Novel Coagulation Test Detects Anticoagulation Resistance and Is Associated With Thrombotic Events in Pediatric Patients Requiring Extracorporeal Membrane Oxygenation.

Bivalirudin, an IV direct thrombin inhibitor, and unfractionated heparin (UFH) are frequently used anticoagulants in the pediatric critical care setting. An accurate, specific, point-of-care test to quantify and detect anticoagulation resistance is not currently available. This study evaluates the ability of a rapid (< 10 min), micro-volume (< 50 uL) coagulation test to detect and quantify the anticoagulation effect of bivalirudin and UFH using a functional, clot time endpoint in pediatric critical care patients. DESIGN: Single-site retrospective laboratory sample analysis and chart review. SETTING: A 105-bed pediatric and cardiac ICUs delivering extracorporeal membrane oxygenation. SUBJECTS: Forty-one citrated, frozen, biobanked plasma specimens comprising 21 with bivalirudin and 20 with UFH from 15 anticoagulated pediatric patients were analyzed. Thirteen patients were on extracorporeal membrane oxygenation, one had a submassive pulmonary embolism, and one was on a left ventricular assist device. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: A Clotting Time Score (CTS) was derived on each sample. The CTS detected patients that had developed a pathologic clotting event with 100% sensitivity and 82% specificity compared with prothrombin time with 25% sensitivity/76% specificity and activated partial thromboplastin time with 0% sensitivity/0% specificity. Additionally, the CTS detected subtherapeutic anticoagulation in response to UFH in patients that were clinically determined to be UFH resistant requiring alternative anticoagulation with bivalirudin. CONCLUSIONS: The CTS appears to be a clinically valuable indicator of coagulation status in patients treated with either UFH or bivalirudin. Results outside of the therapeutic range due to inadequate dosing or anticoagulation resistance appeared to be associated with clot formation. CTS testing may reduce the risk of anticoagulation-related complications via the rapid identification of patients at high risk for pathologic thrombotic events.

MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme.

ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.

Predictive modelling of thrombus formation in diabetic retinal microaneurysms.

Microaneurysms (MAs) are one of the earliest clinically visible signs of diabetic retinopathy (DR). Vision can be reduced at any stage of DR by MAs, which may enlarge, rupture and leak fluid into the neural retina. Recent advances in ophthalmic imaging techniques enable reconstruction of the geometries of MAs and quantification of the corresponding haemodynamic metrics, such as shear rate and wall shear stress, but there is lack of computational models that can predict thrombus formation in individual MAs. In this study, we couple a particle model to a continuum model to simulate the platelet aggregation in MAs with different shapes. Our simulation results show that under a physiologically relevant blood flow rate, thrombosis is more pronounced in saccular-shaped MAs than fusiform-shaped MAs, in agreement with recent clinical findings. Our model predictions of the size and shape of the thrombi in MAs are consistent with experimental observations, suggesting that our model is capable of predicting the formation of thrombus for newly detected MAs. This is the first quantitative study of thrombosis in MAs through simulating platelet aggregation, and our results suggest that computational models can be used to predict initiation and development of intraluminal thrombus in MAs as well as provide insights into their role in the pathophysiology of DR.

Quantifying Fibrinogen-Dependent Aggregation of Red Blood Cells in Type 2 Diabetes Mellitus.

Fibrinogen is regarded as the main glycoprotein in the aggregation of red blood cells (RBCs), a normally occurring phenomenon that has a major impact on blood rheology and hemodynamics, especially under pathological conditions, including type 2 diabetes mellitus (T2DM). In this study, we investigate the fibrinogen-dependent aggregation dynamics of T2DM RBCs through patient-specific predictive computational simulations that invoke key parameters derived from microfluidic experiments. We first calibrate our model parameters at the doublet (a rouleau consisting of two aggregated RBCs) level for healthy blood samples by matching the detaching force required to fully separate RBC doublets with measurements using atomic force microscopy and optical tweezers. Using results from companion microfluidic experiments that also provide in vitro quantitative information on cell-cell adhesive dynamics, we then quantify the rouleau dissociation dynamics at the doublet and multiplet (a rouleau consisting of three or more aggregated RBCs) levels for obese patients with or without T2DM. Specifically, we examine the rouleau breakup rate when it passes through microgates at doublet level and investigate the effect of rouleau alignment in altering its breakup pattern at multiplet level. This study seamlessly integrates in vitro experiments and simulations and consequently enhances our understanding of the complex cell-cell interaction, highlighting the importance of the aggregation and disaggregation dynamics of RBCs in patients at increased risk of microvascular complications.

Quantifying Shear-Induced Deformation and Detachment of Individual Adherent Sickle Red Blood Cells.

Vaso-occlusive crisis, a common painful complication of sickle cell disease, is a complex process triggered by intercellular adhesive interactions among blood cells and the endothelium in all human organs (e.g., the oxygen-rich lung as well as hypoxic systems such as liver and kidneys). We present a combined experimental-computational study to quantify the adhesive characteristics of sickle mature erythrocytes (SMEs) and irreversibly sickled cells (ISCs) under flow conditions mimicking those in postcapillary venules. We employed an in vitro microfluidic cell adherence assay, which is coated uniformly with fibronectin. We investigated the adhesion dynamics of SMEs and ISCs in pulsatile flow under well-controlled hypoxic conditions, inferring the cell adhesion strength by increasing the flow rate (or wall shear stress (WSS)) until the onset of cell detachment. In parallel, we performed simulations of individual SMEs and ISCs under shear. We introduced two metrics to quantify the adhesion process, the cell aspect ratio (AR) as a function of WSS and its rate of change (the dynamic deformability index). We found that the AR of SMEs decreases significantly with the increase of WSS, consistent between the experiments and simulations. In contrast, the AR of ISCs remains constant in time and independent of the flow rate. The critical WSS value for detaching a single SME in oxygenated state is in the range of 3.9-5.5 Pa depending on the number of adhesion sites; the critical WSS value for ISCs is lower than that of SMEs. Our simulations show that the critical WSS value for SMEs in deoxygenated state is above 6.2 Pa (multiple adhesion sites), which is greater than their oxygenated counterparts. We investigated the effect of cell shear modulus on the detachment process; we found that for the same cell adhesion spring constant, the higher shear modulus leads to an earlier cell detachment from the functionalized surface. These findings may aid in the understanding of individual roles of sickle cell types in sickle cell disease vaso-occlusion.

Simultaneous polymerization and adhesion under hypoxia in sickle cell disease.

Polymerization and adhesion, dynamic processes that are hallmarks of sickle cell disease (SCD), have thus far been studied in vitro only separately. Here, we present quantitative results of the simultaneous and synergistic effects of adhesion and polymerization of deoxygenated sickle hemoglobin (HbS) in the human red blood cell (RBC) on the mechanisms underlying vasoocclusive pain crisis. For this purpose, we employ a specially developed hypoxic microfluidic platform, which is capable of inducing sickling and unsickling of RBCs in vitro, to test blood samples from eight patients with SCD. We supplemented these experimental results with detailed molecular-level computational simulations of cytoadherence and biorheology using dissipative particle dynamics. By recourse to image analysis techniques, we characterize sickle RBC maturation stages in the following order of the degree of adhesion susceptibility under hypoxia: sickle reticulocytes in circulation (SRs) → sickle mature erythrocytes (SMEs) → irreversibly sickled cells (ISCs). We show that (i) hypoxia significantly enhances sickle RBC adherence; (ii) HbS polymerization enhances sickle cell adherence in SRs and SMEs, but not in ISCs; (iii) SRs exhibit unique adhesion dynamics where HbS fiber projections growing outward from the cell surface create multiple sites of adhesion; and (iv) polymerization stimulates adhesion and vice versa, thereby establishing the bidirectional coupling between the two processes. These findings offer insights into possible mechanistic pathways leading to vasoocclusion crisis. They also elucidate the processes underlying the onset of occlusion that may involve circulating reticulocytes, which are more abundant in hemolytic anemias due to robust compensatory erythropoiesis.

Synergistic Integration of Laboratory and Numerical Approaches in Studies of the Biomechanics of Diseased Red Blood Cells.

In red blood cell (RBC) disorders, such as sickle cell disease, hereditary spherocytosis, and diabetes, alterations to the size and shape of RBCs due to either mutations of RBC proteins or changes to the extracellular environment, lead to compromised cell deformability, impaired cell stability, and increased propensity to aggregate. Numerous laboratory approaches have been implemented to elucidate the pathogenesis of RBC disorders. Concurrently, computational RBC models have been developed to simulate the dynamics of RBCs under physiological and pathological conditions. In this work, we review recent laboratory and computational studies of disordered RBCs. Distinguished from previous reviews, we emphasize how experimental techniques and computational modeling can be synergically integrated to improve the understanding of the pathophysiology of hematological disorders.

A deep convolutional neural network for classification of red blood cells in sickle cell anemia.

Sickle cell disease (SCD) is a hematological disorder leading to blood vessel occlusion accompanied by painful episodes and even death. Red blood cells (RBCs) of SCD patients have diverse shapes that reveal important biomechanical and bio-rheological characteristics, e.g. their density, fragility, adhesive properties, etc. Hence, having an objective and effective way of RBC shape quantification and classification will lead to better insights and eventual better prognosis of the disease. To this end, we have developed an automated, high-throughput, ex-vivo RBC shape classification framework that consists of three stages. First, we present an automatic hierarchical RBC extraction method to detect the RBC region (ROI) from the background, and then separate touching RBCs in the ROI images by applying an improved random walk method based on automatic seed generation. Second, we apply a mask-based RBC patch-size normalization method to normalize the variant size of segmented single RBC patches into uniform size. Third, we employ deep convolutional neural networks (CNNs) to realize RBC classification; the alternating convolution and pooling operations can deal with non-linear and complex patterns. Furthermore, we investigate the specific shape factor quantification for the classified RBC image data in order to develop a general multiscale shape analysis. We perform several experiments on raw microscopy image datasets from 8 SCD patients (over 7,000 single RBC images) through a 5-fold cross validation method both for oxygenated and deoxygenated RBCs. We demonstrate that the proposed framework can successfully classify sickle shape RBCs in an automated manner with high accuracy, and we also provide the corresponding shape factor analysis, which can be used synergistically with the CNN analysis for more robust predictions. Moreover, the trained deep CNN exhibits good performance even for a deoxygenated dataset and distinguishes the subtle differences in texture alteration inside the oxygenated and deoxygenated RBCs.

Cellular normoxic biophysical markers of hydroxyurea treatment in sickle cell disease.

Hydroxyurea (HU) has been used clinically to reduce the frequency of painful crisis and the need for blood transfusion in sickle cell disease (SCD) patients. However, the mechanisms underlying such beneficial effects of HU treatment are still not fully understood. Studies have indicated a weak correlation between clinical outcome and molecular markers, and the scientific quest to develop companion biophysical markers have mostly targeted studies of blood properties under hypoxia. Using a common-path interferometric technique, we measure biomechanical and morphological properties of individual red blood cells in SCD patients as a function of cell density, and investigate the correlation of these biophysical properties with drug intake as well as other clinically measured parameters. Our results show that patient-specific HU effects on the cellular biophysical properties are detectable at normoxia, and that these properties are strongly correlated with the clinically measured mean cellular volume rather than fetal hemoglobin level.

Superior performance of multilayered fluoropolymer films in low voltage electrowetting.

The requirement for low operational voltage in electrowetting devices, met using thin dielectrics, is usually connected with serious material failure issues. Dielectric breakdown (visible as electrolysis) is frequently evident slightly beyond the onset of the contact angle saturation. Here, plasma-enhanced chemical vapor deposition (PECVD) is used to deposit thin fluorocarbon films prior to the spin-coating of Teflon® amorphous fluoropolymer. The resulting multilayered hydrophobic top coating improves the electrowetting performance of the stack, by showing high resistance to dielectric breakdown at high applied voltages and for continuous long term application of DC and AC voltage. Leakage current measurements during electrowetting experiments with the proposed composite coating showed that current remains fairly constant at consecutive electrowetting tests in contrast to plain Teflon® coating in which material degradation is evident by a progressive increase in the leakage current after multiple electrowetting tests. Since the proposed composite coating demonstrates increased resistance to material failure and to dielectric breakdown even at thin configurations, its integration in electrowetting devices may impact their reliability, robustness, and lifetime.