Use of red cell deformability measurements to assess the impact of pyruvate kinase activators on combined drug and transfusion therapies

Abstract

Abstract Background Sickle cell disease (SCD) is characterized by poorly deformable red blood cells (RBC) that worsen with hypoxia. Some individuals with SCD are placed on chronic transfusion therapy (CTT) to prevent clinical complications like stroke. Despite suppression of HbS to ≤30%, individuals on CTT may still be symptomatic. We propose to test RBC deformability over a range of HbS percentages to determine if there is a detectable population of poorly deformable cells contributing to ongoing clinical complications, using two different devices that measure RBC deformability, oxygen gradient ektacytometry (LoRRca), or by the Microfluidic Impedance Red Cell Assay (MIRCA). The LoRRca measures RBC deformability under controlled deoxygenation with RBC elongated by rotational shear stress; it reports RBC deformability at normoxia (elongation index maximum, EImax) and hypoxia (elongation index minimum, EImin). The MIRCA mechanically measures RBC deformability as they squeeze between ever closer pillars under normoxia and chemical hypoxia, creating impedance when trapped, and provides occlusion values under normoxia (NOI) or sodium metabisulfite-induced hypoxia (HOI). We will then determine if the pyruvate kinase activator, mitapivat, now in phase II/III clinical trials in individuals with SCD, can modify the poorly deformable RBC from SCD individuals on CTT when added in vitro. Methods 117 peripheral blood samples from SCD aged 6 to 74 (94 HbSS/Sβ0, 14 HbSC/Sβ+/SE, and 9 HbAA) were collected in EDTA. Washed RBCs were run on the LoRRca or resuspended to a 20% hematocrit in 1X PBS for normoxia (NOI) measurements or 1.5% MBS for chemical hypoxia (HOI) and analyzed on MIRCA. RBC were treated with 50µM mitapivat dissolved in DMSO or with DMSO alone, incubated at 25°C for 1 hour, then 37°C for 3 hours, resuspended in 1.5% MBS in 1X PBS and run on the MIRCA. Transfusion status was determined by chart review. LOWESS (Locally Weighted Scatterplot Smoothing) were created to analyze the relationship of LoRRca and MIRCA readouts with %HbS and %HbF. HOI values of mitapivat (n=6) were compared with DMSO and HbAA controls (n=4) using a Wilcoxon test. StataNow 19.5 (TX) was used for statistical analysis; a p <0.05 was considered significant. The deformability of two individuals who had undergone gene therapy (GT) were also analyzed on MIRCA. Results LoRRca values obtained from samples with a range of %HbS from 10-95% report optimal RBC deformability (high EImin and EIMax) at the lowest %HbS under normoxia and hypoxia, indicating that the device is averaging the deformability of the HbAA and HbSS RBC populations. In contrast, MIRCA readouts show poor deformability (high HOI and NOI) both in heavily transfused individuals (HbS<30%) and in untreated/high%HbS (HbS>80%), creating a U-shaped curve when plotted using LOWESS. This demonstrates that MIRCA captures endogenous sickle RBCs despite the presence of large amounts of HbAA RBC. HOI values of mitapivat treated RBCs from SCD individuals on CTT were significantly lower compared to DMSO controls, indicating improved deformability (p=0.02, median 11.94 vs.19.21) but HOI and NOI were still significantly higher compared to HbAA individuals (p=0.01, median 11.94 vs. 3.93). GT NOI/HOI ranged from 6-15. Discussion A significant population of poorly deformable RBC were detected by MIRCA at very low %HbS consistent with CTT; LoRRca did not detect this population. This is likely due to the distinct mechanisms of the two devices, and it indicates that the problem of continued clinical complications despite CTT is best assessed with RBC deformability biomarkers measured by MIRCA. In vitro, the addition of mitapivat significantly improved the sickle RBC still present in transfused individuals, as measured by MIRCA. Mitapivat-modified red cells had poorer deformability than HbAA RBC but were comparable to MIRCA measurements of deformability on two individuals who have undergone GT for SCD. Modification of recipient RBCs by mitapivat may be key to preventing SCD related complications while on CTT, and MIRCA is an effective tool to monitor the efficacy of combined CTT and mitapivat.