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During cardiopulmonary bypass blood gases can be analyzed with laboratory equipment or with an in-line monitor giving instant results. The manufacturer of the CDI 500 in-line blood gas monitor recommends gas calibration before use. In acute cases there may not be time to perform a gas calibration. We hypothesized that after calibration against laboratory results, the CDI values of pH, pO2, and pCO2will keep the same level of accuracy, whether the CDI has been gas calibrated or not. We performed a prospective randomized observational study using a study group without gas calibration (29 patients) and a control group with gas calibration (29 patients). Blood sampling was done at the beginning of bypass, and 30 minutes later. After each blood sample the CDI was in-vivo calibrated to the values simultaneously obtained from the ABL. Before in-vivo calibration values from the CDI without gas calibration were significantly different from the ABL-values in accuracy as well as precision, whereas the results from the gas calibrated CDI were largely consistent with the ABL. Before in-vivo calibration, the CDI without gas calibration was completely unreliable. After in-vivo calibration there was no statistical difference between the values of the CDI with and without calibration. We recommend gas calibration of the CDI before use in the period before in-vivo calibration.