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 N2 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 .