The Study Of Collagen Fiber Difference In Pathological Tissue Between Crohn’s Disease And Intestinal Tuberculosis

BackgroundCrohn’s disease (CD) is a chronic non-specific Inflammatory bowel disease (IBD), of which the etiology is complicated and the pathogenesis is unclear. So far there is not a specific way to diagnose CD. CD is a common digestive system disease in Europe and North America countries, while in developing countries are rare relatively. Along with our country industrialization and urbanization process accelerated, incidence of CD is rising. Intestinal tuberculosis (ITB) is a chronic specific infectious disease caused by mycobacterium tuberculosis, one of the common extrapulmonary tuberculosis, which clinical manifestations are diversiform, so some cases diagnose difficultly. On the clinical manifestations of CD and ITB, there are irregular fever, abdominal pain,diarrhea, hematochezia and abdomianal mass. When lesion confined to ileocecus, radiological images and endoscopic are similar, it is difficulty to identify them. According to some reprot, the misdiagnosis rate between them was50%-70%. Due to the etiology, treatment and prognosis between CD with ITB are different, when ITB misdiagnosed as CD using hormones and immunosuppressive treatment could result in speading of tuberculosis, or even death; when CD misdiagnosed as ITB, patients are suffered unnecessary side-effects causing by anti-tb dugs and delayed treatment of CD may cause some avoidable complications. Therefore, it is very necessary to differentiate CD and ITB before treatment.In2007the consensus of the diagnosis and treatment for IBD in China, about the opinions of the differential diagnosis between CD and ITB, only patients with active tuberculosis or caseous necrotic granuloma in biopsy can diagnosis of ITB surely; patients with one or more characteristics complications such as anus disease, parenteral complications, intestinal fistula, abdominal abscess or hematochezia and recurrence after resection of the lesions, are consider CD. At home and abroad doctors identify CD and ITB mainly depending on traditional examination and experience.Looking for predicting indicators to differentiate CD and ITB through summarizing features among clinical manifestations, endoscope and histopathology, aims to improve the diagnostic sensitivity, specificity and accuracy. This is clinical integrated reasoning and probabilistic diagnosis, only clearing etiology or pathogenesis could solve the difficulty of identify them. The etiology of CD and ITB is differernt, so the key to identify them is to find mycobacterium tuberculosis. However, it is extremely low positive rate in the traditional acid fast stain microscopy and bacterial culture, so the value is little. PCR technology provides a new method to identify them. Studies have foud that PCR technique detect mycobacterium tuberculosis DNA positive rate was71.4%in granulomas without caseous necrosis of intestinal tuberculosis biopsy tissues, and there was no1positive case in the same biopsy tissues of CD. It means PCR technique is valuable for differentiate CD and ITB, especially in some ITB case only find out granuloma but without caseous necrosis. PCR technique is difficult to applicate in clinic on large scale, because it’s operation process is complex, the technical requirements is high and it should be carefully to prevent RNA enzyme to contaminate and it is also very complicated to choose the specificity of the primers design. Therefore it is neccessary to find more simple, economic, feasible and suitable for clinical application methods to differentiate CD and ITB.In CD and ITB lesions, intestinal tissue undering chronic inflammation stimulating and irreversible response form fibrosis. Histology is the intestinal muscle overgrowth, excessive collagen deposition and interstitial cells overgrowth. When intestinal damage, lamina propria subepithelial myofibroblast promote healing wound and the secretion of collagen and cell proliferation. Excessive collagen deposition and excessive proliferation of mesenchymal cells can lead to the occurrence of intestinal fibrosis. The study intends to research the different characteristics of intestinal fibrosis between CD and ITB, and set up a new way of diagnosis intestinal fibrosis for CD and ITB. It is helpful for diagnosis and differential diagnosis of CD and ITB, and lay the foundation for further clinical treatment. However, it is difficult to evaluate the intestinal fibrosis, even the intestinal tissue pathology biopsy recognized as the gold standard has limits. Traditional pathology of tissue fibrosis, the evaluation is based on the particular collagen fiber staining method and pathology doctor’s assessment, so the objectivity and repeatability is poor.In recently, nonlinear potics technology provides a new method for researching organization within the collagen fiber. Second harmonic generation (SHG) microscopic imaging technique is the most suitable for non centrosymmetric molecular imaging, because almost all types of collagen fibers can produce SHG signals. SHG can display the collagen fiber and its specificity of three dimensional form. Two-photon excited fluorescence (TPEF) can display cell matrix structure and location of collagen deposition. In1986, Freund applied SHG in rat tail tendon collagen fiber imaging for the first time. Followed, including cornea, rat tail tendon and bovine type I Achilles tendon, skin, are also used in model mice kidney fibrosis, rat model of liver fibrosis, liver fibrosis research and by observing the different collagen structure to identify benign and malignant tumor, early diagnosis of tumor recurrence, etc. SHG image can be obtained by computer processing, accurate interpretation of tissue fibrosis degree. Studies also found that it is significantly different in three-dimensional structure of collagen fibers and deposition methods among different factors lead to fibrosis, and it is a worthy to explore to infer the cause by collagen fiber three-dimensional structure. Jianxin Chen observed normal stomach and gastric mucosal and submucosal tissue characteristics by SHG/TPEF technology, in order to improve early diagnosis rate of gastric cance by the technology. But applying this technology to detect colon lesions fibrosis characteristics has not been reported.At home and abroad, studies showed SHG/TPEF microscopic imaging technique can display organization specificity of fibrous deposition of collagen, to realize to the automated assessment fibrosis of liver and kidney in the non-staining tissue, and SHG is able to display the three-dimensional structure of cellulose deposition, and can be used to speculate that the cause of fibrosis. This study through to clinical features, endoscopic examination and pathological specimens review study of crohn’s disease and intestinal tuberculosis clinical cases, summarizing the two different characteristics, looking for predicting identify the indicators of CD and ITB, aims to improve the sensitivity, specificity and accuracy of the diagnostic. And by applying the traditional pathology (Masson’s dyeing) and SHG/TPEF microscopic imaging, observe cellulose deposition characteristics between CD and ITB, to explore a new method of the differential diagnosis of both.Part1:Compare clinical, endoscopic and pathologic between Crohn’s disease and intestinal tuberculosisObjective Compare clinical, endoscopic and pathological features of crohn’s disease and intestinal tuberculosis, in order to find prediction identify the indicators between CD and ITB, to improve the accuracy of diagnosis.Methods Retrospective method, find our hospital from2006to2012who diagnosed with CD and ITB, there are39cases of CD and34cases of ITB. CD group:23cases of male,13cases of female, mean age:39.13±18.39years old; ITB group:14cases of male,20cases of female, mean age:36.62±14.09years old. Collected the two groups cases datas respectively:(1) general information:name, gender, age and region (rural, urban);(2) clinical symptoms:abdominal pain, diarrhea, constipation, hematochezia, fever, night sweats and lose flesh, ascites;(3) merging complications:lumen stenosis, intestinal obstruction, fistula formation and parenteral tuberculosis;(4) the experimental examination:hemoglobin, blood sedimentation, albumin and PPD test;(5) endoscopic performance:mucosal hyperemia and edema, erosion, ulcer, longitudinal ulcer, circular ulcers, pebbles sign, segmental lesions, lumen stenosis, pseudo polyps, mucosal bridge and ileocecal valve involvement;(6) the pathological histology:inflammation, granuloma formation, caseating granuloma, non-caseating granulomas, crack ulcer, gland hyperplasia and granulation tissue. Compare the two groups cases data of CD and ITB, counts the data performed by chi-square test, measurement data performed by two-sample t-test, and P value<0.05was considered to be significant. All calculations were processed using the SPSS13.0software.Results (1) Region:CD is concentrated in the cities (61.5%), ITB is in rural areas (64.7%), P=0.025).(2) Clinical symptoms:hematochezia supported CD(17.9%, P=0.041), night sweats (CD group7.7%<ITB group29.4%, P=0.016); ascites (CD group7.7%<29.4%, P=0.016) supported the diagnosis of ITB; compared abdominal pain(P=0.562), diarrhea (P=0.743), constipation (P=0.380), fever (P=0.244), thin (P=0.541) in the two groups had no statistical difference.(3) Merging complications:fistula formation (CD group17.9%> ITB group2.9%, P=0.041) support the diagnosis of CD and parenteral tuberculosis (CD group2.6%<ITB group58.8%, P=0.000) support the diagnosis of ITB. However comparing lumen stenosis (P=0.719) and intestinal obstruction (P=0.208) in two groups had no statistical difference.(4) Experimental examination:comparing hemoglobin (108.46of CD group±19.01g/L<111.81±19.17g/L of ITB group, P=0.458), blood sedimentation (37.71±19.98mm/h of CD group>31.91±17.45mm/h of ITB group, P=0.194) and albumin (33.47±7.87g/L of CD group>31.91±17.45g/L of ITB group, P=0.282) in the two groups had no statistical significance.(5) Endoscopic performance:longitudinal ulcer (CD group33.3%, ITB group6.5%, P=0.008), segmental lesions (CD group48.5%, ITB group9.7%, P=0.001), false polyps (CD group48.5%, ITB group16.1%, P=0.006) and mucous bridge (CD group21.2%, ITB group3.2%, P=0.030) were more support to crohn’s disease diagnosis, and pebbles sign (CD group21.2%, ITB group0%, P=0.007) was only found in crohn’s disease; Annular ulcer (CD group12.1%, ITB group41.9%, P=0.007) and ileocecal valve involvement (CD group51.5%, ITB group77.4%, P=0.031) were supported to intestinal tuberculosis diagnosis; Compared mucosal hyperemia edema (P=0.854), erosion, ulcer (P=0.955)(P=0.894) and lumen stenosis (P=0.362) between the two groups had no statistical difference.(6) Pathological histology:crack ulcer (14.3%, P=0.022) was only found in crohn’s disease, and caseating granuloma (17.6%, P= 0.009) was only to intestinal tuberculosis, both comparision between the two groups were statistically significant. Comparision Inflammation (P=0.307), granuloma formation (P=0.113), non-caseating granulomas (P=0.733), gland hyperplasia (P=0.082) and granulation tissue formation(P=0.463), there were no statistical difference.Conclusion Because it is very similar between Crohn’s disease and intestinal tuberculosis on clinical symptoms, endoscopic and histopathological manifestations, they are easy to misdiagnosis. Pebbles sign and crack ulcer are characteristic of Crohn, and caseating granuloma restricted to intestinal tuberculosis, but their occurrence rate is low, therefore, the identification value is limited by reling on endoscopic and histopathological, it Should be combined with clinical symptoms, laboratory tests and others comprehensive judgment. Part2:Analysis of collagen fiber difference in pathological tissue between Crohn’s disease and intestinal tuberculosisObjective There are considerable difficulties to identify CD and ITB through the clinical symptoms, endoscopic and histopathological manifestations. It is obviously different in tissue of collagen fibrous three-dimensional structure caused by different causes. To observe collagen fibers deposition characteristics of CD and ITB by applying the traditional pathology (Masson’s dyeing) and SHG/TPEF microscopic imaging, intend to explore a new method of the differential diagnosis of both.Methods Collected our hospital pathology specimen of who diagnosed CD and ITB. CD group had29cases (25cases of endoscopic biopsy specimens,4cases of the surgical specimens), ITB group had14cases (12cases of endoscopic biopsy samples,2case of surgical specimen) and healthy controls had11cases.of enteroscope biopsy specimens (took the ileocecus tissue). Each pathological specimen cut serial section3, to do HE staining, Masson’s three dyeing and SHG/TPEF imaging respectively. Determination of collagen fibers by Masson trichromatic dyeing imaging, using the Image-Pro Plus6.0software to analysis images, calculating the Average optical density absorbance (AOD), AOD=integral absorbance/(total area-the blank area), to reflect the expression levels of collagen fibers; Measurement integral Area (Area), Average area=integral Area/(total Area-the blank Area), to reflect the collagen deposition Area. Observed the characteristics of collagen deposition SHG/TPEF images. Comparison among multiple groups performed by a standard one-way analysis of variance, compared between groups performed by LSD-t method if the variance is neat, or Dunnett’s T3multiple comparison if the variance is not neat. Comparision between CD and ITB was performed by two-sample t-test, small sample by Fisher’s exact probability method. P value<0.05was considered to be significant. All calculations were processed using the SPSS13.0software.Results (1) Masson’s three dyeing:among CD, ITB group and healthy control group, the ITB group collagen expression quantity highest average absorbance (=560.1772±230.6484), the largest of collagen deposition (1116.115±1116.115). Collagen fibers and collagen deposition area were higher of CD group and ITB group than that of healthy controls, and P<0.05. but the CD group compared with ITB group had no statistical difference., P>0.05. After eliminating surgical specimens, collagen expression quantity and collagen area of ITB group were higher than CD group, comparison between the two groups P<0.05. In endoscopic biopsy specimens, collagen expression quantity in specimens with granuloma (430.2869±187.6046) was higher than the specimens without granuloma (273.6598±243.2126), and the comparision between them was statistically significant (P<0.05); collagen fiber area in specimens with granuloma (902.1117±379.9651) were significantly larger than that without granuloma (557.4849±380.6856), the comparision between them was statistically significant (P<0.05).(2) SHG/TPEF imaging:in surgical specimens of CD and ITB, there were in a large number of collagen fibers deposition in the submucosa, and collagen fibers in CD group looked like clumps and curling, without obvious regularity, and in ITB group collagen fibers rang aroud the caseating granuloma. Both in endoscopic biopsy specimens collagen fibers could be found scattering around the glands. Conclusion It is valuable to use Masson’s dyeing and SHG/TPEF imaging in evaluation of crohn’s disease and intestinal tuberculosis fibrosis. In endoscopic biopsy specimens, intestinal tuberculosis collagen expression quantity higher than crohn’s disease, and it could be a the new basis to identify them.