Significance of Anti-Neutrophil Cytoplasmic Antibodies in Systemic Sclerosis

Jayne Moxey; Molla Huq; Susanna Proudman; Joanne Sahhar; Gene-Siew Ngian; Jenny Walker; Gemma Strickland; Michelle Wilson; Laura Ross; Gabor Major; Janet Roddy; Wendy Stevens; Mandana Nikpour


Arthritis Res Ther. 2019;21(57) 

In This Article



Patients were recruited from the Australian Scleroderma Cohort Study (ASCS), a multicentre study of risk and prognostic factors in SSc across 5 participating Australian centres (St. Vincent's Hospital, Melbourne and Monash Health, Victoria; John Hunter Hospital, New South Wales; Royal Adelaide Hospital, South Australia; Fiona Stanley Hospital, Western Australia). All human research ethics committees of the participating sites have approved the ASCS. Written informed consent was obtained from all patients at recruitment.

Inclusion and Exclusion Criteria

We included patients from the ASCS recruited between January 1, 2007, and May 23, 2016, who fulfilled the 2013 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria[24] for diagnosis of SSc. Patients with mixed connective tissue disease were excluded from the study.

ANCA testing is routinely performed as part of a panel of tests requested at entry to the ASCS. Patients were excluded if they did not have an ANCA result recorded in the database. All positive ANCA results were confirmed by inspection of the original laboratory results. Patients were excluded if original results were not available for review. For statistical analyses involving autoantibodies, patients without a documented autoantibody result were excluded from that particular analysis.

ANCA Testing

The presence of ANCA was defined by a positive result for any one or a combination of p-ANCA, c-ANCA, atypical ANCA, anti-MPO and anti-PR3. Indirect immunofluorescence (IIF) was performed using the local laboratory protocol to detect p-ANCA, c-ANCA and atypical ANCA. IIF results described as "unable to exclude p-ANCA due to the presence of ANA" were defined as ANCA negative if anti-MPO or anti-PR3 were negative. Anti-MPO and anti-PR3 were measured by enzyme-linked immunosorbent assay (ELISA) using the local laboratory commercial test kit and reference range.

For each ANCA-positive patient, a retrospective case note review was performed to assess for clinical and histopathological evidence of vasculitis. Cases of ANCA-negative vasculitis were not explored in this study.

For statistical analysis, patients were divided into categories based on their ANCA results. Demographics and clinical manifestations were compared between ANCA-positive patients and ANCA-negative patients, anti-MPO-positive patients and anti-MPO-negative patients, and anti-PR3 positive patients and anti-PR3 negative patients.

Data Collection

Demographic and disease data were prospectively collected at baseline and at subsequent annual reviews as per a standardised protocol. All disease features and autoantibodies were defined as present if they were ever present from the time of diagnosis. Disease onset and disease duration were defined from the date of onset of the first non-Raynaud's manifestation. Disease subtype was defined as diffuse or limited as per LeRoy criteria.[25] Patients were diagnosed with SSc overlap syndrome at physician discretion if features of RA, SLE, SS, PM or DM were clinically evident. Anti-nuclear antibody (ANA) was detected by indirect immunofluorescence (IIF) using the local laboratory protocol. Extractable nuclear antigen (ENA) testing was performed by ELISA, immunoblot or a combination of these two methods, using the local laboratory commercial test kits. Anti-dsDNA testing was performed by ELISA in most laboratories, with two laboratories using the Farr radioimmunoassay. ILD was diagnosed on high-resolution-computed tomography (HRCT) of the chest, which was performed on the basis of abnormal respiratory function tests or the presence of crepitations on respiratory system examination. Pulmonary arterial hypertension (PAH) was defined by right heart catheterisation as a mean pulmonary artery pressure ≥ 25 mmHg and a pulmonary arterial wedge pressure ≤ 15 mmHg. Scleroderma renal crisis was defined by a combination of any two of three criteria, which include new-onset hypertension in the absence of an alternate aetiology, rising creatinine or microangiopathic haemolytic anaemia. Small intestinal bacterial overgrowth (SIBO) was considered present if a patient described concurrent diarrhoea and the use of cyclical antibiotics. Gastric antral vascular ectasia (GAVE), reflux oesophagitis and oesophageal stricture were defined on endoscopy. Data regarding hospitalisations were collected annually from patient-reported admissions for any reason, for a period of greater than 24 h. Patients were recorded as having had a malignancy if they reported the presence of any skin, solid organ or haematological malignancy. Treatments were defined as use ever from disease onset to the most recent visit. The use of biologics included patients ever being exposed to any of rituximab, abatacept, tumour necrosis factor (TNF) alpha inhibitors and tocilizumab.

Statistical Analysis

Data are presented as the mean ± standard deviation for continuous variables and as a number (percentage) for categorical variables. Univariable analyses of the relationship between positive ANCA, and positive anti-MPO and anti-PR3 were conducted using the chi-squared or Yate's corrected chi-squared/Fisher's exact test (where appropriate) for categorical variables. The t test or rank sum test (where appropriate) was utilised for continuous variables. Summary statistics, univariable and multivariable logistic regression were performed to determine the correlates of ANCA. Multicollinearity and first-order interaction between variables were taken into consideration when selecting variables for inclusion in the regression models. Kaplan-Meier (KM) survival graphs and Cox proportional hazards regression analysis were used to compare survival between the ANCA-positive and ANCA-negative groups. Statistical significance was defined as p ≤ 0.05. All statistical analyses were performed using Stata/SE 15.1 software (StataCorp, College Station, TX, USA).