• 7
    Nov
  • Diagnostic Value and Clinical Significance: MATERIAL AND METHODS

Patients

In this cross-sectional study, two groups were compared: 76 RA patients, according to the RA criteria revised by the ACR in 1987, with disease duration of more than two years, admitted to our outpatient rheumatology clinics consequently were included in the patient group. All patients underwent complete clinical evaluation by the same physician (NS) and the following data were recorded; age, gender, disease duration, duration of morning stiffness, swollen and tender joint count (both by 28 joint), presence of hand deformity, patient’s assessment of pain [visual analog scale (VAS)], disease-modifying antirheumatic drug (DMARD) intake as well as anti-CCP, RF, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), white blood cell (WBC), platelet (PLT), and hemoglobin values (Hb). Daily life function was assessed with the Modified Health Assessment Questionnaire (HAQ).

Eighty-three age-matched non-RA volunteers, recruited over the same period as the RA patients in the outpatient clinic were enrolled in the study as the control group. The control group included both healthy volunteers (n=32) and non-RA patients (n=51), which included those with osteoarthritis, fibromyalgia, myofacial pain, acute lumbar and cervical strain. Additionally, patients with granulamatous, lymphoproliferative, neoplastic and chronic viral diseases, were excluded from the study because of the possibility of positive RF in these diseases. Informed consent was obtained in all study subjects.

Laboratory Analysis

Serum RF and CRP concentrations determined by immuno-nephelometry method on BNII nephelometer (Dade Behring, Marburg, Germany). The concentrations were expressed as IU/ml for RF and mg/dl for CRP. RF concentrations higher than 15 IU/ml were considered positive. Our upper limit for CRP was 0.5 mg/dl.
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Anti-CCP antibodies were tested using commercially available ELISA kit (Euroimmun, Germany). Briefly, 100 ц1 anti-CCP standards (0, 2, 8, 30 and 100 RU/ml), controls and patient samples (1:100 in sample buffer) were distributed into the appropriate wells. The microtiter plates were coated with highly purified synthetic cyclic peptides. After incubation for 60 minutes at room temperature, the wells were washed three times with 300 ц1 wash buffer. The microplates were then incubated for 30 minutes at room temperature with alkaline phosphate-labeled antibody to human IgG and washed again three times. A chromogenic substrate solution (p-nitro-phenylphosphate solution) was added to each well. After 30 minutes, the reaction was stopped using sodium hydroxide stop solution. The absorbancy was read at 405 nm on a microplate reader (Sirios, Seac Radim Group, Italy). Serum anti-CCP concentrations were calculated according to standard curve. Results were expressed as RU/ml. Serum samples were evaluated in duplicate, and the upper normal limit (5 RU/ml) was assumed according to the manufacturer’s recommendations. The anti-CCP and RF measurements were also dichotomized as present or not for statistical analysis.

ESR was measured with a folly automated system working on the basis of Westergren method controlled by a microprocessor (SRS 100, Greiner Bio-one, FL). The complete blood count (WBC, PLT, and Hb) was determined on a GEN-S automated hematology analyzer (Beckman Coulter Inc., Miami, FL).
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Statistical Analysis

Statistical analysis was performed using the SPSS 11.0 for Windows® statistical package. Means, standard deviations (SD) and confidence intervals (CI) were used where appropriate. The significances of the differences between the means were tested using the Student’s t test (two-tailed). Comparison of proportions was performed using % analyses. The relationship between the anti-CCP and RF was assessed by Pearson correlation. The distribution of anti-CCP and RF levels was also converted to a four-cell (2×2) table using the cut-points 350 IU/ml for RF and 30 RU/ml for anti-CCP and analyzed by McNemar test. Receiv­er operating characteristic (ROC) curve was used to calculate cut-off values for sensitivity and specificity. In the logistic regression analysis (forward conditional) for anti-CCP or RF positivity, the independent variables were selected according to the univariate analysis (p<0.05). Presence of hand deformity was taken as the primary outcome measure and dichotomized as present or not. Age, duration of disease, HAQ, swollen joint count, CRP, ESR, RF and anti-CCP were entered as continuous independent variables in the logistic regression analysis for the presence of hand deformity. In all instances, p values of <0.05 were considered significant.

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