Publications by authors named "Wilbur A Lam"

90 Publications

Impact of repeated nasal sampling on detection and quantification of SARS-CoV-2.

Sci Rep 2021 07 21;11(1):14903. Epub 2021 Jul 21.

The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, GA, USA.

The impact of repeated sample collection on COVID-19 test performance is unknown. The FDA and CDC currently recommend the primary collection of diagnostic samples to minimize the perceived risk of false-negative findings. We therefore evaluated the association between repeated sample collection and test performance among 325 symptomatic patients undergoing COVID-19 testing in Atlanta, GA. High concordance was found between consecutively collected mid-turbinate samples with both molecular (n = 74, 100% concordance) and antigen-based (n = 147, 97% concordance, kappa = 0.95, CI = 0.88-1.00) diagnostic assays. Repeated sample collection does not decrease COVID-19 test performance, demonstrating that multiple samples can be collected for assay validation and clinical diagnosis.
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http://dx.doi.org/10.1038/s41598-021-94547-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295385PMC
July 2021

Physical forces regulating hemostasis and thrombosis: Vessels, cells, and molecules in illustrated review.

Res Pract Thromb Haemost 2021 Jul 14;5(5):e12548. Epub 2021 Jul 14.

Department of Bioengineering University of Colorado Denver Anschutz Medical Campus Aurora CO USA.

This illustrated review focuses on the physical forces that regulate hemostasis and thrombosis. These phenomena span from the vessel to the cellular to the molecular scales. Blood is a complex fluid with a viscosity that varies with how fast it flows and the size of the vessel through which it flows. Blood flow imposes forces on the vessel wall and blood cells that dictates the kinetics, structure, and stability of thrombi. The mechanical properties of blood cells create a segmented flowing fluid whereby red blood cells concentrate in the vessel core and platelets marginate to the near-wall region. At the vessel wall, shear stresses are highest, which requires a repertoire of receptors with different bond kinetics to roll, tether, adhere, and activate on inflamed endothelium and extracellular matrices. As a thrombus grows and then contracts, forces regulate platelet aggregation as well as von Willebrand factor function and fibrin mechanics. Forces can also originate from platelets as they respond to the external forces and sense the stiffness of their local environment.
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http://dx.doi.org/10.1002/rth2.12548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8279127PMC
July 2021

Multidisciplinary assessment of the Abbott BinaxNOW SARS-CoV-2 point-of-care antigen test in the context of emerging viral variants and self-administration.

Sci Rep 2021 07 16;11(1):14604. Epub 2021 Jul 16.

The Atlanta Center for Microsystems-Engineered Point-of-Care Technologies, Atlanta, Georgia.

While there has been significant progress in the development of rapid COVID-19 diagnostics, as the pandemic unfolds, new challenges have emerged, including whether these technologies can reliably detect the more infectious variants of concern and be viably deployed in non-clinical settings as "self-tests". Multidisciplinary evaluation of the Abbott BinaxNOW COVID-19 Ag Card (BinaxNOW, a widely used rapid antigen test, included limit of detection, variant detection, test performance across different age-groups, and usability with self/caregiver-administration. While BinaxNOW detected the highly infectious variants, B.1.1.7 (Alpha) first identified in the UK, B.1.351 (Beta) first identified in South Africa, P.1 (Gamma) first identified in Brazil, B.1.617.2 (Delta) first identified in India and B.1.2, a non-VOC, test sensitivity decreased with decreasing viral loads. Moreover, BinaxNOW sensitivity trended lower when devices were performed by patients/caregivers themselves compared to trained clinical staff, despite universally high usability assessments following self/caregiver-administration among different age groups. Overall, these data indicate that while BinaxNOW accurately detects the new viral variants, as rapid COVID-19 tests enter the home, their already lower sensitivities compared to RT-PCR may decrease even more due to user error.
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http://dx.doi.org/10.1038/s41598-021-94055-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285474PMC
July 2021

Pathologically stiff erythrocytes impede contraction of blood clots.

J Thromb Haemost 2021 Jul 7. Epub 2021 Jul 7.

Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

Background: Blood clot contraction, volume shrinkage of the clot, is driven by platelet contraction and accompanied by compaction of the erythrocytes and their gradual shape change from biconcave to polyhedral, with the resulting cells named polyhedrocytes.

Objectives: Here, we examined the role of erythrocyte rigidity on clot contraction and erythrocyte shape transformation.

Methods: We used an optical tracking methodology that allowed us to quantify changes in contracting clot size over time.

Results And Conclusions: Erythrocyte rigidity has been shown to be increased in sickle cell disease (SCD), and in our experiments erythrocytes from SCD patients were 4-fold stiffer than those from healthy subjects. On average, the final extent of clot contraction was reduced by 53% in the clots from the blood of patients with SCD compared to healthy individuals, and there was significantly less polyhedrocyte formation. To test if this reduction in clot contraction was due to the increase in erythrocyte rigidity, we used stiffening of erythrocytes via chemical cross-linking (glutaraldehyde), rigidifying Wright antibodies (Wr ), and naturally more rigid llama ovalocytes. Results revealed that stiffening erythrocytes result in impaired clot contraction and fewer polyhedrocytes. These results demonstrate the role of erythrocyte rigidity in the contraction of blood clots and suggest that the impaired clot contraction/shrinkage in SCD is due to the reduced erythrocyte deformability, which may be an underappreciated mechanism that aggravates obstructiveness of erythrocyte-rich (micro)thrombi in SCD.
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http://dx.doi.org/10.1111/jth.15407DOI Listing
July 2021

Flow-induced segregation and dynamics of red blood cells in sickle cell disease.

Phys Rev Fluids 2020 May 4;5(5). Epub 2020 May 4.

Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706-1691.

Blood flow in sickle cell disease (SCD) can substantially differ from normal blood flow due to significant alterations in the physical properties of the red blood cells (RBCs). Chronic complications, such as inflammation of the endothelial cells lining blood vessel walls, are associated with SCD, for reasons that are unclear. Here, detailed boundary integral simulations are performed to investigate an idealized model flow flow in SCD, a binary suspension of flexible biconcave discoidal fluid-filled capsules and stiff curved prolate capsules that represent healthy and sickle RBCs, respectively, subjected to pressure-driven flow in a planar slit. The stiff component is dilute. The key observation is that, unlike healthy RBCs that concentrate around the center of the channel and form an RBC-depleted layer (i.e. cell-free layer) next to the walls, sickle cells are largely drained from the bulk of the suspension and aggregate inside the cell-free layer, displaying strong margination. These cells are found to undergo a rigid-body-like rolling orbit near the walls. A binary suspension of flexible biconcave discoidal capsules and stiff straight (non-curved) prolate capsules is also considered for comparison, and the curvature of the stiff component is found to play a minor role in the behavior. Additionally, by considering a mixture of flexible and stiff biconcave discoids, we reveal that rigidity difference by itself is sufficient to induce the segregation behavior in a binary suspension. Furthermore, the additional shear stress on the walls induced by the presence of cells is computed for the various cases. Compared to the small fluctuations in wall shear stress for a suspension of healthy RBCs, large local peaks in wall shear stress are observed for the binary suspensions, due to the proximity of the marginated stiff cells to the walls. This effect is most marked for the straight prolate capsules. As endothelial cells are known to mechanotransduce physical forces such as aberrations in shear stress and convert them to physiological processes such as activation of inflammatory signals, these results may aid in understanding mechanisms for endothelial dysfunction associated with SCD.
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http://dx.doi.org/10.1103/physrevfluids.5.053101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8174308PMC
May 2020

Platelet heterogeneity enhances blood clot volumetric contraction: An example of asynchrono-mechanical amplification.

Biomaterials 2021 07 23;274:120828. Epub 2021 Apr 23.

George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, 801 Ferst Drive, Atlanta, GA, 30332-0405, USA. Electronic address:

Physiological processes such as blood clotting and wound healing as well as pathologies such as fibroses and musculoskeletal contractures, all involve biological materials composed of a contracting cellular population within a fibrous matrix, yet how the microscale interactions among the cells and the matrix lead to the resultant emergent behavior at the macroscale tissue level remains poorly understood. Platelets, the anucleate cell fragments that do not divide nor synthesize extracellular matrix, represent an ideal model to study such systems. During blood clot contraction, microscopic platelets actively pull fibers to shrink the macroscale clot to less than 10% of its initial volume. We discovered that platelets utilize a new emergent behavior, asynchrono-mechanical amplification, to enhanced volumetric material contraction and to magnify contractile forces. This behavior is triggered by the heterogeneity in the timing of a population of actuators. This result indicates that cell heterogeneity, often attributed to stochastic cell-to-cell variability, can carry an essential biophysical function, thereby highlighting the importance of considering 4 dimensions (space + time) in cell-matrix biomaterials. This concept of amplification via heterogeneity can be harnessed to increase mechanical efficiency in diverse systems including implantable biomaterials, swarm robotics, and active polymer composites.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184644PMC
July 2021

Vascularized Microfluidics and Their Untapped Potential for Discovery in Diseases of the Microvasculature.

Annu Rev Biomed Eng 2021 Jul 16;23:407-432. Epub 2021 Apr 16.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA; email:

Microengineering advances have enabled the development of perfusable, endothelialized models of the microvasculature that recapitulate the unique biological and biophysical conditions of the microcirculation in vivo. Indeed, at that size scale (<100 μm)-where blood no longer behaves as a simple continuum fluid; blood cells approximate the size of the vessels themselves; and complex interactions among blood cells, plasma molecules, and the endothelium constantly ensue-vascularized microfluidics are ideal tools to investigate these microvascular phenomena. Moreover, perfusable, endothelialized microfluidics offer unique opportunities for investigating microvascular diseases by enabling systematic dissection of both the blood and vascular components of the pathophysiology at hand. We review () the state of the art in microvascular devices and () the myriad of microvascular diseases and pressing challenges. The engineering community has unique opportunities to innovate with new microvascular devices and to partner with biomedical researchers to usher in a new era of understanding and discovery of microvascular diseases.
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http://dx.doi.org/10.1146/annurev-bioeng-091520-025358DOI Listing
July 2021

Future potential of Rapid Acceleration of Diagnostics (RADx Tech) in molecular diagnostics.

Expert Rev Mol Diagn 2021 03 9;21(3):251-253. Epub 2021 Mar 9.

Research Associate Professor, Deputy Director, Center for Innovation in Point-of-Care Technology for HIV/AIDS at Northwestern, Northwestern University, Evanston, IL, USA.

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http://dx.doi.org/10.1080/14737159.2021.1898950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137643PMC
March 2021

Point-of-Care Diagnostic Assays and Novel Preclinical Technologies for Hemostasis and Thrombosis.

Semin Thromb Hemost 2021 Mar 26;47(2):120-128. Epub 2021 Feb 26.

Department of Pediatrics, Aflac Cancer and Blood Disorders Center of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, Georgia.

Hemostasis is a complex wound-healing process involving numerous mechanical and biochemical mechanisms and influenced by many factors including platelets, coagulation factors, and endothelial components. Slight alterations in these mechanisms can lead to either prothrombotic or bleeding consequences, and such hemostatic imbalances can lead to significant clinical consequences with resultant morbidity and mortality. An ideal hemostasis assay would not only address all the unique processes involved in clot formation and resolution but also take place under flow conditions to account for endothelial involvement. Global assays do exist; however, these assays are not flow based. Flow-based assays have been limited secondary to their large blood volume requirements and low throughput, limiting potential clinical applications. Microfluidic-based assays address the aforementioned limitations of both global and flow-based assays by utilizing standardized devices that require low blood volumes, offer reproducible analysis, and have functionality under a range of shear stresses and flow conditions. While still largely confined to the preclinical space, here we aim to discuss these novel technologies and potential clinical implications, particularly in comparison to the current, commercially available point-of-care assays.
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http://dx.doi.org/10.1055/s-0041-1723798DOI Listing
March 2021

AnemoCheck-LRS: an optimized, color-based point-of-care test to identify severe anemia in limited-resource settings.

BMC Med 2020 11 16;18(1):337. Epub 2020 Nov 16.

Division of Hematology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave, MLC 11027, Cincinnati, OH, 45229, USA.

Background: Severe anemia is common and frequently fatal for hospitalized patients in limited-resource settings. Lack of access to low-cost, accurate, and rapid diagnosis of anemia impedes the delivery of life-saving care and appropriate use of the limited blood supply. The WHO Haemoglobin Colour Scale (HCS) is a simple low-cost test but frequently inaccurate. AnemoCheck-LRS (limited-resource settings) is a rapid, inexpensive, color-based point-of-care (POC) test optimized to diagnose severe anemia.

Methods: Deidentified whole blood samples were diluted with plasma to create variable hemoglobin (Hb) concentrations, with most in the severe (≤ 7 g/dL) or profound (≤ 5 g/dL) anemia range. Each sample was tested with AnemoCheck-LRS and WHO HCS independently by three readers and compared to Hb measured by an electronic POC test (HemoCue 201) and commercial hematology analyzer.

Results: For 570 evaluations within the limits of detection of AnemoCheck-LRS (Hb ≤ 8 g/dL), the average difference between AnemoCheck-LRS and measured Hb was 0.5 ± 0.4 g/dL. In contrast, the WHO HCS overestimated Hb with an absolute difference of 4.9 ± 1.3 g/dL for samples within its detection range (Hb 4-14 g/dL, n = 405). AnemoCheck-LRS was much more sensitive (92%) for the diagnosis of profound anemia than WHO HCS (22%).

Conclusions: AnemoCheck-LRS is a rapid, inexpensive, and accurate POC test for anemia. AnemoCheck-LRS is more accurate than WHO HCS for detection of low Hb levels, severe anemia that may require blood transfusion. AnemoCheck-LRS should be tested prospectively in limited-resource settings where severe anemia is common, to determine its utility as a screening tool to identify patients who may require transfusion.
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http://dx.doi.org/10.1186/s12916-020-01793-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667733PMC
November 2020

Hematocrit significantly confounds diffuse correlation spectroscopy measurements of blood flow.

Biomed Opt Express 2020 Aug 29;11(8):4786-4799. Epub 2020 Jul 29.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 1760 Haygood Dr, NE, Atlanta, GA 30322, USA.

Diffuse correlation spectroscopy (DCS) is an optical modality used to measure an index of blood flow in biological tissue. This blood flow index depends on both the red blood cell flow rate and density (i.e., hematocrit), although the functional form of hematocrit dependence is not well delineated. Herein, we develop and validate a novel tissue-simulating phantom containing hundreds of microchannels to investigate the influence of hematocrit on blood flow index. For a fixed flow rate, we demonstrate a significant inverse relationship between hematocrit and blood flow index that must be accounted for to accurately estimate blood flow under anemic conditions.
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http://dx.doi.org/10.1364/BOE.397613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449719PMC
August 2020

A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.

J Biol Chem 2020 11 3;295(46):15438-15453. Epub 2020 Sep 3.

School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA; Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia, USA.

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.
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http://dx.doi.org/10.1074/jbc.RA120.015434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667971PMC
November 2020

Buzz about RT-qPCR: An RT-qPCR formulation for SARS-CoV-2 detection using reagents produced at Georgia Institute of Technology.

medRxiv 2020 Jul 31. Epub 2020 Jul 31.

School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA.

Widespread testing for the presence novel coronavirus SARS-CoV-2 in patients remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. The early testing shortfall in some parts of the US can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can produce the RT-qPCR assay and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. We compare the performance of our in-house kit to a commercial product used for diagnostic testing and describe implementation of environmental testing to monitor surfaces across various campus laboratories for the presence of SARS-CoV-2.
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http://dx.doi.org/10.1101/2020.07.29.20163949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402063PMC
July 2020

Label-free hematology analysis using deep-ultraviolet microscopy.

Proc Natl Acad Sci U S A 2020 06 19;117(26):14779-14789. Epub 2020 Jun 19.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332;

Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screening, diagnosing, and monitoring blood conditions and diseases but requires complex equipment, multiple chemical reagents, laborious system calibration and procedures, and highly trained personnel for operation. Here we introduce a hematological assay based on label-free molecular imaging with deep-ultraviolet microscopy that can provide fast quantitative information of key hematological parameters to facilitate and improve hematological analysis. We demonstrate that this label-free approach yields 1) a quantitative five-part white blood cell differential, 2) quantitative red blood cell and hemoglobin characterization, 3) clear identification of platelets, and 4) detailed subcellular morphology. Analysis of tens of thousands of live cells is achieved in minutes without any sample preparation. Finally, we introduce a pseudocolorization scheme that accurately recapitulates the appearance of cells under conventional staining protocols for microscopic analysis of blood smears and bone marrow aspirates. Diagnostic efficacy is evaluated by a panel of hematologists performing a blind analysis of blood smears from healthy donors and thrombocytopenic and sickle cell disease patients. This work has significant implications toward simplifying and improving CBC and blood smear analysis, which is currently performed manually via bright-field microscopy, and toward the development of a low-cost, easy-to-use, and fast hematological analyzer as a point-of-care device and for low-resource settings.
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http://dx.doi.org/10.1073/pnas.2001404117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334528PMC
June 2020

The biophysics and mechanics of blood from a materials perspective.

Nat Rev Mater 2019 May 28;4(5):294-311. Epub 2019 Mar 28.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.

Cells actively interact with their microenvironment, constantly sensing and modulating biochemical and biophysical signals. Blood comprises a variety of non-adherent cells that interact with each other and with endothelial and vascular smooth muscle cells of the blood vessel walls. Blood cells are further experiencing a range of external forces by the hemodynamic environment and they also exert forces to remodel their local environment. Therefore, the biophysics and material properties of blood cells and blood play an important role in determining blood behaviour in health and disease. In this Review, we discuss blood cells and tissues from a materials perspective, considering the mechanical properties and biophysics of individual blood cells and endothelial cells as well as blood cell collectives. We highlight how blood vessels provide a mechanosensitive barrier between blood and tissues and how changes in vessel stiffness and flow shear stress can be correlated to plaque formation and exploited for the design of vascular grafts. We discuss the effect of the properties of fibrin on blood clotting, and investigate how forces exerted by platelets are correlated to disease. Finally, we hypothesize that blood and vascular cells are constantly establishing a mechanical homeostasis, which, when imbalanced, can lead to hematologic and vascular diseases.
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http://dx.doi.org/10.1038/s41578-019-0099-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238390PMC
May 2019

Platelet-rich plasma as endothelial rocket fuel for engineered in vitro microvasculature.

J Thromb Haemost 2020 06 28;18(6):1239-1241. Epub 2020 Apr 28.

Division of Pediatric Hematology/Oncology, Department of Pediatrics, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA, USA.

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http://dx.doi.org/10.1111/jth.14823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757418PMC
June 2020

In vitro flow based systems to study platelet function and thrombus formation: Recommendations for standardization: Communication from the SSC on Biorheology of the ISTH.

J Thromb Haemost 2020 03;18(3):748-752

Center for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, Moscow, Russia.

Experimental videomicroscopic in vitro assays of thrombus formation based on blood perfusion are instrumental in a wide range of basic studies in thrombosis, screening for hereditary or acquired plateletrelated pathologies, and assessing the effectiveness of novel anti-platelet therapies. Here, we discuss application of the broadly used "in vitro thrombosis model": a frequently used assay to study the formation of 3D aggregates under flow, which involves perfusing anticoagulated whole blood over fibrillar collagen in a flow geometry of rectangular cross-section, such as glass microcapillaries or parallel-plate flow chambers. Major advantaged of this assay are simplicity and ability to reproduce the four main stages of platelet thrombus formation, i.e. platelet tethering, adhesion, activation and aggregation under a wide range of hemodynamic conditions. On the other hand, these devices represent, at best, simple reductive models of thrombosis. We also describe how blood flow assays can be used to study various aspects of platelet function on adhesive proteins and discuss the relevance of such flow models. Finally, we propose recommendations for standardization related to the use of this assay that cover collagen source, coating methods, micropatterning, sample composition, anticoagulation, choice of flow device, hemodynamic conditions, quantification challenges, variability, pre-analytical conditions and other issues.
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http://dx.doi.org/10.1111/jth.14717DOI Listing
March 2020

Getting a good view: imaging of platelets under flow.

Platelets 2020 Jul 28;31(5):570-579. Epub 2020 Feb 28.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University , Atlanta, GA, USA.

As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myriad of biophysical and biochemical stimuli within vasculature and heterogeneous blood clots. Highly controlled, reductionist imaging studies have been instrumental in providing a detailed and quantitative understanding of platelet biology and behavior, and have helped elucidate some surprising functions of platelets. In this review, we highlight the tools and approaches that enable visualization of platelets in conjunction with precise control over the local biofluidic and biochemical microenvironment. We also discuss next generation tools that add further control over microenvironment cell stiffness or enable visualization of the interactions between platelets and endothelial cells. Throughout the review, we include pragmatic knowledge on imaging systems, experimental conditions, and approaches that have proved to be useful to our imaging studies of platelets under flow.
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http://dx.doi.org/10.1080/09537104.2020.1732320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332395PMC
July 2020

Decreased cell stiffness enhances leukemia development and progression.

Leukemia 2020 09 24;34(9):2493-2497. Epub 2020 Feb 24.

Department of Pediatrics, Division of Hematology and Oncology, Aflac Cancer and Blood Disorders Center, Winship Cancer Institute, Children's Healthcare of Atlanta, Emory University, Atlanta, GA, 30322, USA.

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http://dx.doi.org/10.1038/s41375-020-0763-7DOI Listing
September 2020

Diabetes affects endothelial cell function and alters fibrin clot formation in a microvascular flow model: A pilot study.

Diab Vasc Dis Res 2020 Jan-Feb;17(1):1479164120903044

Experimental Haemostasis Group, Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland.

Diabetes is a proinflammatory and prothrombotic condition that increases the risk of vascular complications. The aim of this study was to develop a diabetic microvascular flow model that allows to study the complex interactions between endothelial cells, blood cells and plasma proteins and their effects on clot formation. Primary human cardiac microvascular endothelial cells from donors without diabetes or donors with diabetes (type 1 or type 2) were grown in a microfluidic chip, perfused with non-diabetic or diabetic whole blood, and clot formation was assessed by measuring fibrin deposition in real time by confocal microscopy. Clot formation in non-diabetic whole blood was significantly increased in the presence of endothelial cells from donors with type 2 diabetes compared with cells from donors without diabetes. There was no significant difference in clot formation between non-diabetic and diabetic whole blood. We present for the first time a diabetic microvascular flow model as a new tool to study clot formation as a result of the complex interactions between endothelial cells, blood cells and plasma proteins in a diabetes setting. We show that endothelial cells affect clot formation in whole blood, attributing an important role to the endothelium in the development of atherothrombotic complications.
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http://dx.doi.org/10.1177/1479164120903044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510361PMC
March 2020

Vascularized Microfluidics and the Blood-Endothelium Interface.

Micromachines (Basel) 2019 Dec 23;11(1). Epub 2019 Dec 23.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA.

The microvasculature is the primary conduit through which the human body transmits oxygen, nutrients, and other biological information to its peripheral tissues. It does this through bidirectional communication between the blood, consisting of plasma and non-adherent cells, and the microvascular endothelium. Current understanding of this blood-endothelium interface has been predominantly derived from a combination of reductionist two-dimensional in vitro models and biologically complex in vivo animal models, both of which recapitulate the human microvasculature to varying but limited degrees. In an effort to address these limitations, vascularized microfluidics have become a platform of increasing importance as a consequence of their ability to isolate biologically complex phenomena while also recapitulating biochemical and biophysical behaviors known to be important to the function of the blood-endothelium interface. In this review, we discuss the basic principles of vascularized microfluidic fabrication, the contribution this platform has made to our understanding of the blood-endothelium interface in both homeostasis and disease, the limitations and challenges of these vascularized microfluidics for studying this interface, and how these inform future directions.
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http://dx.doi.org/10.3390/mi11010018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019435PMC
December 2019

Dynamics of deformable straight and curved prolate capsules in simple shear flow.

Phys Rev Fluids 2019 Apr 18;4(4). Epub 2019 Apr 18.

Department of Chemical and Biological Engineering University of Wisconsin-Madison, Madison, WI 53706-1691.

This work investigates the motion of neutrally-buoyant, slightly deformable straight and curved prolate fluid-filled capsules in unbounded simple shear flow at zero Reynolds number using direct simulations. The curved capsules serve as a model for the typical crescent-shaped sickle red blood cells in sickle cell disease (SCD). The effects of deformability and curvature on the dynamics are revealed. We show that with low deformability, straight prolate spheroidal capsules exhibit tumbling in the shear plane as their unique asymptotically stable orbit. This result contrasts with that for rigid spheroids, where infinitely many neutrally stable Jeffery orbits exist. The dynamics of curved prolate capsules are more complicated due to a combined effect of deformability and curvature. At short times, depending on the initial orientation, slightly deformable curved prolate capsules exhibit either a Jeffery-like motion such as tumbling or kayaking, or a non-Jeffery-like behavior in which the director (end-to-end vector) of the capsule crosses the shear-gradient plane back and forth. At long times, however, a Jeffery-like quasiperiodic orbit is taken regardless of the initial orientation. We further show that the average of the long-time trajectory can be well approximated using the analytical solution for Jeffery orbits with an effective orbit constant and aspect ratio . These parameters are useful for characterizing the dynamics of curved capsules as a function of given deformability and curvature. As the capsule becomes more deformable or curved, decreases, indicating a shift of the orbit towards log-rolling motion, while increases weakly as the degree of curvature increases but shows negligible dependency on deformability. These features are not changed substantially as the viscosity ratio between the inner and outer fluids is changed from 1 to 5. As cell deformability, cell shape, and cell-cell interactions are all pathologically altered in blood disorders such as SCD, these results will have clear implications on improving our understanding of the pathophysiology of hematologic disease.
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http://dx.doi.org/10.1103/PhysRevFluids.4.043103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880959PMC
April 2019

Microfluidic Approach for Highly Efficient Viral Transduction.

Methods Mol Biol 2020 ;2097:55-65

Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA, USA.

Lentiviral vectors enable gene transfer into target cells, but manufacturing is complex, scale-limited, and costly. Here, we describe the use of microfluidic devices for efficient ex vivo gene transfer. Up to four- to fivefold reductions in viral vector usage and two- to fourfold reductions in transduction times can be obtained by using this method.
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http://dx.doi.org/10.1007/978-1-0716-0203-4_3DOI Listing
January 2021

Noninvasive optical assessment of resting-state cerebral blood flow in children with sickle cell disease.

Neurophotonics 2019 Jul 19;6(3):035006. Epub 2019 Aug 19.

Georgia Institute of Technology and Emory University, Wallace H. Coulter Department of Biomedical Engineering, Atlanta, Georgia, United States.

Sickle cell disease (SCD) is a genetic blood disorder that has profound effects on the brain. Chronic anemia combined with both macro- and microvascular perfusion abnormalities that arise from stenosis or occlusion of blood vessels increased blood viscosity, adherence of red blood cells to the vascular endothelium, and impaired autoregulatory mechanisms in SCD patients all culminate in susceptibility to cerebral infarction. Indeed, the risk of stroke is 250 times higher in children with SCD than in the general population. Unfortunately, while transcranial Doppler ultrasound (TCD) has been widely clinically adopted to longitudinally monitor macrovascular perfusion in these patients, routine clinical screening of microvascular perfusion abnormalities is challenging with current modalities (e.g., positron emission tomography and magnetic resonance imaging) given their high-cost, requirement for sedation in children year, and need for trained personnel. We assess the feasibility of a low-cost, noninvasive optical technique known as diffuse correlation spectroscopy (DCS) to quantify an index of resting-state cortical cerebral blood flow (BFI) in 11 children with SCD along with 11 sex- and age-matched healthy controls. As expected, BFI was significantly higher in SCD subjects compared to healthy controls ( ). Within SCD subjects, BFI was inversely proportional to resting-state arterial hemoglobin levels ( ), consistent with expected anemia-induced compensatory vasodilation that aims to maintain adequate oxygen delivery to the tissue. Further, in a subset of patients measured with TCD ( ), DCS-measured blood flow was correlated with TCD-measured blood flow velocity in middle cerebral artery ( ), although the trend was not statistically significant ( ). These results are consistent with those of several previous studies using traditional neuroimaging techniques, suggesting that DCS may be a promising low-cost tool for assessment of tissue-level CBF in pediatric SCD.
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http://dx.doi.org/10.1117/1.NPh.6.3.035006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699550PMC
July 2019

Diagnosis of acute serious illness: the role of point-of-care technologies.

Curr Opin Biomed Eng 2019 Sep 16;11:22-34. Epub 2019 Sep 16.

Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA, USA.

Access to rapid diagnostic information is a core value of point-of-care (POC) technology. This is particularly relevant in acute, emergency, and critical care settings where diagnostic speed and precision directly guide the management of patients with potentially life-threatening conditions. Many POC diagnostics described in the literature, however, remain largely unproven and have yet to enter the market entirely. Only a few have traversed the translation and commercialization pathways to reach widespread clinical adoption. Moreover, even technologies that have successfully translated to the patient bedside still frequently lack an evidence base showing improvement of clinical outcomes. In this review, we present aspects of diagnosis of acute life-threatening diseases and describe the potential role of POC technologies, emphasizing the available evidence of clinical outcomes. Finally, we discuss what is needed to identify clinically meaningful new technologies and translate them toward the long-promised goal of better health through rapid POC diagnosis.
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http://dx.doi.org/10.1016/j.cobme.2019.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168915PMC
September 2019

Enabling mesenchymal stromal cell immunomodulatory analysis using scalable platforms.

Integr Biol (Camb) 2019 04;11(4):154-162

Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA.

Human mesenchymal stromal cells (hMSCs) are a promising cell source for numerous regenerative medicine and cell therapy-based applications. However, MSC-based therapies have faced challenges in translation to the clinic, in part due to the lack of sufficient technologies that accurately predict MSC potency and are viable in the context of cell manufacturing. Microfluidic platforms may provide an innovative opportunity to address these challenges by enabling multiparameter analyses of small sample sizes in a high throughput and cost-effective manner, and may provide a more predictive environment in which to analyze hMSC potency. To this end, we demonstrate the feasibility of incorporating 3D culture environments into microfluidic platforms for analysis of hMSC secretory response to inflammatory stimuli and multi-parameter testing using cost-effective and scalable approaches. We first find that the cytokine secretion profile for hMSCs cultured within synthetic poly(ethylene glycol)-based hydrogels is significantly different compared to those cultured on glass substrates, both in growth media and following stimulation with IFN-γ and TNF-α, for cells derived from two donors. For both donors, perfusion with IFN-γ and TNF-α leads to differences in secretion of interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and interleukin-1 receptor antagonist (IL-1ra) between hMSCs cultured in hydrogels and those cultured on glass substrates. We then demonstrate the feasibility of analyzing the response of hMSCs to a stable concentration gradient of soluble factors such as inflammatory stimuli for potential future use in potency analyses, minimizing the amount of sample required for dose-response testing.
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http://dx.doi.org/10.1093/intbio/zyz014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863738PMC
April 2019

Ptpn21 Controls Hematopoietic Stem Cell Homeostasis and Biomechanics.

Cell Stem Cell 2019 04 14;24(4):608-620.e6. Epub 2019 Mar 14.

Division of Hematology/Oncology, Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Winship Cancer Institute, Children's Healthcare of Atlanta, Emory University, Atlanta, GA 30322, USA. Electronic address:

Hematopoietic stem cell (HSC) quiescence is a tightly regulated process crucial for hematopoietic regeneration, which requires a healthy and supportive microenvironmental niche within the bone marrow (BM). Here, we show that deletion of Ptpn21, a protein tyrosine phosphatase highly expressed in HSCs, induces stem cell egress from the niche due to impaired retention within the BM. Ptpn21 HSCs exhibit enhanced mobility, decreased quiescence, increased apoptosis, and defective reconstitution capacity. Ptpn21 deletion also decreased HSC stiffness and increased physical deformability, in part by dephosphorylating Spetin1 (Tyr), a poorly described component of the cytoskeleton. Elevated phosphorylation of Spetin1 in Ptpn21 cells impaired cytoskeletal remodeling, contributed to cortical instability, and decreased cell rigidity. Collectively, these findings show that Ptpn21 maintains cellular mechanics, which is correlated with its important functions in HSC niche retention and preservation of hematopoietic regeneration capacity.
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http://dx.doi.org/10.1016/j.stem.2019.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450721PMC
April 2019

Feeling the Force: Measurements of Platelet Contraction and Their Diagnostic Implications.

Semin Thromb Hemost 2019 Apr 19;45(3):285-296. Epub 2018 Dec 19.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia.

In addition to the classical biological and biochemical framework, blood clots can also be considered as active biomaterials composed of dynamically contracting platelets, nascent polymeric fibrin that functions as a matrix scaffold, and entrapped blood cells. As platelets sense, rearrange, and apply forces to the surrounding microenvironment, they dramatically change the material properties of the nascent clot, increasing its stiffness by an order of magnitude. Hence, the mechanical properties of blood clots are intricately tied to the forces applied by individual platelets. Research has also shown that the pathophysiological changes in clot mechanical properties are associated with bleeding and clotting disorders, cancer, stroke, ischemic heart disease, and more. By approaching the study of hemostasis and thrombosis from a biophysical and mechanical perspective, important insights have been made into how the mechanics of clotting and the forces applied by platelets are linked to various diseases. This review will familiarize the reader with a mechanics framework that is contextualized with relevant biology. The review also includes a discussion of relevant tools used to study platelet forces either directly or indirectly, and finally, concludes with a summary of potential links between clotting forces and disease.
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http://dx.doi.org/10.1055/s-0038-1676315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284283PMC
April 2019
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