Browsing by Author "Piety, Nathaniel Z."
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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 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 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 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.Item Influence of red blood cell aggregation on perfusion of an artificial microvascular network(Microcirculation, 7/1/2018) Reinhart, Walter H.; Piety, Nathaniel Z.; Shevkoplyas, Sergey S.Red blood cells (RBCs) suspended in plasma form multicellular aggregates under low flow conditions, increasing apparent blood viscosity at low shear rates. It has previously been unclear, however, if RBC aggregation affects microvascular perfusion. Here we analyzed the impact of RBC aggregation on perfusion and ‘capillary’ hematocrit in an artificial microvascular network (AMVN) at driving pressures ranging from 5 to 60 cmH2O to determine if aggregation could improve tissue oxygenation. RBCs were suspended at 30% hematocrit in either 46.5 g/L dextran 40 (D40, non-aggregating medium) or 35 g/L dextran 70 (D70, aggregating medium) solutions with equal viscosity. Aggregation was readily observed in the AMVN for RBCs suspended in D70 at driving pressures ? 40 cmH2O. The AMVN perfusion rate was the same for RBCs suspended in aggregating and non-aggregating medium, at both ‘venular’ and ‘capillary’ level. Estimated ‘capillary’ hematocrit was higher for D70 suspensions than for D40 suspensions at intermediate driving pressures (5 – 40 cm H2O). We conclude that although RBC aggregation did not affect the AMVN perfusion rate independently of the driving pressure, a higher hematocrit in the ‘capillaries’ of the network for D70 suspensions suggested a better oxygen transport capacity in the presence of RBC aggregation.Item Microfluidic and Paper-Based Devices for the Analysis of Red Blood Cells: Improving the Quality and Accessibility of Diagnosis, Monitoring and Treatment for Sickle Cell Disease(2017-05) Piety, Nathaniel Z.; Shevkoplyas, Sergey S.; George, Alex; Mohan, Chandra; Majd, Sheereen; Akay, MetinSickle cell disease (SCD) is a common inherited blood disorder which gives rise to life-long health problems. Technologies capable of sensitive and specific early diagnosis of SCD as well as accurate quantification of HbS exist, but are expensive, complex, slow, laborious, and also require stable electricity, specialized equipment, and well trained technicians. Additionally, tools for quantifying stored RBC quality and selecting well-preserved RBC units for transfusion to vulnerable patients (e.g., chronically transfused individuals with SCD) are currently lacking. These limitations have largely prevented implementation of universal newborn screening programs in low-income, developing regions (e.g., sub-Saharan Africa) and have made it difficult for clinicians to personalize care for individuals with SCD even in high-income, developed countries. In this dissertation, we engineered and validated several novel microfluidic and paper-based devices which address the limitations of existing technologies and thereby increase access to quality SCD care while enabling further understanding and optimization of transfusion medicine practices. First, we created a simple, rapid and equipment-free paper-based newborn SCD screening test capable of sensitive and specific detection of sickle hemoglobin (HbS) and SCD. Next, we made a rapid, low-cost paper-based assay for quantifying HbS in blood samples based on the color intensities of bloodstain patterns in paper, which showed high correlation and agreement with ‘gold-standard’ quantification methods. Then, we engineered a simple microfluidic device and associated image analysis algorithm capable of high throughput, automated analysis of stored RBC morphology – a potential metric for assessing the quality of stored blood and selecting well-preserved units for vulnerable patients. Finally, we used these novel devices, in combination with commercially available technologies and previously developed microfluidic devices, to quantify the impact of two novel RBC storage and rejuvenation techniques on stored RBC quality, as well as to quantify the effect of several physiologically relevant processes – i.e., RBC morphology, osmolality, aggregation and hematocrit – on overall blood rheology. If adopted, these novel tools for diagnosis, monitoring and treatment optimization could drastically increase the quality and accessibility of SCD care for millions of affected individuals worldwide, in both high-income, developed countries and resource-limited, developing regions.Item Optimal hematocrit in an artificial microvascular network(Transfusion, 9/1/2018) Piety, Nathaniel Z.; Reinhart, Walter H.; Stutz, Julianne; Shevkoplyas, Sergey S.BACKGROUND Higher hematocrit increases the oxygen?carrying capacity of blood but also increases blood viscosity, thus decreasing blood flow through the microvasculature and reducing the oxygen delivery to tissues. Therefore, an optimal value of hematocrit that maximizes tissue oxygenation must exist. STUDY DESIGN AND METHODS We used viscometry and an artificial microvascular network device to determine the optimal hematocrit in vitro. Suspensions of fresh red blood cells (RBCs) in plasma, normal saline, or a protein?containing buffer and suspensions of stored red blood cells (at Week 6 of standard hypothermic storage) in plasma with hematocrits ranging from 10 to 80% were evaluated. RESULTS For viscometry, optimal hematocrits were 10, 25.2, 31.9, 37.1, and 37.5% for fresh RBCs in plasma at shear rates of 3.2 or less, 11.0, 27.7, 69.5, and 128.5 inverse seconds. For the artificial microvascular network, optimal hematocrits were 51.1, 55.6, 59.2, 60.9, 62.3, and 64.6% for fresh RBCs in plasma and 46.4, 48.1, 54.8, 61.4, 65.7, and 66.5% for stored RBCs in plasma at pressures of 2.5, 5, 10, 20, 40, and 60 cm H2O. CONCLUSION Although exact optimal hematocrit values may depend on specific microvascular architecture, our results suggest that the optimal hematocrit for oxygen delivery in the microvasculature depends on perfusion pressure. Therefore, anemia in chronic disorders may represent a beneficial physiological response to reduced perfusion pressure resulting from decreased heart function and/or vascular stenosis. Our results may help explain why a therapeutically increasing hematocrit in such conditions with RBC transfusion frequently leads to worse clinical outcomes.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 Shape matters: the effect of red blood cell shape on perfusion of an artificial microvascular network(Transfusion, 4/1/2017) Piety, Nathaniel Z.; Reinhart, Walter H.; Pourreau, Patrick H.; Abidi, Rajaa; Shevkoplyas, Sergey S.BACKGROUND The shape of human red blood cells (RBCs) deteriorates progressively throughout hypothermic storage, with echinocytosis being the most prevalent pathway of this morphological lesion. As a result, each unit of stored blood contains a heterogeneous mixture of cells in various stages of echinocytosis and normal discocytes. Here we studied how the change in shape of RBCs following along the path of the echinocytic transformation affects perfusion of an artificial microvascular network (AMVN). STUDY DESIGN AND METHODS Blood samples were obtained from healthy consenting volunteers. RBCs were leukocyte-reduced, re-suspended in saline, and treated with various concentrations of sodium salicylate to induce shape changes approximating the stages of echinocytosis experienced by RBCs during hypothermic storage (e.g. discocyte, echinocyte I, echinocyte II, echinocyte III, sphero-echinocyte and spherocyte). The AMVN perfusion rate was measured for 40% hematocrit suspensions of RBCs with different shapes. RESULTS The AMVN perfusion rates for RBCs with discocyte and echinocyte I shapes were similar, but there was a statistically significant decline in the AMVN perfusion rate between RBCs with shapes approximating each subsequent stage of echinocytosis. The difference in AMVN perfusion between discocytes and spherocytes (the last stage of the echinocytic transformation) was 34%. CONCLUSION The change in shape of RBCs from normal discocytes progressively through various stages of echinocytosis to spherocytes produced a substantial decline in the ability of these cells to perfuse an artificial microvascular network. Echinocytosis induced by hypothermic storage could therefore be responsible for a similarly substantial impairment of deformability previously observed for stored RBCs.Item Substituting Sodium Hydrosulfite with Sodium Metabisulfite Improves Long-Term Stability of a Distributable Paper-Based Test Kit for Point-of-Care Screening for Sickle Cell Anemia(Biosensors, 9/20/2017) Torabian, Kian; Lezzar, Dalia; Piety, Nathaniel Z.; George, Alex; Shevkoplyas, Sergey S.Sickle cell anemia (SCA) is a genetic blood disorder that is particularly lethal in early childhood. Universal newborn screening programs and subsequent early treatment are known to drastically reduce under-five SCA mortality. However, in resource-limited settings, cost and infrastructure constraints limit the effectiveness of laboratory-based SCA screening programs. To address this limitation our laboratory previously developed a low-cost, equipment-free, point-of-care, paper-based SCA test. Here, we improved the stability and performance of the test by replacing sodium hydrosulfite (HS), a key reducing agent in the hemoglobin solubility buffer which is not stable in aqueous solutions, with sodium metabisulfite (MS). The MS formulation of the test was compared to the HS formulation in a laboratory setting by inexperienced users (n = 3), to determine visual limit of detection (LOD), readout time, diagnostic accuracy, intra- and inter-observer agreement, and shelf life. The MS test was found to have a 10% sickle hemoglobin LOD, 21-min readout time, 97.3% sensitivity and 99.5% specificity for SCA, almost perfect intra- and inter-observer agreement, at least 24 weeks of shelf stability at room temperature, and could be packaged into a self-contained, distributable test kits comprised of off-the-shelf disposable components and food-grade reagents with a total cost of only $0.21 (USD).Item Validation of a Low-Cost Paper-Based Screening Test for Sickle Cell Anemia(PLoS One, 1/6/2016) Piety, Nathaniel Z.; Yang, Xiaoxi; Kanter, Julie; Vignes, Seth M.; George, Alex; Shevkoplyas, Sergey S.The high childhood mortality and life-long complications associated with sickle cell anemia (SCA) in developing countries could be significantly reduced with effective prophylaxis and education if SCA is diagnosed early in life. However, conventional laboratory methods used for diagnosing SCA remain prohibitively expensive and impractical in this setting. This study describes the clinical validation of a low-cost paper-based test for SCA that can accurately identify sickle trait carriers (HbAS) and individuals with SCA (HbSS) among adults and children over 1 year of age.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.Item Washing stored red blood cells in an albumin solution improves their morphological and hemorheological properties(Transfusion, 8/1/2016) Reinhart, Walter H.; Piety, Nathaniel Z.; Deuel, JeremyW.; Makhro, Asya; Schulzki, Thomas; Bogdanov, Nikolay; Goede, Jeroen S.; Bogdanova, Anna; Abidi, Rajaa; Shevkoplyas, Sergey S.BACKGROUND Prolonged storage of red blood cells leads to storage lesions, which may impair clinical outcomes after transfusion. A hallmark of storage lesions is progressive echinocytic shape transformation, which can be partially reversed by washing in albumin solutions. Here we have investigated the impact of this shape recovery on biorheological parameters. METHODS Red blood cells stored hypothermically for 6–7 weeks were washed in a 1% human serum albumin solution. Red cell deformability was measured with osmotic gradient ektacytometry. The viscosity of red cell suspensions were measured with a Couette-type viscometer. The flow behaviour of red cells suspended at 40% hematocrit was tested with an artificial microvascular network. RESULTS Washing in 1% albumin reduced higher degrees of echinocytes and increased the frequency of discocytes, thereby shifting the morphological index towards discocytosis. Washing also reduced red cell swelling. This shape recovery was not seen after washing in saline, buffer or plasma. Red cell shape normalisation did not improve cell deformability measured by ektacytometry, but it tended to decrease suspension viscosities at low shear rates and improved the perfusion of an artificial microvascular network. CONCLUSIONS Washing of stored red blood cells in a 1% human serum albumin solution specifically reduces echinocytosis, and this shape recovery has a beneficial effect on microvascular perfusion in vitro. Washing in 1% albumin may represent a new approach to improving the quality of stored red cells, and thus potentially reducing the likelihood of adverse clinical outcomes associated with transfusion of blood stored for longer periods of time.