Methods to Assess the Impact of Combined Pharmacologic and Transfusion Therapies on Red Cell Deformability

Background

Sickle cell disease (SCD) is an inherited blood disorder in which abnormal hemoglobin (HbS) polymerizes in hypoxic environments, creating rigid, or poorly deformable red cells. RBC deformability can be measured by oxygen gradient ektacytometry (LoRRca), or by the Microfluidic Impedance Red Cell Assay (MIRCA). The MIRCA is a microchannel assay which evaluates RBC deformability under mechanical stress and provides occlusion values under normoxia (NOI) or sodium metabisulfite-induced hypoxia (HOI).

Individuals with SCD may be placed on chronic transfusion therapy (CTT) to prevent clinical complications like stroke. Despite suppression of HbS to <=40%, individuals on red cell exchange (RCE) and chronic simple transfusion may still be symptomatic. We propose to test RBC deformability over a range of HbS percentages using the LoRRca and MIRCA to determine if there is a detectable population of poorly deformable cells contributing to ongoing clinical complications. We will then determine if the hemoglobin oxygen affinity modifier GBT021601 can modify the poorly deformable RBC when added in vitro.

Methods

117 peripheral blood samples from individuals aged 6 to 74 (94 HbSS/Sβ0, 14 HbSC/Sβ+/SE, and 9 HbAA) were collected under an Emory University IRB approved protocol in EDTA. Washed red blood cells (RBC) were run on the LoRRca, or resuspended to a 20% hematocrit in 1X PBS for normoxia (normoxia occlusion index, NOI) measurements or 1.5% MBS for chemical hypoxia (hypoxia occlusion index, HOI), and run on the MIRCA. To test the impact of GBT201601 on deformability, RBC were treated with 1.66mM GBT021601 dissolved in DMSO or with DMSO alone, incubated at 25°C for 1 hour, and resuspended in 1.5% MBS in 1X PBS and run on the MIRCA.

Transfusion status and %HbS were determined by chart review. Scatter-plots were utilized and included either NOI or HOI with %HbS and %HbF status. A Wilcoxon signed rank test compared control and GBT021601 HOI values (n=7); GBT021601 data was further compared against HbA individuals (n=2) through a Wilcoxon rank sum test. The association between control, GBT21601-spiked, and %HbS was analyzed through a linear regression. The data was analyzed using StataNow 18.5 (College Station, TX); a p <0.05 was considered significant.

Results

Scatter-plots of %HbS and %HbF against either NOI or HOI showed a non-linear relationship consisting of three segments. HOI and NOI values decreased as HbS increased to 39%. HbS>40% showed increasing HOI and NOI values with a steeper incline from HbS>=90%. %HbF was statistically significantly higher in HbS groups between 40-89% (median 12) compared to compared to HbS<40% (median 0, p<0.001) and HbS<=90% groups (median 3.9, p<0.001). Scatter-plots of %HbS and %HbF against LoRRca were linear.

HOI values for GBT021601 treated RBCs from individuals not on transfusion therapy were significantly lower compared to DMSO controls (p=0.016). HOI values in transfused patients with %HbS<40% declined with GBT021601 exposure (p=0.06). HOI values for GBT021601 exposed samples were not significantly different from that of HbA individuals (p=0.11).

Discussion

We noted a non-linear relationship of %HbS with MIRCA, and a significant population of poorly deformable cells even at very low %HbS typical of RCE, the most aggressive transfusion regimen. Of note, the LoRRca did not detect this population. These poorly deformable, endogenous RBCs also have a very low %HbF. It is not known if these individuals naturally have low %HbF, or if the reduced erythropoietic drive from chronic transfusion therapy has reduced stress erythropoiesis and therefore reduced their %HbF. These unmodified, poor quality recipient cells may still cause VOE and organ damage; reduced given their low percentage, but still present and measurable by the MIRCA.

In vitro addition of GBT021601 modified the poorly deformable cells that were present in transfused individuals. GBT021601-modified red cells had similar MIRCA deformability values to healthy controls; we expect to see a similar benefit in patients on dual transfusion and drug therapy. Future work will involve the use of MIRCA to assess the longitudinal variability of RBC deformability in individuals on chronic transfusion therapy alone and in combination with anti-sickling agents, to determine if transfusions can be reduced in frequency and volume without sacrificing RBC quality.

Suster:XaTek Inc: Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties, Research Funding. Mohseni:XaTek Inc: Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties, Research Funding. Gurkan:Hemex Health Inc: Consultancy, Current Employment, Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties, Research Funding; DxNow Inc: Patents & Royalties; XaTek Inc: Patents & Royalties; BioChip Labs Inc: Consultancy, Current Employment, Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties, Research Funding.