Abstract
Introduction:
Sickle cell disease (SCD) is an inherited blood disorder caused by an amino acid change that produces an abnormal hemoglobin (HbS) which polymerizes under hypoxia, producing rigid red blood cells (RBC). In healthy individuals, whole blood viscosity (WBV) increases in a predictable manner with increases in hemoglobin (Hb); in individuals with SCD, WBV rises more rapidly with increase in Hb under normoxia, and rises even more with RBC changes under hypoxia, further reducing blood flow velocity. Current WBV measurements are typically performed only under normoxia and therefore lack the ability to measure the impact of HbS polymerization on WBV. A hypoxia-based, WBV assay is especially needed in this era of anti-sickling agents that increase hemoglobin and therefore viscosity, and to assess gene-based therapies. Assessing WBV under hypoxic conditions can also provide functional insight into how well the modified or functional hemoglobin perfuses low oxygen regions of the body. Some gene therapy (GT) clinical trials have achieved total hemoglobin levels of 16-18g/dL, raising concerns about WBV, especially under hypoxic conditions. Here, we describe the development of a chemical hypoxia-based strategy to assess WBV using a cone and plate viscometer.
Methods:
Peripheral blood samples (PBS) were collected in EDTA from 27 unique individuals with HbSS, 5 with HbAA, and one individual with HbSS who had undergone GT and analyzed at shear rate 225 s-1 using a cone and plate viscometer (Brookfield), with and without 50mg of sodium metabisulfite (MBS). We investigated the impact of an anti-polymerization agent, osivelotor, on WBV under hypoxia by adding 8.35ul of a 100mM stock solution of osivelotor to the PBS 50 minutes before the addition of MBS. PBS were incubated with MBS for 5 minutes at room temperature to allow deoxygenation to occur. Hemoglobin levels were adjusted to ~ 7 g/dL with 1X phosphate buffer. Statistical analysis was performed using GraphPad Prism 10.5.0. Paired samples were analyzed using a Wilcoxon matched-pairs signed rank test, and unpaired samples were analyzed with a Mann-Whitney test; a p-value<0.05 was considered significant.
Results: Hypoxia significantly increases the WBV compared to normoxic conditions for HbSS samples (median 6.3 vs 3.23, p <0.0001), while there was no statistically significant difference between WBV under hypoxia and normoxia for HbAA samples (median 3.02 vs 2.01, p= 0.06). The change in WBV with hypoxia compared to normoxia was significantly more in HbSS compared to HbAA (median 3.3 vs 1.02, p= 0.0009). The change in WBV with hypoxia was significantly less when HbSS samples were pre-treated with osivelotor compared to HbSS samples not pre-treated with osivelotor (1.94 vs 2.41, p=0.0016). We also observed minimal WBV change with hypoxia for the post GT sample (1.05), comparable to HbAA (median 1.11) and lower than HbSS (median 3.35).
Discussion: WBV of HbSS samples increased significantly with the addition of chemical hypoxia; HbAA WBV did not. HbSS samples treated with osivelotor ex vivo had less of an increase in WBV with the addition of hypoxia than the original, untreated HbSS samples, supporting the effectiveness and safety of osivelotor. An HbSS sample from an individual who had undergone GT also showed minimal change in WBV with the addition of hypoxia. This work highlights the feasibility and importance of testing WBV under hypoxic conditions and indicates that this assay can provide clinically relevant insights into drug and GT therapeutic effects as well as their safety. Integrating hypoxia-based viscosity profiling into clinical and research advancement could improve disease monitoring, risk stratification, and therapeutic optimization in SCD. Future work will standardize thresholds for pathologic WBV under hypoxia, patient-specific response patterns, and how this metric correlates with outcomes across different treatment regimens. As novel therapies such as gene editing and hemoglobin modifiers continue to advance, and produce high total hemoglobin levels, integrating hypoxia-based viscometry will play a critical role in monitoring therapeutic efficacy, and safety.
