A biophysical indicator of vaso-occlusive risk in sickle cell disease

Abstract

The search for predictive indicators of disease has largely focused on molecular markers. However, biophysical markers, which can integrate multiple pathways, may provide a more global picture of pathophysiology. Sickle cell disease affects millions of people worldwide and has been studied intensely at the molecular, cellular, tissue, and organismal level for a century, but there are still few, if any, markers quantifying the severity of this disease. Because the complications of sickle cell disease are largely due to vaso-occlusive events, we hypothesized that a physical metric characterizing the vaso-occlusive process could serve as an indicator of disease severity. Here, we use a microfluidic device to characterize the dynamics of "jamming," or vaso-occlusion, in physiologically relevant conditions, by measuring a biophysical parameter that quantifies the rate of change of the resistance to flow after a sudden deoxygenation event. Our studies show that this single biophysical parameter could be used to distinguish patients with poor outcomes from those with good outcomes, unlike existing laboratory tests. This biophysical indicator could therefore be used to guide the timing of clinical interventions, to monitor the progression of the disease, and to measure the efficacy of drugs, transfusion, and novel small molecules in an ex vivo setting.

Figure 1. Measuring rate of conductance decrease after deoxygenation

Time course of measurement showing oxygen concentration (a) in the gas reservoir and corresponding median velocity (b, left y-scale and circles) of RBCs by video tracking as well as applied pressure bias (right y-scale and dot-dash line). Corresponding channel conductance (c) is measured by dividing instantaneous velocity by the pressure. We see that the rate of change of the conductance as the blood flow stops is effectively constant and independent of both the velocity and pressure. All conductance values are scaled by the mean HbA blood conductance (C*_HbA ~ 1.7 µm/s/mmHg (0.013 µm/s/Pa)), and time is scaled by the time scale for hemoglobin deoxygenation (τ_deox ~ 10s).

Figure 2. Rate of conductance decrease correlates with patient clinical course

(a) Box plot comparing rates of conductance decrease for benign and severe samples. Receiver operating characteristics (b) for rate of conductance decrease (solid line) and a theoretical random prediction (dashed line). The areas under the ROC curves are 0.85 and 0.5 respectively. (c) Box plot comparing HbS fraction for benign and severe samples. HbF fractions (d) and rates of conductance change (e) for benign and severe samples further subdivided by hydroxyurea use. Rates of conductance decrease (f) for patient blood samples before and after addition of ABOand RhD-compatible HbA blood, simulating a blood transfusion. For each sample, data is normalized to unmodified sample. *p < 0.01 as determined by Mann-Whitney non-parametric analysis [56]. Bar heights in (f) represent means, and error bars represent standard deviations of at least 5 deoxygenation cycles. In box plots (a,b-e) red line is the median, blue box shows interquartile range (IQR), and dashed lines show extent of data within 1.5 times IQR. Rates of conductance decrease are scaled by mean HbA blood conductance (C*_HbA ~ 1.7 µm/s/mmHg (0.013 µm/s/Pa)) divided by the time scale for hemoglobin deoxygenation (τ_deox ~ 10s).

Figure 3. Rate of conductance decrease is modulated by a small molecule

Oxygen data as measured in the gas reservoir (top graph) and conductance data (bottom graph) are shown for an untreated severe sample (a) and the same sample treated (b) with 10mM 5-hydroxymethyl furfural (5HMF). Oxygen data (top graph) are shown as measured in the gas reservoir (dashed line) and in the blood channel (open circles, as measured by RBC intensity). Rates of conductance decrease (open circles) are quantified in (c). *p<0.05 as determined by Mann-Whitney non-parametric analysis [56]. Bar heights in (c) represent means, and error bars represent standard deviations of at least 5 independent deoxygenation cycles. All conductance values are scaled by the mean HbA blood conductance (C*_HbA ~ 1.7 µm/s/mmHg (0.013 µm/s/Pa)), and time is scaled by the time scale for hemoglobin deoxygenation (τ_deox ~ 10s). Rates of conductance decrease are scaled by mean HbA blood conductance divided by the time scale for hemoglobin deoxygenation (C*_HbA /τ_deox).

Scheme 1. Microfluidic device for studying sickle cell blood flow conductance

The device comprises 3 layers (inset): artificial capillary for blood flow, hydration layer with PBS, and gas reservoir. Blood is flowed under constant pressure bias, controlled by a digital pressure regulator. Two solenoid valves control the gas in the top chamber. A fiber optic probe is used to measure the oxygen concentration in the gas reservoir. The device is illuminated through an optical filter whose transmission band (434+/−17 nm) is centered on an absorption peak for deoxy-hemoglobin (Fig. S1). The absorption peak of oxy-hemoglobin shifts making deoxygenated RBCs appear dark and oxygenated RBCs transparent. Qualitative measurements of hemoglobin oxygen saturation are made using the intensity of transmitted light.