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

Abstract

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.

Effects of Carbon Dioxide and Temperature on the Oxygen-Hemoglobin Dissociation Curve of Human Blood: Implications for Avalanche Victims

“…Unfortunately, raw data are not published by Hlastala, so a direct comparison by recalculating their results using our equation is not possible. Hlastala also used an oxygenation protocol, whereas both Callaghan (15) and our group worked with deoxygenation for the determination of ODCs (17). As the solubility of CO 2 increases with a decrease of temperature, and CO 2 and O 2 are competing agents in binding to hemoglobin (Haldane effect), the way of the oxygen ramp might be of importance as this could interfere with the measurement results.…”

“…Oxygen dissociation curve experiments were performed with a modification of a recently published method (17). In this method, PO 2 is continuously decreased from 140 mmHg to 0 mmHg, and data points of PO 2 and SO 2 are acquired every minute to record the ODC.…”

“…To address the increase of water vapor saturation pressure with temperature, gas mixtures were prepared volumetrically from dry, pure gases (CO 2 , N 2 and O 2 ) in gas-tight sampling bags, according to the Magnus equation (21). The temperature management of the fluorescence plate reader instrument (Tecan 200 Infinite Pro, Tecan Genios Corp., Switzerland) ensured correct temperature in the measurement chamber, whereas temperature of the conducting Viton tubes, gas mixtures, humidifiers, and temperature of the whole experimental setup (17) for further descriptions of plate design and the measurement principles]. The gas supply was randomized throughout the study, here exemplary shown for increasing PCO 2 levels from left to right.…”

“…The average of triplet measurements for every combination and sample (Figure 1) was used for analysis. Additionally, an internal standard Hb solution (Equil QC 463 Level 2, RNA medical, USA) was analyzed in each test to check and adjust for accuracy of the measurements as described before (17).…”

Dose- and Sex-Dependent Changes in Hemoglobin Oxygen Affinity by the Micronutrient 5-Hydroxymethylfurfural and α-Ketoglutaric Acid

“…We used aa novel in-vitro high-throughput method for the recording of ODCs, which is based on a modified, gas-perfused 96-well microplate and a fluorescence microplate reader [21]. Duplicate measurements were performed for every concentration in the thin films of unbuffered whole blood samples.…”