Background
In theory, quantitative measurements of UC-related auto-antibodies would be helpful for the clinical diagnosis and treatment assessment of UC. Anti-neutrophil cytoplasmic antibodies (ANCA) are a group of auto-antibodies with cytoplasmic components of the neutrophils as the target antigens, which can release lysozymes through capillaries, damage blood vessels and intestine tissues, and also cause tissue damages through T cell-mediated cellular immune synergy [
1,
2].
In recent years, studies have demonstrated that these ANCAs specifically mediate several diseases such as glomerulonephritis, systemic vasculitis, nodular granulomatosis and autoimmune hepatitis. An antibody named as atypical nuclear ANCA (atypical p-ANCA) has been related to UC [
3,
4]. Another antibody highly associated with IBD is anti-
Saccharomyces cerevisiae antibody (ASCA) which is directed against the yeast genus. ASCA mainly targets at peptidemimetic polysaccharide on the cell wall of the yeast. The mechanisms of these antibodies involved in IBD may be related to the increased intestinal permeability of the disease and the exposure to immune response cells of yeast antibodies [
5]. By detecting serum levels of ANCA-IgG, ASCA-IgA and ASCA-IgG, we have investigated the clinical significance of these antibodies in UC diagnosis, differential diagnosis, and possible correlations of antibody levels to the disease state and to the treatment efficacy.
Patients and methods
Patients
Blood samples from 105 UC patients (52 males, 53 females, average age of 47.33 ± 15.43 years old) diagnosed by the outpatient and inpatient of the First Hospital of Shanxi Medical University from July 2015 to October 2016 were included in this study. The diagnosis of UC was based on clinical, endoscopic and histopathological findings in accordance with the IBD diagnostic criteria determined by the Chinese Medical Association meeting in Guangzhou [
6]). Blood samples from 52 non-UC patients (28 males, 24 females, average age of 50.9 ± 14.0 years old), which were diagnosed as other intestinal disease (i.e. colitis, terminal ileitis, intestinal tuberculosis, intestinal polyps, white’s disease and crohn’s disease) according to the colonoscopyexamination, were selected as disease control. Meanwhile, 100 blood samples from healthy volunteers who came from our health examination center (54 males, 46 females, average age of 53.2 ± 14.9 years old) used in this study. There were no significant differences in both gender and average age among the three groups. According to Mayo Score System, the 105 UC serum samples were divided into three subgroups based on the disease severity: mild group (49 cases), moderate group (40 cases) and severe group (16 cases) [
7]. The basic clinical data of the selected samples were shown in Table
1.
Table 1Basic clinical information, serum levels and positive rates of ANCA and ASCA of the patients and controls
Healthy controls | 100 | 54 | 46 | 1:0.85 | 53.19 ± 14.85 | 3.15 ± 2.24 | 1.16 ± 1.49 | 3.41 ± 5.41 |
6 (6) | 7 (7) | 8 (8) |
Disease controls | 52 | 28 | 24 | 1:0.86 | 50.88 ± 14.01 | 4.03 ± 5.96 | 0.95 ± 1.94 | 2.38 ± 3.16 |
7 (13.5) | 3 (5.77) | 1 (1.92) |
UC | 105 | 52 | 53 | 1:1.02 | 47.33 ± 15.43 | 17.5 ± 30.6a,b | 2.06 ± 9.18 | 4.05 ± 6.42 |
65 (61.9)a,b | 11 (10.5) | 13 (12.4) |
Serologic measurements
Five mL of blood samples were drawn from UC patients, disease controls and healthy controls and then centrifuged at 3000 r/min for 10 min. The serum samples were collected and stored at − 80 °C until used. Three serum antibodies (ANCA-IgG, ASCA-IgA and ASCA-IgG) were detected using ANCA-IgG, ASCA-IgA and ASCA-IgG ELISA kits manufactured by Shanxi Ruihao Biotechnology company (Shanxi, China), and all the assays were conducted according to the kit instructions. Based on the kit instruction, the cut-off values of ANCA-IgG, ASCA-IgA and ASCA-IgG were 5.03, 1.91 and 7.31 U/mL, respectively. Antibody concentration levels greater than the cut-off value were regarded as positive.
Statistical analysis
SPSS 19.0 software package was used for statistical analysis. All the results were presented as the mean ± standard error (\(\bar{x}\) ± S). The comparisons were conducted using variance analysis, and the comparison between each pair of the three groups was conducted using SNK test. The enumeration data were expressed by percentage, and the data comparison between two groups used Fisher Exact Probability test. The difference was regarded as statistically significant when p < 0.05.
Discussion
Ulcerative colitis (UC) is a chronic inflammatory bowel disease with onset most frequently in young adulthood. Many patients with UC have a lifetime of debilitating physical symptoms (e.g., urgent diarrhea, rectal bleeding, abdominal pain). UC is diagnosed by the combination of clinical pattern, colonoscopy, CT, MR and histological findings (basal plasmacytosis, diffuse crypt atrophy and distortion, villous surface irregularity and mucus depletion) [
6]. mild-moderate fewer than 4–6 bowel movements per day, mild-moderate rectal bleeding, absence of constitutional symptoms, and absence of features suggestive of high inflammatory activity based upon Truelove and Witt’s criteria 3 and the Mayo Clinic score. Patients with mild-moderate disease activity generally are at low risk of requiring colectomy. However, certain disease features may predict an aggressive disease course. These include extensive disease, severe endoscopic activity, extra-intestinal manifestations, and elevated inflammatory markers [
9]. ANCA-associated vasculitides (AAV)includes granulomatosis with polyangiitis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA) and microscopic polyangiitis (MPA). Few cases of vasculitis were found associated with IBD [
10].
Searching for laboratory bio-indicators related to UC diagnosis and providing a simple, objective and accurate serum marker for diagnosis, differential diagnosis and efficacy assessment are the ultimate goal in clinical gastroenterology. It has been well documented that ANCA is associated with UC. At present, the most widely used technologies for ANCA detection are indirect immunofluorescence (IIF) assay and ELISA. Since it is quite common that antinuclear antibodies (ANA) can be present at the same time when ANCA is detected with the qualitative IIF assay. It has been documented that ANCA+ may be missed through IIF test due to the interference of ANA, while this can be overcome when a specific ELISA method is used.
In our previous studies, we have demonstrated that quantitative ELISA assaying ANCA levels has a good consistency with IIF and yet has a sensitivity superior to IIF [
11]. According to a large number of reports, ANCA is present in 40%–85% of UC patients, 5%–10% of CD patients, and 0–5% of healthy controls [
12]. Bansi et al. have reported that ANCA is detected in 42.4% of UC patients but only in 5% of CD with a specificity of 98% and a sensitivity of 42%, respectively, for diagnosing UC [
13]. Liu et al. have also reported that ANCA is detected in 48.6% of UC patients and in 25% of CD patients and the sensitivity of single test of ANCA for diagnosing UC is 48.6% with a specificity of 92.9% [
14]. Qiu et al. have demonstrated that ANCA was detected in 68.3% of UC patients and only in 1.7% of healthy controls with a sensitivity of 65.9% and a specificity of 92.7%, respectively, in a single test of ANCA for the diagnosis of UC [
15]. In this report, our results indicate that ANCA positivity is of a good clinical reference value for UC diagnosis and, the augment of ANCA levels may improve the diagnostic accuracy of UC.
At present, it is still controversial whether ANCA levels in UC patient is associated with the severity of the disease. He et al. have reported that ANCA levels are associated with the severity of the disease. Their results have shown that ANCA positive UC patients have a higher rate of intestinal mucosal vasculitis than ANCA negative UC patients. In addition, the UC patients with high ANCA levels also have a severe pathological inflammation at the intestinal mucosa [
16]. However, Yang et al. have reported conflicting results [
17]. In order to evaluate the correlation, UC patients have been divided into mild, moderate and severe groups based on the disease severity. Following the aggravation of the disease, levels of ANCA-IgG show upward tendency and the serum ANCA-IgG levels are indeed higher in patients in the severe group than those in the moderate and mild groups (p < 0.001). Similarly, the serum ANCA-IgG levels are also significantly higher in moderate patients than those in the mild group (p < 0.05).
A study reported that the positive rate of ASCA in CD patients was 39%–72%, higher than that of UC and disease control patients (4%–14% and 4%–14% respectively). In this study, the positive rates of ASCA -IgG and ASCA -IgA in UC patients were 13% and 11%, respectively, with no significant difference from this research data. Studies have shown that the presence of ASCA is related to small bowel involvement (especially ileum) [
18], while the disease site of UC is mainly located in the colorectal, which may be one reason for the low positive rate of ASCA in UC patients. In a prospective comparative study of ASCA in IBD patients in China and whites, it was mentioned that there was a significant difference in the mutation frequency of NOD2 gene (NOD2 gene is considered to be a strongly correlated gene of CD) in CD among different ethnic groups. Currently, the sensitivity of Chinese people to ASCA detection is lower than that of white people [
19].
Conclusions
Serum ANCA+ levels are helpful in the diagnosis of UC, while combined tests of serum ANCA-IgG, ASCA-IgG, and ASCA-IgA levels are useful in differential diagnosis of UC. Furthermore, in UC patients, serum ANCA levels is correlated with the severity of the disease and, therefore, a dynamic monitoring of serum ANCA levels during treatment may relieve patients from repeated use of invasive diagnostic methods. The dynamic testing of serum antibody levels as mentioned above may also help monitor disease progression, guide for more effective clinical medications and reduce the recurrence of UC.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.