Single‐cell Measurement of Red Blood Cell Oxygen Delivery Rate

Abstract

Oxygen (O2) is supplied throughout the body by hemoglobin (Hb) bounded in red blood cells (RBCs). The ability of RBCs to deliver O2 has been routinely assessed by P50 using clinical hematology analyzers, defined as the partial pressure of O2 (PO2) required to saturate the Hb in RBCs to 50%. Although this defines the ability of RBCs to carry O2 under equilibrium states, it cannot determine the efficacy of O2 delivery in the dynamic blood flow, which could occur over the span of ~3 s in the microcirculation. Furthermore, while various factors such as pH, temperature and 2,3‐diphosphoglycerate (2,3‐DPG) affecting the O2 dissociation curve (ODC) and P50 have been established, it is unclear how, and the variations of these factors, may influence the RBC O2 delivery on the cellular level. Therefore, in this study, we developed a microwell array oxygen‐sensing chip for measuring the rate of O2 delivery from RBCs at a single‐cell level. Washed RBCs were re‐suspended in an oxygen‐sensitive fluorescence dye solution (ruthenium tris(2,2′‐dipyridyl) dichloride hexahydrate, RTDP) and introduced into the microfluidic device. After trapping of single RBCs in the microwells, heavy mineral oil is used to flush excess RBCs and to isolate single RBCs in the microwell array. Subsequently, the fluorescence intensity of the oxygen‐sensitive dye in the microwell was measured as it gets deoxygenated by N2 gas separated by a PDMS membrane. RTDP fluorescence is quenched in the presence of O2, the fluorescence intensities in the microwells increase as they get deoxygenated by the continuous flow of N2 gas. The rate of O2 release from the single RBC trapped in the microwells, however, would alter the rate of change of microwell PO2. Accordingly, RBCs with higher rates of O2 release would reduce the overall rate of deoxygenation in the microwell, thus reducing the rate of decrease in the PO2. To demonstrate the sensitivity of the device, we deoxygenated the microwells containing RBCs and RBC ghosts (RBCs without Hb). As expected, the PO2 in the microwells containing RBCs was significantly higher (p < 0.0001) than that containing RBC ghosts, due to O2 release from Hb bounded in the RBCs. Additionally, we investigated the potential difference in the O2 delivery rate between density‐fractionated young (top 5% fraction) and old RBCs (bottom 5% fraction). The obtained results showed an apparent difference in the PO2 in microwells with young and old RBCs is statistically significant (p < 0.0001), suggesting that senescent RBCs have a lower O2 delivery rate.Support or Funding InformationNMRC/CBRG/0078/2014 and Biomedical Institute for Global Health Research and TechnologyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.