Patients and controls.
Consecutive patients at the outpatient Department of Rheumatology were selected for inclusion in this descriptive, cross-sectional study. All patients had RA based on American College of Rheumatology criteria (21). Ten laboratory technicians served as healthy controls. Patients had to be between the ages of 18 and 80 years. Exclusion criteria included use of anticoagulants and/or corticosteroids. Only those patients with either very active RA or inactive disease were included. Active RA was defined as erythrocyte sedimentation rate (ESR) ≥28 mm/hour and/or C-reactive protein (CRP) level ≥28 mg/liter, combined with ≥9 painful joints and ≥6 swollen joints. Criteria for inactive RA were ESR ≤27 mm/hour, CRP ≤27 mg/liter, and the absence of painful and swollen joints. For all RA patients, a disease activity score (DAS) comprising the ESR, the tender joint count (TJC; 28 joints), the swollen joint count (SJC; 28 joints), and the patient's assessment of global well-being (on a 100-mm visual analog scale [VAS]) was calculated using the following formula: (0.56 × [√TJC]) + (0.28 × [√SJC]) + (0.70 × ln-ESR) + (0.014 × VAS). This DAS 28-joint count has been shown to be valuable in RA patients with both early and late disease (22). A DAS >3.8 indicates active disease, and a DAS ≤2.4 indicates that RA is in remission. All patients gave informed consent, and the study was approved by the local ethics committee.
Laboratory methods. EDTA-anticoagulated blood was collected. Blood cells were removed by centrifugation at 1,550g for 20 minutes at room temperature. Aliquots of cell-free plasma (first snap frozen in liquid nitrogen) were stored at −70°C until the time of analysis.
Isolation of microparticles. Cell-free plasma (250 μl) was centrifuged at 17,570g for 30 minutes at 20°C, after which 225 μl of microparticle-free plasma was removed. Phosphate buffered saline (PBS; 225 μl), 154 mmoles/liter of NaCl, 1.4 mmoles/liter of phosphate, and 10.9 mmoles/liter of trisodium citrate (pH 7.4) was added to the microparticle pellet and the remaining plasma (25 μl). The microparticles were resuspended and centrifuged at 17,570g for 30 minutes at 20°C. Again, 225 μl of the microparticle-free supernatant was removed, and microparticles were resuspended in the remaining 25 μl. For flow cytometric analysis, the 25-μl microparticle suspension was diluted 4-fold with PBS/citrate, of which 5 μl per incubation was used.
Flow cytometric analysis. The samples were analyzed in a FACScan flow cytometer using CellQuest software (BD Biosciences, San Jose, CA). Forward scatter (FS) and side scatter (SS) were set at logarithmic gain. Earlier studies showed that upon addition of a calcium ionophore (A23187) to platelets, particles (PMP) are released that are smaller (lower FS) and less dense (lower SS) than platelets (23). These in vitro–generated microparticles can be easily stained with annexin V, and antibodies can be directed against proteins present on the platelet surface, such as glycoprotein (GP) IIIa (CD61). Annexin V is a protein that in the presence of calcium ions binds with high affinity and high specificity to negatively charged phospholipids. GPIIIa is part of the integrin GPIIb–IIIa complex and is the most abundant receptor on the platelet surface.
To exclude events caused by noise, the microparticles were labeled with annexin V in the absence of calcium ions and an IgG1 control antibody to set the fluorescence thresholds (24, 25). Because >75% of the events generated in vitro bind both annexin V and CD61, we and other investigators assume that, by far, the majority of microparticles in vivo expose negatively charged phospholipids. For instance, Aupix et al used annexin V–coated enzyme-linked immunosorbent assay plates to capture cell-derived microparticles from plasma, which after washings were identified by cell-specific antibodies (26). The mixtures were incubated in the dark for 15 minutes at room temperature. Subsequently, 200 μl of PBS/calcium was added, and the suspensions were centrifuged at 17,570g for 30 minutes at 20°C. Finally, 200 μl of the microparticle-free supernatant was removed. The microparticles were resuspended with 300 μl of PBS/calcium buffer before flow cytometry. All samples were analyzed for 1 minute, during which time the flow cytometer analyzed ∼150 μl of the microparticle suspension. To estimate the number of microparticles per liter of plasma, the number of microparticles (N) found in the upper right quadrant of the flow cytometry analysis was used in the following formula: N/liter = N × (100/5) × (355/150) × (106/250).
Statistical analysis. Nonparametric tests (Mann-Whitney) were used to compare the values of PMPs in the various groups. Because data were not normally distributed, Spearman's test was applied to correlate the number of PMPs with disease activity. All data were expressed as the median (range).