High-throughput oxygen dissociation assay enables rapid screening of hemoglobin oxygen affinity modifying therapies Free

Introduction The oxygen dissociation curve (ODC) quantitatively describes the affinity of hemoglobin (Hb) to bind and release oxygen (O₂) as the partial pressure of oxygen (pO₂) changes. Precise determination of the ODC and its key parameter p50 (the pO₂tension at which Hb is 50% saturated) is fundamental for characterizing red blood cell (RBCs) physiology, diagnosing or monitoring hemoglobinopathies such as sickle cell disease (SCD), and evaluating emerging therapeutics that modulate Hb-O₂ affinity such as pyruvate-kinase activators (PKR activators), voxelotor, or GBT021601. Conventional assays and instruments require highly elaborate, specialized, and costly technical equipment, such as Hemox analyzer. Moreover, these methods do not operate in a high-throughput, multiplexed sense using low sample volumes that would otherwise facilitate and expedite routine clinical or research analyses. To address these limitations, we developed the Hydrogel-Based Rapid Hemoglobin Oxygen Dissociation Assay (RHODA), a 96-well microplate platform that couples a hydrogel for controlled release of an O₂scavenger with a soluble ruthenium fluorophore to report the pO₂ in each microwell.

Methods The high-throughput RHODA leverages hydrogel chemistry and fluorescence-based O₂ sensing in a microplate reader format. HODA comprises a chemical crosslinkable hydrogel, a ruthenium-based O₂ sensor, and a buffered medium of pH 7.4. The hydrogel is synthesized by crosslinking polyvinyl alcohol (PVA) and polyethylene glycol (PEG), incorporating sodium metabisulfite (Na₂S₂O₅) as a chemical O₂ scavenger. This scavenger slowly and predictably deoxygenates the buffered medium containing 25 μL of whole blood sample over the assay time. Absorbance measurements are performed at dual wavelengths of 414/430 nm to determine oxygen saturation (SO₂) directly from the conformational changes of Hb. Fluorescence measurements are conducted with Ex 462 nm/Em 620 nm, allowing determination of pO₂ via luminescence quenching. By combining pO₂ data from fluorescence and SO₂ data from Hb absorbance measurements, ODCs of samples in all wells can be generated rapidly, within 15 minutes for a 96-microwell plate. Additionally, we have designed a hydrogel insert attachable to commercially available 96-microwell plates to facilitate synchronous introduction of PVA/PEG pellets to each well. We validated RHODA by comparing its performance to the reference standard Hemox analyzer using blood samples from healthy individuals (HbAA) and SCD (HbSS). We also tested the sensitivity of RHODA in assessing the potency of Hb-O₂ modifying drugs that were treated with HbSS RBCs.

Results Validation studies demonstrated a strong correlation between the RHODA-generated p50 values and those obtained from the Hemox analyzer across all tested blood types (HbAA (n=5) and HbSS(n=15)) (Pearson's coefficient, PCC = 0.91, p = 0.004). RHODA provided accurate and reproducible measurements with a minimal 25ul of whole blood sample. The assay showed high consistency, with intra-assay and inter-assay variability well within acceptable limits (CV = 2.5). Moreover, RHODA significantly reduced analysis time, completing ODC analysis for 96 samples within 15 minutes compared to the 2 hours typically required by conventional methods per sample. Hb-O₂ affinity-modifying drugs, including GBT021601, voxelotor, and mitapivat demonstrated distinct shifts in ODC profiles and changes in p50, consistent with their known mechanisms of action, confirming RHODA's sensitivity for drug potency assessment. Additionally, RHODA detected a ~6% decrease in p50, indicating increased Hb-O₂ affinity, in stored blood over a one-week storage period.

Conclusion RHODA delivers rapid, reliable, and high-throughput quantification of Hb-O₂ affinity using minimal blood volume. Its strong correlation with the Hemox analyzer, sensitivity to Hb-O₂ affinity modulators, and ability to track storage-related changes highlight its versatility. The distinct and reproducible shifts in ODCs observed with mitapivat, voxelotor, and GBT021601 emphasize RHODA's potential as a powerful tool for evaluating therapeutic efficacy and optimizing dosage strategies for novel Hb-O₂ affinity-modifying drugs. By enabling routine ODC assessment in a microplate format, RHODA stands to accelerate drug development, facilitate genome-editing-based therapies, support blood bank quality control, and inform personalized management of hemoglobinopathies such as SCD.