Abstract Background Reproducibility of the calibration curve within the analytical measurement range (AMR) is crucial for ensuring reliable patient values within and between laboratory instruments to detect shifts and trends of clinical significance. In this practical example for ionized calcium (Ca²⁺) assayed with two cobas b221® instruments, the equality of calibration lines and the verification with patient specimen comparison study was assessed with regression analysis techniques. Methods Instruments: Two cobas b 221 (Roche Diagnostics). Calibration material: COOX/MSS™ (Five levels, Roche Diagnostics). This was independently assayed either three or five times in parallel with both instruments. The patient specimens were obtained by venipuncture in Na/Li-heparin BD™ tubes (Becton-Dickinson). Upon receipt, specimens were assayed with both instruments within two minutes. The data were transferred to Minitab® (Version 21, Minitab Inc) statistical software and analyzed with polynomial ordinary least squares (POLS), orthogonal and locally weighted scatterplot smoother (lowess) regression analysis models. The aptness of the model was assessed with standardized residuals (std. res.) diagnostics. For acceptable total error the CLIA’s criterion target value ± 10% was adopted. Results While the POLS model did not show probabilistically significant differences between instruments (P=0.8), it showed probabilistically significant differences between years (P=0.003). However, this was due to a small MSE (= 0.003 mg/dL), which is not clinically significant. The test for linearity showed probabilistically significant lack-of-fit (P<0.0001) due to a small MS pure error (<0.0001 mg/dL). The lowess for the standardized deleted residuals by the fitted value showed a quasi-linear relationship. The multiple comparisons (P=0.3) and the Levene’s (P=0.4) tests did not detect probabilistically significant heteroscedasticity of the variance by reference value. The plot of the std. res. by the leverage showed two possible outliers (std. res.>|3|) and no influential observations (Hi<0.5). These diagnostics proved the aptness of the POLS model. The harmonization between the two instruments was verified with patient specimens. The orthogonal regression model showed in the interval 3-6 mg/dL a quasi-linear relationship between the values as obtained with the two instruments. The orthogonal regression line (y=0.1+1.02 n) and the OLS regression line (y=0.03+601 n) were very similar. There was no probabilistically significant lack-of-fit (P=0.5). The plot of the std. res. by the leverage showed one possible outlier (std.res.>|3|) and no influential observations. Additionally, the plot of the relative differences between the values as obtained with b221#9371 and b221#24796 by the values as obtained with b221#9371 showed only one value exceeding half of the total allowable error. Conclusions The POLS model clearly showed that the calibration lines for the two instruments were quasi-linear in the interval 2-10 mg/dL and reproducible within and between instruments. Additionally, this reliable performance was verified with patient specimens in the interval 3-6 mg/dL. The differences between values as obtained with each instrument were within half of the CLIA’s total allowable error. This clearly showed that equality of calibration lines ensured interchangeable patient values between instruments. Finally, electronic transfer of data and the availability of statistical software like Minitab was crucial for conducting this study.