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).