Browsing by Author "Shevkoplyas, Sergey S."
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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 Paper-Based Test for Screening Newborns for Sickle Cell Disease(Scientific Reports, 4/3/2017) Piety, Nathaniel Z.; George, Alex; Serrano, Sonia; Lanzi, Maria R.; Patel, Palka R.; Noli, Maria P.; Kahan, Silvina; Nirenberg, Damian; Camanda, João F.; Airewele, Gladstone; Shevkoplyas, Sergey S.The high cost, complexity and reliance on electricity, specialized equipment and supplies associated with conventional diagnostic methods limit the scope and sustainability of newborn screening for sickle cell disease (SCD) in sub-Saharan Africa and other resource-limited areas worldwide. Here we describe the development of a simple, low-cost, rapid, equipment- and electricity-free paper-based test capable of detecting sickle hemoglobin (HbS) in newborn blood samples with a limit of detection of 2% HbS. We validated this newborn paper-based test in a cohort of 159 newborns at an obstetric hospital in Cabinda, Angola. Newborn screening results using the paper-based test were compared to conventional isoelectric focusing (IEF). The test detected the presence of HbS with 81.8% sensitivity and 83.3% specificity, and identified SCD newborns with 100.0% sensitivity and 70.7% specificity. The use of the paper-based test in a two-stage newborn screening process could have excluded about 70% of all newborns from expensive confirmatory testing by IEF, without missing any of the SCD newborns in the studied cohort. This study demonstrates the potential utility of the newborn paper-based test for reducing the overall cost of screening newborns for SCD and thus increasing the practicality of universal newborn SCD screening programs in resource-limited settings.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 A rapid paper-based test for quantifying sickle hemoglobin in blood samples from patients with sickle cell disease(American Journal of Hematology, 6/1/2016) Piety, Nathaniel Z.; Yang, Xiaoxi; Lezzar, Dalia; George, Alex; Shevkoplyas, Sergey S.Quantification of sickle hemoglobin (HbS) in patients with sickle cell disease (SCD) undergoing hydroxyurea or chronic transfusion therapy is essential to monitoring the effectiveness of these therapies. The clinical monitoring of %HbS using conventional laboratory methods is limited by high per-test costs and long turnaround times usually associated with these methods. Here we demonstrate a simple, rapid, inexpensive paper-based assay capable of quantifying %HbS in blood samples from patients with SCD. A 20 ?L droplet of whole blood and hemoglobin solubility buffer was deposited on chromatography paper. The relative color intensities of regions of the resulting blood stain, determined by automated image analysis, are used to estimate %HbS. We compared the paper-based assay with hemoglobin electrophoresis (comparison method) using blood samples from 88 subjects. The test shows high correlation (R2 = 0.86) and strong agreement (standard deviation of difference = 7 %HbS) with conventional Hb electrophoresis measurement of %HbS, and closely approximates clinically predicted change in %HbS with transfusion therapy (mean difference 2.6 %HbS, n = 4). The paper-based assay can be completed in less than 35 minutes and has a per-test cost less than $0.25. The assay is accurate across a wide range of HbS levels (10–97%) and hemoglobin concentrations (5.6–12.9 g/dL) and is unaffected by high levels of HbF (up to 80.6%). This study demonstrates the feasibility of the paper-based %HbS assay. The paper-based test could improve clinical care for SCD, particularly in resource-limited settings, by enabling more rapid and less expensive %HbS monitoring.Item Anaerobic storage of red blood cells(Blood Transfusion, 2010-10) Yoshida, Tatsuro; Shevkoplyas, Sergey S.Item Anaerobic storage of red blood cells: the need for caution regarding donor red cells with sickle cell trait(Blood Transfusion, 2011-07) Yoshida, Tatsuro; Shevkoplyas, Sergey S.Item Bedside Washing of Stored Red Blood Cells: A Simple Apparatus Based on Microscale Sedimentation in Normal Gravity(2015-05) Huynh, Rose Ann A.; Shevkoplyas, Sergey S.; Mohan, Chandra; George, Alex; Wu, TianfuThe aim of this study was to develop and evaluate a simple, inexpensive bedside RBC washer with the capability to process multiple pRBC units within a short period without any alteration to current blood collection and storage practices. Washing pRBC units prior to transfusion is recommended for hypersensitive patients who have a higher susceptibility towards Allergic Transfusion Reactions (ATR) due to residual plasma proteins. Unfortunately, conventional washing is bulky, expensive and laborious, due to concentrating washed units through centrifugation. In response, we developed a bedside RBC washer that utilizes gravitational sedimentation of RBCs within a saline suspension. We evaluated its ability to concentrate washed pRBC units to a therapeutic hematocrit of 65%, but it optimally concentrated to a physiological hematocrit of 41%. Concentration was found to be dependent on initial morphology of the unit. In terms of improved quality, the gravity-driven washing system performed comparably with conventional washing.Item Biomarker Panel Array Systems for Disease Detection(2023-05-11) Tang, Chenling; Wu, Tianfu; Mohan, Chandra; Ning, Jing; Shevkoplyas, Sergey S.; Li, ZhengweiLupus nephritis (LN) is a devastating chronic kidney disease (CKD) caused by Systemic lupus erythematosus (SLE), an autoimmune disease that involves a loss of immune tolerance to endogenous materials and causes inflammatory responses and multiple organ damage. Currently, invasive renal biopsy as the gold standard diagnosis for LN may cause kidney injury, especially for multiple biopsy tests. Moreover, the histopathological morphology classification system doesn’t stratify the heterogenous nature of lupus patients. Fortunately, serum and urine biomarkers could potentially serve as a surrogate of disease activity, and a biomarker panel composed of multiple biomarkers could largely improve the diagnostic or prognostic sensitivity and specificity. By applying a LN biomarker panel on a multiplexable protein microarray platform, we aim to build a robust, highly sensitive, multiplexed, and high-throughput point-of-care system for lupus nephritis. This research covers four sections: (1) Discovery of novel circulating immune complexes (ICx) in the serum of lupus nephritis as potential biomarkers. Immunoproteomics-based discovery studies combined with Bioinformatics-assisted selection have enabled us to identify circulating immune complexes as potential biomarkers of LN. (2) Discovery of novel serum biomarkers that have diagnostic or predictive value in lupus nephritis. A novel serum biomarker VSIG4 has been discovered using proteomics and further validated as a promising novel serum biomarker of lupus nephritis and renal pathology activity. (3) Development of a Point-of-Care serum Biomarker-Panel Mini-Array (BPMA) system for the diagnosis, disease monitoring, and flare prediction of lupus nephritis; BPMA-S6 has been developed as a novel promising POC device for LN diagnosis, disease monitoring and flare prediction of LN (4) Discovery of novel urine biomarkers and biomarker-panel that have diagnostic or predictive values or disease monitoring capability for lupus nephritis. Collectively, this study has led to the discovery of novel biomarkers, the identification of a novel 6-plex biomarker panel, and the development of a novel biomarker panel detection system for autoimmune kidney disease. These findings hold great promise for the next generation of home care and community medicine.Item Biomimetic autoseparation of leukocytes from whole blood in a microfluidic device(Analytical Chemistry, 1/18/2011) Shevkoplyas, Sergey S.; Yoshida, Tatsuro; Munn, Lance L.; Bitensky, Mark W.Leukocytes comprise less than 1% of all blood cells. Enrichment of their number, starting from a sample of whole blood, is the required first step of many clinical and basic research assays. We created a microfluidic device that takes advantage of the intrinsic features of blood flow in the microcirculation, such as plasma skimming and leukocyte margination, to separate leukocytes directly from whole blood. It consists of a simple network of rectangular microchannels designed to enhance lateral migration of leukocytes and their subsequent extraction from the erythrocyte-depleted region near the sidewalls. A single pass through the device produces a 34-fold enrichment of the leukocyte-to-erythrocyte ratio. It operates on microliter samples of whole blood, provides positive, continuous flow selection of leukocytes, and requires neither preliminary labeling of cells nor input of energy (except for a small pressure gradient to support the flow of blood). This effortless, efficient, and inexpensive technology can be used as a lab-on-a-chip component for initial whole blood sample preparation. Its integration into microanalytical devices that require leukocyte enrichment will enable accelerated transition of these devices into the field for point-of-care clinical testing.Item Biomolecular Detection Assays(2022-05-10) Hlavinka, Victoria; Willson, Richard C.; Orman, Mehmet A.; Kourentzi, Katerina; Shevkoplyas, Sergey S.; Mohan, ChandraIn the present work, three methods for the detection or monitoring of biomolecules are outlined in detail. The first study describes the development of a saliva-based enzymatic screening assay for diabetes mellitus. In the second study, lateral flow assays (LFAs) utilizing commercial colloidal gold and blue latex nanoparticle reporters are compared to persistent luminescence nanoparticle (PLNP; “nanophosphor”) LFAs to assess the sensitivity of nanophosphors as a reporter system. The final study demonstrates the utility of a nanophosphor-based LFA for detecting low concentrations of dengue virus (DENV) biomarker non-structural protein 1 (NS1). 1,5-Anhydroglucitol (AHG) is a naturally occurring monosaccharide and a clinically validated blood biomarker for diabetes. The blood concentration of AHG falls during periods of hyperglycemia, as glucose outcompetes AHG for kidney reuptake. Salivary AHG quantification has been suggested to be useful for diabetes screening but has not been implemented in any widely applicable fashion. We have developed a chemiluminescence assay to quantify AHG in saliva and demonstrated that the assay could distinguish between healthy and diabetic individuals (N = 265; p < 0.0001, ROC AUC = 0.82). These findings suggest that, with further validation, this approach may serve as the basis of a non-invasive tool for diabetes screening. Commercially-available LFAs commonly use colloidal gold or blue latex nanoparticles reporter systems that lack sensitivity and are prone to human error when interpreted visually. We have developed nanophosphors that can detect low levels of antigen. In a comparison study, a nanophosphor-based human immunoglobulin G (IgG) LFA had a limit of detection of 0.625 ng/mL, an 81-fold and 58-fold increase in sensitivity over colloidal gold and blue latex nanoparticles, respectively. Current DENV diagnostic methods are commonly unspecific and cannot detect early infection. We have developed an inexpensive, rapid LFA to detect DENV NS1, a known marker of early dengue infection. Using strontium aluminate nanophosphors as reporters, we achieved a limit of detection of 1 ng/mL DENV serotype 1 NS1 antigen. Our assay is comparable to a laboratory-based NS1 ELISA with a 1 ng/mL limit of detection.Item C4d Deposits on the Surface of Red Blood Cells in Trauma Patients and Interferes with their Function(Critical Care Medicine, 5/1/2015) Muroya, Takashi; Kannan, Lakshmi; Ghiran, Ionita C.; Shevkoplyas, Sergey S.; Paz, Ziv; Tsokos, Maria; Dalle Lucca, Jurandir J.; Shapiro, Nathan I.; Tsokos, George C.Objective Complement system is activated in patients with trauma. Although complement activation is presumed to contribute to organ damage and constitutional symptoms, little is known about the involved mechanisms. Because complement components may deposit on red blood cells (RBC), we asked whether complement deposits on the surface of RBC in trauma and whether such deposition alters RBC function. Design A prospective experimental study Setting Research laboratory Subjects Blood samples collected from 42 trauma patients and 21 healthy donors Intervention None Measurements and Main Results RBC and sera were collected from trauma patients and control donors. RBC from trauma patients (n=40) were found to display significantly higher amounts of C4d on their surface by flow cytometry compared to normal RBC (n=17) (P<0.01). Increased amounts of iC3b were found in trauma sera (n=27) (vs. 12 controls, P<0.01) by ELISA. Incubation of RBC from universal donors (O,Rh-) with trauma sera (n=10) promoted C4d deposition on their surface (vs. 6 controls, P<0.05). Complement-decorated RBC (n=6) displayed limited their deformability (vs. 6 controls, P<0.05) in 2-dimensional microchannel arrays. Incubation of RBC with trauma sera (n=10) promoted the phosphorylation of band 3, a cytoskeletal protein important for the function of the RBC membrane (vs. 8 controls, P<0.05), and also accelerated calcium influx (n=9) and enhanced nitric oxide production (n=12) (vs. 4 and 8 controls respectively, P<0.05) in flow cytometry. Conclusions Our study found the presence of extensive complement activation in trauma patients and presents new evidence in support of the hypothesis that complement activation products deposit on the surface of RBC. Such deposition could limit RBC deformability and promote the production of nitric oxide. Our findings suggest that RBC in trauma patients malfunction, which may explain organ damage and constitutional symptoms that is not accounted for otherwise by previously known pathophysiologic mechanisms.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 Deep Learning Enables High-Precision Classification of Morphology of Stored Red Blood Cells(2018-10-18) Villarreal, NataliaAbout 15 million unit of stored red blood cells are transfused to nearly 5 million patients each year in the United States alone. However, the key properties of red blood cells progressively deteriorate during hypothermic storage, and this reduction in quality contributes to adverse outcomes that have been associated with blood transfusions, including serious infections and multiple organ failures. Recently, red blood cell shape (morphology) has emerged as a novel quantitative marker of the functional quality of stored blood units. Red blood cell morphology is currently measured via manual observation and classification of morphology of only ~100-200 individual cells by a technician, which is a notoriously tedious and highly subjective process. Building on prior work from Dr. Sergey Shevkoplyas’s Blood Microfluidics Lab, we trained an AlexNet Deep Learning CNN with the objective of creating a simple, robust, and automated system for high throughput microscopy that classifies heterogeneous cell morphology. The foundation of this project was successful at predicting morphological classifications with an overall low-resolution accuracy of 97.9% and an overall high-resolution accuracy of 95.3%.Item Deterioration of red blood cell mechanical properties is reduced in anaerobic storage(Blood Transfusion, 1/14/2016) Burns, Jennie M.; Yoshida, Tatsuro; Dumont, Larry J.; Yang, Xiaoxi; Piety, Nathaniel Z.; Shevkoplyas, Sergey S.Background Hypothermic storage of red blood cells (RBCs) results in progressive deterioration of the rheological properties of the cells, which may reduce the efficacy of RBC transfusions. Recent studies have suggested that storing RBC units under anaerobic conditions may reduce this storage-induced deterioration. Materials and methods The aim of this study was to compare the rheological properties of conventionally and anaerobically stored RBC and provide a measure of the relationship between oxidative damage to stored RBC and their ability to perfuse microvascular networks. Three different microfluidic devices were used to measure the ability of both types of stored RBC to perfuse artificial microvascular networks. Flow rates of the RBC passing through the entire network (bulk perfusion) and the individual capillaries (capillary perfusion) of the devices were measured on days 2, 21, 42, and 63 of storage. Results The bulk perfusion rates for anaerobically stored RBC were significantly higher than for conventionally stored RBCs over the entire duration of storage for all devices (up to 10% on day 42; up to 14% on day 63). Capillary perfusion rates suggested that anaerobically stored RBC units contained significantly fewer non-deformable RBC capable of transiently plugging microfluidic device capillaries. The number of plugging events caused by these non-deformable RBC increased over the 63 days of hypothermic storage by nearly 16- to 21-fold for conventionally stored units, and by only about 3- to 6-fold for anaerobically stored units. Discussion The perfusion measurements suggest that anaerobically stored RBC retain a greater ability to perfuse networks of artificial capillaries compared to conventionally (aerobically) stored RBC. It is likely that anaerobic storage confers this positive effect on the bulk mechanical properties of stored RBC by significantly reducing the number of non-deformable cells present in the overall population of relatively well-preserved RBC.Item Effect of osmolality on erythrocyte rheology and perfusion of an artificial microvascular network(Microvascular Research, 3/1/2016) Reinhart, Walter H.; Piety, Nathaniel Z.; Goede, Jeroen S.; Shevkoplyas, Sergey S.Plasma sodium concentration is normally held within a narrow range. It may, however, vary greatly under pathophysiological conditions. Changes in osmolality lead to either swelling or shrinkage of red blood cells (RBCs). Here we investigated the influence of suspension osmolality on biophysical properties of RBCs and their ability to perfuse an artificial microvascular network (AMVN). Blood was drawn from healthy volunteers. RBC deformability was measured by osmotic gradient ektacytometry over a continuous range of osmolalities. Packed RBCs were suspended in NaCl solutions (0.45, 0.6, 0.9, 1.2, and 1.5 g/dL), resulting in supernatant osmolalities of 179±4, 213±1, 283±2, 354±3, and 423±5 mOsm/kg H2O. MCV (mean corpuscular volume) and MCHC (mean corpuscular hemoglobin concentration), were determined using centrifuged microhematocrit. RBC suspensions at constant cell numbers were used to measure viscosity at shear rates ranging from 0.11 to 69.5 s?1 and the perfusion rate of the AMVN. MCV was inversely and MCHC directly proportional to osmolality. RBC deformability was maximized at isosmotic conditions (290 mOsm/kg H2O) and markedly decreased by either hypo- or hyperosmolality. The optimum osmolality for RBC suspension viscosity was shifted towards hyperosmolality, while lower osmolalities increased suspension viscosity exponentially. However, the AMVN perfusion rate was maximized at 290 mOsm/kg H2O, and changed by less than 10% over a wide range of osmolalities. These findings contribute to the basic understanding of blood flow in health and disease, and may have significant implications for the management of osmotic homeostasis in clinical practice.Item Histamine reduces GPIb?-mediated adhesion of platelets to TNF-?-activatedvascular endothelium(Thrombosis Research, 2013) Brown, Theodore P.; Forouzan, Omid; Shevkoplyas, Sergey S.; Khismatullin, Damir B.Histamine and tumor necrosis factor-? (TNF-?) are critical mediators of acute and chronic inflammation that are generated by mast cells and macrophages in atherosclerotic lesions or systemically during allergic attacks. Both of them induce activation of vascular endothelium and thus may play a role in thrombosis. Here we studied the interplay between histamine and TNF-? in glycoprotein (GP) Ib?-mediated platelet adhesion to cultured human vascular endothelial cells under static and shear flow conditions. The stimulation of endothelial cells with histamine or TNF-? increased the number of adherent or slow rolling GP Ib?-coated microbeads or washed human platelets. However, the application of histamine to endothelium pre-activated by TNF-? inhibited GP Ib?-mediated platelet adhesion. These effects were found to be associated with changes in the concentration of ultra large von Willebrand factor (ULVWF) strings anchored to endothelium. The results of this study indicate that histamine released during mast cell degranulation may cause or inhibit thrombosis, depending on whether it acts on resting endothelial cells or on cells pre-activated by other inflammatory stimuli.Item How Pure is Pure Enough: Effects of Red Blood Cell and Platelet Contamination on T-cell Culture(2020-12) Tedesco, Victor E.; Shevkoplyas, Sergey S.; Mohan, Chandra; Wu, TianfuRed blood cells (RBCs) and platelets (PLTs) have historically been considered immunologically inert. Recently, enough evidence has accumulated to suggest an important immunoregulatory role for these cells, both in vivo and in vitro. The latter is particularly important in the context of manufacturing novel cellular immunotherapies, including chimeric antigen receptor (CAR) T-cell therapies. T-cells are a sub-set of lymphocytes, a type of white blood cells that play a major role in the adaptive immune response. By reprogramming these cells to seek out and destroy target malignant cells, CAR T-cell therapies for cancer achieve remission rates as high as 90%. To manufacture such a highly effective treatment, T-cells must first be separated from whole blood and ultimately expanded in culture. RBCs serve as dynamic reservoirs of immunological signaling proteins, able to absorb and release at least 46 different cytokines. The presence of RBCs during culture has a significant impact on T-cell proliferation and survival. Contamination of T-cell culture with residual PLTs results in significant suppression of CD4+ cells and upregulation of regulatory T-cells, a type of T-cell that suppresses the function of other T-cells to prevent autoimmune responses. Additionally, residual PLTs modulate a variety of cytokines including IL-2, IL-5, IL-10, IL-17, IFN-γ and TNF-α. There is a wide range of cell separation methods currently employed in both research and clinical settings. And the number of residual RBCs and PLTs present during T-cell culture is almost entirely determined by the method used to isolate T-cells from whole blood. Given their significant immunoregulatory effects, the level of RBC and PLT contamination necessitates careful consideration when choosing a T-cell isolation method, in addition to the typical performance metrics such as T-cell recovery and purity, separation throughput, and operating costs. This thesis examines the known effects of RBCs and PLTs on expansion of T-cells in culture to help evaluate the allowable number of these contaminating cells, and facilitate a well-informed decision when selecting a separation method for manufacturing CAR T-cell therapies.Item Identification and Quantitation of Novel Urinary Biomarkers of Lupus Nephritis(2018-08) Stanley, Samantha; Mohan, Chandra; Buhrman, Jonathan; Majd, Sheereen; Shevkoplyas, Sergey S.; Wu, TianfuSystemic lupus erythematosus (SLE) is a chronic autoimmune disorder in which the body produces anti-ds DNA and anti-nuclear antibodies and leads to multi-system inflammation and tissue damage. One of the leading causes of mortality and morbidity of SLE is lupus nephritis (LN), a condition where SLE begins to manifest within the renal tissue; it has been estimated that more than half of all SLE patients will develop LN and 10-15% of those patients will experience end-stage renal failure. Early-stage disease diagnosis could allow for earlier treatment and preventative measures to be taken but is not presently possible because the currently used laboratory tests only detect disease activity at a severe stage, and the current gold standard of care to analyze renal involvement in SLE is a renal biopsy. While renal biopsies are highly informative, they cannot be performed routinely, and come with several risks to the patient, including the highly invasive nature of the procedure, the risk of infection, and the possibility of sampling error that the biopsy taken is not representative of the entire kidney. The need for earlier diagnostic tools for LN has sparked a large research interest in urine proteins to identify a noninvasive biomarker specific to LN, but insofar no ideal biomarker has been identified. Thus, the overall goal of this thesis is to identify new urinary biomarkers for lupus nephritis and assess various detection technologies and their effectiveness at measuring urinary proteins.Item Incorporation of prefabricated screw, pneumatic, and solenoid valves into microfluidic devices(Lab on a Chip, 3/28/2011) Hulme, Elizabeth S.; Shevkoplyas, Sergey S.; Whitesides, George M.This paper describes a method for prefabricating screw, pneumatic, and solenoid valves and embedding them in microfluidic devices. This method of prefabrication and embedding is simple, requires no advanced fabrication, and is compatible with soft lithography. Because prefabrication allows many identical valves to be made at one time, the performance across different valves made in the same manner is reproducible. In addition, the performance of a single valve is reproducible over many cycles of opening and closing: an embedded solenoid valve opened and closed a microfluidic channel more than 100,000 times with no apparent deterioration in its function. It was possible to combine all three types of prefabricated valves in a single microfluidic device to control chemical gradients in a microfluidic channel temporally and spatially.Item Influence of feeding hematocrit and perfusion pressure on hematocrit reduction (Fåhræus effect) in an artificial microvascular network(Microcirculation, 11/1/2018) Reinhart, Walter H.; Piety, Nathaniel Z.; Shevkoplyas, Sergey S.Objective Hct in narrow vessels is reduced due to concentration of fast?flowing RBCs in the center, and of slower flowing plasma along the wall of the vessel, which in combination with plasma skimming at bifurcations leads to the striking heterogeneity of local Hct in branching capillary networks known as the network Fåhræus effect. We analyzed the influence of feeding Hct and perfusion pressure on the Fåhræus effect in an AMVN. Methods RBC suspensions in plasma with Hcts between 20% and 70% were perfused at pressures of 5?60 cm H2O through the AMVN. A microscope and high?speed camera were used to measure RBC velocity and Hct in microchannels of height of 5 ?m and widths of 5?19 ?m. Results Channel Hcts were reduced compared with Hctfeeding in 5 and 7 ?m microchannels, but not in larger microchannels. The magnitude of Hct reduction increased with decreasing Hctfeeding and decreasing ?P (flow velocity), showing an about sevenfold higher effect for 40% Hctfeeding and low pressure/flow velocity than for 60% Hctfeeding and high pressure/flow velocity. Conclusions The magnitude of the network Fåhræus effect in an AMVN is inversely related to Hctfeeding and ?P.
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