Materials and Methods
Blood samples were drawn from two healthy male (M1, M2) and female (F1, F2) donors with informed consents according to a protocol approved by the Institutional Review Board (IRB) from The Ohio State University (protocol number 38334). These donors’ blood was collected into EDTA-coated vacutainers. Samples were then diluted 1:100 in PBS, pH 7.4. After dilution, RBC concentration and size were measured in a Multisizer 4e Coulter Counter (Beckman Coulter). Unlysed, oxygenated RBCs or oxyHb were converted to the deoxygenated state by adding Oxyrase (Oxyrase Inc.) with 60 wt.% Sodium DL-lactate to maintain a pH suitable for a rapid deoxygenation rate. For comparison, deoxyRBCs or deoxyHb were obtained using 3.7 mM sodium dithionite (without passing an inert gas) and 5 mM sodium nitrite was used for oxidation to the metHb form. Oxyrase and lactate were mixed by pipette and left in an aerobic environment for 30 minutes, and dissolved oxygen was confirmed to be near zero using a ThermoFisher Orion 083005MD dissolved oxygen probe. Free Hb and RBCs were added to PBS containing either Oxyrase/lactate, sodium dithionite or sodium nitrite while exposed to the atmosphere. RBCs from SCD patients was provided by Dr. Irwin at University of Colorado Anschutz Medical Campus as apheresis waste from three patients undergoing scheduled blood transfusions. These SCD blood samples were considered waste products and deidentified. Both normal and SCD samples were prepared identically.
Free Hb for characterization and kinetics studies was prepared by lysing healthy RBCs in a -86°C freezer and thawing in a bath of room temperature water. This method of lysis was chosen over hypotonic membrane rupture because an anaerobic environment could not be maintained using a typical lysis buffer containing dissolved oxygen. Cell lysate was centrifuged at 2,000 RCF for 5 minutes in an Eppendorf 5415c centrifuge. Spectrophotometry was performed by adding the supernatant to cuvettes containing the appropriate salt or enzyme in an Eppendorf BioSpectrometer Basic while reaction times were followed manually using a stopwatch.
Cell tracking velocimetry (CTV) was performed as previously described (Zborowski et al. , 2003; Moore et al. , 2006; Mooreet al. , 2000; Zhang et al. , 2005). During this experiment, CTV samples were measured at a position within the magnet with a Sm value of 224.9 TA/mm2 (6.47mm from the converging axis of the pole pieces). Hb on a cellular basis was calculated using the Winterbourne equation with spectroscopy and calculated from CTV data using the method presented by Chalmers et al . (2017).
For free Hb samples prepared with dithionite and Oxyrase, the percentage of oxyHb and deoxyHb was calculated using the method presented by Tsao, Sethna, Sloan & Wyngarden (1955). A linear correlation of the percentage of oxygen saturation as a function of a ratio of the extinction coefficients ε575505 was obtained by measuring oxygen-saturated Hb and a completely anaerobic sample (<1% DO) of deoxyHb. Using equal hemoglobin concentrations, the extinction coefficients in the Beer-Lambert law can be replaced with the absorbance reported by a spectrophotometer. The 100% oxyHb sample was diluted with PBS exposed to air because if left in an anaerobic buffer, the deoxygenation would take place. The first measurement after adding oxyHb took place at 10 seconds because mixing shortly after hemoglobin addition caused scattering and made the result unreadable. Due to the first order rate of reaction, the 100% oxygenated point was extrapolated and a correction factor was applied to match the 100% oxygenation point and the final point, at 0% oxygenation. OxyHb samples and those treated with sodium nitrite (suspected of also containing metHb and hemichrome) were evaluated using the equation reported by Winterbourn (1990).
Oxygen dissociation curves were determined by using a Hemox Analyzer (TCS Scientific Corp) while controlling temperature at 37°C. For samples using Oxyrase, a temperature-controlled sample without Oxyrase and lactate was bubbled to 100% oxygenation using air, flushed out and refilled with a sample containing Oxyrase that was placed in a separate 37°C temperature bath and the measurements proceeded without bubbling nitrogen. During these experiments the sample temperature slightly deviated from the setpoint.