Reactions with RBCs and CTV
Figure 4A presents the magnetic velocity characteristics of paramagnetic RBCs from healthy donors. These graphs include the settling velocity, us, versus the magnetic velocity, um, scatter plot with appropriate histograms aligned along the side and top, respectively. The range in distribution presented here is consistent with what we have reported earlier in a study of 17 healthy donors (Kim et al. 2020). Ongoing studies are investigating the significance of these distributions in healthy, anemic and patients with sickle cell disease.
The histograms indicate that the distributions in both velocities nearly overlap for all three treatment methods across all four donors. However, due to increased viscosity of the lactate added to the Oxyrase-deoxygenated samples, the magnetic and settling velocities appear offset from the dithionite and nitrite samples, which can clearly be seen by following the smooth cumulative curves. This trend is more pronounced in the y-axis (settling velocity) than in the x-axis (magnetic velocity) and can be attributed to side reactions producing diamagnetic hemichrome (around 540nm) due to over-oxidation in the case of nitrite. For example, the spectra of Hb in suspension,Figure1A , indicates high amounts of hemichrome contaminating metHb solution produced from exposure to nitrite. There are no other derivative forms of hemoglobin present in these samples.
Figure 4B represents the same data for the three SCD donors. The scatter data for SCD RBCs are significantly wider than the um-us scatter data for healthy RBCs. A large distribution in RBC size is indicative of hypoxia and iron deficiency (Ycas, Horrow & Horne, 2015; Sultana, Haque, Sultana & Ahmed, 2013), however this metric does not differentiate these healthy and SCD donors using CTV. Data in Figure 4 suggest similar distributions between us (which can be converted to cell size, assuming a constant cell density) histograms for healthy and SCD donors but shows that CTV is able to recognize a much wider um (analogous to pgHb/cell, potentially more clinically relevant) histogram for SCD donors compared to heathy ones. Scatter plot data for SCD donors also have a stronger fit along a line with positive correlation between um and us, indicating that smaller/less dense cells have lower mobility, and therefore less hemoglobin than larger/denser cells.
Although the gender and heterozygous/homozygous status of the sickle cell patients are unknown, scatter plot data in Figure 4Bsuggest that the SCD RBCs have a similar size to healthy RBCs, and not the rigid, elongated, polymerized homozygous HbSS RBCs.
Converting um data from Figure 4 to pgHb per cell (Chalmers et al ., 2017), Figure 5A, 5B calculate the amount of hemoglobin per cell for healthy and sickle cell donors, respectively. In this analysis, we are able to take into account the increased viscosity of Oxyrase and lactate in the reaction medium as well as us variations due to size. For each donor, the viscosity of the Oxyrase/lactate solution is calculated to match the average us between the dithionite and nitrite samples was calculated while using 0.89 mPa*s for water at room temperature for dithionite and nitrite. The experimentally calculated viscosities for Oxyrase/lactate CTV samples for all donors are presented inTable 1 along with average and standard error of the donor’s iron status (Morison, K. R., & Mackay, F. M., 2001). Most calculated viscosities are within 10% of that of PBS and the added viscous transport limitation to the reaction is assumed to be negligible compared to pH dependence.
Figure 6 compares average intracellular masses of Hb between preparations methods for each healthy and sickle cell donor. Interestingly, the average iron content between healthy and SCD donors is quite similar. Sickle cell disease originating from a β-globin chain mutation polymerizes into HbS and the charts suggest that HbS cells retain their intracellular hemoglobin in this form. Lastly, it is suggested that CTV results for deoxygenated RBCs, induced by eliminating DO (as is the function of RBCs in vivo ) reflect the amount of iron that is available for oxygen binding and dissociation. If this is the case, magnetic mobility measured this way may reflect overall cell health and performance better than total iron (which may be influenced by chemical reactions) or total hemoglobin (intracellular and free Hb).
The deoxygenation enzyme is active at high pH and subsequent experiments reveal improved methods to deoxygenate RBC buffer without significantly altering the viscosity. Most notably, laboratory prepared AS-3 (pH 5.8) with NaOH (added until the desired pH is achieved) is able to completely remove DO in 15 minutes. This AS-3 buffered media is chosen due to its RBC preservation properties and the enzyme is able to consume H+ from citric acid while NaOH maintains the desired basic pH. Figure 7 shows CTV data from donor F1 (on a different date) with AS-3 as the buffer at five different pH. Settling velocity cumulative curves reveal that buffers with high pH have greater settling velocities than AS-3 with low pH. This result is due to NaOH diluting the AS-3 and decreasing viscosity while equilibrium DO remains at zero. Using the same analysis to correct for viscosity, the pgHb/cell between the five buffers closely match (average 26.7, SD 1.1).
Figures 8A presents the oxygen dissociation curves of healthy donor hemoglobin deoxygenated with N and two samples deoxygenated with Oxyrase (all samples 2 mg/mL Hb). The Hemox-Analyzer was used per protocol to obtain a normal curve controlled at 37°C. The other two curves were obtained by adding hemoglobin to a completely deoxygenated buffer (maintained at 37°C by a separate water bath) and quickly filling the measurement chamber while keeping the other settings consistent. The first point in these data is the point of maximum oxygenation, which was matched with the control sample. The three curves are nearly identical, suggesting that the ability of Oxyrase-deoxygenated RBCs to bind to oxygen is identical to unaltered RBCs. The samples with Oxyrase and lactate have a small leftward shift from the control, which is consistent with that of a sample at lower temperature due to unconventional instrument use, shown inFigure 8B .