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Intra- and inter-observer (K.S. and H.N.) variability was evaluated for all tissue Doppler-derived variables in 20 randomly selected recordings. Variability was determined as a mean percent error derived from the absolute difference between two observations divided by the mean of the two observations. On the day of echocardiography, blood samples were obtained to determine BNP, white blood cell count (WBC), and CRP in the KDG and to determine BNP in the CG. In the KDG, an additional portion of blood samples (46 in the KDG) were directly centrifuged at 1500?g for 10?min and the sera were stored at ?80��C until assay of IL-6. The concentration of IL-6 were measured by using flow cytometric assay (BD Cytometric Bead Array Human Inflammation Kit; BD Biosciences, San Jose, CA, USA).26,27 Fifty microliters [http://www.selleckchem.com/products/epz-5676.html EPZ5676 order] of serum or cytokine standards were added to precoated microbeads with capture antibody specific for IL-6 in 12 �� 75-mm Falcon tubes (BD Biosciences), and then were incubated for 1.5?h in the dark at room temperature. These mixtures were washed and centrifuged at 200?g for 5?min, and the supernatant was discarded. [http://www.selleckchem.com/products/ly2109761.html LY2109761] Then, after the addition of 50??L of a phycoerythrin-conjugated antibody against IL-6, these mixtures were processed in the same way to get pellet resuspended in 300??L of wash buffer. A flow cytometer (BD FACScan) was calibrated with setup beads; 3000 events were acquired for each of the samples. The concentration of IL-6 was determined by measuring fluorescence intensity (FL-2) and computing with the standard reference curve of the software (BD Cell Quest Software; BD Bioscience, San Jose, CA, USA). Variables were expressed as mean and SD or range as appropriate. We compared echocardiographic parameters between the KDG and the CG using the Mann�CWhitney U-test. We converted BNP to common logarithmic expression [https://en.wikipedia.org/wiki/Oxacillin Oxacillin] and determined correlation between log (BNP) and echocardiographic parameters, WBC, CRP, or IL-6 as well as correlation between IL-6 and echocardiographic parameters in the KDG using univariate analysis. In addition, we identified possible factors that correlate with BNP production in the acute phase of KD using forward stepwise regression analysis. Level of statistical difference was set at P