Browsing by Author "Gifford, Sean C."
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Item A high-throughput microfluidic approach for 1000-fold leukocyte reduction of platelet-rich plasma(Scientific Reports, 10/24/2016) Xia, Hui; Strachan, Briony C.; Gifford, Sean C.; Shevkoplyas, Sergey S.Leukocyte reduction of donated blood products substantially reduces the risk of a number of transfusion-related complications. Current ‘leukoreduction’ filters operate by trapping leukocytes within specialized filtration material, while allowing desired blood components to pass through. However, the continuous release of inflammatory cytokines from the retained leukocytes, as well as the potential for platelet activation and clogging, are significant drawbacks of conventional ‘dead end’ filtration. To address these limitations, here we demonstrate our newly-developed ‘controlled incremental filtration’ (CIF) approach to perform high-throughput microfluidic removal of leukocytes from platelet-rich plasma (PRP) in a continuous flow regime. Leukocytes are separated from platelets within the PRP by progressively syphoning clarified PRP away from the concentrated leukocyte flowstream. Filtrate PRP collected from an optimally-designed CIF device typically showed a ~1000-fold (i.e. 99.9%) reduction in leukocyte concentration, while recovering >80% of the original platelets, at volumetric throughputs of ~1?mL/min. These results suggest that the CIF approach will enable users in many fields to now apply the advantages of microfluidic devices to particle separation, even for applications requiring macroscale flowrates.Item A portable system for processing donated whole blood into high quality components without centrifugation(PLoS One, 1/18/2018) Gifford, Sean C.; Strachan, Briony C.; Xia, Hui; Vörös, Eszter; Torabian, Kian; Tomasino, Taylor A.; Griffin, Gary D.; Lichtiger, Benjamin; Aung, Fleur M.; Shevkoplyas, Sergey S.Background The use of centrifugation-based approaches for processing donated blood into components is routine in the industrialized world, as disparate storage conditions require the rapid separation of ‘whole blood’ into distinct red blood cell (RBC), platelet, and plasma products. However, the logistical complications and potential cellular damage associated with centrifugation/apheresis manufacturing of blood products are well documented. The objective of this study was to evaluate a proof-of-concept system for whole blood processing, which does not employ electromechanical parts, is easily portable, and can be operated immediately after donation with minimal human labor. Methods and findings In a split-unit study (n = 6), full (~500mL) units of freshly-donated whole blood were divided, with one half processed by conventional centrifugation techniques and the other with the new blood separation system. Each of these processes took 2–3 hours to complete and were performed in parallel. Blood products generated by the two approaches were compared using an extensive panel of cellular and plasma quality metrics. Comparison of nearly all RBC parameters showed no significant differences between the two approaches, although the portable system generated RBC units with a slight but statistically significant improvement in 2,3-diphosphoglyceric acid concentration (p < 0.05). More notably, several markers of platelet damage were significantly and meaningfully higher in products generated with conventional centrifugation: the increase in platelet activation (assessed via P-selectin expression in platelets before and after blood processing) was nearly 4-fold higher for platelet units produced via centrifugation, and the release of pro-inflammatory mediators (soluble CD40-ligand, thromboxane B2) was significantly higher for centrifuged platelets as well (p < 0.01). Conclusion This study demonstrated that a simple, passive system for separating donated blood into components may be a viable alternative to centrifugation—particularly for applications in remote or resource-limited settings, or for patients requiring highly functional platelet product.Item Controlled incremental filtration: a simplified approach to design and fabrication of high-throughput microfluidic devices for selective enrichment of particles.(Lab on a Chip, 12/7/2015) Gifford, Sean C.; Spillane, Angela M.; Vignes, Seth M.; Shevkoplyas, Sergey S.The number of microfluidic strategies aimed at separating particles or cells of a specific size within a continuous flow system continues to grow. The wide array of biomedical and other applications that would benefit from successful development of such technology has motivated the extensive research in this area over the past 15 years. However, despite promising advancements in microfabrication capabilities, a versatile approach that is suitable for a large range of particle sizes and high levels of enrichment, with a volumetric throughput sufficient for large-scale applications, has yet to emerge. Here we describe a straightforward method that enables the rapid design of microfluidic devices that are capable of enriching/removing particles within a complex aqueous mixture, with an unprecedented range of potential cutoff diameter (below 1µm to above 100µm) and an easily scalable degree of enrichment/filtration (up to 10-fold and well beyond). A simplified model of a new approach to crossflow filtration – controlled incremental filtration – was developed and validated for its ability to generate microfluidic devices that efficiently separate particles on the order of 1–10µm, with throughputs of tens of µL/min, without the use of a pump. Precise control of the amount of fluid incrementally diverted at each filtration “gap” of the device allows for the gap size (~20µm) to be much larger than the particles of interest, while the simplicity of the model allows for many thousands of these filtration points to be readily incorporated into a desired device design. This new approach should enable truly high-throughput microfluidic particle-separation devices to be generated, even by users only minimally experienced in fluid mechanics and microfabrication techniques.Item Quantifying morphological heterogeneity: a study of more than 1 000 000 individual stored red blood cells(Vox Sanguinis, 10/1/2016) Piety, Nathaniel Z.; Gifford, Sean C.; Yang, Xiaoxi; Shevkoplyas, Sergey S.Background and Objectives The morphology of red blood cells (RBCs) deteriorates progressively during hypothermic storage. The degree of deterioration varies between individual cells, resulting in a highly heterogeneous population of cells contained within each RBC unit. Current techniques capable of categorizing the morphology of individual stored RBCs are manual, laborious, error-prone procedures that limit the number of cells that can be studied. Our objective was to create a simple, automated system for high-throughput RBC morphology classification. Materials and Methods A simple microfluidic device, designed to enable rapid, consistent acquisition of images of optimally oriented RBCs, was fabricated using soft lithography. A custom image analysis algorithm was developed to categorize the morphology of each individual RBC in the acquired images. The system was used to determine morphology of individual RBCs in several RBC units stored hypothermically for 6–8 weeks. Results The system was used to automatically determine the distribution of cell diameter within each morphological class for >1,000,000 individual stored RBCs (speed: >10,000 cells/hour; accuracy: 91.9% low-resolution, 75.3% high-resolution). Diameter mean and standard deviation by morphology class: discocyte 7.80±0.49?m, echinocyte 1 7.61±0.63?m, echinocyte 2 7.02±0.61?m, echinocyte 3 6.47±0.42?m, sphero-echinocyte 6.01±0.26?m, spherocyte 6.02±0.27?m, stomatocyte 1 6.95±0.61?m, stomatocyte 2 7.32 ± 0.47?m. Conclusion The automated morphology classification procedure described in this study is significantly simpler, faster and less subjective than conventional manual procedures. The ability to evaluate the morphology of individual RBCs automatically, rapidly and in statistically significant numbers enabled us to perform the most extensive study of stored RBC morphology to date.Item Washing in hypotonic saline reduces the fraction of irreversibly-damaged cells in stored blood: a proof-of-concept study(Blood Transfusion, 2017-09) Xia, Hui; Khanal, Grishma; Strachan, Briony C.; Vörös, Eszter; Piety, Nathaniel Z.; Gifford, Sean C.; Shevkoplyas, Sergey S.Background During hypothermic storage, a substantial fraction of red blood cells (RBCs) transforms from flexible discocytes to rigid sphero-echinocytes and spherocytes. Infusion of these irreversibly-damaged cells into the recipient during transfusion serves no therapeutic purpose and may contribute to adverse outcomes in some patients. In this proof-of-concept study we describe the use of hypotonic washing for selective removal of the irreversibly-damaged cells from stored blood. Materials and methods Stored RBCs were mixed with saline of various concentrations to identify optimal concentration for inducing osmotic swelling and selective bursting of spherical cells (sphero-echinocytes, spherocytes), while minimising indiscriminate lysis of other RBCs. Effectiveness of optimal treatment was assessed by measuring morphology, rheological properties, and surface phosphatidylserine (PS) exposure for cells from several RBCs units (n=5, CPD>AS-1, leucoreduced, 6 weeks storage duration) washed in hypotonic vs isotonic saline. Results Washing in mildly hypotonic saline (0.585 g/dL, osmolality: 221.7±2.3 mmol/kg) reduced the fraction of spherical cells 3-fold from 9.5±3.4% to 3.2±2.8%, while cutting PS exposure in half from 1.48±0.86% to 0.59±0.29%. Isotonic washing had no effect on PS exposure or the fraction of spherical cells. Both isotonic and hypotonic washing increased the fraction of well-preserved cells (discocytes, echinocytes 1) substantially, and improved the ability of stored RBCs to perfuse an artificial microvascular network by approximately 25%, as compared with the initial sample. Discussion This study demonstrated that washing in hypotonic saline could selectively remove a significant fraction of the spherical and PS-exposing cells from stored blood, while significantly improving the rheological properties of remaining well-preserved RBCs. Further studies are needed to access the potential effect from hypotonic washing on transfusion outcomes.