| Abstract|| |
Background: Inflammatory bowel diseases (IBDs) mainly consist of Crohn's Disease (CD) and Ulcerative Colitis (UC). These two categories have overlapping histopathological features and sometimes it is difficult to diagnose them into distinct category and such biopsies are categorised as Inflammatory Bowel Disease (IBD-U). Recently, there has been an increase in interest to discover new biomarkers of IBD to differentiate UC and CD and predict their prognosis. Method: In the present study, 273 non-neoplastic colonic biopsies with clinicoendoscopic features of IBD were studied and categorized into UC (88; 32.3%) and CD (03; 1.1%) but a major chunk remained in category of IBD-U (182; 66.6%). 161 (58.9%) of these biopsies were then subjected to IHC for RB protein and β-catenin and Serology for pANCA and ASCA was done in only 85 (31.13%) of these selected cases for identification of UC and CD on colonic biopsies. Result: 161 biopsies that were subjected to IHC analysis included 57 cases of UC, 03 cases of CD, and rest 101 cases of IBD-U. Out of 101 cases of IBD-U, 87 (86.13%) cases were reclassified as UC (61; 60.3%) and CD (14; 13.86%) on the basis of results of IHC and Serology. Conclusion: The two major tools IHC for β-catenin and RB protein and the assay of serum ASCA and p-ANCA along with proper history and clinical presentation can act as a good adjunct to conventional H and E in subclassifying cases of IBD-U into UC and CD.
Keywords: ASCA, β-catenin, colonic biopsies, Crohn's disease, inflammatory bowel diseases, inflammatory bowel diseases- unclassified, p-ANCA, RB protein, ulcerative colitis-catenin
|How to cite this article:|
Paridhi, Singh K, Kumar V, Tewari S, Misra V, Misra SP, Dwivedi M. Role of Immunohistochemistry and serology in subclassifying the Inflammatory Bowel Disease cases diagnosed as Inflammatory Bowel Diseases-unclassified on colonic biopsies. Indian J Pathol Microbiol 2022;65:558-64
|How to cite this URL:|
Paridhi, Singh K, Kumar V, Tewari S, Misra V, Misra SP, Dwivedi M. Role of Immunohistochemistry and serology in subclassifying the Inflammatory Bowel Disease cases diagnosed as Inflammatory Bowel Diseases-unclassified on colonic biopsies. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Aug 15];65:558-64. Available from: https://www.ijpmonline.org/text.asp?2022/65/3/558/351617
| Introduction|| |
Crohn's disease (CD) and ulcerative colitis (UC) are the two main forms of inflammatory bowel diseases (IBDs), characterized by intestinal inflammation and ulceration of unknown etiology. Although CD and UC share similar pathophysiological mechanisms, such as immune activation, leukocyte infiltration, and increased colonic vascular density, they usually present important differences regarding anatomical localization, histopathological findings, disease progression, and therapeutic response.
The prevalence of IBD rapidly increased in Europe and North America in the second half of the 20th century and is becoming more common in the rest of the world as different countries adopt a Western lifestyle and is now seen commonly in India.
IBD-U is characterized by clinical and endoscopic signs of chronic colitis without specific features for UC or CD but rather subtle features of both. Initially, it was considered a temporary diagnosis or a provisional descriptive term; it may actually represent a separate subgroup of IBD, although the lack of a specific diagnostic test or marker makes it a diagnosis of exclusion.
It is this particular point in the diagnostic algorithm of IBD where serological testing has the greatest benefit. The measurement of serological markers has been used as a method of differentiating between UC and CD. Two kinds of antibodies have been extensively studied for this purpose: perinuclear antineutrophil cytoplasmic antibodies (pANCA) and antibodies against the cell wall of Saccharomyces Cerevisiae (ASCA). Although most cases of indeterminate colitis eventually “evolve” into definite UC or CD, a substantial number of patients remain with a diagnosis of IC (Indeterminate Colitis). Due to their lack of sensitivity, serological markers are not advisable for use in the diagnosis of IBD but rather in differentiating CD from UC, particularly with the use of a wide panel of antibodies. Serological markers such as acute phase reactants, cytokines and adhesion molecules, and faecal markers such as calprotectin and lactoferrin have been studied for assessment of disease activity. However, considerable effort has been devoted to the development of an accurate panel of non-invasive biomarkers that have increased diagnostic sensitivity and specificity. IHC along with serological biomarkers in differentiating IBD and in evaluating disease activity, prognosis, and treatment selection for IBD will be presented in this study.
Till date, no well documented study on this topic from India with immunohistochemical and serological correlation is available in literature; hence this study on lower gastrointestinal biopsies with special reference to inflammatory bowel disease was undertaken. As the cases of CD are increasing in India and that can sometimes be missed due to lack of proper histological criteria, the present study was taken to assess the role of IHC for RB protein and β-catenin and Serological assay for pANCA and ASCA into identifying cases of UC and CD from those labelled as IBD-U.
| Materials and Methods|| |
Patients: 273 non-neoplastic lower GI biopsies were included in the present study during a period of about 4 years (September 2016 to August 2020), of these 121 were collected prospectively and 152 were taken from the archives of the department along with clinicoensoscopic details of patients (retrospective). 15 controls were taken from the resected margins of colectomy and hemicolectomy specimens sent to the department for non-neoplastic causes like intussuceptions and perforations.
Colonoscopic examination: The subjects in prospective group (121 cases) underwent full length colonoscopy and the entire lower gastrointestinal tract was thoroughly examined. Details of lesions were noted and 4-5 biopsy pieces were taken from areas showing maximum changes. Details of retrospective biopsies were taken from the archives of the department.
Histopathology: The biopsies of prospective cases were fixed in 10% formal saline, processed routinely and stained with H and E for morphological details and retrospective cases were archived which were already stained with H and E. Thereafter, final diagnosis was made after proper correlation with patient's clinicoendoscopic findings with special attention to histological features of IBD. These biopsies were categorised into UC and CD as per the features described by (Pignatelli and Gallagher); 2014 [Table 1]. Diagnosis of IBD-U was kept when there were overlapping features of UC and CD and a definite diagnosis could not be made. Ethical clearance was taken on 31/07/2018.
|Table 1: Histological features of UC and CD for their definite categorization on biopsies|
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IHC was done in 161 cases of IBD that included 57 cases of UC, 03 cases of CD, and 101 cases of IBD-U and the expression of β -catenin and RB gene was studied to categories IBD-U cases into UC and CD. IHC study was carried out using streptavidin ABC labelling technique which was visualized using DAB. Sections were dewaxed, washed in alcohol, and antigen retrieval was carried out by pressure cooker [Bio SB] with Tris Buffer at pH of 10 for 35 min. Monoclonal mouse Anti-β-catenin, Clone 14 (Bio SB, Santa Barbara, USA) was used for β-catenin and Recombinant human Rb protein, Clone 1F8 (Bio SB, Santa Barbara, USA) was used for RB protein. A total of up to 20 crypts were examined and β-catenin and RB protein expression was scored as described by Jass et al.; (2003) for β -catenin and Goldhoff et al.; (2012) for RB protein.,
β-catenin scoring was based upon the distribution and intensity of β-catenin within the cell membrane (0-1), cytoplasm (0-2) and nuclei (0-2) as below.
Score 1 reflects cell membrane staining only similar to that seen in normal colonic mucosa. Total scores were then collapsed into three groups, that is, with score of 4 and 5 as true positive β-catenin expression, 3 being border line and 0, 1, 2, and negative β-catenin expression [Figure 1].
|Figure 1: β -catenin Scoring and expression by IHC- 1a) Biopsy from control expressing membranous β -catenin expression (Score 1) (IHC x100), 1b) Biopsy from a case of CD showing cytoplasmic and membranous expression of β -catenin (Score 2) (IHC × 400), 1c) Biopsy from a case of UC showing diffuse cytoplasmic and membranous expression of β -catenin (Score 3) (IHC × 400), 1d) β -catenin expression in nucleus and cytoplasm from a case of UC (Score 4) (IHC × 100), 1e) Biopsy from a case of UC showing diffuse β -catenin expression in nucleus and cytoplasm (Score 5) (IHC × 100)|
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RB protein score was based on distribution as below [Figure 2].
|Figure 2: Retinoblastoma protein Expression and Scoring by IHC- 2a) Biopsy from a case of UC expressing < 20% RB protein expression (Score 0) (IHC × 400), 2b) Biopsy from control expressing 20-50% RB protein expression (Score 1) (IHC × 400), 2c) Biopsy from a case of Crohn's disease expressing RB protein expression in >50% cells (Score 2) (IHC × 400)|
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β-catenin and RB expression by IHC were compared with similarly scored controls.
Serology: 2 ml of blood was obtained from patients diagnosed with UC, IBD-U, and CD at the time of collection of reports in a plain vial. Serum was then frozen at -20°C. Serum of 85 IBD patients along with 5 control sera that were obtained from healthy donors coming to blood banks as well as provided by the kit were assayed for MPO-ANCA and ASCA (Aesku Diagnostics, Wendelsheim, Germany) by solid phase ELISA (Enzyme Linked Immunosorbent Assays) with quantitative interpretation. Standard curve was established by plotting the Optical Density values of each calibrator on y-axis with respect to the corresponding concentration values in U/ml on x-axis. The obtained ODs of each sample were plotted on the graph and corresponding concentration expressed in U/ml were obtained as patient's Serum p-ANCA or ASCA values. The serum concentration of pANCA and ASCA then categorised into positive, equivocal, and negative values on the basis of the values provided by the kit (i.e., Normal range <12 U/ml, Equivocal Range 12-18 U/ml, and Positive Result >18 U/ml)
Statistical analysis using 2021 Graph Pad Prism software was carried out using Fisher's exact Test, unpaired t-test and Pearson test to calculate “P” value. “P” value of ≤ 0.05 was considered statistically significant.
| Result|| |
IBD cases were further categorized into three groups as UC constituting 88 (32.23%) cases, CD with 03 (1.10%) cases and IBD-U constituting up to 182 (66.66%).
Spectrum of IBD
A. Ulcerative Colitis: It constituted about 32.23% of IBD cases. Male: female ratio was 2.03:1 with most of the cases belonging to 21-30 year age group. It was observed that 57 (64.78%) of biopsies were obtained from mucosal ulcers alone, 8 (9.09%) from strictures and 21 (23.86%) from areas with loss of vascular pattern with mucosal friability and the rest of the cases, i.e., 2 (2.27%) were from polypoidal lesions with ulcers. Most common site affected was descending colon 33 (37.51%), followed by rectosigmoid 25 (28.41%), pancolitis 21 (23.86%), ascending colon 7 (7.95%), and backwash ileitis 2 (2.27%) in the decreasing order. Histologically, 100% of cases had chronic inflammatory infiltrate, 97.72% cases had features of cryptitis and 81.82% cases had crypt distortion, 54.54% showed crypt abscess, and 27.27% showing acute inflammatory cells. 18.18% of the cases showed loss of goblet cells and 11.36% showed Paneth cell metaplasia.
B. Crohn's Disease: It accounted for 1.10% of all IBD cases with a male: female ratio of 1:2 belonging to 21-30 year age group. Histological features showed deep fissuring ulcers with cryptitis, crypt distortion, and non-cryptolytic microgranulomas along with transmural inflammation.
C. Inflammatory Bowel Disease-Unclassified: This category accounted for 66.66% of the IBD cases. The most common presenting age group was 21-30 years with mean age being 25.27 years contributing to 33.52% of the cases with a male: female ratio of 2.14:1. Most common presentation was bleeding per rectum in 77 cases (42.31%), followed by diarrhea in 52 cases (28.57%) and abdominal pain in 41 cases (22.53%). Loss of weight/appetite was seen in only 12 (6.59%) cases. It was seen that pancolic involvement (49.51%) was most common, followed by descending colon (16.83%) and terminal ileum (16.83%) equally, rectum (9.90%), ascending colon (4.95%), and transverse colon (1.98%) in the decreasing order.
Histological features like Chronic inflammation (100%) was seen most commonly followed by deep ulcers (70.3%), cryptitis, presence of granuloma, crypt distortion, muscular hypertrophy, acute inflammation, crypt abscess, and others as mentioned in Table 2.
IHC: IHC expression of β-catenin was significantly higher in cases of UC and IBD-U when compared to the control group, (P < 0.0001). Also, it was observed that the total score of β-catenin in UC was extremely statistically significant when compared to the IBD-U (P < 0.0001) and CD (P = 0.0004) as well. Significance was also seen in comparing IBD-U with CD group (P = 0.0177). However, there was no statistical significance on comparing Control with CD as no β-catenin over expression was seen in patients with CD as mentioned in Table 3.
|Table 3: Statistical Analysis of β-catenin Score in different IBD groups|
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Immunohistochemical expression of nuclear RB protein was highly statistically significant on comparing UC with IBD-U (0.0001) and UC with CD (0.0094) due to positive expression of pRB in CD and few of IBD-U cases which were later confirmed by serum ASCA levels. However, no statistical significance was noted on comparing UC and IBD-U with control as no expression of pRB was seen in UC and only few cases of CD were present in IBD-U category as mentioned in Table 4.
|Table 4: Statistical Analysis of RB protein Score in different IBD groups|
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Serum levels of MPO ANCA was significantly higher with P value < 0.0001 in the IBD-U as well as in UC (P value < 0.0001) but insignificant in CD and ASCA levels were significant in cases of IBD-U and UC alone (P < 0.001) but not significant in CD. This may be attributed to the smaller sample size and that these patients had early disease as mentioned in Table 5.
|Table 5: Mean (SD) u/ml values of p-ANCA and ASCA in the different Diagnostic Subsets|
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The expression for β-catenin in biopsies from UC showed positive correlation with serum p-ANCA (P = 0.016343) too, indicating that with increase in degree of inflammation in UC, there is corresponding increase in β-catenin expression [Figure 3]. It was also found that the percentage of RB expression was also increased with increasing ASCA levels (P = 0.026) [Figure 4]. Hence both can be used to distinguish CD from other inflammatory colitides in cases where either the patient serum or IHC with pRB was not available.
|Figure 3: Scatter plot of serum ANCA levels vs β-catenin score showing positive correlation amongst them.|
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|Figure 4: Scatter plot of serum ASCA levels vs Percentage of RB expression showing positive correlation amongst them.|
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Following IHC and Serology, 87 out of 101 IBD-U cases accounting for 86.13% were reclassified into UC and CD category [Figure 5] and [Figure 6]. 26 cases were added to CD category and 61 cases to UC and rest 14 cases still remained unclassifiable as these 14 had inconclusive results on IHC and Serology [Table 6].
|Figure 5: Case of a IBD-U showing histology and IHC pattern favouring UC- 5a) Biopsy of IBD-U favouring UC (H&E × 400), 5b) IHC for b -catenin (Score 4) (H&E × 400) and 5c) IHC for RB protein (Score 0) (H&E × 400)|
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|Figure 6: Case of a IBD-U showing histology and IHC pattern favouring CD- 6a) Biopsy of IBD-U favouring CD (H&E × 400), 6b) IHC for b -catenin (Score2) (H&E × 400) and 6c) IHC for RB protein (Score 2) (H&E × 400)|
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|Table 6: IHC scores and serology levels in 14 patients of IBD-U that shows inconclusive IHC score 0r Serology|
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After reclassification, tests for significance was reapplied and it was observed that β-catenin score of UC was extremely significant when compared with CD and IBD-U as well as RB protein expression of CD was extremely significant when compared with UC and IBD-U (P < 0.0001). Final UC: CD ratio was 5.13: 1
| Discussion|| |
The most commonly affected age group was 21-30 years. It was in concordance with the study done by Karve et al., (2015) which also showed the most commonly affected age group to be 21-30 years accounting for 32.35% of the cases. IBD was second to the most common diagnosis of non-specific Colitis which was exactly similar to Geetha et al. (2018), who documented 29.6% cases of IBD. Nearly 50-70% of the patients of IBD are presented with predominant complain of pain in abdomen similar to the present study.,
In 2005, the Working Party of the World Congress of Gastroenterology in Montreal decided that the terminology of Indeterminate colitis be reserved to resected specimens and advised that IBD-U be used for such cases in biopsies. There are several overlapping features of IBD-U and emphasized on the need of correlation of clinical, endoscopic, and histopathological findings to correctly classify the two spectrums UC and CD.,
The distinct and divergent T-cell cycle characteristics underlie the pathogenesis the two major IBD, Ulcerative Colitis, and CD. A similar study by Soletti et al. (2013) showed that β-catenin was significantly increased in UC compared with CD (P < 0.005) samples. Our study supported this finding also with a finding that differential nuclear and increased cytoplasmic staining was noted in UC. RB phosphorylation was significantly greater in colon samples from patients with CD compared with UC.
Telakis and Tsironi, 2008 concluded that “The distinction between ulcerative colitis (UC) and CD (CD) affecting the colon is of paramount importance in inflammatory bowel disease (IBD) patients “as the decision to perform an Ileal pouch-anal anastomosis (IPAA) would be totally dependent on this differentiation. IPAA will be of great use in UC while it is contraindicated in CD which was seconded by a study implying the need of reclassification of IBD-U to provide benefits for both the decision-making of the correct treatment and the prognosis and understanding of disease course., Therefore, immunohistochemical expression of β -catenin can be very useful in cases of where there is uncertainty regarding diagnosis UC and IBD-U too as this will effectively separate UC and IBD-U in favor of UC from CD and hence enable the clinician to go ahead with appropriate treatment protocol.
It was observed in the present study that the serum levels of MPO ANCA was significantly higher with P value < 0.0001 in the IBD-U as well as in UC (P value < 0.0001) but insignificant in CD. This was in accordance to the study done by various studies who concluded that anti p-ANCA (for MPO) is markedly increased in cases with UC., ASCA levels were significant in cases of IBD-U and UC alone (P < 0.001) but not significant in CD. This may be attributed to the smaller sample size and that these patients had early disease.,
The expression for β-catenin in biopsies from UC showed positive correlation with serum p-ANCA (against MPO) too, indicating that with increase in degree of inflammation in UC, there is corresponding increase in β -catenin expression. (P = 0.016343). It was also found that the percentage of RB expression was also increased with increasing ASCA levels (P = 0.026). Hence both can be used to distinguish CD from other inflammatory colitides in cases where either the patient serum or IHC with pRB is not available.
Finally, the UC: CD ratio of 5.13: 1 was in accordance to the recent study conducted by Sood et al. (2020) who also showed UC: CD ratio of 5.1:1 in India, deciphering the rise of CD cases in recent years.
On follow-up of the patients with UC showed good response to the therapy. Majority of the patients with CD were lost for follow-up as the surgical specimens were not sent. Main limitation of the study was lack of follow-up of the patients to find the treatment response and/or diagnostic confirmation by resected specimens as most of the patients didn't report back due to COVID-19 epidemic.
| Conclusion|| |
The pathologist should be well aware of the important histological features of IBD, to identify, define, and subclassify them into two distinct categories UC or CD in order to reduce the morbidity due to these disorders. Despite well described colonoscopic and histopathological features to differentiate UC and CD, a subset of colonoscopic biopsies may still pose a problem in categorization. Use of IHC for β-catenin and RB protein and the assay of serum ASCA and p-ANCA along with proper history and clinical presentation can act as a good adjunct to HPE of conventional H&E-stained slides in subclassifying cases of IBD-U.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Hendrickson BA, Gokhale R, Cho JH. Clinical aspects and pathophysiology of inflammatory bowel disease. Clin Microbiol Rev 2002;15:79-94.
Makharia GK, Ramakrishna BS, Abraham P, Choudhuri G, Misra SP, Ahuja V, et al
. Indian society of gastroenterology task force on inflammatory bowel disease. Survey of inflammatory bowel diseases in India. Indian J Gastroenterol 2012;31:299-306.
Telakis E and Tsironi E. Indeterminate colitis-Definition, diagnosis, characteristics and management. Ann Gastroenterol 2008;21:173-9.
Kuna AT. Serological markers of inflammatory bowel disease. Biochem Med (Zagreb) 2013;23:28-42.
Shen J, Qiao Y, Ran Z, Wang T, Xu J, Feng J. Intestinal protein expression profile identifies inflammatory bowel disease and predicts relapse. Int J Clin Exp Pathol 2013;6:917-25.
Derkacz A, Olczyk P, Komosinska-Vassev K. Diagnostic markers for nonspecific inflammatory bowel diseases. Dis Markers 2018;2018:7451946.
Sood A, Midha V, Sood N, Bhatia AS, Avasthi G. Incidence and prevalence of ulcerative colitis in Punjab, North India. Gut 2003;52:1587-90.
Pignatelli M and Gallagher P. Recent advances in histopathology. J Clin Pathol 2014;23:122-3.
Jass JR, Barker M, Fraser L, Walsh MD, Whitehall VL, Gabrielli B, et al
. APC mutation and tumour budding in colorectal cancer. J Clin Pathol 2003;56:69-73.
Goldhoff P, Clarke J, Smirnov I, Berger MS, Prados MD, James CD, et al
. Clinical stratification of glioblastoma based on alterations in retinoblastoma tumor suppressor protein (RB1) and association with the proneural subtype. J Neuropathol Exp Neurol 2012;71:83-9.
Karve SH, Vidya K, Shivarudrappa AS, Prakash CJ. The Spectrum of colonic lesions: A clinico-pathological study of colonic biopsies. Indian J Pathol Oncol 2015;2:189-209.
Geetha C, Pavani M, Prabhala S, Deshpande A, Histomorphological spectrum of colonic biopsies: A two year study. Indian J Pathol Oncol 2018;5:242-8.
Bielefeldt K, Davis B, Binion DG. Pain and inflammatory bowel disease. Inflamm Bowel Dis 2009;15:778-88.
Islam S, Agarwal M, Talukdar AJ, Dutta S, Kalita PA. Study on clinical profile of patients with inflammatory bowel disease. J Med Res 2016;6:233-6.
Zhou N, Chen WX, Chen SH, Xu CF, Li YM. Inflammatory bowel disease unclassified. J Zhejiang Univ Sci B 2011;12:280-6.
Feakins RM, British society of gastroenterology. Inflammatory bowel disease biopsies: Updated British society of gastroenterology reporting guidelines. J Clin Pathol 2013;66:1005-26.
Sturm A, Leite AZ, Danese S, Krivacic KA, West GA, Mohr S, et al
. Divergent cell cycle kinetics underlie the distinct functional capacity of mucosal T cells in Crohn's disease and ulcerative colitis. Gut 2004;53:1624-31.
Soletti RC, Rodrigues NA, Biasoli D, Luiz RR, de Souza HS, Borges HL. Immunohistochemical analysis of retinoblastoma and β--catenin as an assistant tool in the differential diagnosis between Crohn's disease and ulcerative colitis. PLoS One 2013;8:e70786.
Soubières AA, Poullis A. Emerging role of novel biomarkers in the diagnosis of inflammatory bowel disease. World J Gastrointest Pharmacol Ther 2016;7:41-50.
Mitsuyama K, Niwa M, Takedatsu H, Yamasaki H, Kuwaki K, Yoshioka S, et al
. Antibody markers in the diagnosis of inflammatory bowel disease. World J Gastroenterol 2016;22:1304-10.
Sood A, Kaur K, Singh A, et al. Trends of inflammatory bowel disease at a tertiary care center in northern India. Intest Res. 2021;19:282-90. doi:10.5217/ir.2020.00010.
Department of Pathology, M.L.N. Medical College, Prayagraj - 211 001, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]