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Aug 17, 2012

Blood Flow as Diagnostic Marker

Jamming, or vaso-occlusion, could be used to distinguish sickle cell disease patients with poor outcomes from those with good outcomes.

Blood Flow as Diagnostic Marker

In an era focused on molecular markers, the authors revisit often-overlooked biophysical markers, which may provide a more global picture of pathophysiology in disorders like sickle-cell disease. [© Dario Bajurin - Fotolia.com]

  • ASSAY & Drug Development Technologies offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents one article that was analyzed in the "Literature Search and Review" column, a paper published in Science Translational Medicine titled "A biophysical indicator of vaso-occlusive risk in sickle cell disease." Authors of the paper are Wood DK, Soriano A, Mahadevan L, Higgins JM, and Bhatia SN.

  • Abstract from Science Translational Medicine

    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.

  • Commentary

    Click Image To Enlarge +
    Figure 1. Microfluidic device for studying sickle cell blood flow conductance. (A) The device comprised three layers (inset): artificial capillaries for blood flow, a hydration layer with PBS, and a gas reservoir. Blood was driven under constant pressure bias, controlled by a digital pressure regulator. Two solenoid valves controlled the gas (N2 and air) in the top chamber. A fiber optic probe was used to measure the oxygen concentration in the gas reservoir. The device was illuminated through an optical filter whose transmission band (434 ± 17 nm) was centered on an absorption peak for deoxyhemoglobin. (B) The absorption peak of hemoglobin shifts in deoxygenated conditions, making deoxygenated RBCs appear dark and oxygenated RBCs appear transparent. Qualitative measurements of hemoglobin oxygen saturation were made using the intensity of transmitted light. PBS, phosphate-buffered saline; RBC, red blood cell.

    The article by Wood and colleagues highlights the validation of a simple physical parameter associated with blood-flow properties as a diagnostic marker for increased risk in sickle cell disease. In an era when low-abundance protein analytes and combinations of single-nucleotide polymorphisms are increasingly being developed for diagnostic purposes, simpler (bio)physical parameters risk being overlooked. Examples of such parameters already driving clinical decisions include blood pressure, heart beat, and basic imaging. The present study of a sickle cell disease prognostic parameter marks a mini-revival in this area, with the authors presenting a microfluidic device that measures the change in blood flow characteristics upon deoxygenation.

    Typically, the status of sickle cell patients is evaluated through the measurement of the ratio of hemoglobin S to fetal hemoglobin, but this blood chemistry result does not provide for a strong enough prognostic marker to assess the risk of the patient developing vaso-occlusion crisis, a state associated with a dramatic change in blood flow characteristics due to the “jamming” of capillaries through the change in shape and rigidity of red blood cells. Instead of looking for molecular markers to assess the risk of vaso-occlusion, the authors opted to measure the blood flow properties directly.

  • Click Image To Enlarge +
    Figure 2. Rate of conductance decrease is modulated by a small molecule. (A, B) Oxygen data from gas reservoir (top) and conductance data (bottom) are shown for an untreated severe sample (A) and the same sample treated with 10 mM 5HMF (B). Oxygen data are shown as measured in the gas reservoir (dashed line) and in the blood channel (open circles), the latter measured by RBC intensity. (C) Rates of conductance decrease [open circles in (A, B)] are quantified. *P < 0.05, Mann–Whitney nonparametric analysis. Data are means ± SD of at least five independent deoxygenation cycles for both samples. All conductance values are scaled by the mean HbA blood conductance [~1.7 µm s-1 mm Hg-1 (0.013 µm s-1 Pa-1)], and time is scaled by the time scale for hemoglobin deoxygenation (~10 s). Rates of conductance decrease are scaled by mean HbA blood conductance divided by the time scale for hemoglobin deoxygenation (C*HbA/tdeox). 5HMF, 5-hydroxymethyl furfural; HbA, hemoglobin A.

    In the device, capillary channels carrying the blood sample were coupled with gas reservoirs (Figure 1). When whole blood was flowed through the device, the gas composition of the adjacent channel could be controlled to provide different levels of dissolved gasses in the bloodstream, imitating the transition of the blood cells from large blood vessels to the capillaries; in turn, the changes in hemoglobin polymerization state (driven by the changing level of dissolved oxygen) and the concomitant change in red blood cell rigidity could be measured through the changes in the resistance of the blood sample to pressure-driven flow.

    The device was initially validated by comparing blood flow resistance changes as a function of oxygen percentage for samples from healthy and sickle cell disease donors. The testing was then extended to compare samples from patients considered to have different levels of sickle cell disease severity: samples from patients who had been treated extensively (through transfusions and hydroxyurea administrations) during the past 12 months showed dramatically different flow resistance profiles when compared to those from patients considered to have a mild case of sickle cell disease (i.e., not having been treated intensively during the past 12 months). This patient stratification through flow characteristics measurement was then used in expanded survey of 23 severe-case and 6 mild-case patients which also showed that the new biophysical parameter correlated well with clinical outcome for the two patient strata. Finally, a new experimental treatment to manage sickle cell disease with the small molecule agent 5-hydroxymethyl furfural was evaluated through the use of the new microfluidic device (Figure 2).

    Patient samples treated with the agent showed a large and rapid improvement in their blood flow characteristics, indicating that the device is likely to be of utility in monitoring the treatment progress with new drugs.

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