Diagnostic utility and phenotype correlation of serum ASCA and ANCA in pediatric inflammatory bowel disease

doi:10.21203/rs.3.rs-1658007/v1

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Diagnostic utility and phenotype correlation of serum ASCA and ANCA in pediatric inflammatory bowel disease

https://doi.org/10.21203/rs.3.rs-1658007/v1

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Background

Both anti-Saccharomyces cerevisiae antibody (ASCA) and anti-neutrophil cytoplasmic antibody (ANCA) are associated with inflammatory bowel disease (IBD); however, the clinical role of these serological biomarkers in pediatric IBD has been poorly investigated. Therefore, this study aimed to evaluate the diagnostic utility and phenotypic correlation of serological biomarkers in pediatric IBD.

Methods

A retrospective review was performed on all pediatric subjects diagnosed with IBD between March 2004 and July 2021. Pediatric patients were recruited and classified as Crohn’s disease (CD), ulcerative colitis (UC), and IBD unclassified (IBD-U) based on the revised Porto criteria. The Paris classification was used to evaluate disease phenotypes. Laboratory markers including fecal calprotectin were also evaluated. Serum levels of ASCA IgG and IgA, anti-proteinase-3 with cytoplasmic ANCA (PR3-ANCA), and anti-myeloperoxidase with perinuclear ANCA (MPO-ANCA) using ELISA, and serum ANCA titer using immunofluorescent test were measured.

Results

In all, 229 pediatric patients with IBD (CD: 147, UC: 53, IBD-U: 29) were included. The ASCA IgG seropositivity was significantly different among the three groups (CD: 75.4%, UC: 17.5%, and IBD-U: 60.0%; p < 0.001) and ASCA IgA seropositivity was also significantly different among the three groups (CD: 23.8%, UC: 2.5%, and IBD-U: 20.0%; p < 0.001). Serum titers of ASCA IgG were significantly difference among the 3 IBD subgroups (CD 32.7 U/mL vs. UC 11.3 U/mL vs. IBD-U 25.30 U/mL; p < 0.001), and ASCA IgA also revealed significant difference (CD 10.2 U/mL vs. UC 6.8 U/mL vs. IBD-U 9.0 U/mL; p < 0.001). Three ANCA antibodies were positive in 1.4%, 32.1%, and 37.9% of CD, UC, and IBD-U patients, respectively (p < 0.001). Perianal disease modifier P1 revealed higher ASCA IgG titers (P1 40.0 U/mL vs. P0 29.4 U/mL, p = 0.001), while showing no differences in age, disease location, and behavior. Using optimal cutoffs determined by ROC analysis, serum IgG and IgA ASCAs showed 68.0% and 63.1% sensitivity, and 67.7% and 64.6% specificity, respectively, in distinguishing CD.

Conclusions

Serological biomarkers of ASCAs IgG and IgA, pANCA, and PR3-ANCA may be effective in the diagnosis and clinical evaluation of disease phenotypes in an Asian pediatric IBD cohort.

inflammatory bowel disease

diagnosis

biomarkers

serologic tests

child

Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory intestinal disease classified as Crohn’s disease (CD), ulcerative colitis (UC), and unclassified IBD (IBD-U). Current diagnostic techniques for IBD include clinical, endoscopic, radiological, and pathological evaluations. If there is uncertainty about the diagnosis of IBD, it is called IBD-U, and about 10–15% of pediatric patients with IBD are classified as IBD-U [1]. Therefore, a non-invasive supportive test for pediatric patients with IBD would be desirable. In the diagnosis and evaluation of IBD, the anti-Saccharomyces cerevisiae antibody (ASCA) and anti-neutrophil cytoplasmic antibody (ANCA) [2–5] are shown to be useful. ASCAs IgG and IgA are directed against the mannan cell wall of Saccharomyces, which shares homology with intestinal bacteria [6]. ANCAs are autoantibodies directed against neutrophil granules and classified as perinuclear ANCA (pANCA), anti-proteinase-3 with cytoplasmic ANCA (PR3-ANCA), and anti-myeloperoxidase with perinuclear ANCA (MPO-ANCA), which are commonly associated with IBD, Wegener granulomatosis, systemic vasculitis, etc [7].

To date, the diagnostic accuracies of serological markers of IBD have not been evaluated for a large Asian pediatric IBD cohort. Moreover, very few studies have examined the association between serological titers of those markers and the clinical phenotype and disease activities of different IBD subtypes (CD, UC, and IBD-U).

Therefore, this study aimed to evaluate the diagnostic accuracy of ASCA IgG, ASCA IgA, pANCA, PR3-ANCA, and MPO-ANCA in pediatric patients with IBD and determine the association between the serological titers of these markers and their clinical manifestations, including disease phenotype, disease activity, and inflammatory markers.

Study subjects

Pediatric IBD patients who were diagnosed and treated at a single tertiary institution were recruited between March 2004 and July 2021. Data were obtained retrospectively from the medical records of all participants. We collected the data of clinical and demographic characteristics (age, sex, IBD disease activity, IBD phenotype, serum and fecal inflammatory markers, and serological biomarkers of IBD, such as ASCA IgG, ASCA IgA, pANCA, PR3-ANCA, and MPO-ANCA). The following exclusion criteria were applied on the cohort: a) infectious colitis, b) diagnosis of primary immune deficiency, c) previous bowel resection, or d) infantile IBD (diagnosed in children before 2 years of age).

Diagnosis of pediatric IBD

The diagnosis of IBD was confirmed based on clinical, endoscopic, radiologic, and pathological evaluations and according to the revised Porto criteria [8]. Upper endoscopy, lower endoscopy, and small intestine radiological imaging were performed in the same procedure for the first diagnostic evaluation. Small bowel lesions were examined with magnetic resonance enterography (MRE), capsule endoscopy, or a small bowel series when MRE failed due to claustrophobia.

The subjects were divided into three subgroups representing CD, UC, and IBD-U, based on the revised Porto criteria. Ulcerative colitis was defined as a continuous transmural inflammatory lesion in the entire colon without small bowel involvement and no granulomas on biopsy [8]. Crohn's disease was identified as having mucosal aphthous ulcers and noncaseating granulomas in the ileum or colon and granulomatous or chronic localized inflammation on biopsy [8]. Patients in whom the symptomatic distinctions between UC and CD could not be identified were considered as IBD-U cases, even after a careful evaluation per the revised Porto criteria; all the characteristics that were consistent with UC but were rectal sparing, duodenal, or esophageal ulcers and not attributable to other causes (Non-steroidal anti-inflammatory drugs (NSAIDs), Helicobacter pylori, and celiac disease) are examples of IBD-U [8].

Disease phenotype

The Paris classification was used to categorize pediatric patients with IBD based on their disease phenotypes [9]. CD was categorized into different phenotypes by considering the age at diagnosis (A), disease location (L), and disease behavior (B), including the perianal disease modifier (P) and growth (G) [9]. Age was classified as A0 (< 10 years), A1 (10–17 years), and A2 (> 17 years). Disease location was classified as L1 (distal 1/3 ileum ± limited cecal disease), L2 (colonic), L3 (ileocolonic), or L4 (upper gastrointestinal involvement). Disease behavior was classified as B1 (nonstricturing and nonpenetrating), B2 (stricturing), and B3 (penetrating). For UC, the phenotypic categories consisted of disease extent (E) and disease severity (S) [9]. The UC disease extent was classified as E1 (ulcerative proctitis), E2 (left-sided UC), E3 (extensive colitis), and E4 (pancolitis). The disease severity was considered as ‘positive’ (S1) if the pediatric ulcerative colitis activity index (PUCAI) score exceeded 65.

Disease activity

To assess the disease activity of IBD in children, the pediatric Crohn’s disease activity index (PCDAI) was used for CD, and the PUCAI was used for UC [10, 11]. In pediatric patients with IBD-U, both PCDAI and PUCAI criteria were applied for respective disease phenotypes.

Laboratory tests

Blood sampling was performed for all included subjects and the following markers were measured: C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), total white blood cell count (WBC), absolute neutrophil count (ANC), hemoglobin (Hb), hematocrit (Hct), platelet count, serum total protein, and serum total albumin. Fecal calprotectin was measured using a fluorescence enzyme immunoassay (Calprotectin, Phadia AB, Sweden).

Serological markers of IBD

Serum samples were collected at the time of diagnosis and the serum ASCA IgG and IgA levels were analyzed by enzyme-linked immunosorbent assay (ELISA) (QUANTA Lite® kit, INOVA Diagnostics Inc., San Diego, CA, USA) following the manufacturer’s protocol. The cutoff values for both serum ASCA IgG and IgA provided by the manufacturer were set at ≥ 1:20 IU/mL. Serum pANCA levels were analyzed using an immunofluorescence antibody test (Axioskop, Carl ZEISS®, Jena, Germany) with the cut-off value set at ≥ 1:40 IU/mL. Serum PR3-ANCA and MPO‐ANCA levels were analyzed using ELISA (Immunocap 250 kit, Aloka®, Tokyo, Japan) with cutoff values set to ≥ 2.0 IU/mL for PR3‐ANCA and ≥ 3.5 IU/mL for MPO-ANCA.

Statistical analysis

Statistical analyses were performed in a non-parametric manner. The Mann–Whitney U test was used for continuous variables between the two groups, and the Kruskal-Wallis test was used for continuous variables among the three groups. The χ² test or Fisher’s exact test was used for numeric variables. Bivariate correlation analyses were performed using Pearson correlation for parametric tests and Spearman correlation for nonparametric tests. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic accuracy of the serological markers. Statistical analyses were performed using SPSS version 25.0 (IBM SPSS Statistics for Windows, version 25.0. Armonk, NY: IBM Corp.) A p values below 0.05 were considered statistically significant.

Ethical Considerations

We conducted this study following the Helsinki Declaration (1975, revised in 2013). This study was approved by the Institutional Review Board of the Seoul National University Hospital (SNUH) Institutional Review Board (IRB approval number: B-2111-720-101). The requirement for informed consent from enrolled patients and/or parents was waived by the institutional review board of SNUH (Seoul, Republic of Korea).

Patient characteristics

A total of 229 participants (151 boys, 78 girls; mean age: 14.0 ± 3.3 years) were included (CD: 147, UC: 53, and IBD-U: 29). The demographics, clinical characteristics, and laboratory findings of the study participants are shown in Table 1. There were no statistically significant differences between the three groups regarding sex, age at diagnosis, and PCDAI or PUCAI scores that indicate disease activity. However, the CRP, ESR, ANC, Hb, Hct, and serum albumin levels showed statistically significant differences among the 3 groups (Table 1).

Table 1

Demographic data, disease activity, and laboratory findings of the study subjects
	TOTAL (n = 229)	CD (n = 147)	UC (n = 53)	IBD-U (n = 29)	p
Boys, n (%)	151 (65.9%)^*	100 (68.0%)	33 (62.3%)	18 (62.1%)	0.671
Age at diagnosis (yrs)	14.5 (3.1–18.9)	14.6 (3.5–18.9)	13.1 (3.1–18.6)	14.7 (5.2–18.6)	0.075
PCDAI	30.0 (17.5–40.0)	32.5 (20.0-37.5)	-	30.0 (15.0–40.0)	0.957
PUCAI	40.0 (20.0–60.0)	-	30.0 (20.0-55.5)	50.0 (25.0–60.0)	0.054
Calprotectin (mg/kg)	1753.3 (477.6–2479.0)	1709.1 (364.7–2000.0)	1481.4 (298.6–2780.0)	2000.0 (1460.8-2991.5)	0.328
CRP (mg/L)	0.53 (0.20–2.78)	1.84 (0.38–4.20)	0.30 (0.04–0.56)	0.40 (0.30–2.44)	< 0.001
ESR (mm/hr)	25.0 (11.0–52.0)	33.5 (11.0-72.5)	17.0 (7.0–30.0)	28.0 (15.3–64.3)	0.001
WBC count (/µL)	8680.0 (6600.0-10670.0)	8330.0 (6197.5-10385.0)	8080.0 (6580.0-9900.0)	9670.0 (7432.5-11632.5)	0.322
ANC (/µL)	5340.0 (3593.0-7392.0)	5133.5 (3675.8-7301.3)	4283.0 (3279.0-6648.0)	6608.5 (4452.8-8841.8)	0.047
Hb (g/dL)	12.1 (10.2–13.7)	12.2 (10.8–13.8)	12.6 (10.7–14.0)	11.6 (9.5–13.4)	0.041
Hct (%)	37.1 (33.0-41.2)	38.3 (34.9–41.4)	38.2 (33.5–41.6)	37.0 (30.2–40.1)	0.036
Platelet (x10³/µL)	379,500 (307,250–497,750)	397,500 (321,750 − 501,250)	348,000 (287,000-464,000)	401,000 (318,250–517,750)	0.095
Protein (g/dL)	7.1 (6.7–7.5)	7.2 (6.7–7.5)	7.2 (6.9–7.4)	7.2 (6.7–7.7)	0.682
Albumin (g/dL)	4.1 (3.5–4.4)	3.8 (3.2–4.3)	4.3 (3.8–4.5)	4.0 (3.6–4.2)	< 0.001
Values are presented as number of patients (%) or median (range). For numeric variables, the Chi-square test was used, and for continuous variables, the Kruskal-Wallis test was used. A p value of less than 0.05 was considered statistically significant.
CD = Crohn’s disease; UC = ulcerative colitis; IBD-U = inflammatory bowel disease unclassified; PCDAI = Pediatric Crohn’s disease activity index; PUCAI = Pediatric ulcerative colitis activity index; CRP = C-reactive protein; ESR = erythrocyte sedimentation rate; WBC = white blood cell; ANC = absolute neutrophil count; Hb = hemoglobin; Hct = hematocrit

Serological markers compared among different subgroups

The serological results of ASCA IgG and IgA are reported in Tables 2 and 3. Serum ASCA IgG and IgA titers were significantly higher in CD (32.7 U/mL and 10.2 U/mL, respectively) than in IBD-U (25.3 U/mL and 9.0 U/mL, respectively) and UC (11.3 U/mL and 6.8 U/mL, respectively) (p < 0.001 for both ASCA IgG and IgA) (Table 3). The serological results of pANCA, PR3-ANCA, and MPO-ANCA are presented in Table 2. The ANCA positivity rates were significantly higher in IBD-U (37.9%) and UC (32.1%) than in CD (1.4%) (p < 0.001).

Table 2

Serologic results of anti-*Saccharomyces cerevisiae* antibody and anti-neutrophil cytoplasmic antibody according to disease spectrum of inflammatory bowel disease
	TOTAL (n = 229)	CD (n = 147)	UC (n = 53)	IBD-U (n = 29)	p
ASCA positive rate, n (%)	114 (61.0%)	92 (75.4%)	7 (17.5%)	15 (60.0%)	< 0.001
ASCA IgG positive rate, n (%)	114 (61.0%)	92 (75.4%)	7 (17.5%)	15 (60.0%)	< 0.001
ASCA IgA positive rate, n (%)	35 (18.7%)	29 (23.8%)	1 (2.5%)	5 (20.0%)	0.011
ANCA positive rate, n (%)	30 / 227 (13.2%)	2 / 145 (1.4%)	17 / 53 (32.1%)	11 / 29 (37.9%)	< 0.001
pANCA positive rate, n (%)	24 / 218 (11.0%)	2 / 139 (1.4%)	14 / 50 (28.0%)	8 / 29 (33.6%)	< 0.001
PR3-ANCA positive rate, n (%)	9 / 93 (9.7%)	0 / 51 (0.0%)	6 / 25 (24.0%)	3 / 17 (17.6%)	0.002
MPO-ANCA positive rate, n (%)^†	0 / 116 (0.0%)	0 / 67 (0.0%)	0 / 29 (0.0%)	0 / 20 (0.0%)	-
Values are presented as number of patients (%) or median (range). For numeric variables, the Chi-square test was used, and for continuous variables, the Kruskal-Wallis test was used. A p value of less than 0.05 was considered statistically significant. Cut-off values provided by the manufacturer were ≥ 1:20 IU/mL for serum ASCA IgG and IgA and ≥ 1:40 IU/mL for pANCA, ≥ 2.0 IU/mL for PR3-ANCA, and ≥ 3.5 IU/mL for MPO-ANCA.
ASCA = anti-Saccharomyces cerevisiae antibody; ANCA = anti-neutrophil cytoplasmic antibody; CD = Crohn’s disease; UC = ulcerative colitis; IBD-U = inflammatory bowel disease unclassified
† None of the MPO-ANCA was positive.

Table 3

Serologic results of anti-*Saccharomyces cerevisiae* antibody according to disease spectrum of inflammatory bowel disease
	Total (n = 229)	CD (n = 147)	UC (n = 53)	IBD-U (n = 29)	p
Serum ASCA IgG titer (U/mL)	22.4 (12.3–41.2)	32.7 (19.3–55.0)	11.3 (7.0-17.5)	25.3 (14.2–41.2)	< 0.001
Serum ASCA IgA titer (U/mL)	8.2 (5.5–13.5)	10.2 (5.8–18.8)	6.8 (4.4–8.4)	9.0 (6.6–15.3)	< 0.001
Values are presented as median (range). For continuous variables, the Kruskal-Wallis test was used. A p value of less than 0.05 was considered statistically significant.
ASCA = anti-Saccharomyces cerevisiae antibody; CD = Crohn’s disease; UC = ulcerative colitis; IBD-U = inflammatory bowel disease unclassified

Laboratory and serological markers compared across disease phenotypes

Regarding the laboratory markers of CD, the A1a, A1b, and A2 subgroups displayed statistically significant differences in the Hct levels (p = 0.016). Similarly, the L1, L2, L3, and L4 subgroups had significant differences in the fecal calprotectin (p < 0.001), ESR (p < 0.001), WBC (p = 0.009), ANC (p = 0.002), Hb (p < 0.001), Hct (p = 0.005), platelet (p = 0.001), and albumin (p < 0.001) levels. In terms of disease behavior (B1 ~ B3) of CD, only serum albumin (p = 0.032) showed a significant difference. Regarding the existence of perianal disease (P0, P1) in CD patients, there were significant differences in Hb (p = 0.015) and Hct (p = 0.038).

In the subgroup analysis of laboratory markers based on disease extent (E1–E4) in UC, there were significant differences in calprotectin (p = 0.049), CRP (p < 0.001), ESR (p < 0.001), Hb (p < 0.001), Hct (p < 0.001), platelets (p = 0.003), protein (p = 0.003), and albumin (p < 0.001). The severity of UC (S0–S1), calprotectin (p < 0.001), CRP (p = 0.027), ESR (p = 0.008), WBC (p = 0.028), ANC (p = 0.019), Hb (p = 0.004), Hct (p = 0.008), platelets (p = 0.005), protein (p = 0.005), and albumin (p < 0.001) were significantly different.

In CD and IBD-U, serum ASCA IgG titers were significantly higher in the P1 subgroup than in the P0 subgroup (p = 0.019) and significantly higher ASCA IgA titers were shown in the L3 subgroup than in other colonic disease subgroups (p = 0.025) (Fig. 1). The serum ASCA IgG and IgA titers had no significant differences when compared among phenotype subgroups classified according to age at diagnosis (A) and disease behavior (B) (Fig. 1). For UC and IBD-U, there were no significant differences in serum ASCA IgG and IgA titers among phenotype subgroups classified according to disease extent (E) and disease severity (S) (all p > 0.05).

Correlation of serum ASCA titers with disease activity

The correlation analysis of serum ASCA titers with disease activity and other inflammatory markers in pediatric patients with IBD is reported in Table 4. Correlation analysis was also conducted in each IBD subgroup (CD, UC, and IBD-U). In pediatric patients with CD, serum ASCA IgG titers correlated positively with PCDAI (r = 0.197, p = 0.029), fecal calprotectin (r = 0.311, p = 0.002), CRP (r = 0.222, p = 0.014), ESR (r = 0.222, p = 0.015), WBC count (r = 0.200, p = 0.027), ANC (r = 0.194, p = 0.032), and platelet count (r = 0.231, p = 0.010) and negatively correlated with Hb (r = -0.225, p = 0.013) and serum albumin (r = -0.185, p = 0.042). Serum ASCA IgA titers were positively correlated with PCDAI (r = 0.180, p = 0.047), platelet (r = 0.216, p = 0.017), and albumin (r = 0.180, p = 0.047), and negatively correlated with Hb (r = -0.228, p = 0.012) and Hct (r = -0.195, p = 0.031).

Table 4

Correlation analyses of serum ASCA titers with disease activity and other laboratory findings
		PCDAI	PUCAI	Calprotectin	CRP	ESR	WBC	ANC	Hb	Hct	PLT	Protein	Albumin
Serum ASCA IgG titer	r	0.167	-0.035	0.203	0.290	0.288	0.171	0.189	-0.086	-0.035	0.181	0.023	-0.268
Serum ASCA IgG titer	p	0.019	0.665	0.012	< 0.001	< 0.001	0.020	0.010	0.241	0.633	0.013	0.758	< 0.001
Serum ASCA IgA titer	r	0.115	-0.044	0.004	0.093	0.139	0.079	0.092	-0.166	-0.138	0.137	0.029	-0.158
Serum ASCA IgA titer	p	0.109	0.583	0.959	0.206	0.059	0.284	0.208	0.023	0.060	0.061	0.695	0.031
Bivariate correlation analysis was performed on the variables using the Pearson correlation. A p value of less than 0.05 was considered statistically significant.
ASCA = anti-Saccharomyces cerevisiae antibody; PCDAI = Pediatric Crohn’s disease activity index; PUCAI = Pediatric ulcerative colitis activity index; CRP = C-reactive protein; ESR = erythrocyte sedimentation rate; WBC = white blood cell; ANC = absolute neutrophil count; Hb = hemoglobin; Hct = hematocrit; PLT = platelet

In UC, serum ASCA IgG titers were positively correlated with serum protein only (r = 0.367, p = 0.001). Serum ASCA IgA titers correlated positively with protein (r = 0.358, p = 0.001), Hb (r = 0.232, p = 0.038), and Hct (r = 0.251, p = 0.025), and negatively correlated with fecal calprotectin (r = -0.407, p < 0.001).

In the IBD-U group, no correlation between serum ASCA titers and disease activity or other laboratory findings was present.

Diagnostic accuracy of serum ASCA IgG and IgA titers

We analyzed the diagnostic accuracy of serum ASCA IgG and IgA titers for pediatric Crohn’s disease by performing ROC analysis and calculating the area under the ROC (AUC) (Fig. 2). The serum ASCA IgG and IgA titers were compared between pediatric CD and non-CD (UC and IBD-U) patients. By ROC analysis, we found the optimal cutoff value for serum ASCA IgG was 23.7 IU/mL for pediatric CD (sensitivity: 68.0%, specificity: 67.7%, positive predictive value (PPV): 80.7%, and negative predictive value (NPV): 58.9%) (Fig. 2). The optimal cutoff value for serum ASCA IgA was 8.3 IU/mL for pediatric CD (sensitivity: 63.1%, specificity 64.6%, PPV: 82.8%, and NPV: 38.8%) (Fig. 2).

In this single-center retrospective cohort study of pediatric patients with IBD in South Korea, we aimed to procure accurate serological markers to distinguish pediatric IBD subgroups and reflect disease activity and clinical phenotypes. This study revealed that pediatric CD patients had greater seropositivity rates and higher titers for both serum ASCA IgG and IgA than IBD-U and UC patients. In contrast, patients with pediatric IBD-U and UC showed greater serum ANCA positivity rates than those with pediatric CD. Especially, pANCA-positive rates were the highest in IBD-U, and PR3-ANCA positive rates were the highest in UC.

Our findings are concordant with the findings of a longitudinal report by Birimberg-Schwartz et al., where serum ASCA positivity was most prevalent in CD, followed by IBD-U, and then UC (40%, 26%, and 6%, respectively; p < 0.001). Similarly, serum ANCA positivity was most prevalent in UC, followed by IBD-U, and then CD (64%, 43%, and 30%, respectively; p < 0.001) [12]. The serological features of IBD-U were reported to be similar as of UC than those of CD [1], which may explain the high serum pANCA-positive rates in IBD-U. Although the serum ANCA-positive rates were greater in IBD-U than in UC, further research is needed since the number of patients examined was small, especially with pANCA and PR3-ANCA, because the positive rate of MPO-ANCA is very low [13, 14]. Overall, our results suggest that serum ASCA IgG, IgA, pANCA, and PR3-ANCA may be helpful in the diagnosis and differentiation of pediatric IBD subgroups.

Regarding disease phenotypes, a previous study on adult IBD patients reported a significant association between serum ASCA titers and age of onset, disease behavior, and disease severity of IBD [15]. However, unlike our study, this report classified disease behavior only as fibrostenosing disease, internal penetrating disease, perianal penetrating disease, and UC-like features, and the investigators measured the disease severity based on the prevalence and number of surgeries per patient among those with small bowel involvement, instead of using a standardized index as in our study. Chandrakumar et al. examined the associations between ASCA-positive status and disease location and behavior in a pediatric cohort, and suggested that ASCA-positive patients did not have a higher risk of perianal disease [16]. In contrast, our results showed a positive correlation between the ASCA IgG and IgA titers and the frequency of anal CD lesions. The difference might be because the previous study focused only on seropositivity instead of serological titers of ASCAs. Therefore, our results suggest the clinical utility of measuring serum ASCA IgG titers in pediatric CD-related perianal disease.

Regarding disease activity, Canani et al. reported a relationship between ASCA titers and disease activity in pediatric patients with CD as measured by PCDAI [17]. However, this study did not investigate the correlations with other inflammatory markers, including fecal calprotectin, which indirectly reflect disease activity. When we conducted bivariate analysis, serum ASCA IgG titers correlated positively with PCDAI and with CRP, ESR, WBC, ANC, platelets, and fecal calprotectin. Moreover, a negative correlation between serum levels of ASCA IgG and serum albumin levels were found. A negative correlation between serum ASCA IgA titer and Hb and serum albumin was noted. CRP, ESR, and platelets are acute-phase reactants that positively reflect disease activity, and fecal calprotectin is a well-established marker of intestinal inflammation in IBD [18]. In contrast, serum albumin negatively reflects the inflammatory response. Based on our results, serum IgG and IgA titers may be useful as disease activity markers in the evaluation of pediatric IBD. Besides, our study found a strong correlation between serum albumin levels and quantitative values of IgG and IgA ASCAs, which may indicate that greater quantitative levels reflect more severe illness, similar to earlier studies demonstrating that serum albumin might indicate disease severity [19].

In a meta-analysis of 60 studies, the ASCA+/pANCA– test distinguished adult CD with 55% sensitivity and 93% specificity, whereas the ASCA IgG + test was slightly less sensitive and more specific (42.6%, 95.7%) [20]. In pediatric CD, Mizuochi et al. found that ASCA had 26.7% sensitivity and 95.0% specificity [21], although they analyzed sensitivity and specificity using cutoff values provided by the manufacturer and determined only seropositivity instead of serological titers. In the present study, using the optimal cutoff values determined by ROC analysis, serum ASCA IgG titers could distinguish pediatric CD from UC and IBD-U with sensitivity of 68.0% and specificity of 67.7%, and serum ASCA IgA titers with sensitivity of 63.1% and specificity of 64.6%, respectively.

In conclusion, we found serological markers, including ASCA IgG, ASCA IgA, pANCA, PR3-ANCA, and MPO-ANCA to be useful biomarkers. This is the first study to comprehensively cover the diagnostic and clinical values of serum ASCA and ANCA antibodies and the first study in Asian children and adolescents to include PR3-ANCA and MPO-ANCA along with existing pANCA. The analysis of serological titers is unique to this study, in addition to the previously studied seropositivity, and the study provides several noteworthy insights. Nevertheless, several limitations were present in this study. First, this was a retrospective study; hence, our analyses were limited to the data available from the medical records. Second, all patients included in this study were East Asian children, and data were collected from a single center. Further prospective multicenter studies of serological markers for pediatric IBD should be conducted in multiple ethnic groups and countries.

ASCA

Anti-Saccharomyces cerevisiae antibody

ANCA

Anti-neutrophil cytoplasmic antibody

IBD

Inflammatory bowel disease

Crohn’s disease

Ulcerative colitis

IBD-U

IBD unclassified

PR3-ANCA

Anti-proteinase-3 with cytoplasmic ANCA

MPO-ANCA

Anti-myeloperoxidase with perinuclear ANCA

MRE

Magnetic resonance enterography

NSAIDs

Non-steroidal anti-inflammatory drugs

PUCAI

Pediatric ulcerative colitis activity index

PCDAI

Pediatric Crohn’s disease activity index

CRP

C-reactive protein

ESR

Erythrocyte sedimentation rate

WBC

White blood cell count

ANC

Absolute neutrophil count

Hemoglobin

Hct

Hematocrit

ELISA

Enzyme-linked immunosorbent assay

ROC

Receiver operating characteristic

AUC

Area under the ROC

Acknowledgements

This article was consulted by the Medical Research Collaborating Center (MRCC) of Seoul National University Bundang Hospital, and the authors would like to thank Seung Hyun Won for her contributions in statistical analysis.

Author contributions

Jeong Min Kim contributed to the study design, data collection, data analysis and manuscript drafting, and critical revision of the manuscript for important academic content. Young Min Choi and Seung A Jung contributed to the minor revision of the manuscript. Jin Soo Moon and Jae Sung Ko contributed to the study's concept and design and its supervision. Hye Ran Yang contributed to the study design, data analysis, and critical manuscript revision for important academic content.

Funding

This work was supported by the New Faculty Startup Fund from Seoul National University (no.800-20210277).

Seoul National University College of Medicine

Gwanak Main Campus 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea

Availability of data and materials

The datasets used during the current study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

We conducted this study following the Helsinki Declaration (1975, revised in 2013). This study was approved by the Institutional Review Board of the Seoul National University Bundang Hospital (SNUBH) Institutional Review Board (IRB approval number: B-2111-720-101). The requirement for informed consent from enrolled patients and/or parents was waived by the institutional review board of SNUBH (Seongnam, Republic of Korea).

Consent for publication

Not applicable

Competing interests

The authors declare no potential conflicts of interests.

Author details

¹Department of Pediatrics, Seoul National University Bundang Hospital, 82, Gumi-ro 173 Beon-gil, Bundang-gu, Seongnam, Gyeonggi-do, 13620, Korea. ²Seoul National University College of Medicine

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No competing interests reported.

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Diagnostic utility and phenotype correlation of serum ASCA and ANCA in pediatric inflammatory bowel disease

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Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Methods

Study subjects

Diagnosis of pediatric IBD

Disease phenotype

Disease activity

Laboratory tests

Serological markers of IBD

Statistical analysis

Ethical Considerations

Results

Patient characteristics

Serological markers compared among different subgroups

Laboratory and serological markers compared across disease phenotypes

Correlation of serum ASCA titers with disease activity

Diagnostic accuracy of serum ASCA IgG and IgA titers

Discussion

Abbreviations

Declarations

References

Additional Declarations

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