High-throughput determination of oxygen dissociation curves in a microplate reader-A novel, quantitative approach

In vitro determination of the hemoglobin oxygen dissociation curve (ODC) requires highly elaborate, specialized, and costly technical equipment. In addition, there is a lack of methods that combine reliable ODC recordings with high throughput in small blood samples for routine analysis. We here introduce a modified, commercial 96-well plate with an integrated unidirectional gas flow system specifically adapted for use in fluorescence microplate readers. Up to 92 samples of whole or hemolyzed, buffered or unbuffered blood, including appropriate controls or internal standard hemoglobin solutions, can be analyzed within ~25 min. Oxygen saturation is measured in each well with dual wavelength spectroscopy, and oxygen partial pressure using fluorescence lifetime of commercial oxygen sensors at the in- and outlet ports of the gas-flow system. Precision and accuracy of this method have been determined and were compared with those of a standard method. We further present two applications that exemplarily highlight the usefulness and impact of this novel approach for clinical diagnostics or basic research.