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Single‐cell Measurement of Red Blood Cell Oxygen Delivery Rate
Author(s) -
Kim Sangho
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.684.14
Subject(s) - chemistry , oxygen , red blood cell , hemoglobin , oxygen delivery , oxygen–haemoglobin dissociation curve , fluorescence , biophysics , microfluidics , blood cell , analytical chemistry (journal) , chromatography , nanotechnology , biochemistry , materials science , immunology , physics , organic chemistry , quantum mechanics , biology
Oxygen (O 2 ) is supplied throughout the body by hemoglobin (Hb) bounded in red blood cells (RBCs). The ability of RBCs to deliver O 2 has been routinely assessed by P50 using clinical hematology analyzers, defined as the partial pressure of O 2 (P O2 ) required to saturate the Hb in RBCs to 50%. Although this defines the ability of RBCs to carry O 2 under equilibrium states, it cannot determine the efficacy of O 2 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 O 2 dissociation curve (ODC) and P50 have been established, it is unclear how, and the variations of these factors, may influence the RBC O 2 delivery on the cellular level. Therefore, in this study, we developed a microwell array oxygen‐sensing chip for measuring the rate of O 2 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 N 2 gas separated by a PDMS membrane. RTDP fluorescence is quenched in the presence of O 2 , the fluorescence intensities in the microwells increase as they get deoxygenated by the continuous flow of N 2 gas. The rate of O 2 release from the single RBC trapped in the microwells, however, would alter the rate of change of microwell P O2 . Accordingly, RBCs with higher rates of O 2 release would reduce the overall rate of deoxygenation in the microwell, thus reducing the rate of decrease in the P O2 . To demonstrate the sensitivity of the device, we deoxygenated the microwells containing RBCs and RBC ghosts (RBCs without Hb). As expected, the P O2 in the microwells containing RBCs was significantly higher (p < 0.0001) than that containing RBC ghosts, due to O 2 release from Hb bounded in the RBCs. Additionally, we investigated the potential difference in the O 2 delivery rate between density‐fractionated young (top 5% fraction) and old RBCs (bottom 5% fraction). The obtained results showed an apparent difference in the P O2 in microwells with young and old RBCs is statistically significant (p < 0.0001), suggesting that senescent RBCs have a lower O 2 delivery rate. Support or Funding Information NMRC/CBRG/0078/2014 and Biomedical Institute for Global Health Research and Technology This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .