Contribution of Red Blood Cells and Platelets to Blood Clot Computed Tomography Imaging and Compressive Mechanical Characteristics.

Thrombus computed tomography (CT) imaging characteristics may correspond with thrombus mechanical properties and thus predict thrombectomy success. The impact of red blood cell (RBC) content on these properties (imaging and mechanics) has been widely studied. However, the additional effect of platelets has not been considered. The objective of the current study was to examine the individual and combined effects of blood clot RBC and platelet content on resultant CT imaging and mechanical characteristics. Human blood clot analogues were prepared from a combination of preselected RBC volumes and platelet concentrations to decouple their contributions. The resulting clot RBC content (%) and platelet content (%) were determined using Martius Scarlet Blue and CD42b staining, respectively. Non-contrast and contrast-enhanced CT (NCCT and CECT) scans were performed to measure the clot densities. CECT density increase was taken as a proxy for clinical perviousness. Unconfined compressive mechanics were analysed by performing 10 cycles of 80% strain. RBC content is the major determinant of clot NCCT density. However, additional consideration of the platelet content improves the association. CECT density increase is influenced by clot platelet and not RBC content. Platelet content is the dominant component driving clot stiffness, especially at high strains. Both RBC and platelet content contribute to the clot's viscoelastic and plastic compressive properties. The current in vitro results suggest that CT density is reflective of RBC content and subsequent clot viscoelasticity and plasticity, and that perviousness reflects the clot's platelet content and subsequent stiffness. However, these indications should be confirmed in a clinical stroke cohort.

Polycarbonate Ultracentrifuge Tube Re-use in Proteomic Analyses of Extracellular Vesicles.

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins. This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples. Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (≤20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs.

The association between human blood clot analogue computed tomography imaging, composition, contraction, and mechanical characteristics.

BACKGROUND: Clot composition, contraction, and mechanical properties are likely determinants of endovascular thrombectomy success. A pre-interventional estimation of these properties is hypothesized to aid in selecting the most suitable treatment for different types of thrombi. Here we determined the association between the aforementioned properties and computed tomography (CT) characteristics using human blood clot analogues. METHODS: Clot analogues were prepared from the blood of 4 healthy human donors with 5 red blood cell (RBC) volume suspensions: 0%, 20%, 40%, 60% and 80% RBCs. Contraction was measured as the weight of the contracted clots as a percentage of the original suspension. The clots were imaged using CT with and without contrast to quantify clot density and density increase. Unconfined compression was performed to determine the high strain compressive stiffness. The RBC content was analysed using H&E staining. RESULTS: The 5 RBC suspensions formed only two groups of clots, fibrin-rich (0% RBCs) and RBC-rich (>90% RBCs), as determined by histology. The density of the fibrin-rich clots was significantly lower (31-38HU) compared to the RBC-rich clots (72-89HU), and the density increase of the fibrin-rich clots was significantly higher (82-127HU) compared to the RBC-rich clots (3-17HU). The compressive stiffness of the fibrin-rich clots was higher (178-1624 kPa) than the stiffness of the RBC-rich clots (6-526 kPa). Additionally, the degree of clot contraction was higher for the fibrin-rich clots (89-96%) compared to the RBC-rich clots (11-77%). CONCLUSIONS: CT imaging clearly reflects clot RBC content and seems to be related to the clot contraction and stiffness. CT imaging might be a useful tool in predicting the thrombus characteristics. However, future studies should confirm these findings by analysing clots with intermediate RBC and platelet content.

Tensile and Compressive Mechanical Behaviour of Human Blood Clot Analogues.

Endovascular thrombectomy procedures are significantly influenced by the mechanical response of thrombi to the multi-axial loading imposed during retrieval. Compression tests are commonly used to determine compressive ex vivo thrombus and clot analogue stiffness. However, there is a shortage of data in tension. This study compares the tensile and compressive response of clot analogues made from the blood of healthy human donors in a range of compositions. Citrated whole blood was collected from six healthy human donors. Contracted and non-contracted fibrin clots, whole blood clots and clots reconstructed with a range of red blood cell (RBC) volumetric concentrations (5-80%) were prepared under static conditions. Both uniaxial tension and unconfined compression tests were performed using custom-built setups. Approximately linear nominal stress-strain profiles were found under tension, while strong strain-stiffening profiles were observed under compression. Low- and high-strain stiffness values were acquired by applying a linear fit to the initial and final 10% of the nominal stress-strain curves. Tensile stiffness values were approximately 15 times higher than low-strain compressive stiffness and 40 times lower than high-strain compressive stiffness values. Tensile stiffness decreased with an increasing RBC volume in the blood mixture. In contrast, high-strain compressive stiffness values increased from 0 to 10%, followed by a decrease from 20 to 80% RBC volumes. Furthermore, inter-donor differences were observed with up to 50% variation in the stiffness of whole blood clot analogues prepared in the same manner between healthy human donors.

Identification of Nanoparticle Properties for Optimal Drug Delivery across a Physiological Cell Barrier.

Nanoparticles (NPs) represent an attractive strategy to overcome difficulties associated with the delivery of therapeutics. Knowing the optimal properties of NPs to address these issues could allow for improved in vivo responses. This work investigated NPs prepared from 5 materials of 3 sizes and 3 concentrations applied to a cell barrier model. The NPs permeability across a cell barrier and their effects on cell barrier integrity and cell viability were evaluated. The properties of these NPs, as determined in water (traditional) vs. media (realistic), were compared to cell responses. It was found that for all cellular activities, NP properties determined in media was the best predictor of the cell response. Notably, ZnO NPs caused significant alterations to cell viability across all 3 cell lines tested. Importantly, we report that the zeta potential of NPs correlates significantly with NP permeability and NP-induced changes in cell viability. NPs with physiological-based zeta potential of -12 mV result in good cell barrier penetration without considerable changes in cell viability.

The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells.

Assessing the ability of pharmaceutics to cross biological barriers and reach the site-of-action requires faithful representation of these barriers in vitro. Difficulties have arisen in replicating in vivo resistance in vitro. This paper investigated serum starvation as a method to increase Caco-2 barrier stability and resistance. The effect of serum starvation on tight junction production was examined using transwell models; specifically, transendothelial electrical resistance (TEER), and the expression and localization of tight junction proteins, occludin and zonula occludens-1 (ZO-1), were studied using western blotting and immunofluorescence. Changing cells to serum-free media 2 days post-seeding resulted in TEER readings of nearly 5000 Ω cm2 but the TEER rapidly declined subsequently. Meanwhile, exchanging cells to serum-free media 4-6 days post-seeding produced barriers with resistance readings between 3000 and 4000 Ω cm2, which could be maintained for 18 days. This corresponded to an increase in occludin levels. Serum starvation as a means of barrier formation is simple, reproducible, and cost-effective. It could feasibly be implemented in a variety of pre-clinical pharmaceutical assessments of drug permeability across various biological barriers with the view to improving the clinical translation of novel therapeutics.

In vitro and in silico modeling of endovascular stroke treatments for acute ischemic stroke.

Acute ischemic stroke occurs when a thrombus obstructs a cerebral artery, leading to sub-optimal blood perfusion to brain tissue. A recently developed, preventive treatment is the endovascular stroke treatment (EVT), which is a minimally invasive procedure, involving the use of stent-retrievers and/or aspiration catheters. Despite its increasing use, many critical factors of EVT are not well understood. In this respect, in vitro, and in silico studies have the great potential to help us deepen our understanding of the procedure, perform further device and procedural optimization, and help in clinical training. This review paper provides an overview of the previous in vitro and in silico evaluations of EVT treatments, with a special emphasis on the four main aspects of the adopted experimental and numerical set-ups: vessel, thrombus, device, and procedural settings.

On the association between circulating biomarkers and atherosclerotic calcification in a cohort of arterial disease participants.

BACKGROUND AND AIMS: Atherosclerotic calcification is a powerful predictor of cardiovascular disease. This study aims to determine whether circulating levels of a local/systemic calcification inhibitor or a marker of bone formation correlate with measures of coronary or extracoronary calcification. METHODS AND RESULTS: Clinical computed tomography (CT) was performed on 64 arterial disease participants undergoing carotid and lower extremity endarterectomy. Coronary artery calcium (CAC) scores and volumes were acquired from the CT scans (n = 42). CAC scores and volumes were used to derive CAC density scores. Micro-CT was performed on excised carotid (n = 36) and lower extremity (n = 31) plaques to quantify the volume and volume fraction of extracoronary calcification. Circulating levels of dephospho-uncarboxylated Matrix Gla Protein (dp-ucMGP), fetuin-A, carboxylated and uncarboxylated osteocalcin (ucOC) were quantified using commercial immunoassays. Carotid participant CAC density scores were moderately negatively correlated with plasma dp-ucMGP (rs = -0.592, P = 0.008). A weak negative association was found between CAC scores and %ucOC for all participants (rs = -0.335, P = 0.040). Another weak negative correlation was observed between fetuin-A and the volume of calcification within excised carotid specimens (rs = -0.366, P = 0.031). Despite substantial differences in coronary and extracoronary calcium measurements, the levels of circulating biomarkers did not vary significantly between carotid and lower extremity subgroups. CONCLUSION: Correlations identified between circulating biomarkers and measures of coronary and extracoronary calcium were not consistent among participant subgroups. Further research is required to determine the association between circulating biomarkers, coronary and extracoronary calcium.

Bevel angle study of flexible hollow needle insertion into biological mimetic soft-gel: Simulation and experimental validation.

BACKGROUND: A thorough understanding of cutting-edge geometry and cutting forces of hollow biopsy needles are required to optimise needle tip design to improve fine needle aspiration procedures. OBJECTIVES: To incorporate the dynamics of needle motion in a model for flexible hollow bevel tipped needle insertion into a biological mimetic soft-gel using parameters obtained from experimental work. Additionally, the models will be verified against corresponding needle insertion experiments. METHODS: To verify simulation results, needle deflection and insertion forces were compared with corresponding experimental results acquired with an in-house developed needle insertion mechanical system. Additionally, contact stress distribution on needles from agar gel for various time scales were also studied. RESULTS: For the 15°, 30°, 45°, 60° bevel angle needles, and 90° blunt needle, the percentage error in needle deflection of each needle compared to experiments, were 7.3%, 9.9%, 8.6%, 7.8%, and 9.7% respectively. Varying the bevel angle at the needle tip demonstrates that the needle with a lower bevel angle produces the largest deflection, although the insertion force does not vary too much among the tested bevel angles. CONCLUSION: This experimentally verified computer-based simulation model could be used as an alternative tool for better understanding the needle-tissue interaction to optimise needle tip design towards improved biopsy efficiency.

Correlating Ex Vivo Carotid Calcification Measurements With Cerebrovascular Symptoms: A Proof-of-Concept Study.

BACKGROUND AND PURPOSE: The purpose of this study is to examine the ability of ex vivo derived Agatston, Volume, and Density-Volume calcium scores or calcium density measurements to differentiate between carotid plaques based on preoperative cerebrovascular symptomatology. METHODS: Thirty-eight carotid plaques were acquired from standard endarterectomy. Micro-computed tomography was performed on the ex vivo samples. Image series were downsampled to represent the resolution of clinical multidetector computed tomography. Agatston, Volume, and Density-Volume carotid calcium scores were then calculated using coronary methodologies. The fractions of low- and high-density calcification were also determined. RESULTS: The coronary calcium scores could not differentiate between carotid plaques from asymptomatic versus symptomatic patients. However, plaques from asymptomatic patients contained significantly lower fractions of low-density calcification and higher fractions of high-density calcification. CONCLUSIONS: Screening for carotid calcium density in noncontrast computed tomography could reflect plaque stability.

A blood biomarker and clinical correlation cohort study protocol to diagnose sports-related concussion and monitor recovery in elite rugby.

INTRODUCTION: In professional rugby, sports-related concussion (SRC) remains the most frequent time loss injury. Therefore, accurately diagnosing SRC and monitoring player recovery, through a multi-modal assessment process, is critical to SRC management. In this protocol study, we aim to assess SRC over multiple time points post-injury to determine the value of multi-modal assessments to monitor player recovery. This is of significance to minimise premature return-to-play and, ultimately, to reduce the long-term effects associated with SRC. The study will also establish the logistics of implementing such a study in a professional setting to monitor a player's SRC recovery. METHODS AND ANALYSIS: All players from the participating professional rugby club within the Irish Rugby Football Union are invited to participate in the current study. Player assessment includes head injury assessment (HIA), neuropsychometric assessment (ImPACT), targeted biomarker analysis and untargeted biomarker analysis. Baseline HIA, ImPACT, and blood draws are performed prior to the start of playing season. During the baseline tests, player's complete consent forms and an SRC history questionnaire. Subsequently, any participant that enters the HIA process over the playing season due to a suspected SRC will be clinically assessed (HIA and ImPACT) and their blood will be drawn within 3 days of injury, 6 days post-injury, and 13 days post-injury. ETHICS AND DISSEMINATION: Ethical approval was attained from the Science and Engineering Research Ethics Committee, University of Limerick (Approval Code: 2018_06_11_S&E). On completion of the study, further manuscripts will be published to present the results of the tests and their ability to measure player recovery from SRC. TRIAL REGISTRATION NUMBER: NCT04485494.

Comparative analysis of calcification parameters with Agatston Score approximations for ex vivo atherosclerotic lesions.

BACKGROUND: The Agatston Calcium Score is a predictor of major adverse cardiovascular events but it is unable to identify high-risk lesions. Recent research suggests that examining calcification phenotype could be more indicative of plaque stability. OBJECTIVE: To examine the Agatston score's ability to determine atherosclerotic calcification phenotype. METHODS: Micro-Computed Tomography was performed on 20 carotid and 20 peripheral lower limb lesions. ImageJ pixel histogram analysis quantified the non-calcified (≥30HU, <130HU) and calcified (≥130HU) tissue volumes. ImageJ '3D Objects Counter' plugin determined the calcified particle count, volumes and maximum attenuation density of each particle. Image stacks were subsequently downsampled to a resolution of 0.7 × 0.7 × 3 mm and an approximation for the Extra-Coronary Calcium Scores (ECCS) were calculated. Spearman's correlation examined the relationships between ECCS approximations and calcification parameters. RESULTS: ECCS has a strong positive correlation with the Calcified Volume Fraction (CVF) (rs = 0.865, p < 0.0005), weak positive correlations with Calcified Particle Fraction (CPF) (rs = 0.422, p = 0.007) and Microcalcification Fraction (micro-CF) (rs = 0.361, p = 0.022). There is no correlation evident between ECCS and Calcified Particle Index (CPI) (rs = -0.162, p = 0.318). It is apparent that there is a high prevalence of microcalcifications in both carotid and peripheral lower limb lesions. Additionally, an inverse relationship exists between calcified particle volume and maximum-recorded attenuation density. CONCLUSION: The density-weighted Agatston calcium scoring methodology needs to be reviewed. Calcium scoring which differentiates between critical calcification morphologies, rather than presenting a density-weighted score, is required to direct high-risk plaques towards tailored treatment.

Relating the mechanical properties of atherosclerotic calcification to radiographic density: A nanoindentation approach.

Calcification morphology can determine atherosclerotic plaque stability and is associated with increased failures rates for endovascular interventions. Computational efforts have sought to elucidate the relationship between calcification and plaque rupture in addition to predicting tissue response during aggressive revascularisation techniques. However, calcified material properties are currently estimated and may not reflect real tissue conditions. The objective of this study is to correlate calcification mechanical properties with three radiographic density groups obtained from corresponding Computed Tomography (CT) images. Seventeen human plaques extracted from carotid (n = 10) and peripheral lower limb (n = 7) arteries were examined using micro-computed tomography (µCT), simultaneously locating the calcified deposits within their internal structure and quantifying their densities. Three radiographic density groups were defined based on the sample density distribution: (A) 130-299.99 Hounsfield Units (HU), (B) 300-449.99 HU and (C) >450 HU. Nanoindentation was employed to determine the Elastic Modulus (E) and Hardness (H) values within the three density groups. Results reveal a clear distinction between mechanical properties with respect to radiographic density groups (p < 0.0005). No significant differences exist in the density-specific behaviours observed between carotid and peripheral samples. Previously defined calcification classifications indicate an association with specific radiographic density patterns. Scanning Electron Microscopy (SEM) examination revealed that density group A regions consist of both calcified and non-calcified tissues. Further research is required to define the radiographic thresholds which identify varying degrees of tissue calcification. This study demonstrates that the mechanical properties of fully mineralised atherosclerotic calcification emulate that of bone tissues (17-25 GPa), affording computational models with accurate material parameters. STATEMENT OF SIGNIFICANCE: Global mechanical characterisation techniques disregard the heterogeneous nature of atherosclerotic lesions. Previous nanoindentation results for carotid calcifications have displayed a wide range. This study evaluates calcification properties with respect to radiographic density obtained from Micro-CT images. This is the first work to characterise calcifications from peripheral lower limb arteries using nanoindentation. Results demonstrate a strong positive correlation between radiographic density and calcification mechanical properties. Characterising calcifications using their density values provides clarity on the variation in published properties for calcified tissues. Furthermore, this study confirms the hypothesis that fully calcified plaque tissue behaviour similar to that of bone. Appropriate material parameters for calcified tissues can now be employed in computational simulations.