Methods
After obtaining the approval of the clinical research ethics committees, we reviewed all ANCA determinations performed by the Immunology Service of Parc Taulí Hospital Universitari (PTHU) and by the Immunology Service at Hospital Universitari Vall d’Hebron (HUVH) from January 2014 through December 2015.
Patients were eligible for inclusion if they met the following three criteria: 1) had a biopsy compatible with the diagnosis of MPA, GPA, or EGPA, or met the American College of Rheumatology classification criteria for GPA (8) or EGPA (9) without an alternative clinical diagnosis; 2) had positive anti-MPO or anti-PR3 antibodies with titers ≥20 U/ml at least once; and 3) had at least 5 serial ANCA determinations from the date of diagnosis until December 2017. We excluded patients who did not complete 6 months of follow-up, patients with vasculitis limited to the skin or associated with cocaine abuse, and patients with insufficient clinical information.
ANCA titers were determined in serum by indirect immunofluorescence both on ethanol and on formalin-fixed human neutrophils smeared onto glass slides (Granulocyte Mosaic 13 EUROIMMUN, Lübeck, Germany). All ANCA-positive samples were analysed for anti-MPO and anti-PR3 antibodies by capture ELISA techniques (ImmunoCAP® 250 Phadia AB, Thermo Fisher Scientific, Uppsala, Sweden) or chemiluminescence (QUANTA Flash MPO / PR3, INOVA diagnostics, San Diego, California, USA).
Full details of the presenting illness, clinical relapses, and treatments carried out during follow-up were collected from case records. Clinical relapse was defined as a new or worsened manifestation of AAV that required a change in therapy.
Worsening renal function was defined as an increase in serum creatinine ≥1.5 times from baseline during follow-up. End-stage renal disease (ESRD) was defined as the need for sustained renal replacement therapy.
Four longitudinal patterns of ANCA levels were defined: 1) Monophasic pattern: initial peak of anti-PR3 or anti-MPO antibodies with subsequent negative results; 2) Remitting pattern: initial peak of anti-PR3 or anti-MPO antibodies followed by persistent low ANCA levels (<20 U/ml); 3) Recurrent ANCA pattern: at least two successive peaks of anti-PR3 or anti-MPO antibodies (≥20 U/ml) on follow-up, with periods of negative results; and 4) Persistent ANCA pattern: sustained positive anti-PR3 or anti-MPO antibodies (> 20 U/ml) throughout follow-up.
Associations between ANCA patterns and clinical variables were analysed using univariate statistics (Student’s t or Mann–Whitney U tests, as appropriate, for continuous variables and Chi-square or Fisher’s exact test for dichotomous variables). The association between ANCA patterns and clinical relapses or worsening renal function were analysed using Kaplan Meier curves and Cox proportional hazards model. P-values < 0.05 were considered statistically significant. All statistical analyses were carried out using SPSS version 23.0 for Windows (SPSS, Chicago, IL, USA).