RBCs Regulate Platelet Function and Hemostasis under Shear Conditions through Biophysical and Biochemical Means.

Red blood cells (RBCs) have been hypothesized to support hemostasis by facilitating platelet margination and releasing platelet-activating factors such as adenosine diphosphate (ADP). Significant knowledge gaps remain regarding how RBCs influence platelet function, especially in (patho)physiologically relevant hemodynamic conditions. Here we present results showing how RBCs affect platelet function and hemostasis in conditions of anemia, thrombocytopenia, and pancytopenia, and how the biochemical and biophysical properties of RBCs regulate platelet function at the blood-vessel wall interface and in the fluid phase under flow conditions. We found that RBCs promoted platelet deposition to collagen under flow conditions in moderate (50  103/L) but not severe (10  103/L) thrombocytopenia in vitro. Reduction in hematocrit by 45% led to increased bleeding in mice with hemolytic anemia. In contrast, bleeding diathesis was observed in mice with a 90% but not with a 60% reduction in platelet counts. RBC transfusion improved hemostasis by enhancing fibrin clot formation at the site of vascular injury in mice with severe pancytopenia induced by total body irradiation. Altering membrane deformability changed the ability of RBCs to promote platelet aggregation. RBC-derived ADP contributed to platelet activation and aggregation in vitro under pathologically high shear stresses, as observed in patients supported by left ventricular assist devices. These findings demonstrate that RBCs support platelet function and hemostasis through multiple mechanisms, both at the blood-vessel wall interface and in the fluidic phase of circulation.

Clots reveal anomalous elastic behavior of fiber networks.

The adaptive mechanical properties of soft and fibrous biological materials are relevant to their functionality. The emergence of the macroscopic response of these materials to external stress and intrinsic cell traction from local deformations of their structural components is not well understood. Here, we investigate the nonlinear elastic behavior of blood clots by combining microscopy, rheology, and an elastic network model that incorporates the stretching, bending, and buckling of constituent fibrin fibers. By inhibiting fibrin cross-linking in blood clots, we observe an anomalous softening regime in the macroscopic shear response as well as a reduction in platelet-induced clot contractility. Our model explains these observations from two independent macroscopic measurements in a unified manner, through a single mechanical parameter, the bending stiffness of individual fibers. Supported by experimental evidence, our mechanics-based model provides a framework for predicting and comprehending the nonlinear elastic behavior of blood clots and other active biopolymer networks in general.

Prospection of Peptide Inhibitors of Thrombin from Diverse Origins Using a Machine Learning Pipeline.

Thrombin is a key enzyme involved in the development and progression of many cardiovascular diseases. Direct thrombin inhibitors (DTIs), with their minimum off-target effects and immediacy of action, have greatly improved the treatment of these diseases. However, the risk of bleeding, pharmacokinetic issues, and thrombotic complications remain major concerns. In an effort to increase the effectiveness of the DTI discovery pipeline, we developed a two-stage machine learning pipeline to identify and rank peptide sequences based on their effective thrombin inhibitory potential. The positive dataset for our model consisted of thrombin inhibitor peptides and their binding affinities (KI) curated from published literature, and the negative dataset consisted of peptides with no known thrombin inhibitory or related activity. The first stage of the model identified thrombin inhibitory sequences with Matthew's Correlation Coefficient (MCC) of 83.6%. The second stage of the model, which covers an eight-order of magnitude range in KI values, predicted the binding affinity of new sequences with a log room mean square error (RMSE) of 1.114. These models also revealed physicochemical and structural characteristics that are hidden but unique to thrombin inhibitor peptides. Using the model, we classified more than 10 million peptides from diverse sources and identified unique short peptide sequences (<15 aa) of interest, based on their predicted KI. Based on the binding energies of the interaction of the peptide with thrombin, we identified a promising set of putative DTI candidates. The prediction pipeline is available on a web server.

Hyperbaric treatment of platelets is comparable to cold storage alone over 14 days.

BACKGROUND: Platelets stored at room temperature (22-24°C) for transfusion purposes have a shelf life of 5-7 days, or 72 h when stored refrigerated (1-6°C). The limited shelf life of platelet products severely compromises platelet inventory. We hypothesized that cold storage of platelets in 100% plasma using xenon gas under high pressure would extend shelf life to 14 days. STUDY DESIGN AND METHODS: Double apheresis platelet units were collected and split equally between two bags. One unit was placed in a hyperbaric chamber, pressurized to 4 bars with a xenon/oxygen gas mixture, and placed in a refrigerator for 14 days (Xe). The remaining unit was aliquoted into mini-bags (10 ml) for storage at room temperature (RTP) or in cold (CSP). Samples were assayed on days 5 (RTP) or 14 (Xe and CSP) for count, metabolism, clot strength, platelet aggregation, and activation markers. RESULTS: The platelet count in Xe samples was lower than that of RTP but significantly higher than CSP. Despite similar levels of glucose and lactate, the pH of Xe samples was significantly lower than CSP. Glycoprotein expression was better preserved by Xe storage compared to CSP, but no differences in activation were observed. Thromboelastography and aggregometry results were comparable between all groups. DISCUSSION: Cold storage of platelets in plasma with hyperbaric xenon provides no significant improvement in platelet function over cold storage alone. The use of a hyperbaric chamber and the slow off-gassing of Xe-stored units complicate platelet storage and delivery logistics.

A low-cost, high-throughput microfluidic nano-culture platform for functional metagenomics.

Functional metagenomics is an attractive culture-independent approach for functional screening of diverse microbiomes to identify known and novel genes. Since functional screening can involve sifting through tens of thousands of metagenomic library clones, an easy high-throughput screening approach is desirable. Here, we demonstrate a proof-of-concept application of a low-cost, high-throughput droplet based microfluidic assay to the selection of antibiotic resistance genes from a soil metagenomic library. Metagenomic library members encapsulated in nanoliter volume water-in-oil droplets were printed on glass slides robotically, and cell growth in individual drops in the presence of ampicillin was imaged and quantified to identify ampicillin-resistant clones. From the hits, true positives were confirmed by sequencing and functional validation. The ease of liquid handling, ease of set-up, low cost, and robust workflow makes the droplet-based nano-culture platform a promising candidate for screening and selection assays for functional metagenomic libraries.

The interaction of vortical flows with red cells in venous valve mimics.

The motion of cells orthogonal to the direction of main flow is of importance in natural and engineered systems. The lateral movement of red blood cells (RBCs) distal to sudden expansion is considered to influence the formation and progression of thrombosis in venous valves, aortic aneurysms, and blood-circulating devices and is also a determining parameter for cell separation applications in flow-focusing microfluidic devices. Although it is known that the unique geometry of venous valves alters the blood flow patterns and cell distribution in venous valve sinuses, the interactions between fluid flow and RBCs have not been elucidated. Here, using a dilute cell suspension in an in vitro microfluidic model of a venous valve, we quantified the spatial distribution of RBCs by microscopy and image analysis, and using micro-particle image velocimetry and 3D computational fluid dynamics simulations, we analyzed the complex flow patterns. The results show that the local hematocrit in the valve pockets is spatially heterogeneous and is significantly different from the feed hematocrit. Above a threshold shear rate, the inertial separation of streamlines and lift forces contribute to an uneven distribution of RBCs in the vortices, the entrapment of RBCs in the vortices, and non-monotonic wall shear stresses in the valve pockets. Our experimental and computational characterization provides insights into the complex interactions between fluid flow, RBC distribution, and wall shear rates in venous valve mimics, which is of relevance to understanding the pathophysiology of thrombosis and improving cell separation efficiency.

Nicotine Affects Murine Aortic Stiffness and Fatigue Response During Supraphysiological Cycling.

Nicotine exposure is a major risk factor for several cardiovascular diseases. Although the deleterious effects of nicotine on aortic remodeling processes have been studied to some extent, the biophysical consequences are not fully elucidated. In this investigation, we applied quasi-static and dynamic loading to quantify ways in which exposure to nicotine affects the mechanical behavior of murine arterial tissue. Segments of thoracic aortas from C57BL/6 mice exposed to 25 mg/kg/day of subcutaneous nicotine for 28 days were subjected to uniaxial tensile loading in an open-circumferential configuration. Comparing aorta segments from nicotine-treated mice relative to an equal number of control counterparts, stiffness in the circumferential direction was nearly twofold higher (377 kPa ± 165 kPa versus 191 kPa ± 65 kPa, n = 5, p = 0.03) at 50% strain. Using a degradative power-law fit to fatigue data at supraphysiological loading, we observed that nicotine-treated aortas exhibited significantly higher peak stress, greater loss of tension, and wider oscillation band than control aortas (p ≤ 0.01 for all three variables). Compared to simple stress relaxation tests, fatigue cycling is shown to be more sensitive and versatile in discerning nicotine-induced changes in mechanical behavior over many cycles. Supraphysiological fatigue cycling thus may have broader potential to reveal subtle changes in vascular mechanics caused by other exogenous toxins or pathological conditions.

peri-Adventitial delivery of smooth muscle cells in porous collagen scaffolds for treatment of experimental abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is associated with the loss of vascular smooth muscle cells (SMCs) within the vessel wall. Direct delivery of therapeutic cells is challenging due to impaired mechanical integrity of the vessel wall. We hypothesized that porous collagen scaffolds can be an effective vehicle for the delivery of human-derived SMCs to the site of AAA. The purpose was to evaluate if the delivery of cell-seeded scaffolds can abrogate progressive expansion in a mouse model of AAA. Collagen scaffolds seeded with either primary human aortic SMCs or induced pluripotent stem cell derived-smooth muscle progenitor cells (iPSC-SMPs) had >80% in vitro cell viability and >75% cell penetrance through the scaffold's depth, while preserving smooth muscle phenotype. The cell-seeded scaffolds were successfully transplanted onto the murine aneurysm peri-adventitia on day 7 following AAA induction using pancreatic porcine elastase infusion. Ultrasound imaging revealed that SMC-seeded scaffolds significantly reduced the aortic diameter by 28 days, compared to scaffolds seeded with iPSC-SMPs or without cells (acellular scaffold), respectively. Bioluminescence imaging demonstrated that both cell-seeded scaffold groups had cellular localization to the aneurysm but a decline in survival with time. Histological analysis revealed that both cell-seeded scaffold groups had more SMC retention and less macrophage invasion into the medial layer of AAA lesions, when compared to the acellular scaffold treatment group. Our data suggest that scaffold-based SMC delivery is feasible and may constitute a platform for cell-based AAA therapy.

Cold-stored platelets have better preserved contractile function in comparison with room temperature-stored platelets over 21 days.

Although it is well established that transfusion of platelets in cases of severe bleeding reduces mortality, the availability of platelets is hampered by harsh restrictions on shelf life due to elevated risks of microbial contamination and functional losses with room temperature-stored platelets (RTP) kept at 22°C. In contrast, many recent studies have shown that 4°C cold-stored platelets (CSP) are able to overcome these shortcomings leading to the recent Food and Drug Administration licensure for 14-day stored CSP when conventional platelets are unavailable. This work expands the evidence supporting superiority of CSP function by assaying the less explored platelet-mediated clot retraction of RTP and CSP in either autologous plasma (AP) or platelet additive solution (PAS) for up to 21 days. The results demonstrate that CSP have better preservation of contractile function, exhibiting retraction for up to 21 days in both AP and PAS and forming highly ordered fibrin scaffolds similar to those of fresh platelets. In contrast, RTP stored in AP showed impaired contractile function by Day 5 with no retraction after 10 days, whereas PAS-stored RTP retained contractile function for up to 21 days. Collectively, these findings support extended storage of CSP and suggest that storage in PAS can mitigate functional losses in RTP.

A Facile High-Throughput Model of Surface-Independent Staphylococcus aureus Biofilms by Spontaneous Aggregation.

Many microbes in their natural habitats are found in biofilm ecosystems attached to surfaces and not as free-floating (planktonic) organisms. Furthermore, it is estimated that nearly 80% of human infections are associated with biofilms. Biofilms are traditionally defined as three-dimensional, structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. While this view of biofilm largely arises from in vitro studies under static or flow conditions, in vivo observations have indicated that this view of biofilms is essentially true only for foreign-body infections on catheters or implants where biofilms are attached to the biomaterial. In mucosal infections such as chronic wounds or cystic fibrosis or joint infections, biofilms can be found unattached to a surface and as three-dimensional aggregates. In this work, we describe a high-throughput model of aggregate biofilms of methicillin-resistant Staphylococcus aureus (MRSA) using 96-well plate hanging-drop technology. We show that MRSA forms surface-independent biofilms, distinct from surface-attached biofilms, that are rich in exopolymeric proteins, polysaccharides, and extracellular DNA (eDNA), express biofilm-related genes, and exhibit heightened antibiotic resistance. We also show that the surface-independent biofilms of clinical isolates of MRSA from cystic fibrosis and central catheter-related infections demonstrate morphological differences. Overall, our results show that biofilms can form by spontaneous aggregation without attachment to a surface, and this new in vitro system can model surface-independent biofilms that may more closely mimic the corresponding physiological niche during infection.IMPORTANCE The canonical model of biofilm formation begins with the attachment and growth of microbial cells on a surface. While these in vitro models reasonably mimic biofilms formed on foreign bodies such as catheters and implants, this is not the case for biofilms formed in cystic fibrosis and chronic wound infections, which appear to present as aggregates not attached to a surface. The hanging-drop model of biofilms of methicillin-resistant Staphylococcus aureus (MRSA), the major causative organism of skin and soft tissue infections, shows that these biofilms display morphological and antibiotic response patterns that are distinct from those of their surface-attached counterparts, and biofilm growth is consistent with their in vivo location. The simplicity and throughput of this model enable adoption to investigate other single or polymicrobial biofilms in a physiologically relevant setting.

Identification of Distinct Characteristics of Antibiofilm Peptides and Prospection of Diverse Sources for Efficacious Sequences.

A majority of microbial infections are associated with biofilms. Targeting biofilms is considered an effective strategy to limit microbial virulence while minimizing the development of antibiotic resistance. Toward this need, antibiofilm peptides are an attractive arsenal since they are bestowed with properties orthogonal to small molecule drugs. In this work, we developed machine learning models to identify the distinguishing characteristics of known antibiofilm peptides, and to mine peptide databases from diverse habitats to classify new peptides with potential antibiofilm activities. Additionally, we used the reported minimum inhibitory/eradication concentration (MBIC/MBEC) of the antibiofilm peptides to create a regression model on top of the classification model to predict the effectiveness of new antibiofilm peptides. We used a positive dataset containing 242 antibiofilm peptides, and a negative dataset which, unlike previous datasets, contains peptides that are likely to promote biofilm formation. Our model achieved a classification accuracy greater than 98% and harmonic mean of precision-recall (F1) and Matthews correlation coefficient (MCC) scores greater than 0.90; the regression model achieved an MCC score greater than 0.81. We utilized our classification-regression pipeline to evaluate 135,015 peptides from diverse sources for potential antibiofilm activity, and we identified 185 candidates that are likely to be effective against preformed biofilms at micromolar concentrations. Structural analysis of the top 37 hits revealed a larger distribution of helices and coils than sheets, and common functional motifs. Sequence alignment of these hits with known antibiofilm peptides revealed that, while some of the hits showed relatively high sequence similarity with known peptides, some others did not indicate the presence of antibiofilm activity in novel sources or sequences. Further, some of the hits had previously recognized therapeutic properties or host defense traits suggestive of drug repurposing applications. Taken together, this work demonstrates a new in silico approach to predicting antibiofilm efficacy, and identifies promising new candidates for biofilm eradication.

Fibrin prestress due to platelet aggregation and contraction increases clot stiffness.

Efficient hemorrhagic control is attained through the formation of strong and stable blood clots at the site of injury. Although it is known that platelet-driven contraction can dramatically influence clot stiffness, the underlying mechanisms by which platelets assist fibrin in resisting external loads are not understood. In this study, we delineate the contribution of platelet-fibrin interactions to clot tensile mechanics using a combination of new mechanical measurements, image analysis, and structural mechanics simulation. Based on uniaxial tensile test data using custom-made microtensometer and fluorescence microscopy of platelet aggregation and platelet-fibrin interactions, we show that integrin-mediated platelet aggregation and actomyosin-driven platelet contraction synergistically increase the elastic modulus of the clots. We demonstrate that the mechanical and geometric response of an active contraction model of platelet aggregates compacting vicinal fibrin is consistent with the experimental data. The model suggests that platelet contraction induces prestress in fibrin fibers and increases the effective stiffness in both cross-linked and noncross-linked clots. Our results provide evidence for fibrin compaction at discrete nodes as a major determinant of mechanical response to applied loads.

Cold storage of platelets in platelet additive solution maintains mitochondrial integrity by limiting initiation of apoptosis-mediated pathways.

BACKGROUND: Cold storage of platelets in plasma maintains hemostatic function and is an attractive alternative to room temperature platelets (RTPs). We have recently shown that functional differences between cold-stored platelets (CSPs) and RTPs after 5-day storage are associated with mitochondrial respiration and that CSPs in platelet (PLT) additive solution (PAS) can maintain hemostatic function for at least 15 days. STUDY DESIGN AND METHODS: This study tested the hypothesis that cold storage in PAS preserves mitochondrial integrity by reducing PLT apoptosis. CSPs and RTPs in plasma or PAS were stored and assayed for up to 15 days for mitochondrial function and integrity, mitochondrial-associated mRNA transcript expression, apoptotic proteins, and apoptotic flow cytometry metrics. RESULTS: CSP preserved mitochondria-associated mRNA comparable to baseline levels, improved mitochondrial respiration, and minimized depolarization to Day 15. Additionally, CSPs had minimal induction of caspases, preservation of plasma membrane integrity, and low expression of pro-apoptotic Bax. Storage in PAS appeared to be protective for RTPs in some parameters and enhanced the effects of CSPs. CONCLUSION: Mitochondrial function and molecular analyses defined CSP priming as distinctly different from the well-documented RTP storage lesion. While current blood bank storage at room temperature is limited to 5 to 7 days, refrigeration and storage in PAS for up to 15 days may represent an opportunity to enhance inventories and access to PLT hemostatic support for bleeding patients.

Automated Motion Tracking and Data Extraction for Red Blood Cell Biomechanics.

Red blood cell biomechanics can provide us with a deeper understanding of macroscopic physiology and have the potential of being used for diagnostic purposes. In diseases like sickle cell anemia and malaria, reduced red blood cell deformability can be used as a biomarker, leading to further assays and diagnoses. A microfluidic system is useful for studying these biomechanical properties. We can observe detailed red blood cell mechanical behavior as they flow through microcapillaries using high-speed imaging and microscopy. Microfluidic devices are advantageous over traditional methods because they can serve as high-throughput tests. However, to rapidly analyze thousands of cells, there is a need for powerful image processing tools and software automation. We describe a workflow process using Image-Pro to identify and track red blood cells in a video, take measurements, and export the data for use in statistical analysis tools. The information in this protocol can be applied to large-scale blood studies where entire cell populations need to be analyzed from many cohorts of donors. © 2020 The Authors. Basic Protocol 1: Enhancing raw video for motion tracking Basic Protocol 2: Extracting motion tracking data from enhanced video.

Image-based analysis and simulation of the effect of platelet storage temperature on clot mechanics under uniaxial strain.

Optimal strength and stability of blood clots are keys to hemostasis and in prevention of hemorrhagic or thrombotic complications. Clots are biocomposite materials composed of fibrin network enmeshing platelets and other blood cells. We have previously shown that the storage temperature of platelets significantly impacts clot structure and stiffness. The objective of this work is to delineate the relationship between morphological characteristics and mechanical response of clot networks. We examined scanning electron microscope images of clots prepared from fresh apheresis platelets, and from apheresis platelets stored for 5 days at room temperature or at 4 °C, suspended in pooled plasma. Principal component analysis of nine different morphometric parameters revealed that a single principal component (PC1) can distinguish the effect of platelet storage on clot ultrastructure. Finite element analysis of clot response to uniaxial strain was used to map the spatially heterogeneous distribution of strain energy density for each clot. At modest deformations (25% strain), a single principal component (PC2) was able to predict these heterogeneities as quantified by variability in strain energy density distribution and in linear elastic stiffness, respectively. We have identified structural parameters that are primary regulators of stress distribution, and the observations provide insights into the importance of spatial heterogeneity on hemostasis and thrombosis.

Design Considerations and Assays for Hemocompatibility of FDA-Approved Nanoparticles.

Nanoparticles have numerous biomedical applications including, but not limited to, targeted drug delivery, diagnostic imaging, sensors, and implants for a wide range of diseases including cancer, diabetes, heart disease, and tuberculosis. Although the mode of delivery of the nanoparticles depends on the application and the disease, the nanoparticles are often in immediate contact with the systemic circulation either because of intravenous administration or their ability to enter the bloodstream with relative ease or their longer survival time in circulation. Once in circulation, the nanoparticles may elicit unintended hemostatic and inflammatory responses, and hence the design of nanoparticles for therapeutic applications should take broad hemocompatibility concerns into consideration. In this review, we present the principles underlying the structural and functional design of various classes of nanoparticles that are currently approved by the US Food and Drug Administration, categorize these particles based on their interactions with cardiovascular tissues and ensuing adverse events, and also describe various in vitro assays that may be used evaluate their hemocompatibility.

CgSTE11 mediates cross tolerance to multiple environmental stressors in Candida glabrata.

Candida glabrata is a human commensal and an opportunistic human fungal pathogen. It is more closely related to the model yeast Saccharomyces cerevisiae than other Candida spp. Compared with S. cerevisiae, C. glabrata exhibits higher innate tolerance to various environmental stressors, including hyperthermal stress. Here we investigate the molecular mechanisms of C. glabrata adaptation to heat stress via adaptive laboratory evolution. We show that all parallel evolved populations readily adapt to hyperthermal challenge (from 47 °C to 50 °C) and exhibit convergence in evolved phenotypes with extensive cross-tolerance to various other environmental stressors such as oxidants, acids, and alcohols. Genome resequencing identified fixation of mutations in CgSTE11 in all parallel evolved populations. The CgSTE11 homolog in S. cerevisiae plays crucial roles in various mitogen-activated protein kinase (MAPK) signaling pathways, but its role is less understood in C. glabrata. Subsequent verification confirmed that CgSTE11 is important in hyperthermal tolerance and the observed extensive cross-tolerance to other environmental stressors. These results support the hypothesis that CgSTE11 mediates cross-talks between MAPK signaling pathways in C. glabrata in response to environmental challenges.

Nano-biofilm Arrays as a Novel Universal Platform for Microscale Microbial Culture and High-Throughput Downstream Applications.

Biofilms are the predominant mode of microbial growth and it is now fully accepted that a majority of infections in humans are associated with a biofilm etiology. Biofilms are defined as attached and structured microbial communities surrounded by a protective exopolymeric matrix. Importantly, sessile microorganisms growing within a biofilm are highly resistant to antimicrobial agents. Thus, there is an urgent need to develop new and improved anti-biofilm therapies. Unfortunately, most of the current techniques for in-vitro biofilm formation are not compatible with high throughput screening techniques that can speed up discovery of new drugs with anti-biofilm activity. To try to overcome this major impediment, our group has developed a novel technique consisting of micro-scale culture of microbial biofilms on a microarray platform. Using this technique, hundreds to thousands of microbial biofilms, each with a volume of approximately 30-50 nanolitres, can be simultaneously formed on a standard microscope slide. Despite more than three orders of magnitude of miniaturization over conventional biofilms, these nanobiofilms display similar growth, structural and phenotypic properties, including antibiotic drug resistance. These nanobiofilm chips are amenable to automation, drastically reducing assay volume and costs. This technique platform allows for true high-throughput screening in search for new anti-biofilm drugs.

An Automated Algorithm to Quantify Collagen Distribution in Aortic Wall.

Arterial diseases including abdominal aortic aneurysm and atherosclerosis are biomechanical diseases characterized by significant changes in the structure and strength of the vessel wall. It is now established that local variations in fibrillar collagen and elastin matrix turnover is critical to arterial stiffening and progression of the disease. The collagen content in the aortic wall has nominally been quantified by biochemical assays and immunohistochemical analysis as the total amount because of the difficulty in separating the media and adventitia. In this work, we have developed an algorithm for automatic quantification of layer-specific collagen content from bright-field and polarized microscopic images of histological sections of mouse aorta stained with Picrosirius red (PSR) stain. The images were processed sequentially including separation of layers, erosion, segregation of regions, binarization, and quantification of pixel intensities to obtain collagen content in the media and adventitia separately. We observed that the automated algorithm rapidly and accurately quantified collagen content from a wide range of image quality compared with manual measurements particularly when the medial and adventitial layers overlap. Together, our algorithm will be of significant impact in the rapid, reliable, and accurate analyses of collagen distribution in histological sections of connective tissues.

Red blood cell deformability is diminished in patients with Chronic Fatigue Syndrome.

BACKGROUND: Myalgic encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a poorly understood disease. Amongst others symptoms, the disease is associated with profound fatigue, cognitive dysfunction, sleep abnormalities, and other symptoms that are made worse by physical or mental exertion. While the etiology of the disease is still debated, evidence suggests oxidative damage to immune and hematological systems as one of the pathophysiological mechanisms of the disease. Since red blood cells (RBCs) are well-known scavengers of oxidative stress, and are critical in microvascular perfusion and tissue oxygenation, we hypothesized that RBC deformability is adversely affected in ME/CFS. METHODS: We used a custom microfluidic platform and high-speed microscopy to assess the difference in deformability of RBCs obtained from ME/CFS patients and age-matched healthy controls. RESULTS AND CONCLUSION: We observed from various measures of deformability that the RBCs isolated from ME/CFS patients were significantly stiffer than those from healthy controls. Our observations suggest that RBC transport through microcapillaries may explain, at least in part, the ME/CFS phenotype, and promises to be a novel first-pass diagnostic test.