In the last decade, advances in microchip-based technologies have provided useful, inexpensive, a... more In the last decade, advances in microchip-based technologies have provided useful, inexpensive, and easily reproducible microfluidic devices for conducting microscale biological and biochemical experiments. Similar to a computer chip but with plumbing, a microfluidic chip comprises a set of microchannels etched or molded into a material (typically, a polymer such as polydimethylsiloxane) into which fluids can be perfused via syringe pumps or hydrostatic pressure. The ability to tightly control biological conditions and the dynamic shear environment within these devices have enabled microfluidics to be ideal tools for quantitatively analyzing hematologic and vascular processes such as thrombosis and hemostasis. To that end, several groups including our own have recently incorporated the live culture of endothelial cells into these devices, thereby developing microfluidic systems that accurately recapitulate and integrate the myriad of interactions among blood cells, endothelial cells, and soluble factors that occur in vivo and are vital in studying hemostasis and thrombosis. As such, these "endothelialized" microfluidic devices hold several novel and key advantages as research-enabling systems, including the capabilities to: include or subtract different blood cell subpopulations (platelets, red cells, leukocyte subsets) and/or soluble factors (coagulation proteins, inflammatory mediators, etc.) for mechanistic studies, tightly control shear conditions, incorporate human and patient blood samples, modulate endothelial function and activation, culture different endothelial cell phenotypes (e.g., various anatomic beds, different species), and directly visualize clot formation in real time via brightfield and fluorescence videomicroscopy. With these physiologically relevant features not commonly found in existing in vitro assays of clot formation, endothelialized microfluidics therefore aptly complement in vivo studies of thrombosis and under certain circumstances, may even serve as alternatives for murine thrombosis models. Indeed, recently published seminal studies that leveraged endothelialized microfluidics studies have quantitatively demonstrated the relationship between shear rate and thrombosis via endothelial secretion of von Willebrand factor as well as answered questions related to clot formation that were technologically infeasible to resolve with existing in vivo and in vitro methods. Moreover, these thrombosis-on-a-chip systems have also been recently utilized as physiologic in vitro drug discovery platforms for novel antithrombotic therapeutics or novel applications of existing pharmacologic agents. In addition, numerous efforts to apply thrombosis-on-a-chip systems as point-of-care diagnostics to determine thrombosis risk in patients are also currently underway. However, as no in vitro device can fully recapitulate all in vivo conditions, thrombosis-on-a-chip systems are not without limitations and shortcomings, which often involve the material and geometric properties of these devices. To address those issues, more recent studies have further advanced the endothelialized microfluidic technology to comprise microdevices that are either hydrogel-based or incorporate complex vascular geometries that occur in vivo . With mounting evidence that murine models do not exactly recapitulate all aspects of thrombosis in humans, thrombosis-on-a-chip technologies provide ample opportunities to apply the benefits of microfluidic technology to investigate, diagnose, and treat clotting disorders. Disclosures Lam: Sanguina, LLC: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: ... more The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The “coagulome” as a critical driver of cardiovascular disease. Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow, and kidney. Four investigators shared their views on these organ-specific topics. Theme 2: Novel mechanisms of thrombosis. Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infection-associated coagulopathies perturb the hemostatic balance resulting in thrombosis and/or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies. This theme included state-of-the-art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of met...
BackgroundRapid and accurate testing for SARS-CoV-2 is an essential tool in the medical and publi... more BackgroundRapid and accurate testing for SARS-CoV-2 is an essential tool in the medical and public health response to the COVID-19 pandemic. An ideal test for COVID-19 would combine the sensitivity of laboratory-based PCR combined with the speed and ease of use of point-of-care (POC) or home-based rapid antigen testing.MethodsTo evaluate the performance of the Diagnostic Analyzer for Selective Hybridization (DASH) SARS-CoV-2 POC PCR (sample insertion to result time of 16 minutes), we conducted a cross-sectional study of adults with and without symptoms of COVID-19 at four clinical sites. We collected two bilateral anterior nasal swabs from each participant and information on COVID-19 symptoms, vaccination, and exposure. One swab was tested with the DASH SARS-CoV-2 POC PCR and the second in a central laboratory using Cepheid Xpert Xpress SARS-CoV-2 PCR. We assessed test concordance and calculated sensitivity, specificity, negative and positive predictive values using Xpert as the “go...
Biochemical aspects of cellular process are well characterized, but more recently, it has been sh... more Biochemical aspects of cellular process are well characterized, but more recently, it has been shown that cells dynamically sense and respond to biophysical cues such as substrate stiffness and geometrical constraints; physical cues even direct cell differentiation and stem cell lineage (Discher et al, Science, 2005). In hematology, we know that platelets are shear activated and attenuate force based on substrate stiffness, and that endothelial cells align with flow and are activated by shear stress. Blood cells pass through, and interact with, biological matrices such as fibrin clots and the vascular wall, but the physical and biochemical aspects of these interactions are indistinguishable from one another in vivo. As such, there is a gap in knowledge as to how blood cells respond to matrices as they transit through them. To decouple the physical and biochemical interactions of blood cells and biological matrices, we sought to recreate the physical geometry of a fibrin network in a...
Blood cells circulate in a dynamic fluidic environment, and during hematologic processes such as ... more Blood cells circulate in a dynamic fluidic environment, and during hematologic processes such as hemostasis, thrombosis, and inflammation, blood cells interact biophysically with a myriad of vascular matrices-blood clots and the subendothelial matrix. While it is known that adherent cells physiologically respond to the mechanical properties of their underlying matrices, how blood cells interact with their mechanical microenvironment of vascular matrices remains poorly understood. To that end, we developed microfluidic systems that achieve high fidelity, high resolution, single-micron PDMS features that mimic the physical geometries of vascular matrices. With these electron beam lithography (EBL)-based microsystems, the physical interactions of individual blood cells with the mechanical properties of the matrices can be directly visualized. We observe that the physical presence of the matrix, in and of itself, mediates hematologic processes of the three major blood cell types: platel...
Rapid antigen tests (RATs) have become an invaluable tool for combating the COVID-19 pandemic. Ho... more Rapid antigen tests (RATs) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of 10 commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using both individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR cycle threshold (C T ) value (a rough proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subgenomic RNA (sgRNA) may indicate ... more Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subgenomic RNA (sgRNA) may indicate actively replicating virus, but sgRNA abundance has not been systematically compared between SARS-CoV-2 variants. sgRNA was quantified in 169 clinical samples by real-time reverse-transcription polymerase chain reaction, demonstrating similar relative abundance among known variants. Thus, sgRNA detection can identify individuals with active viral replication regardless of variant.
Viability of saliva samples stored for longer than 28 days has not been reported in the literatur... more Viability of saliva samples stored for longer than 28 days has not been reported in the literature. The COVID-19 pandemic has spawned new research evaluating various sample types, thus large biobanks have been started. Residual saliva samples from university student surveillance testing were retested on SalivaDirect and compared with original RT-PCR (cycle threshold values) and quantitative antigen values for each month in storage. We conclude that saliva samples stored at -80°C are still viable in detecting SARS-CoV-2 after 12 months of storage, establishing the validity of these samples for future testing.
Context.— Diagnostic testing for SARS-CoV-2 in symptomatic and asymptomatic children remains inte... more Context.— Diagnostic testing for SARS-CoV-2 in symptomatic and asymptomatic children remains integral to care, particularly for supporting return to and attendance in schools. The concordance of SARS-CoV-2 detection in children, using various specimen types, has not been widely studied. Objective.— To compare 3 sample types for SARS-CoV-2 polymerase chain reaction (PCR) testing in children, collected and tested at a single facility. Design.— We prospectively recruited 142 symptomatic and asymptomatic children/young adults into a sample comparison study performed in a single health care system. Each child provided self-collected saliva, and a trained health care provider collected a mid-turbinate nasal swab and nasopharyngeal (NP) swab. Specimens were assayed within 24 hours of collection by using reverse transcription–polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 on a single testing platform. Results.— Concurrently collected saliva and mid-turbinate swabs had greater than ...
Blood flow in sickle cell disease (SCD) can substantially differ from normal blood flow due to si... more 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.
Physiological processes such as blood clotting and wound healing as well as pathologies such as f... more 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.
Neutrophil extracellular traps (NETs) are antimicrobial cobweb‐structured materials produced by i... more Neutrophil extracellular traps (NETs) are antimicrobial cobweb‐structured materials produced by immune cells for clearance of pathogens in the body, but are paradoxically associated with biofilm formation and exacerbated lung infections. To provide a better materials perspective on the pleiotropic roles played by NETs at diverse compositions/concentrations, a NETs‐like material (called “microwebs”, abbreviated as μwebs) is synthesized for decoding the antimicrobial activity of NETs against Staphylococcus aureus in infection‐relevant conditions. It is shown that μwebs composed of low‐to‐intermediate concentrations of DNA‐histone complexes successfully trap and inhibit S. aureus growth and biofilm formation. However, with growing concentrations and histone proportions, the resulting microwebs appear gel‐like structures accompanied by reduced antimicrobial activity that can even promote the formation of S. aureus biofilms. The simplified model of NETs provides materials‐based evidence ...
Proceedings of the National Academy of Sciences, 2020
Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screenin... more 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 c...
Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vi... more Widespread testing for the presence of the novel coronavirus 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 reverse transcription polymerase chain reaction (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 compare...
Background: Originally described as a monogenic hemoglobin disorder resulting in increased red bl... more Background: Originally described as a monogenic hemoglobin disorder resulting in increased red blood cell (RBC) stiffness leading to vaso-occlusion, sickle cell disease (SCD) is now known to be a vasculopathic disease with some semblance to cardiovascular disease in which the endothelium is inflamed. While adhesive RBC-endothelial interactions, inflammatory cytokines, and hemolysis all contribute to SCD vasculopathy, whether the increased stiffness of sickle RBCs directly contributes to endothelial inflammation is unknown. Endothelial cells are now known to mechanotransduce shear forces into biological signals. Pathological alteration of such forces leads to proinflammatory endothelial cell signaling including upregulation of VCAM-1 and E-selectin, which contribute to atherosclerotic plaques leading to myocardial infarction and stroke (Abe, ATVB, 2014). In addition, under normal homeostatic conditions, RBCs do not come into contact with the endothelium due to a cell-free layer creat...
In the last decade, advances in microchip-based technologies have provided useful, inexpensive, a... more In the last decade, advances in microchip-based technologies have provided useful, inexpensive, and easily reproducible microfluidic devices for conducting microscale biological and biochemical experiments. Similar to a computer chip but with plumbing, a microfluidic chip comprises a set of microchannels etched or molded into a material (typically, a polymer such as polydimethylsiloxane) into which fluids can be perfused via syringe pumps or hydrostatic pressure. The ability to tightly control biological conditions and the dynamic shear environment within these devices have enabled microfluidics to be ideal tools for quantitatively analyzing hematologic and vascular processes such as thrombosis and hemostasis. To that end, several groups including our own have recently incorporated the live culture of endothelial cells into these devices, thereby developing microfluidic systems that accurately recapitulate and integrate the myriad of interactions among blood cells, endothelial cells, and soluble factors that occur in vivo and are vital in studying hemostasis and thrombosis. As such, these "endothelialized" microfluidic devices hold several novel and key advantages as research-enabling systems, including the capabilities to: include or subtract different blood cell subpopulations (platelets, red cells, leukocyte subsets) and/or soluble factors (coagulation proteins, inflammatory mediators, etc.) for mechanistic studies, tightly control shear conditions, incorporate human and patient blood samples, modulate endothelial function and activation, culture different endothelial cell phenotypes (e.g., various anatomic beds, different species), and directly visualize clot formation in real time via brightfield and fluorescence videomicroscopy. With these physiologically relevant features not commonly found in existing in vitro assays of clot formation, endothelialized microfluidics therefore aptly complement in vivo studies of thrombosis and under certain circumstances, may even serve as alternatives for murine thrombosis models. Indeed, recently published seminal studies that leveraged endothelialized microfluidics studies have quantitatively demonstrated the relationship between shear rate and thrombosis via endothelial secretion of von Willebrand factor as well as answered questions related to clot formation that were technologically infeasible to resolve with existing in vivo and in vitro methods. Moreover, these thrombosis-on-a-chip systems have also been recently utilized as physiologic in vitro drug discovery platforms for novel antithrombotic therapeutics or novel applications of existing pharmacologic agents. In addition, numerous efforts to apply thrombosis-on-a-chip systems as point-of-care diagnostics to determine thrombosis risk in patients are also currently underway. However, as no in vitro device can fully recapitulate all in vivo conditions, thrombosis-on-a-chip systems are not without limitations and shortcomings, which often involve the material and geometric properties of these devices. To address those issues, more recent studies have further advanced the endothelialized microfluidic technology to comprise microdevices that are either hydrogel-based or incorporate complex vascular geometries that occur in vivo . With mounting evidence that murine models do not exactly recapitulate all aspects of thrombosis in humans, thrombosis-on-a-chip technologies provide ample opportunities to apply the benefits of microfluidic technology to investigate, diagnose, and treat clotting disorders. Disclosures Lam: Sanguina, LLC: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: ... more The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The “coagulome” as a critical driver of cardiovascular disease. Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow, and kidney. Four investigators shared their views on these organ-specific topics. Theme 2: Novel mechanisms of thrombosis. Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infection-associated coagulopathies perturb the hemostatic balance resulting in thrombosis and/or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies. This theme included state-of-the-art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of met...
BackgroundRapid and accurate testing for SARS-CoV-2 is an essential tool in the medical and publi... more BackgroundRapid and accurate testing for SARS-CoV-2 is an essential tool in the medical and public health response to the COVID-19 pandemic. An ideal test for COVID-19 would combine the sensitivity of laboratory-based PCR combined with the speed and ease of use of point-of-care (POC) or home-based rapid antigen testing.MethodsTo evaluate the performance of the Diagnostic Analyzer for Selective Hybridization (DASH) SARS-CoV-2 POC PCR (sample insertion to result time of 16 minutes), we conducted a cross-sectional study of adults with and without symptoms of COVID-19 at four clinical sites. We collected two bilateral anterior nasal swabs from each participant and information on COVID-19 symptoms, vaccination, and exposure. One swab was tested with the DASH SARS-CoV-2 POC PCR and the second in a central laboratory using Cepheid Xpert Xpress SARS-CoV-2 PCR. We assessed test concordance and calculated sensitivity, specificity, negative and positive predictive values using Xpert as the “go...
Biochemical aspects of cellular process are well characterized, but more recently, it has been sh... more Biochemical aspects of cellular process are well characterized, but more recently, it has been shown that cells dynamically sense and respond to biophysical cues such as substrate stiffness and geometrical constraints; physical cues even direct cell differentiation and stem cell lineage (Discher et al, Science, 2005). In hematology, we know that platelets are shear activated and attenuate force based on substrate stiffness, and that endothelial cells align with flow and are activated by shear stress. Blood cells pass through, and interact with, biological matrices such as fibrin clots and the vascular wall, but the physical and biochemical aspects of these interactions are indistinguishable from one another in vivo. As such, there is a gap in knowledge as to how blood cells respond to matrices as they transit through them. To decouple the physical and biochemical interactions of blood cells and biological matrices, we sought to recreate the physical geometry of a fibrin network in a...
Blood cells circulate in a dynamic fluidic environment, and during hematologic processes such as ... more Blood cells circulate in a dynamic fluidic environment, and during hematologic processes such as hemostasis, thrombosis, and inflammation, blood cells interact biophysically with a myriad of vascular matrices-blood clots and the subendothelial matrix. While it is known that adherent cells physiologically respond to the mechanical properties of their underlying matrices, how blood cells interact with their mechanical microenvironment of vascular matrices remains poorly understood. To that end, we developed microfluidic systems that achieve high fidelity, high resolution, single-micron PDMS features that mimic the physical geometries of vascular matrices. With these electron beam lithography (EBL)-based microsystems, the physical interactions of individual blood cells with the mechanical properties of the matrices can be directly visualized. We observe that the physical presence of the matrix, in and of itself, mediates hematologic processes of the three major blood cell types: platel...
Rapid antigen tests (RATs) have become an invaluable tool for combating the COVID-19 pandemic. Ho... more Rapid antigen tests (RATs) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of 10 commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using both individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR cycle threshold (C T ) value (a rough proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subgenomic RNA (sgRNA) may indicate ... more Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subgenomic RNA (sgRNA) may indicate actively replicating virus, but sgRNA abundance has not been systematically compared between SARS-CoV-2 variants. sgRNA was quantified in 169 clinical samples by real-time reverse-transcription polymerase chain reaction, demonstrating similar relative abundance among known variants. Thus, sgRNA detection can identify individuals with active viral replication regardless of variant.
Viability of saliva samples stored for longer than 28 days has not been reported in the literatur... more Viability of saliva samples stored for longer than 28 days has not been reported in the literature. The COVID-19 pandemic has spawned new research evaluating various sample types, thus large biobanks have been started. Residual saliva samples from university student surveillance testing were retested on SalivaDirect and compared with original RT-PCR (cycle threshold values) and quantitative antigen values for each month in storage. We conclude that saliva samples stored at -80°C are still viable in detecting SARS-CoV-2 after 12 months of storage, establishing the validity of these samples for future testing.
Context.— Diagnostic testing for SARS-CoV-2 in symptomatic and asymptomatic children remains inte... more Context.— Diagnostic testing for SARS-CoV-2 in symptomatic and asymptomatic children remains integral to care, particularly for supporting return to and attendance in schools. The concordance of SARS-CoV-2 detection in children, using various specimen types, has not been widely studied. Objective.— To compare 3 sample types for SARS-CoV-2 polymerase chain reaction (PCR) testing in children, collected and tested at a single facility. Design.— We prospectively recruited 142 symptomatic and asymptomatic children/young adults into a sample comparison study performed in a single health care system. Each child provided self-collected saliva, and a trained health care provider collected a mid-turbinate nasal swab and nasopharyngeal (NP) swab. Specimens were assayed within 24 hours of collection by using reverse transcription–polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 on a single testing platform. Results.— Concurrently collected saliva and mid-turbinate swabs had greater than ...
Blood flow in sickle cell disease (SCD) can substantially differ from normal blood flow due to si... more 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.
Physiological processes such as blood clotting and wound healing as well as pathologies such as f... more 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.
Neutrophil extracellular traps (NETs) are antimicrobial cobweb‐structured materials produced by i... more Neutrophil extracellular traps (NETs) are antimicrobial cobweb‐structured materials produced by immune cells for clearance of pathogens in the body, but are paradoxically associated with biofilm formation and exacerbated lung infections. To provide a better materials perspective on the pleiotropic roles played by NETs at diverse compositions/concentrations, a NETs‐like material (called “microwebs”, abbreviated as μwebs) is synthesized for decoding the antimicrobial activity of NETs against Staphylococcus aureus in infection‐relevant conditions. It is shown that μwebs composed of low‐to‐intermediate concentrations of DNA‐histone complexes successfully trap and inhibit S. aureus growth and biofilm formation. However, with growing concentrations and histone proportions, the resulting microwebs appear gel‐like structures accompanied by reduced antimicrobial activity that can even promote the formation of S. aureus biofilms. The simplified model of NETs provides materials‐based evidence ...
Proceedings of the National Academy of Sciences, 2020
Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screenin... more 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 c...
Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vi... more Widespread testing for the presence of the novel coronavirus 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 reverse transcription polymerase chain reaction (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 compare...
Background: Originally described as a monogenic hemoglobin disorder resulting in increased red bl... more Background: Originally described as a monogenic hemoglobin disorder resulting in increased red blood cell (RBC) stiffness leading to vaso-occlusion, sickle cell disease (SCD) is now known to be a vasculopathic disease with some semblance to cardiovascular disease in which the endothelium is inflamed. While adhesive RBC-endothelial interactions, inflammatory cytokines, and hemolysis all contribute to SCD vasculopathy, whether the increased stiffness of sickle RBCs directly contributes to endothelial inflammation is unknown. Endothelial cells are now known to mechanotransduce shear forces into biological signals. Pathological alteration of such forces leads to proinflammatory endothelial cell signaling including upregulation of VCAM-1 and E-selectin, which contribute to atherosclerotic plaques leading to myocardial infarction and stroke (Abe, ATVB, 2014). In addition, under normal homeostatic conditions, RBCs do not come into contact with the endothelium due to a cell-free layer creat...
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Papers by Wilbur Lam