Investigator: Gregory Kato Univ. of Pitts Medical Center
Dr. Gregory Kato, Director of Adult Sickle Cell Center of Excellence at Pittsburgh, is a hematologist with an active translational research interest in Sickle cell disease (SCD). He leads a vibrant clinical and laboratory bench research team that focuses on the causes of various forms of vascular dysfunction associated with SCD. Dr. Kato is also interested in understanding the mechanisms of existing therapies as well as developing new treatments to treat accute syndromes of SCD or prevent these crisis altogether.
Sickle cell disease (SCD) is a genetic blood disorder resulted from a single point mutation in the β-globin gene. The mutation causes sickle hemoglobin (HbS) to bind and form long chains when deoxygenated. The HbS polymerization is believed to decrease RBC deformability and trigger RBC sickling (Fig. 1), which leads to vaso-occlusion in capillaries and small venules [1]. Each year, over 180,000 children are born with SCD worldwide. In the US alone, 90,000 to 100,000 people - mostly African Americans - have SCD, and about 1 in 12 African Americans carry the sickle cell trait. While the molecular details of the sickle hemoglobin (HbS) polymerization as well as the clinical heterogeneity of SCD are now reasonably well understood, the proper understanding of RBC deformability and shape changes when deoxygenated especially on individual cell basis remains elusive in SCD investigations primarily due to the lack of appropriate measurement techniques. The best attempts to understand sickle cell biomechanics have been made using micropipette aspiration at fully stabilized oxygenation / deoxygenation conditions. Other methods to quantify mechanical properties of sickle RBCs including atomic force microscopy (AFM), optical and magnetic tweezers, and electric field deformation are limited to study fully stabilized RBC mechanical properties. Overall, these low throughput methods inevitably perturb the samples and provide poor statistics at the population level.
Interferometric microscopy has also been used to study sickle cell biomechanics at single cell level. In collaboration with Dr. Kato, the LBRC is interested in understanding the mechanisms of existing therapies for SCD as well as developing new treatments to treat accute syndromes of SCD or to prevent these crisis altogether. In a collaborative study, LBRC researchers have recently investigated blood samples from patients that were on and off Hydroxyurea (HU) treatment using transmission type interferometric microscopy systems [2]. To improve the accuracy of measured cellular biophysical properties, blood samples of sickle patients were sorted into four density categories (I-IV). The researchers also managed to show the beneficial effects of the HU treatment on a range of biophysical properties even under normoxic condition. Our study further concluded that the measured biophysical parameters such as the shear modulus of sickle RBCs correlates with clinically measured mean cell volume (MCV) (see Fig. 2) rather than fetal hemoglobin levels, which provides insight on long-debated mechanism of HU treatment [2]. We note, however, that interferometric microscopy based studies have so far been conducted only under fully oxygenated conditions. Recently, Dr. Dao's Lab has studied population level adhesion and polymerization characteristics of deoxygenated sickle hemoglobin (HbS) in human RBCs using microfluidic platforms [3]. The researchers found that hypoxia significantly enhances the sickle RBC cytoadherence compared with normoxia. Development of new non-contact interferometric optical assays will enable sickle cell biomechanical studies at single cell level under hypoxic conditions as well.
The interferometric microscopy and spectroscopy techniques developed at the LBRC will provide quantitative tools for SCD investigation. For instance, High throughput tomographic microscopy will be used to study fast shape changes in the individual RBCs during hypoxia via three-dimensional refractive index (RI) mapping. Interferometric 3D-resolved biomechanical assays will be used to quantify biomechanical properties at normoxia and hypoxic conditions when the cytosol refractive index may be heterogeneous. Both of these technologies upon further developments can be used in more complex microfluidic environment such as ones with endothelial vasculature. Common-path interferometers with molecular-specific capability will greatly increase the precision of the biomechnical measurements at normaxic and hypoxic conditions. This technology will also provide additional valuable information on the concentration of the various proteins such as oxy / deoxy hemoglobin in the cytosol together with the morphological measurements of individual cells during the sickling process.
Development of new non-contact optical assays is needed to study the biomechanics of sickle RBCs during hypoxia. Depth-resolved interferometric biomechanical assays will be developed to support this collaboration with Dr. Kato (U. of Pitts) that focuses on sickle cell biomechanics as well as understanding mechanisms / efficacy of existing FDA-approved and new drugs in the pipeline. In collaboration with Dr. Dao (MIT), finite element-based biomechanical models will also be developed to extract biomechanical parameters from membrane fluctuations of sickle red cells in hypoxic states.