Improvement of indirect immunofluorescence technique to detect antineutrophil cytoplasmic antibodies and its impact on test positivity rate.

The indirect immunofluorescence (IIF) technique for antineutrophil cytoplasmic antibodies (ANCA) detection is subject to substantial differences across laboratories. This study aimed to assess the impact of improvements in the IIF-ANCA technique on the positivity rate of ANCA tests. A cross-sectional study was performed with serum samples from patients with ANCA-associated vasculitis (AAV), autoimmune hepatitis (AIH), and ulcerative colitis (UC). A paired analysis was performed for IIF-ANCA results using the traditional method and a modified protocol after a series of specific adjustments in the technique based on the protocol of IIF-ANCA test performed at a nation-wide private laboratory in Brazil. ANCA specificity was assessed by ELISA for anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) antibodies. Sixty-one patients were evaluated. The positivity rate of IIF-ANCA tests at disease presentation performed at the University reference laboratory was 32.3% in AAV, AIH, and UC patients, whereas the positivity rates of IIF-ANCA and ELISA tests in other laboratories were 75.0 and 72.7%, respectively. After modifications in the IIF-ANCA technique, there was a significant increase in the positivity rate (14.8 vs 34.3%; P=0.0002) and in median titers [1/40 (1/30-1/160) vs 1/80 (1/40-1/80); P=0.0003] in AAV, AIH, and UC patients. UC had the highest increment in positive results from 5.3 to 36.8%. There was poor agreement between MPO- or PR3-ANCA and both IIF-ANCA techniques. In conclusion, modifications in the IIF-ANCA protocol led to a significant improvement in its positivity rate and titers.

Improvement of indirect immunofluorescence technique to detect antineutrophil cytoplasmic antibodies and its impact on test positivity rate

The indirect immunofluorescence (IIF) technique for antineutrophil cytoplasmic antibodies (ANCA) detection is subject to substantial differences across laboratories. This study aimed to assess the impact of improvements in the IIF-ANCA technique on the positivity rate of ANCA tests. A cross-sectional study was performed with serum samples from patients with ANCA-associated vasculitis (AAV), autoimmune hepatitis (AIH), and ulcerative colitis (UC). A paired analysis was performed for IIF-ANCA results using the traditional method and a modified protocol after a series of specific adjustments in the technique based on the protocol of IIF-ANCA test performed at a nation-wide private laboratory in Brazil. ANCA specificity was assessed by ELISA for anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) antibodies. Sixty-one patients were evaluated. The positivity rate of IIF-ANCA tests at disease presentation performed at the University reference laboratory was 32.3% in AAV, AIH, and UC patients, whereas the positivity rates of IIF-ANCA and ELISA tests in other laboratories were 75.0 and 72.7%, respectively. After modifications in the IIF-ANCA technique, there was a significant increase in the positivity rate (14.8 vs 34.3%; P=0.0002) and in median titers [1/40 (1/30-1/160) vs 1/80 (1/40-1/80); P=0.0003] in AAV, AIH, and UC patients. UC had the highest increment in positive results from 5.3 to 36.8%. There was poor agreement between MPO- or PR3-ANCA and both IIF-ANCA techniques. In conclusion, modifications in the IIF-ANCA protocol led to a significant improvement in its positivity rate and titers.

Transcranial Doppler findings in antiphospholipid syndrome.

INTRODUCTION: Transcranial Doppler is a method that enables the assessment of different cerebral hemodynamic parameters. It also allows for the evaluation of the presence of right-to-left circulation shunts (RLS) and for the detection of microembolic signals (MESs), which might be associated with an increased risk of cerebrovascular events. For instance, the presence of MESs on transcranial Doppler in patients with systemic lupus erythematous (SLE) and antiphospholipid syndrome (APS) is associated with an increased risk of stroke. Therefore, transcranial Doppler could be a useful tool for stroke risk stratification in these patients. OBJECTIVE: Our objective was to evaluate transcranial Doppler cerebral mean blood flow velocities as well as the presence of MESs and RLS in patients with antiphospholipid syndrome and SLE. PATIENTS AND METHODS: Twenty-two patients with primary APS (PAPS), 24 patients with secondary APS (SAPS), 27 patients with SLE without APS and 21 healthy controls were evaluated. Clinical and epidemiological data were compiled from medical charts, and all subjects underwent transcranial Doppler examination with breath-holding index calculation. Both middle cerebral arteries were monitored for 60 min for the detection of MESs. RLS was investigated with agitated saline injected as a bolus. RESULTS: There were no significant differences in mean blood flow velocities among the groups. MESs were more frequently found in patients with SLE when compared with controls and patients with APS (SLE: 17.4%, SAPS: 4.3%, PAPS: 0%, controls: 0%, p = 0.03). Anticoagulant therapy was more frequently used in the APS group (PAPS: 81.8%, SAPS: 75.2%, SLE: 1.7%, p < 0.001). Patients with APS had a higher frequency of RLS when compared with volunteers (63.6% versus 38.1%, p = 0.05). Breath-holding index values tended to be lower in patients with SAPS than in control subjects and patients with PAPS and SLE ( p = 0.06). CONCLUSIONS: Patients with APS had a higher frequency of RLS than healthy controls. This finding alerts to the importance of cardiac investigation in patients with stroke and APS, because further therapies such as RLS occlusion might eventually add protection. The higher frequency of MES in patients with SLE could suggest an effect of anticoagulant therapy on MES prevention, more frequently used in patients with APS.