292 research outputs found
Influence of feeding hematocrit and perfusion pressure on hematocrit reduction (FĂ„hrĂŠus effect) in an artificial microvascular network
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 FĂ„hrĂŠus effect. We analyzed the influence of feeding Hct and perfusion pressure on the FĂ„hrĂŠus 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 FĂ„hrĂŠus effect in an AMVN is inversely related to Hctfeeding and ?P
Influence of red blood cell aggregation on perfusion of an artificial microvascular network
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 âcapillaryâ 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 âvenularâ and âcapillaryâ level. Estimated âcapillaryâ hematocrit was higher for D70 suspensions than for D40 suspensions at intermediate driving pressures (5 â 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 âcapillariesâ of the network for D70 suspensions suggested a better oxygen transport capacity in the presence of RBC aggregation
Quantifying morphological heterogeneity: a study of more than 1 000 000 individual stored red blood cells
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 6â8 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
Optimal hematocrit in an artificial microvascular network
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
A high-throughput microfluidic approach for 1000-fold leukocyte reduction of platelet-rich plasma
Leukocyte reduction of donated blood products substantially reduces the risk of a number of transfusion-related complications. Current âleukoreductionâ 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 âdead endâ filtration. To address these limitations, here we demonstrate our newly-developed âcontrolled incremental filtrationâ (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
Controlled incremental filtration: a simplified approach to design and fabrication of high-throughput microfluidic devices for selective enrichment of particles.
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 1”m to above 100”m) 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 â controlled incremental filtration â was developed and validated for its ability to generate microfluidic devices that efficiently separate particles on the order of 1â10”m, with throughputs of tens of ”L/min, without the use of a pump. Precise control of the amount of fluid incrementally diverted at each filtration âgapâ of the device allows for the gap size (~20”m) 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
Histamine reduces GPIb?-mediated adhesion of platelets to TNF-?-activatedvascular endothelium
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
A rapid paper-based test for quantifying sickle hemoglobin in blood samples from patients with sickle cell disease
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 (10â97%) and hemoglobin concentrations (5.6â12.9 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
Incorporation of prefabricated screw, pneumatic, and solenoid valves into microfluidic devices
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
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
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)
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