Cell Velocity Research Articles

A Guide to Trajectory Inference and RNA Velocity.

Technological developments have led to an explosion of high-throughput single-cell data, which are revealing unprecedented perspectives on cell identity. Recently, significant attention has focused on investigating, from single-cell RNA-sequencing (scRNA-seq) data, cellular dynamic processes, such as cell differentiation, cell cycle and cell (de)activation. In particular, trajectory inference methods, by ordering cells along a trajectory, allow estimating a differentiation tree of cells. While trajectory inference tools typically work with gene expression levels, common scRNA-seq protocols allow the identification and quantification of unspliced pre-mRNAs and mature spliced mRNAs for each gene. By exploiting the abundance of unspliced and spliced mRNA, one can infer the RNA velocity of individual cells, i.e., the time derivative of the gene expression state of cells. Whereas traditional trajectory inference methods reconstruct cellular dynamics given a population of cells of varying maturity, RNA velocity relies on a dynamical model describing splicing dynamics. Here, we initially discuss conceptual and theoretical aspects of both approaches, then illustrate how they can be combined together, and finally present an example use case on real data.

Inhibition of proliferative vitreoretinopathy by a newly developed methotrexate loaded drug carrier in vitro

PurposeRepeated intravitreal injections of methotrexate for proliferative vitreoretinopathy, a rare ocular condition that can cause vision loss, have shown beneficial effects in recent clinical studies. The purpose of this study was to develop a slow-release, long-term drug carrier composed of the polymer polylactide-co-glycolide and methotrexate that can be injected intravitreally. MethodsThe required composition of the drug carrier was modeled using pharmacokinetic parameters based on current literature. Release kinetics were determined using an ocular pharmacokinetic model. Epiretinal PVR-membranes were harvested during pars plana vitrectomy and subsequently transferred to cell culture. The effect of the drug carrier on cell migration was investigated using time-lapse microscopy and a scratch-induced migration assay. The colorimetric WST-1-assay and a live-dead-assay were performed to determine viability, and the BrdU-assay was applied for proliferation. ResultsThe release profile showed an initial and a final burst of methotrexate with an intervening steady state that lasted 9–11 weeks. It showed inhibitory effects on pathobiological processes in human PVR-cells in vitro. Cell velocity in the time-lapse assay, migration in the scratch assay (p = 0.001), and proliferation in the BrdU assay (p = 0.027) were reduced after addition of the drug carrier. These effects occurred without causing a reduction in viability in the WST-1 assay (p > 0.99) and the live-dead assay. ConclusionThe methotrexate-loaded drug carrier can maintain a stable concentration for 9–11 weeks and influence the pathobiological process of PVR cells in vitro. Therefore, it represents a potential therapeutic orphan drug for PVR.

Adhesion molecules and cerebral microvascular hemodynamic abnormalities in sickle cell disease.

Cerebrovascular abnormalities are a common feature of sickle cell disease that may be associated with risk of vaso-occlusive pain crises, microinfarcts, and cognitive impairment. An activated endothelium and adhesion factors, VCAM-1 and P-selectin, are implicated in sickle cell vasculopathy, including abnormal hemodynamics and leukocyte adherence. This study examined the association between cerebral expression of these adhesion factors and cortical microvascular blood flow dynamics by using in-vivo two-photon microscopy. We also examined the impact of blood transfusion treatment on these markers of vasculopathy. Results showed that sickle cell mice had significantly higher maximum red blood cell (RBC) velocity (6.80 ± 0.25 mm/sec, p ≤ 0.01 vs. 5.35 ± 0.35 mm/sec) and more frequent blood flow reversals (18.04 ± 0.95 /min, p ≤ 0.01 vs. 13.59 ± 1.40 /min) in the cortical microvasculature compared to controls. In addition, sickle cell mice had a 2.6-fold (RFU/mm2) increase in expression of VCAM-1 and 17-fold (RFU/mm2) increase in expression of P-selectin compared to controls. This was accompanied by an increased frequency in leukocyte adherence (4.83 ± 0.57 /100 μm/min vs. 2.26 ± 0.37 /100 μm/min, p ≤ 0.001). We also found that microinfarcts identified in sickle cell mice were 50% larger than in controls. After blood transfusion, many of these parameters improved, as results demonstrated that sickle cell mice had a lower post-transfusion maximum RBC velocity (8.30 ± 0.98 mm/sec vs. 11.29 ± 0.95 mm/sec), lower frequency of blood flow reversals (12.80 ± 2.76 /min vs. 27.75 ± 2.09 /min), and fewer instances of leukocyte adherence compared to their pre-transfusion imaging time point (1.35 ± 0.32 /100 μm/min vs. 3.46 ± 0.58 /100 μm/min). Additionally, we found that blood transfusion was associated with lower expression of adhesion factors. Our results suggest that blood transfusion and adhesion factors, VCAM-1 and P-selectin, are potential therapeutic targets for addressing cerebrovascular pathology, such as vaso-occlusion, in sickle cell disease.

DeepVelo: Single-cell transcriptomic deep velocity field learning with neural ordinary differential equations.

Recent advances in single-cell sequencing technologies have provided unprecedented opportunities to measure the gene expression profile and RNA velocity of individual cells. However, modeling transcriptional dynamics is computationally challenging because of the high-dimensional, sparse nature of the single-cell gene expression measurements and the nonlinear regulatory relationships. Here, we present DeepVelo, a neural network-based ordinary differential equation that can model complex transcriptome dynamics by describing continuous-time gene expression changes within individual cells. We apply DeepVelo to public datasets from different sequencing platforms to (i) formulate transcriptome dynamics on different time scales, (ii) measure the instability of cell states, and (iii) identify developmental driver genes via perturbation analysis. Benchmarking against the state-of-the-art methods shows that DeepVelo can learn a more accurate representation of the velocity field. Furthermore, our perturbation studies reveal that single-cell dynamical systems could exhibit chaotic properties. In summary, DeepVelo allows data-driven discoveries of differential equations that delineate single-cell transcriptome dynamics.

Simulation-based inference of differentiation trajectories from RNA velocity fields

We report Cytopath, a method for trajectory inference that takes advantage of transcriptional activity information from the RNA velocity of single cells to perform trajectory inference. Cytopath performs this task by defining a Markov chain model, simulating an ensemble of possible differentiation trajectories, and constructing a consensus trajectory. We show that Cytopath can recapitulate the topological and molecular characteristics of the differentiation process under study. In our analysis, we include differentiation trajectories with varying bifurcated, circular, convergent, and mixed topologies studied in single-snapshot as well as time-series single-cell RNA sequencing experiments. We demonstrate the capability to reconstruct differentiation trajectories, assess the association of RNA velocity-based pseudotime with actually elapsed process time, and identify drawbacks in current state-of-the art trajectory inference approaches.

Experimental characterization of extreme temperature granular flows for solar thermal energy transport and storage

High-temperature, dense granular flows along an inclined plane were considered for solar thermal energy transport and storage with sintered bauxite particles. A series of experiments was performed for particle inlet temperatures of ∼ 200, 400, 600, and 800 °C to understand the mechanisms of granular flows at extreme temperatures. Mass flow rates were measured using a load cell and free-surface velocities were measured and computed using particle image velocimetry. Surface temperatures were measured using infrared cameras. A significant decrease in steady-state particle mass flow rate was observed with increasing temperature due to changing flow properties. A decrease in bulk particle free-surface velocities was observed at higher temperatures. Free-surface velocity measurement error between experiments were within 20% of the average. The particle surface temperatures decreased from inlet to outlet with larger gradients at higher temperatures observed due to increasing convection and radiative heat losses. A decrease in temperature was observed along the side walls due to a decrease in particle velocities. • Refractory granular media enable high temperature solar thermal energy storage. • Granular flow experiments on inclined plane explored at elevated temperatures. • Significant changes in flow behavior observed at elevated temperatures. • Larger heat loss to surroundings observed at side walls due to decrease in velocity.

Dopamine is involved in regulating vascular tone in retinal arterioles

AbstractPurpose: There is clinical evidence suggesting that the neurotransmitter dopamine may play a role in regulating retinal vascular function. Patients with Parkinson's disease tend to have a narrower retinal vessel diameter in fundus images and reduced perfusion density1. Healthy volunteers given dopamine IV show increased retinal blood cell velocity (by 37%) and fundus pulsation amplitude (by 24%)2. However, it is not clear if these observed effects may be due to direct actions of dopamine on retinal vessels, independent of any systemic actions, and if so through which subtype of dopamine receptors. We therefore measured the effects of dopamine ligands on isolated segments of porcine retinal arterioles by small wire myography.Methods: Dissected segments of porcine retinal arterioles and surrounding retinal tissue were mounted in a DMT630MA small wire myograph system for measurement of contractile activity, and pre‐contracted with 10−6 MU‐46619, a thromboxane analog, added to the organ bath. With the vascular tone stabilized, all dopamine ligands tested were applied separately to the bath with the vessel, and the effects of each on the wall tension recorded. Results are presented as wall tension (in mN/mm) normalized for vessel length.Results: In isolated vessel segments pre‐contracted with U‐46619, dopamine induced a vasodilation in a dose‐dependent manner, which was complete at 2 mM. The dopamine D1 receptor agonist SKF38393 induced dose‐dependent vasodilation, complete at 0.7 mM. The D2 receptor agonist sumanirole (10−4 M) had no effect on wall tension in pre‐contracted retinal arterioles. The D2 receptor antagonist S‐sulpiride (10−3 M) had no effect on wall tension and did not block the vasodilation induced by either dopamine or SKF38393.Conclusions: Dopamine induces vasodilation in pre‐contracted retinal arterioles, primarily via D1 receptors.

Coupling cell shape and velocity leads to oscillation and circling in keratocyte galvanotaxis

During wound healing, fish keratocyte cells undergo galvanotaxis where they follow a wound-induced electric field. In addition to their stereotypical persistent motion, keratocytes can develop circular motion without a field or oscillate while crawling in the field direction. We developed a coarse-grained phenomenological model that captures these keratocyte behaviors. We fit this model to experimental data on keratocyte response to an electric field being turned on. A critical element of our model is a tendency for cells to turn toward their long axis, arising from a coupling between cell shape and velocity, which gives rise to oscillatory and circular motion. Galvanotaxis is influenced not only by the field-dependent responses, but also cell speed and cell shape relaxation rate. When the cell reacts to an electric field being turned on, our model predicts that stiff, slow cells react slowly but follow the signal reliably. Cells that polarize and align to the field at a faster rate react more quickly and follow the signal more reliably. When cells are exposed to a field that switches direction rapidly, cells follow the average of field directions, while if the field is switched more slowly, cells follow a “staircase” pattern. Our study indicated that a simple phenomenological model coupling cell speed and shape is sufficient to reproduce a broad variety of different keratocyte behaviors, ranging from circling to oscillation to galvanotactic response, by only varying a few parameters.

BIOM-41. IDENTIFICATION OF GLIOMA CELLS BY PROFILING SINGLE-CELL BIOPHYSICAL PROPERTIES THROUGH MULTICONSTRICTION MICROFLUIDIC CHANNELS

Abstract This study introduces a multicontraction microfluidic channel that can differentiate glioma cells from normal glio cells. As cells pass through successive constriction channels, the incremental velocity and varying size profiles of the cells will be collected, reflecting their biophysical properties. The data of high-dimensional variables were analyzed , including the cell sizes, velocities, and velocity increments. The prediction value is used to represent the difference between two groups using the established classification model from high-dimensional variables. At the same time, we prepared three groups of primary tumor cells from patients with different grades of glioma to verify the efficacy of this classification method. The results show that in this microfluidic channel, the diagnostic model made of cell biophysical properties can well identify glioma cells, which gives a novel method for efficient identification of circulating tumor cells and rapid pathological diagnosis.

Ameboid cell migration through regular arrays of micropillars under confinement

Migrating cells often encounter a wide variety of topographic features—including the presence of obstacles—when navigating through crowded biological environments. Unraveling the impact of topography and crowding on the dynamics of cells is key to better understand many essential physiological processes such as the immune response. We study the impact of geometrical cues on ameboid migration of HL-60 cells differentiated into neutrophils. A microfluidic device is designed to track the cells in confining geometries between two parallel plates with distance h, in which identical micropillars are arranged in regular pillar forests with pillar spacing e. We observe that the cells are temporarily captured near pillars, with a mean contact time that is independent of h and e. By decreasing the vertical confinement h, we find that the cell velocity is not affected, while the persistence reduces; thus, cells are able to preserve their velocity when highly squeezed but lose the ability to control their direction of motion. At a given h, we show that by decreasing the pillar spacing e in the weak lateral confinement regime, the mean escape time of cells from effective local traps between neighboring pillars grows. This effect, together with the increase of cell-pillar contact frequency, leads to the reduction of diffusion constant D. By disentangling the contributions of these two effects on D in numerical simulations, we verify that the impact of cell-pillar contacts on cell diffusivity is more pronounced at smaller pillar spacing.

Glioma Cell Invasion Is Dependent Upon the DNA Damage Response Kinase ATR

Abstract AIMS Gliomas have high levels of DNA replication stress and clinical trials of inhibitors of the key replication stress response protein ATR (ataxia telangiectasia and rad 3 related protein) as radiosensitisers are planned. We aimed to investigate the effect of ATR inhibition on the ability of glioma cells to infiltrate and invade the brain. METHOD Live cell imaging in a panel of primary glioma cultures following siRNA or pharmacological inhibition of ATR. Invasion following treatment of murine orthotopic gliomas was determined by immunohistochemistry. Intravital imaging of GFP expressing murine orthotopic xenografts via an intracranial window model of glioma was undertaken. RESULTS Invading margins of human glioma samples demonstrated increased pATR expression relative to core. Live cell imaging demonstrated reduced cell velocity following ATR inhibition (Berzosertib/BAY1895344) or siRNA. Cytoplasmic vacuolation occurred following ATRi or siRNA which were single walled structures which engulf high molecular weight dextran, compatible with blockade of macropinosome processing. Live cell imaging with GFP-integrin α5 and integrin recycling assays showed sequestration of integrins within macropinosomes and reduced integrin cycling. Intravital in vivo imaging of murine xenograft tumours confirmed vacuolation and dextran uptake following ATRi, whilst a further in vivo study demonstrated a reduction in invading tumour cells. CONCLUSION We demonstrate a novel role for ATR in facilitating macropinocytic vesicle trafficking and integrin recycling in GBM cells which results in a profound motility defect in vitro and in vivo. ATR inhibitors are entering early phase trials as radiation sensitisers and we propose that therapeutic benefit will extend beyond DNA damage potentiation.

Exercise with blood flow restriction improves muscle strength and mass while preserving the vascular and microvascular function and structure of older adults.

Changes in muscle mass, strength, vascular function, oxidative stress, and inflammatory biomarkers were compared in older adults after resistance training (RT) performed with low-intensity without blood flow restriction (RT-CON); low-intensity with BFR (RT-BFR); and high-intensity without BFR (RT-HI). Thirty-two untrained individuals (72±7 y) performed a 12-week RT after being randomized into three groups: RT-CON -30% of 1 repetition maximum (RM); RT-BFR -30% of 1RM and mild BFR (50% of arterial occlusion pressure); RT-HI -70% of 1 RM. Improvements in handgrip strength were similar in RT-BFR (17%) and RT-HI (16%) vs. RT-CON (-0.1%), but increases in muscle mass (6% vs. 2% and -1%) and IGF-1 (2% vs. -0.1% and -1.5%) were greater (p < 0.05) in RT-BFR vs. RT-HI and RT-CON. Changes in vascular function, morphology, inflammation, and oxidative stress were similar between groups, except for time to reach maximum red blood cell velocity which showed a greater reduction (p < 0.05) in RT-BFR (-55%) vs. RT-HI (-11%) and RT-CON (-4%). RT with low intensity and mild BFR improved muscle strength and mass in older individuals while preserving vascular function. This modality should be considered an adjuvant strategy to improve muscle function in older individuals with poor tolerance to high loads.

Pulsations and flows in tissues as two collective dynamics with simple cellular rules

SummaryCollective motions of epithelial cells are essential for morphogenesis. Tissues elongate, contract, flow, and oscillate, thus sculpting embryos. These tissue level dynamics are known, but the physical mechanisms at the cellular level are unclear. Here, we demonstrate that a single epithelial monolayer of MDCK cells can exhibit two types of local tissue kinematics, pulsations and long range coherent flows, characterized by using quantitative live imaging. We report that these motions can be controlled with internal and external cues such as specific inhibitors and substrate friction modulation. We demonstrate the associated mechanisms with a unified vertex model. When cell velocity alignment and random diffusion of cell polarization are comparable, a pulsatile flow emerges whereas tissue undergoes long-range flows when velocity alignment dominates which is consistent with cytoskeletal dynamics measurements. We propose that environmental friction, acto-myosin distributions, and cell polarization kinetics are important in regulating dynamics of tissue morphogenesis.

SIMULATION OF HOMOGENEOUS HYDROGEN-AIR DETONATION INTERACTION WITH POROUS FILTER

Calculations of the interaction of a cellular detonation wave (DW) with an inert porous filter were carried out based on the developed physical and mathematical model of the mechanics of heterogeneous medium which takes into account the detailed chemical kinetics of chemical reactions in the gas phase. Under an inert porous filter, a motionless lattice of inert particles is considered. The following flow regimes were revealed: propagation of attenuated cellular DW at velocities less than the Chapman-Jouguet velocity and detonation failure with the destruction of the cellular structure. Critical volume concentrations of filter particles corresponding to the detonation failure regime were calculated. Dependences of the normalized DW velocity and detonation cell size on the volume concentration of particles in filters were calculated. The concentration limits of detonation (critical volume concentrations of particles lead to detonation failure) in filters with diameters of 50, 100, and 200 µm were determined. The dependences of the normalized DW velocity and size of detonation cell on the volume concentration and diameter of particles in filters were calculated.

Relationship of Effective Circulating Volume with Sublingual Red Blood Cell Velocity and Microvessel Pressure Difference: A Clinical Investigation and Computational Fluid Dynamics Modeling.

The characteristics of physiologic hemodynamic coherence are not well-investigated. We examined the physiological relationship between circulating blood volume, sublingual microcirculatory perfusion, and tissue oxygenation in anesthetized individuals with steady-state physiology. We assessed the correlation of mean circulatory filling pressure analogue (Pmca) with sublingual microcirculatory perfusion and red blood cell (RBC) velocity using SDF+ imaging and a modified optical flow-based algorithm. We also reconstructed the 2D microvessels and applied computational fluid dynamics (CFD) to evaluate the correlation of Pmca and RBC velocity with the obtained pressure and velocity fields in microvessels from CFD (pressure difference, (Δp)). Twenty adults with a median age of 39.5 years (IQR 35.5–44.5) were included in the study. Sublingual velocity distributions were similar and followed a log-normal distribution. A constant Pmca value of 14 mmHg was observed in all individuals with sublingual RBC velocity 6–24 μm s−1, while a Pmca < 14 mmHg was observed in those with RBC velocity > 24 μm s−1. When Pmca ranged between 11 mmHg and 15 mmHg, Δp fluctuated between 0.02 Pa and 0.1 Pa. In conclusion, the intact regulatory mechanisms maintain a physiological coupling between systemic hemodynamics, sublingual microcirculatory perfusion, and tissue oxygenation when Pmca is 14 mmHg.

Persistent capillary rarefication in long COVID syndrome

BackgroundRecent studies have highlighted Coronavirus disease 2019 (COVID-19) as a multisystemic vascular disease. Up to 60% of the patients suffer from long-term sequelae and persistent symptoms even 6 months after the initial infection.MethodsThis prospective, observational study included 58 participants, 27 of whom were long COVID patients with persistent symptoms > 12 weeks after recovery from PCR-confirmed SARS-CoV-2 infection. Fifteen healthy volunteers and a historical cohort of critically ill COVID-19 patients (n = 16) served as controls. All participants underwent sublingual videomicroscopy using sidestream dark field imaging. A newly developed version of Glycocheck™ software was used to quantify vascular density, perfused boundary region (PBR-an inverse variable of endothelial glycocalyx dimensions), red blood cell velocity (VRBC) and the microvascular health score (MVHS™) in sublingual microvessels with diameters 4–25 µm.Measurements and main resultsAlthough dimensions of the glycocalyx were comparable to those of healthy controls, a µm-precise analysis showed a significant decrease of vascular density, that exclusively affected very small capillaries (D5: − 45.16%; D6: − 35.60%; D7: − 22.79%). Plotting VRBC of capillaries and feed vessels showed that the number of capillaries perfused in long COVID patients was comparable to that of critically ill COVID-19 patients and did not respond adequately to local variations of tissue metabolic demand. MVHS was markedly reduced in the long COVID cohort (healthy 3.87 vs. long COVID 2.72 points; p = 0.002).ConclusionsOur current data strongly suggest that COVID-19 leaves a persistent capillary rarefication even 18 months after infection. Whether, to what extent, and when the observed damage might be reversible remains unclear.

Assaying Macrophage Chemotaxis Using Fluid‐Walled Microfluidics

AbstractWhile many tools exist to study immune‐cell chemotaxis in vitro, current methods often lack desirable features. Using fluid‐walled microfluidics, circuits are built around primary murine macrophages deposited in pre‐defined patterns on Petri dishes or microplates. Concentration gradients of complement component 5a (C5a) are established in flow‐free or flowing environments, cell migration imaged, and cell directionality and velocity correlated to calculated local C5a concentrations. In flow‐free circuits built around patterned macrophages, only cells nearest the C5a source migrate regardless of local attractant concentration. Conversely, in flowing circuits free from intercellular signaling and attractant degradation, only cells distant from the source migrate. In both systems, cells respond to lower C5a concentrations than previously reported (≈0.1 pM). Finally, macrophages follow instantly‐shifted gradients better than slowly‐shifting ones, suggesting that migration depends on both spatial and temporal responses to concentration.

In-Vivo PIV Measurement Of The Blood Flow In The Heart And Main Vein Of Medaka Embryo

In order to examine the generation mechanism of the vertebrates, including humans, and to explore the diverging point of the genetic abnormalities, it is effective to investigate the growth of the embryo of an artificial mutant. As the embryos of Medaka hatch outside of the body and their egg membrane are transparent, it is quite easy to carry out the in-vivo observation of their organs. Moreover, the development and the structure of their major organs of Medaka are quite similar to those of human. Thus, the observation of the developmental abnormalities of Medaka embryos may serve as a clue to the pathologic investigation of the genetic disease and hopefully give some insight to the future effective treatment. Presently, the intracardiac blood flows of Medaka embryos have been observed by a high-speed camera under microscope. The obtained images are pre-processed and the velocity fields in the heart and in the main vein of Medaka embryo are measured by PIV method. The red blood cells flowing inside the heart and in the main vein of Medaka embryos are used as tracer particles. The image pre-processing was applied to capture the image of red blood cells and then the cross-correlation method can be applied. As a result, it is found that the velocity defect caused by the valve closing is clearly observed in the main vein and the amplitude of the velocity defect is decreasing with the distance to the heart. It is also found that the beating of the heart corresponds to the pulsatio of the flow velocity of the red blood cell in the heart. In conclusion, the authors observed the Medaka embryo under microscope with a high-speed camera and measured the velocities of the red blood cells in the heart and in the main vein by PIV measurement using the pre-processed images.

A computational approach to model gliding motion of an organism on a sticky slime layer over a solid substrate.

Bacteria are microscopic single-celled microbes that can only be spotted via a microscope. They occur in a variety of shapes and sizes, and their dimensions are measured in micrometers (one-millionth of a meter). Bacterial categorization is based on a variety of features such as morphology, DNA sequencing, presence of flagella, cell structure, staining techniques, oxygen, and carbon-dioxide requirements. Due to these classifications, gliding bacteria are a miscellaneous class of rodlike microorganisms that cling and propel over ooze slime connected with a substrate. Without the assistance of flagella, which are essential parts of bacterial motility, the organism movement is adopted by waves streaming down the outer layer of this microorganism. To simulate the locomotion of such gliding microorganisms, a wavy sheet over Oldroyd-4 constant fluid is utilized. Under the long wavelength assumption, the equations regulating the flow of slime (modeled as Oldroyd-4 constant slime) beneath the cell/organism are developed. The quantities such as slime flow rate, cell speed, and propulsion power are computed by using bvp4c (MATLAB routine) integrated with the modified Newton-Rasphson technique. Furthermore, the flow patterns and velocity of the slime are graphically shown and thoroughly described using precise (calculated) values of the cell speed and velocity of the slime.

A meta-analysis of variability in conjunctival microvascular hemorheology metrics.

Conjunctival hemorheology has been used analytically to assess qualities of blood flow associated with various forms of cardiovascular disorders including diabetes mellitus, stroke, and sickle cell disease. Although conjunctival axial red blood cell velocity (Vax) has been demonstrated in varying disease states, benchmark measures of Vax are not well-defined. Due to various methodologic differences in assessment of Vax, interstudy consistency of hemorheological metrics is susceptible to both systematic and random error. Our study examines interstudy heterogeneity of Vax as measured in the conjunctival microvasculature of healthy subjects and assesses the overall perturbation of Vax based on disease state. Furthermore, our study aims to establish a potential range of normative Vax by comparing inter-study measurements in healthy patients. The most widely employed analytic approach to assess Vax was space-time analysis (n=30). Using a meta-analytic approach, the prediction interval for Vax in healthy subjects among 20 studies ranged from 0.32-2.60mm/s with a combined effect size of 0.52±0.03 (CI: 0.46-0.59) mm/s. Inter-study comparison of Vax in healthy patients showed a high degree of variability (I2: 98.96%), due to studies with low measurement precision and/or dissimilar analytic methodology. Neither age nor diameter was a clinically significant moderator of Vax measurements in healthy patients. The combined effect size, defined as the composite Hedge's g of studies comparing healthy and disease state mean Vax, was 0.21±0.13. High heterogeneity (I2: 80.48%) was observed in studies analyzing the difference between mean Vax in healthy and disease state patients. This heterogeneity was also observed when the difference in mean Vax between healthy and disease state patients was assessed in subgroups based on disease condition (I2: vascular disease 33%, sickle cell disease 62.22%, other 83.43%). Age was found to be a significant moderator (p=0.048, β=-0.40) of Hedge's g while diameter was not. No significant publication bias was observed in studies presenting healthy patient Vax or in studies comparing Vax between healthy and disease state patients. In summary, although homogeneity can be seen in healthy group Vax measurements, a high degree of statistical heterogeneity is found in Vax assessment comparing healthy and disease conditions that is not fully explained by methodologic variability.