Oxygen affinity assessment

The ability to bind oxygen in the lungs and offload oxygen to the tissues is the major role of red blood cells. The hemox analyzer provides data on the binding and release of oxygen at different oxygen tensions. 

The Oxygen Dissociation Curve (ODC) plots the percentage of hemoglobin saturated with oxygen (HbO %) versus  the partial pressure of oxygen (PO₂). The p50 value is the PO₂ (mmHg) at which hemoglobin is 50% oxygen saturated. The ODC measured at 37°C and pH 7.4 depends on cytosolic conditions (2,3 DPG) and type of hemoglobin. The normal P50 range is 25-29 mmHg.

Typical ODC curves of two normal samples (red and blue), an untreated sickle cell disease (SCD) subject with increased p50 = decreased oxygen affinity (green), and a treated SCD subject showing a left-shifted curve (purple) to a lower p50 and increased oxygen affinity. 

While this technology provides an important assessment, the Hemox analyzer is not practical to evaluate/screen large sets of compounds (libraries) with the potential to shift RBC oxygen affinity. The RBC lab has developed a rapid high throughput screen test to define the oxygen affinity shifting of large numbers of potential compounds. The figure shows the status of RBC hemoglobin oxygenation in two wells of a 96 well plate. The spectroscopic characteristics of hemoglobin is monitored while the environmental availability of oxygen is changed. As expected, treatment with voxelotor shows a clear shift to an increased oxygen affinity as compared to untreated RBC. Using this microplate assay allows the evaluation of large numbers of compounds in their ability to shift RBC oxygen affinity in a single experiment. The Hemox analyzer can be used to confirm the change in the oxygen affinity curve of potential compounds identified by the high throughput screening.

LEFT: Hemox analyzer assessment of RBC incubated with Voxelotor or Etapovipat, a pyruvate kinase stimulator. 

RIGHT: HTS Comparison of small amounts of RBC, either Voxelotor treated (red) or untreated (grey) in two wells of a 96 well plate.