Use of Red Blood Cell Phenotypes for Second Line Therapy Selection in Sickle Cell Disease

INTRODUCTION

In sickle cell disease (SCD), abnormal red blood cells (RBCs) sickle upon deoxygenation due to polymerization of hemoglobin S (HbS). Sickle RBCs exhibit poor deformability and increased viscosity, density, and microvascular adhesion. These rheological properties can be measured using existing devices. An oxygen-gradient ektacytometer measures the deformability of sickle RBCs under normoxic (maximum elongation index, or EImax) and hypoxic conditions (minimum elongation index, or EImin), and the pO ₂ level at which sickling beings (point of sickling, or PoS). Dense RBCs are measured using a commercially available hematology analyzer. The hematocrit-to-viscosity ratio (HVR), an oxygen delivery index, is calculated based on the viscosity as measured by a cone and plate viscometer. RBC adhesion in the microvasculature can be modeled using a laminin-lined microfluidics device.

These rheological biomarkers correlate with clinical complications such as pain events and acute chest syndrome, and are modified by known, clinically effective therapies such as hydroxyurea (HU) and transfusion (TF). HU is the standard of care for most individuals with SCD and positively modifies EImin, EImax, PoS, HVR, adhesion, and %DRBC. Recently, new agents to treat SCD have emerged including voxelotor, crizanlizumab, and pyruvate kinase activators like etavopivat, which have more targeted effects. It is essential to pair the appropriate novel agent to the patient, addressing their most prominent RBC abnormality remaining after HU therapy.

We hypothesize that there is significant variability of rheology biomarkers between individuals with SCD on standard of care therapy, and that the most severe aspects of their RBC pathophysiology can be identified and targeted by novel second line therapies for clinical optimization.

METHODS

We collected peripheral blood in EDTA under an IRB approved protocol from 312 pediatric patients with SCD ranging in age from 2 to 21 years, 70% on HU. Subjects on chronic TF therapy were excluded. We measured whole blood viscosity at 45s ^-1 shear and calculated the HVR. %DRBC and complete blood counts were obtained using an ADVIA hematology analyzer (Siemens) and EImin, EImax, and PoS obtained using oxygen gradient ektacytometry (Lorrca, RR Mechatronics). RBCs from 17 HbSS subjects were analyzed for adhesion index to a laminin-lined microfluidics device. Values were assembled for each biomarker into histograms to demonstrate distribution, and quartile ranked. Venn diagrams were constructed comparing overlap between top 25% most severe rheology biomarkers to demonstrate effectiveness of a targeted, precision medicine approach to adding second line therapies to individuals with SCD.

RESULTS

Distribution of biomarkers in a typical pediatric SCD population in a US academic center are shown in Figure 1. High PoS, adhesion index, %DRBC, and low EImin and EImax, low HVR, are associated with disease severity and clinical complications in SCD; biomarker values were stratified from high to low severity association. The most severe quartile subjects from each biomarker were compared, and percentage of overlap noted (Figure 2).

CONCLUSION

Our rheologic assessment of a large pediatric cohort heavily treated with HU indicates a broad distribution of RBC phenotypes. Even on HU, patients exhibited loss of deformability, sickling, adhesion, or RBC density abnormalities, with little overlap of unrelated biomarkers associated with disease severity in an individual, i.e. the individuals with very high PoS did not have low HVR (16% of highest severity quartile subjects in common), compared to related biomarkers PoS and EImin or EImax (48% of highest severity quartile subjects in common). Only four subjects, all on HU, were in the quartile associated with highest severity for all biomarkers. Given the lack of overlapping pathology between different red cell abnormalities, selection of the appropriate agent should be straightforward. With three new FDA approved therapies for SCD and novel therapies in clinical trials, it is possible to choose the appropriate second agent to be added to HU based on individual patient RBC phenotype according to the principles of precision medicine. Future goals include CLIA certification for novel devices like the oxygen gradient ektacytometer and adhesive microfluidics at major academic SCD centers and use of these biomarkers in routine patient care.

Figure 1

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Figure 1

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