary LD without any comorbidity there is no increase of mast cells and lymphocytic cells in the colonic mucosa.     

Negative prognostic value of intra-ductal fat infiltrate in breast cancer

BackgroundRecent studies showed a correlation between Body Mass Index and both breast cancer occurrence and progression. Nevertheless, no study reported an accurate evaluation of intra-ductal fat infiltrate. Therefore, the main aim of this study was to evaluate the putative association between intra-ductal fat infiltrate (IDFi) and breast cancer subtypes by using digital pathology.MethodsWe retrospectively collected 220 breast biopsies. Paraffin serial sections were used for haematoxylin and eosin staining and immunohistochemical evaluation of the following markers: estrogen receptor (ER), progesterone receptor (PR), Ki67 and c-erb2. Three haematoxylin and eosin sections for each paraffin block were digitalized. Digital slides were used to evaluate the areas of IDFi. Five randomized areas were evaluated for each slide. By using GraphPad software IDFi areas was correlated with a) breast cancer histotype, b) presence of microcalcifications and c) biomarkers expression.ResultsBreast biopsies were classified as follow: 20 normal breast, 50 benign lesions, and 150 malignant lesions (85 ductal in situ carcinomas; 65 ductal infiltrating carcinomas). Statistical analysis showed a significant increase of IDFi in malignant lesions as compared to both normal breast and benign lesions. We noted higher IDFi in breast ductal carcinomas as compared to lobular lesions. Significant differences were observed between breast lesions with microcalcifications respect to lesions without calcifications. Noteworthy, we also found a positive association between IDFi and the expression of both ER and Ki67.ConclusionResults of our study highlighted the possible role of fat in breast cancer progression suggesting a negative prognostic value of IDFi.     

Specific histological abnormalities are more likely in biopsies of endoscopically normal large bowel after the age of 60 years

O’Mahony O H, Burgoyne M & Going J J
(2012) Histopathology Specific histological abnormalities are more likely in biopsies of endoscopically normal large bowel after the age of 60 years Aims: Colonic and rectal biopsies, often taken from an endoscopically normal large bowel, form a significant proportion of the histopathology workload. The aim of this study was to determine diagnostic yield from mucosal biopsies in patients with normal colonoscopy or sigmoidoscopy, and whether or not diarrhoea is predictive of abnormal histology. Methods and results: A retrospective analysis of pathology requests, endoscopy and pathology reports taken during 1 year was undertaken in a tertiary care hospital for all biopsies from endoscopically normal ileal, colonic and rectal mucosa. Of 626 patients fulfilling inclusion criteria, 602 had at least one colonic or rectal biopsy. Colorectal histology was abnormal in 65 (14.5%) of 447 patients with diarrhoea, while of 155 patients without diarrhoea, histology was abnormal in 17 (11%; P = 0.41). Patients older than 60 years had a markedly increased likelihood of a specific histological abnormality [odds ratio 2.76 (1.30–5.79); P = 0.0045]. Diagnoses included microscopic colitis, distorted mucosal architecture consistent with inflammatory bowel disease, ischaemia, polyps, mucosal prolapse and schistosomiasis. Conclusions: Biopsy of an endoscopically normal large bowel, and of the normal terminal ileum in isolation, yields little abnormal histology. Diarrhoea per se does not identify patients at higher risk of abnormal histology. Increased age, however, does, and mucosal biopsy in the endoscopically normal colon and rectum may be more cost‐effective in patients aged more than 60 years.     

Automated quantification and architectural pattern detection of hepatic fibrosis in NAFLD

Accurate detection and quantification of hepatic fibrosis remain essential for assessing the severity of non-alcoholic fatty liver disease (NAFLD) and its response to therapy in clinical practice and research studies. Our aim was to develop an integrated artificial intelligence-based automated tool to detect and quantify hepatic fibrosis and assess its architectural pattern in NAFLD liver biopsies. Digital images of the trichrome-stained slides of liver biopsies from patients with NAFLD and different severity of fibrosis were used. Two expert liver pathologists semi-quantitatively assessed the severity of fibrosis in these biopsies and using a web applet provided a total of 987 annotations of different fibrosis types for developing, training and testing supervised machine learning models to detect fibrosis. The collagen proportionate area (CPA) was measured and correlated with each of the pathologists semi-quantitative fibrosis scores. Models were created and tested to detect each of six potential fibrosis patterns. There was good to excellent correlation between CPA and the pathologist score of fibrosis stage. The coefficient of determination (R²) of automated CPA with the pathologist stages ranged from 0.60 to 0.86. There was considerable overlap in the calculated CPA across different fibrosis stages. For identification of fibrosis patterns, the models areas under the receiver operator curve were 78.6% for detection of periportal fibrosis, 83.3% for pericellular fibrosis, 86.4% for portal fibrosis and >90% for detection of normal fibrosis, bridging fibrosis, and presence of nodule/cirrhosis. In conclusion, an integrated automated tool could accurately quantify hepatic fibrosis and determine its architectural patterns in NAFLD liver biopsies.     

Reduced number of Langerhans cells in oesophageal mucosa from AIDS patients

AIMS: The oesophageal mucosa is a frequent target of opportunistic infections in human immunodeficiency virus (HIV) infection. Langerhans cells (LC) are known as a target and reservoir of HIV in the skin. The aim of this study was to characterize oesophageal LC in HIV-infected patients. METHODS AND RESULTS: Thirty oesophageal biopsies were obtained from 29 patients (median age 35.5), all in stage IV of the HIV Center of Disease Control Classification. We performed histological assessment of the oesophageal mucosa and immunohistochemical detection of oesophageal LC using an anti-CD1a antibody, followed by morphometric analysis. Biopsies from 17 noninfected patients were studied using the same procedure. LC in oesophageal mucosa of the HIV positive patients showed a significantly and dramatically decreased number (LC(N) median = 5.85/mm2) and surface/epithelial surface (LC (S) ratio = 0.09) when compared with HIV-negative controls (LC(N) median = 29.7/mm2, LC(S) ratio = 1.83) with P = 0.003 for LC(N) and P < 0.0001 for LC(S). CONCLUSION: These data suggest that oesophageal LC are, like their epidermal counterparts, a preferential target for HIV infection. Their alterations may provide a clue to the pathogenesis of the decreased local oesophageal immunity and to the occurrence of opportunistic infections.     

Quantitation of intraepithelial lymphocytes in human duodenum: what is normal?

BACKGROUND: An increase in intraepithelial lymphocytes (IELs) is mandatory for the histological diagnosis of coeliac disease (CD). Currently, duodenal biopsies are used almost exclusively to establish the diagnosis, yet published work continues to cite an upper limit of 40 lymphocytes/100 epithelial cells, a figure derived from jejunal biopsies over 30 years ago. Aim: To establish the normal range for IEL counts in distal duodenal biopsies. MATERIALS/METHODS: Twenty subjects (seven men, 13 women; median age, 34 years; range, 20-65) with a normal sugar permeability test and concurrent distal duodenal biopsies were identified. The number of IELs and epithelial cell nuclei in an uninterrupted length of surface (villous) epithelium (> 500 cells) was counted. An image analysis system was used to assess villous architecture by calculating the villous height to crypt depth ratio. RESULTS: The range of IEL counts in 20 subjects was 1.8-26/100 villous epithelial cells, with a mean value of 11 and SD of 6.8. The mean villous to crypt ratio was 1.82 (SD, 0.38; range, 1.22-2.46). There was no correlation between IEL counts and villous to crypt ratio (Spearman rank correlation, -0.066; p = 0.80). CONCLUSIONS: These results suggest that 25 IELs/100 epithelial cells (mean +2 SD) should be taken as the upper limit of the normal range for duodenal mucosa.     

Subepithelial myofibroblasts and tenascin expression in microscopic colitis

AIMS: To assess differences in the pattern of subepithelial myofibroblasts and the expression of tenascin as a marker of extracellular matrix production in collagenous and lymphocytic colitis. METHODS AND RESULTS: Colorectal biopsies were studied from 122 patients with chronic diarrhoea and normal colonoscopy. The pathological diagnoses were collagenous colitis (n = 35), lymphocytic colitis (n = 37), mild non-specific chronic inflammation (n = 28) and normal mucosa (n = 18). Four cases showed features of collagenous colitis but with collagen bands <10 micro m thick. Normal mucosa from 14 patients without diarrhoea served as healthy control tissue. Immunohistochemical expression of alpha-smooth muscle actin (myofibroblast marker) and tenascin was evaluated in well-orientated sections. The expression of alpha-smooth muscle actin was significantly increased in collagenous colitis compared with all the other groups. Strong tenascin subepithelial expression was seen in all cases of collagenous colitis, including the four without full-blown features. The mean thickness of tenascin bands was greater than that obtained by conventional stains. CONCLUSIONS: There are clear differences, with respect to extracellular matrix remodelling, between collagenous and lymphocytic colitis. These results support the theory of matrix overproduction in the genesis of collagenous colitis.     

Reproducibility in the diagnosis of needle core biopsies of non‐palpable breast lesions: an international study using virtual slides published on the world‐wide web

Zito F A, Verderio P, Simone G, Angione V, Apicella P, Bianchi S, Conde A F, Hameed O, Ibarra J, Leong A, Pennelli N, Pezzica E, Vezzosi V, Ventrella V, Pizzamiglio S, Paradiso A & Ellis I
(2010) Histopathology 56, 720–726
Reproducibility in the diagnosis of needle core biopsies of non‐palpable breast lesions: an international study using virtual slides published on the world‐wide web Aims: To conduct an internet‐based study using virtual slides (VS) of sterotactic core biopsy specimens of non‐palpable breast lesions in order to evaluate interobserver reproducibility between pathologists. Methods and results: A total of 18 breast lesions, determined to be histologically complex by two pathologists, were selected. Digitized VSs were then created using QuickTime Virtual Reality technology (Apple, Cupertino, CA, USA) and posted on the world‐wide web. In all, 10 pathologists completed the evaluations of 18 VSs using the five diagnostic categories (B1–B5) from the European guidelines for quality assurance in breast cancer screening and diagnosis. Their results were compared with those of every other participating pathologist, and were then individually compared with the results of a highly experienced breast pathologist (referee). Of the 18 cases, 10 (56%) were classified by the referee as borderline (B3 and B4). Comparisons with reference values showed a less than satisfactory level of reproducibility (median κ_w = 0.60). As regards interobserver reproducibility, results showed that, in general, the level of agreement was not satisfactory (median κ_w = 0.53). Conclusions: Overall, the findings are comparable to those quality control studies using circulating slides when analysis is done on borderline cases.     

Cytokeratin immunohistochemistry improves interobserver variability between unskilled pathologists in the evaluation of tumor budding in T1 colorectal cancer

Tumor budding is a major risk factor for T1 colorectal cancer. Quality control of the pathological diagnosis of budding is crucial, irrespective of the pathologist's experience. This study examines the interobserver variability according to pathologists' experience and evaluates the influence of cytokeratin (CK) immunostaining in the assessment of budding. Hematoxylin‐eosin (HE) and CK‐immunostained slides of 40 cases with T1 primary colorectal cancer were examined. Budding grades were individually evaluated by 12 pathologists who we categorized into three groups by their experience (expert, with >10 years of experience (n = 4), senior, with 5–10 years (n = 4), and junior, < 5 years (n = 4)). The results revealed a tendency for the more experienced pathologists to assign higher budding grades compared to the less‐experienced pathologists. In the junior group, the interobserver variability obtained with HE slides was poor, but it was markedly improved in the evaluation using CK‐immunostained slides. The benefit of CK immunostaining was only slight in the expert group. CK immunostaining would be useful when a pathologist is not experienced enough or does not have enough confidence in the assessment of budding.     

Flow cytometric measurement of intracellular migration inhibition factor and tumour necrosis factor alpha in the mucosa of patients with coeliac disease

There is increasing evidence that proinflammatory cytokines contribute to many of the small intestinal features in coeliac disease. The aim of the study was to investigate the expression of two proinflammatory cytokines, migration inhibition factor (MIF) and tumour necrosis factor alpha (TNF-alpha) in duodenal biopsy specimens from patients with coeliac disease on a gluten-free diet and normal control subjects. A flow cytometric system was used to analyse intracellular protein levels of MIF and TNF-alpha in freshly isolated cells from duodenal biopsies taken from 12 patients with treated coeliac disease and 10 healthy control subjects. From the biopsy specimens, single cell suspensions of the epithelium and lamina propria were prepared using EDTA/DTT and enzymes. Intracellular cytokine expression was studied in intraepithelial lymphocytes (IELs), lamina propria T cells (LP T) and intestinal epithelial cells using different surface labelling antibodies. MIF protein was constitutively expressed in IELs, LP T cells and epithelial cells from normal intestinal mucosa. In contrast, although TNF-alpha was found in LP T cells, this cytokine was virtually undetectable in either IELs or epithelial cells. In coeliac disease, intracellular levels of MIF were significantly higher in epithelial cells compared with control subjects (P = 0.005). Raised levels of TNF-alpha were found in epithelial cells (P = 0.03) as well as IELs (P = 0.045) from coeliac patients compared with controls. The findings from this study show up-regulated expression of MIF and TNF-alpha in IELs and epithelial cells of histologically normal mucosa in patients with coeliac disease. Increased expression of proinflammatory cytokines in cells occupying the epithelial layer could help explain the rapidity with which the coeliac mucosa may respond to gluten challenge.     

Digital image analysis of pan-cytokeratin stained tumor slides for evaluation of tumor budding in pT1/pT2 colorectal cancer: Results of a feasibility study

Tumor budding is an independent prognostic factor in colorectal cancer. However, varying degrees of interobserver agreement and reproducibility challenges the use of tumor budding in diagnostics. Immunohistochemical staining of tumor slides with pan-cytokeratin visualizes the budding tumor cells and has been suggested to improve reproducibility. Here we demonstrate the methodology of tumor budding assessment using digital image analysis based on tumor slides stained for pan-cytokeratin, and investigate interobserver agreement, agreement between manual and digital assessment methods and digital reproducibility between users.Tumor slides from 126 patients with pT1/pT2 colorectal cancer were stained with pan-cytokeratin and tumor budding at the invasive tumor front was assessed by conventional manual microscopy. A digital image analysis algorithm for identification and quantification of budding tumor cells was developed and tested on the pan-cytokeratin stained slides.Manual assessment of tumor budding using pan-cytokeratin stained tumor slides exhibited high correlations (Spearman Rank 0.84-0.89, p?<?0.001),excellent agreement between observers (Intra-class correlation coefficient (ICC): 0.86 -0.87) and 2.20 higher odds for regional metastases with increasing budding counts (p?=?0.017). Digital image analysis correlated well to manual assessment (Spearman Rank 0.71-0.88) and agreement between the two methods was good (ICC 0.62-0.82). However, only a trend towards increased odds for metastatic progression was found for the adjusted digital estimates (p?=?0.076). Digital estimates were higher than manual estimates, demonstrated by a systematic median difference of 3–4.5 buds. Image analysis was highly reproducible between users of the algorithm (ICC 0.98). In conclusion, assessment of tumor budding using pan-cytokeratin stained tumor slides is a method with high correlation and agreement between observers. Digital image analysis quantifies budding tumor cells in high agreement with manual estimates, but approval of the digital slides by a pathologist is mandatory. The method qualifies for further investigation.     

溃疡性结肠炎相关性结直肠癌的危险因素及预防

溃疡性结肠炎是一种直肠及结肠的慢性非特异性炎症性疾病,临床以反复发作的腹泻、腹痛和黏液脓血便为特征。溃疡性结肠炎相关性结直肠癌是溃疡性结肠炎严重的并发症及重要死亡原因,其危险因素包括病程、病变范围、原发性硬化性胆管炎、结直肠癌家族史、发病年龄及炎症严重程度等。不典型增生是溃疡性结肠炎相关性结直肠癌发生的重要环节,早期发现不典型增生,积极化学预防是预防溃疡性结肠炎癌变的关键。