Multiscale imaging holds particular relevance to neuroscience, where it helps integrate the cellular and molecular biological scale, which is most accessible to interventions, with holistic organ-level evaluations, most relevant with respect to function. Being inextricably interdisciplinary, multiscale imaging benefits substantially from incremental technology adoption, and a detailed overview of the state-of-the-art is vital to an informed application of imaging methods.In this article, we lay out the background and methodological aspects of multimodal approaches combining functional magnetic resonance imaging (fMRI) with simultaneous optical measurement or stimulation.We focus on optical techniques as these allow, in conjunction with genetically encoded proteins , unprecedented read-out and control specificity for individual cell-types during fMRI experiments, while leveraging non-interfering modalities.A variety of different solutions for optical/fMRI methods has been reported ranging from bulk fluorescence recordings via fiber photometry to high resolution microscopy. In particular, the plethora of optogenetic tools has enabled the transformation of stimulus-evoked fMRI into a cell biological interrogation method. We discuss the capabilities and limitations of these genetically encoded molecular tools in the study of brain phenomena of great methodological and neuropsychiatric interest—such as neurovascular coupling (NVC) and neuronal network mapping. We provide a methodological description of this interdisciplinary field of study, and focus in particular on the limitations of the widely used blood oxygen level dependent (BOLD) signal and how multimodal readouts can shed light on the contributions arising from neurons, astrocytes, or the vasculature.We conclude that information from multiple signaling pathways must be incorporated in future forward models of the BOLD response to prevent erroneous conclusions when using fMRI as a surrogate measure for neural activity. Further, we highlight the potential of direct neuronal stimulation via genetically defined brain networks towards advancing neurophysiological understanding and better estimating effective connectivity. Among such imaging modalities, blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) is probably the most widely used surrogate measure of neural activity in humans.,in vivo microscopy techniques allow the deconvolution of macroscopically lumped FH effects into contributions of individual tissue components  resolved in space and time.Animal models play an important role in addressing these questions. While most of the pioneering fMRI research has been performed in primates and rats,,in vivo microscopy techniques to the study of deep brain structures may become challenging.Further researchAlternatively, information on the contribution of individual cell-types to FH may be obtained using cell-specific labeling strategies in combination with an imaging readout that collects data from a volume comparable to that of the noninvasive fMRI imaging method. The hybrid setup can be designed such that multiple signals can be sampled simultaneously, which becomes critical under conditions in which scan-to-scan variability contains relevant biological information (see below). Early hybrid experiments combining fMRI with well-established electrophysiological recordings have provided valuable insights into the type of neuronal activity that is driving FH.,in vivo targeting of node-like systems  and imaging of effective connectivity patterns associated with specific neuronal populations. For the latter part, we will particularly focus on serotonergic and dopaminergic signaling elicited by stimulation of the respective deep brain nuclei.22.1The BOLD fMRI signal is determined by both neurovascular and neurometabolic adaptation, whereas pure hemodynamic readouts such as activity-induced CBF and CBV changes reflect only volume integrated FH responses. Combining the different fMRI readouts with alternative (invasive) measures of stimulus-evoked activity within the neurovascular unit (NVU) provides important insight regarding the interpretation of fMRI signals. Readouts tightly linked to neuronal activity include optical imaging using voltage-sensitive dyes, measurements of calcium  modalities may be advantageous if the objects of study are, e.g., evoked brain responses that are highly stereotypic and constant over time. Given a stimulus paradigm that excludes refractory or adaptation effects, one might consider trial-by-trial variability of neural responses to identical stimuli as random noise, meaning that it does not contain relevant biological information. Contributions of random noise can be accounted for by averaging over multiple trials. Under these circumstances, sequential acquisition would be advantageous to avoid trade-offs, which, as discussed below, inherently arise in either modality when using a hybrid measurement setup.The first important question in hybrid setup design is: However, recent experimentalFurther, the majority of the studies mentioned in this review are conducted in rodents, which typically require anesthesia. This affects multimodal experiments twofold: first, the anesthetic modulates spontaneous Does the scientific question ask for an optimal method providing cellular resolution? There are two strategies to gather cell-specific information using optical methods: microscopy-based methods, which provide sufficient spatial resolution to identify individual cells; and bulk sampling methods, which permit identification of specific cell population using selective labeling techniques. The fMRI signal of a voxel represents the integrated activity of thousands of neurons. Hence, when investigating the contribution of a specific cell population to the lumped fMRI signal, it may be more appropriate to acquire the bulk signal of a volume comparable to that giving rise to the fMRI signal rather than monitoring the behavior of individual cells within a much smaller FOV. Nevertheless, as already discussed, high spatiotemporal resolution is of relevance for analyzing mechanisms underlying NVC and its dynamics.–The second major consideration is: Signals reflecting brain activity are typically weak, and the signal-to-noise ratio (SNR) is commonly a limiting factor for true hybrid measurements, i.e., simultaneous recording of signals with two (or more) modalities. Adapting modalities to work in parallel exacts a toll on their performance. Boosting up spatial resolution would further compromise SNR, as the signal is proportional to the volume of the individual voxel sampled. This can be accounted for using high magnetic field strengths and a small MRI receiver coil yielding a high filling factor  have to be considered.A third major question related to hybrid setup design is: ,Optical access to deeper brain structures is limited by the nature of light photon interaction with biological tissue . There are two approaches to study deep-lying structures with optical techniques: the use of diffuse optical tomography (including fluorescence tomography), which reconstructs light source distribution in 3D from projections measured at the tissue surface, but which yields spatial information significantly inferior to MRI.2.1.2To provide adequate solutions for multiple of the use case variants delineated above, multiple variations of integrating light modalities with fMRI have been developed, which include the following:in-vivo hybrid recordings in rodents  vectors or lentiviral vectorsA critical aspect for the success of opto-fMRI experiments is the elicited signal amplitude, as the intrinsically low SNR of the method2.2.2,,,,,,,,The foremost consideration in applying opto-fMRI is selecting the correct biological features for both stimulation and imaging. While the breadth of neurosicentific applications cannot be sufficiently detailed without a review of all neurobiology, a series of guidelines can be formulated based on the characteristics of the method. Conceptually, opto-fMRI uses targeted, invasive, and high-temporal-resolution stimulation to drive a set of neurons, combined with a whole-brain, noninvasive, medium-temporal-resolution read-out method. Thus, an important question is whether targeted stimulation is relevant with respect to whole brain imaging. As a consequence, the ideal setting for leveraging the full potential of the technology is targeting widely projecting neurotransmitter systems to investigate their effects on neuronal activity at the whole-brain level. This is substantiated by extant opto-fMRI literature, in which the stimulation of wide efferent spectrum structures such as monoaminergic systems,While fMRI can be freely combined with any optogenetic technologies, the exigences imposed by MRI scanner access specifically encourage the use of the most established and extensively characterized constructs. As such, constructs of great clinical interest, such as anion-permeable channelrhodopsins and step-function opsins, have respectively seen no or little,A key constraint to leveraging the full versatility of optogenetics, and thus of opto-fMRI, is the application of the technology in species with low or no availability of transgenic lines. The highly flexible Cre-LoxP/AAV vector delivery method, which allows the decoupling of signal transducer variants from cell-type selection, is contingent on transgenic lines expressing Cre recombinase under the desired cell-type-specific promoter. Large libraries for such lines are available for the mouse, and increasingly, but to a lesser extent, for the rat. Other model animals, and particularly higher primates, do not offer any comparable level of access, and consequently, opto-fMRI application in these settings presupposes the use of more unwieldy custom lentiviral vectors, as well as close consideration of the trade-off between promoter length and expression characteristics.2.2.3,,,Optogenetics offers considerable flexibility with regard to the stimulation protocol, apt usage of which can considerably enhance the SNR for opto-fMRI. A fundamental distinction is made in the field of fMRI between “event-related” and “block” designs, which refer to the distribution of stimulation events over the duration of the experiment. Nominally, a design is deemed event-related if the stimulation (ON) period consists of a single event or is otherwise equal to or shorter than the acquisition TR—with longer ON durations being deemed block designs. Event-related designs lend themselves to sequence randomization, as they provide low but similar contrast upon variation, more closely resembling physiological activation. While such protocols can be employed in opto-fMRI , which is the analysis method most commonly used to resolve spatial response patterns based on a stimulation time course. Experimental parameters that bear on the fitness of a stimulation time course include experiment length, temporal frequency band reliability (lower frequency bands in particular may be considered unreliable in fMRI due to drift), as well as the impulse response function, which differs greatly with respect to contrasts . These parameters can be submitted to genetic-algorithm optimization workows,,,,,,,,,a priori optimized protocols across brain areas, stimulus structure may also be leveraged as a flexible tool for the exploration of differential signal propagation.The internal structure of stimulus blocks can also be adapted to increase the contrast generated in fMRI, though this may produce varying degrees of physiological comparability in the elicited activity mode. The intrinsic SNR constraints of fMRI, particularly when performed without cryogenic coils, however, prompt stimulus train optimization toward evoking the maximal activity level compatible with tissue preservation. Stimulation protocols for opto-fMRI, based predominantly on in-house empirical optimization, generally span 10 to 100 Hz in frequency and 2 to 20 ms in pulse width.,,A ceiling for the maximization of optogenetically evoked signal can be estimated with regard to heating artefacts, which can arise following light stimulation.An important consideration throughout stimulation protocols is that optogenetics can drive, but cannot clamp activity. This means that while both excitation and inhibition can be elicited, this does not happen in a complementary manner. In the absence of activation-inducing stimuli, cells are not inactivated, but rebound toward endogenous network activity. Similarly, in the absence of inactivation-inducing stimuli, cells will also rebound toward normal endogenous network activity. This constraint even applies to step-function opsins,2.3A common though easily overlooked constraint in small animal applications, which affects both multimodal recording and optogenetic stimulation experiments, is the potential for setup incompatibilities between optical tools and the cutting edge of fMRI technologies. This manifests itself in implant/coil incompatibility, whereby surface coils—cryogenic coils in particular—constrain the positioning of animals with optical implants as are canonically used for light-based measurement or stimulation. Manufacturing as well as operation protocol developments permit pitch and yaw variable targeting of structures,33.1,The basic NVU consists of neuron, astrocyte, and vascular cells  and is schematically summarized in 3.1.1The involvement of astrocytes in NVC has long eluded electrophysiological recordings, as they are electrically inexcitable. However, given their key location within the NVU, engulfing the synapses and covering the vasculature with their end-feet, it appears plausible that astrocytes are involved in FH to an extent beyond the mere role of an energy reservoir. Glutamate mediated signaling leads to activation of neuronal N-methyl-D-aspartate receptors (NMDA-R) as well as astrocytic metabotropic glutamate receptors (mGluR) and ion channels.Investigating the intricate role of astrocytes in the generation of BOLD signals is a prime example of a research question that strongly benefits from hybrid optical/MRI approaches. Their involvement could be demonstrated by combining neuronal and astrocytic response . Such firesponse —i.e., th3.1.2,Electrical forepaw stimulation was shown to prompt a local dilation of the capillary bed and an increase in the velocity of red blood cells indicative of decreased vascular resistance.,While neuronal and astrocytic vasodilatory signaling is focused to the site of activity, spatiotemporal analysis of vascular responses revealed involvement of vessels at distances larger than 1 mm,,Taken together, FH arises as combination of different mechanisms affecting vascular segments in a differential manner.3.1.3,,The aim of noninvasive functional brain imaging is to provide an accurate estimate of the underlying neuronal activity. Employing optical recordings at multiple stages of NVC provides relevant information for optimizing forward modeling. To generate a more comprehensive picture of the observed BOLD response, we suggest extending the well-known balloon model, which predicts the changing venous blood volume and dHb content (thereby prompting the BOLD response), by including non-neuronal mechanisms shown to display stimulus-associated signal transients . The oriAnimals studies revealed interference of anesthesia with CNS physiology at various levels. It was shown to affect neuronal excitability per se without affecting NVC,,A forward model of the neuronal-astrocytic-vascular 4In preclinical neuropsychiatric applications, opto-fMRI has prominently been used to map the effective connectivity of neurotransmitter systems, as illustrated by the landmark results of mapping the dopaminergic,,ceteris paribus , as shown in 5Recent advances in the development of hybrid optical/fMRI systems enabled tackling difficult research questions using the best of two worlds: The capabilities of fMRI to record brain functional parameters, and the plethora of genetic tools in optical neuroscience to investigate the role of individual cell types. Integrating signals at different spatial scales along the neurovascular pathway opens avenues to validate forward models of hemodynamic readouts. The usage of cell type-specific stimulation lends the capacity to bypass sensory signal input mechanisms and drive individual network nodes in the brain. Both in stimulation and complementary spatial scale measurements, correlations with cell biological phenomena enhance the causal understanding of neuronal function.Such insights facilitate both conceptual advances in the accurate interpretation of fMRI data—thereby reflecting on extant results spanning a breadth of human and animal studies—as well as a stark increase in scope for fMRI capabilities.


Escherichia coli (ETEC) are ubiquitous diarrheal pathogens that thrive in areas lacking basic human needs of clean water and sanitation. These genetically plastic organisms cause tremendous morbidity among disadvantaged young children, in the form of both acute diarrheal illness and sequelae of malnutrition and growth impairment. The recent discovery of additional plasmid-encoded virulence factors and elucidation of their critical role in the molecular pathogenesis of ETEC may inform new approaches to the development of broadly protective vaccines. Although the pathogens have been closely linked epidemiologically with nondiarrheal sequelae, these conditions remain very poorly understood. Similarly, while canonical effects of ETEC toxins on cellular signaling promoting diarrhea are clear, emerging data suggest that these toxins may also drive changes in intestinal architecture and associated sequelae. Elucidation of molecular events underlying these changes could inform optimal approaches to vaccines that prevent acute diarrhea and ETEC-associated sequelae.Enterotoxigenic  Escherichia coli are common pathogens that comprise a genetically diverse diarrheagenic E. coli pathovar defined by the production of heat-labile toxin (LT) and/or heat-stable toxin (ST). It is estimated that worldwide hundreds of millions of symptomatic enterotoxigenic E. coli (ETEC) infections occur annually [The enterotoxigenic annually , a burdeannually , 3 and sannually , 5.Vibrio cholera in India and Bangladesh soon revealed the presence of E. coli that produced a heat-labile toxin (LT) LT similar to cholera toxin, and subsequently the small peptide STs, as well as the first of many plasmid-encoded fimbrial colonization factors (CFs). A canonical view of ETEC virulence soon evolved, in which these pathogens simply adhere to intestine via CFs to deliver their toxin payloads, and much of the ensuing investigation of ETEC related to discovery and characterization of additional plasmid-encoded CFs or coli surface (CS) antigens. To date, >25 antigenically distinct CF/CS antigens have been defined. Elucidation of the structural biology and biogenesis of these antigens suggests that development of a broadly protective vaccine would require a multivalent approach incorporating multiple adhesins and toxoid molecules [These pathogens were originally identified now >5 decades ago in patients presenting with severe choleralike diarrheal syndromes . Early iolecules . ApproacE. coli chromosome and the ETEC pathovar-specific features encoded on virulence plasmids [In effect, ETEC molecular pathogenesis can be viewed as the summary of events that enable these bacteria to reach the small intestine, traffic to the mucosal surface, engage epithelial cell receptors and ultimately deliver their LT and/or ST payloads. This process ultimately requires a coordinated division of labor between highly conserved core elements encoded the plasmids .E. coli [E. coli (which happened to lack the type II secretion system) failed to export the toxin. Fortunately, we now know that these chromosomally encoded features work in a highly orchestrated and cooperative fashion [With time, it has become clear that ETEC rely on a number of chromosomally encoded features. Virtually all ETEC are motile and can be serotyped according to their flagellar H antigens, and flagellar motility is essential to successful delivery of both LT and ST. The flagellar assembly apparatus is encoded on the chromosome, as are genes encoding type 1 pili common to all E. coli , and thoE. coli . LikewisE. coli , and theE. coli  are both fashion  with plaetpBAC 2-partner secretion locus responsible for export of EtpA, an extracellular adhesin [The identification of additional surface-expressed antigens has expanded the repertoire of ETEC virulence factors and contributed to our understanding of ETEC molecular pathogenesis. Two plasmid-encoded loci, discovered in a search for novel secreted molecules, have emerged as important virulence factors. These include EatA, a member of the serine protease autotransporter of the Enterobactericiae family , and the adhesin .  molecule to target in a polyvalent vaccine.Seminal studies by Hans Clevers and his laboratory in the Netherlands defined the signals required to maintain and propagate intestinal epithelial cells from stem cells . Stem ceThese small-intestinal “enteroid” systems have already proved to be extraordinarily useful in exploring pathogen-host interactions. Their ability to recapitulate important features of human biology is illustrated in experiments showing that ETEC deliver toxins more efficiently to blood group A enterocytes, reflecting increased disease severity in human volunteers and naturally infected children who express blood group A antigens .In addition, goblet cells present in the enteroids produce significant amounts of MUC2 mucin, which normally serves as a protective barrier to mitigate interaction of ETEC with enterocytes goblet cells . Data emenvironmental enteric dysfunction (EED) or environmental enteropathy, remain very poorly understood.While the mortality rate associated with acute diarrheal diseases has declined appreciably since the introduction of oral rehydration therapy and other measures, even less severe episodes of ETEC diarrhea can exert lasting effects on children, including growth faltering, and malnutrition . AlthougE. coli, tropical sprue, remains the most common cause of chronic small-intestinal malabsorption and diarrhea in areas of high ETEC prevalence including India [E. coli” in luminal aspirates from patients with tropical sprue [Interestingly, another enigmatic condition linked to toxin-producing ng India . Notablyal sprue . UnfortuBoth EED and tropical sprue are characterized by shortening of the small-intestinal villi, lengthened crypts, mucosal inflammation, and nutrient malabsorption . DespiteNotably, both ETEC enterotoxins target intestinal epithelia to activate the production of cyclic nucleotides that serve as major cellular “second messengers” involved in the modulation of multiple cellular pathways. Soon after the discovery of LT, it was recognized that it stimulates the production of cyclic adenosine monophosphate (cAMP) by adenylate cyclase . IntraceSimilarly, ST, binds to guanylate cyclase C on the surface of intestinal epithelial cells, where it activates production of cyclic guanosine monophosphate, to activate protein kinase G, which in turn can phosphorylate multiple cellular proteins in addition to those directly related to modulation of ion channels and development of diarrhea. Remarkably, however, despite the capacity for these toxins to induce pleiotropic effects on the host cell via their impact on critical “second messenger” , pathwayInterestingly, LT was previously shown to play an important role in promoting ETEC adhesion to intestinal epithelial cells in vitro  and smaltransiently benefit the pathogen, these architectural changes may come at the expense of the host, as they are occurring on the surface of enterocytes, the major site of nutrient absorption in the human small intestine. Enterocytes are propagated from stem cells in the small-intestinal crypts, and they migrate to the distal end of small-intestinal villi as they mature, eventually being released from the extracellular matrix and shed from the villus tip to undergo programmed cell death, or anoikis [While these events  anoikis , a proce anoikis . Alterat anoikis  could seAt a microscopic level, both tropical sprue and EED are characterized morphologically by substantial alterations in the small-intestinal villous architecture, characterized by blunting of the villi. The epithelial surface of intestinal villi is comprised of enterocytes, goblet cells, tuft cells, and enteroendocrine cells that differentiate as they propagate up the villus surface from progenitor cells in the crypts of Lieberkuhn, where intestinal stem cells and Paneth cells reside. Enterocytes are the most abundant cells in intestinal epithelia, and the major site of nutrient absorption. At an ultrastructural level, the intestinal “brush border” is normally formed by densely packed regular arrays of microvilli at the surface of enterocytes. In essence, the microvilli are extensions of the enterocyte apical plasma membrane surrounding a central core of actin microfilaments. These protrusions greatly increase the available surface area at luminal surface of the intestine, allowing for maximal nutrient absorption. As such, insults that interfere with the development of microvilli can potentially result in malnutrition.Intriguingly, transmission electron microscopy of small-intestinal biopsy specimens from patients with tropical sprue, obtained by Mathan et al in the 1970s , demonstNot surprisingly, given the importance of cAMP as a second messenger in the cell, we found by RNA sequencing that treatment of small-intestinal enteroids with LT, a close homologue of cholera toxin, results in the modulation of many cellular genes. Among the hundreds of genes altered by LT were those encoding proteins needed for the biogenesis of microvilli , and traUnfortunately, in the absence of a broadly protective vaccine, young children in low-middle-income countries will likely continue to bear the burden of acute diarrheal illness imposed on them by ETEC. Resolving the role of more recently discovered virulence proteins in the molecular pathogenesis of ETEC may focus vaccine development on antigens that encompass the breadth of ETEC.While ETEC have been epidemiology linked to development of sequelae including malnutrition, additional study is needed at the molecular level to understand how these pathogens might promote changes in the small-intestinal architecture that underlie the sequelae. It is becoming clear, however, that one or both toxins secreted by ETEC can affect a variety of cellular pathways that govern normal homeostasis and ultimately absorptive capacity of the small intestine in addition to causing diarrhea. An ideal ETEC vaccine would mitigate or prevent both the acute diarrheal illness caused by ETEC, as well as the substantial burden of nondiarrheal morbidity linked to these pathogens.


Rapid methodological advances in statistical and computational genomics have enabled researchers to better identify and interpret both rare and common variants responsible for complex human diseases. As we continue to see an expansion of these advances in the field, it is now imperative for researchers to understand the resources and methodologies available for various data types and study designs. In this review, we provide an overview of recent methods for identifying rare and common variants and understanding their roles in disease etiology. Additionally, we discuss the strategy, challenge, and promise of gene therapy. As computational and statistical approaches continue to improve, we will have an opportunity to translate human genetic findings into personalized health care. Over the past decade, genome sequencing technology has been one of the fastest growing fields in biomedical science. Thanks to the progress in sequencing automation, the cost of sequencing has dropped dramatically. As a result, an enormous amount of genomic data has been generated, providing an informative profiling of human genetic variations, disease-related mutations, and association between genotype and phenotype ,2,3,4.With the achievement of the Human Genome Project and the HapMap Project in the early 2000s, human genetic research in complex diseases started a new chapter: genome-wide association studies (GWAS). In 2005, a landmark GWAS found two single nucleotide polymorphisms (SNPs) associated with age-related macular degeneration , obesityWith rapid advances in DNA sequencing technologies, assessment of rare genetic variants in complex traits has become feasible. In particular, whole-exome sequencing (WES) and whole-genome sequencing (WGS) have gained popularity in recent studies on gene discovery. Herein, we review the recent analytical approaches for identifying disease-associated rare variants in population-based or family-based studies based on WES or WGS. We also discuss recent advances in common variant association analysis and polygenic risk score methods. Finally, we discuss how to translate genetic discovery into effective therapeutics or treatments. The flow diagram is illustrated in −8 and a relative risk of 3, the power to detect a variant with minor allele frequency (MAF) equal to 0.5% is around 0.05. To boost statistical power, most rare-variant association methods combine association signals across multiple rare variants in pre-defined variant sets  and generally assume the presence of multiple trait-associated variants in the same variant set . Al. Al35]. The recent development of very large WES cohorts such as the Deciphering Develop-mental Disorders project, coupled with the improved understanding of the germline mutation rate, have enabled more robust estimation of the absolute and relative fraction of inherited variants and DNVs for complex diseases. Martin et al. conducted sex-specific burden analyses of damaging DNVs to identify an enrichment of specific classes of X-linked variants in probands and estimated the fraction of probands attributable to those variants . They fohttp://dida.ibsquare.be/, accessed on 17 November 2021). This can be attributed to several factors, including difficulties in establishing a genotype–phenotype correlation, reduced penetrance, phenotypic and expression variability, and most importantly, the lack of efficient and robust methods for detecting gene–gene interaction due to the overall small effect of each variant on disease risk. The genetic linkage analysis method was successful in detecting digenic diseases in some families [Digenic inheritance (DI) refers to the simplest form of oligogenic inheritance . Individfamilies , but othfamilies . The appfamilies . NowadayThe case-only design provides an estimation of gene–gene interactions without requiring negative control samples  and demoAlthough the aforementioned methods have contributed significantly to unraveling oligogenic diseases, they are often met with limitations and criticism, predominantly due to their inability to deal with high dimensional data and non-linear regression tests. For these reasons, machine learning methods started to gain recognition and popularity in the field of genetics, particularly supervised machine learning where the algorithm predicts potential gene–gene interaction as an output depending on the input data and the set of rules obtained through model training. Among the supervised machine learning models, random forests (RFs), neural networks, cellular automata, and multifactor dimensionality reduction are the most used . RFs, a It is understandable that traditional methods alone are unable to detect digenic variants due to the limitations imposed by the used statistical tests and the often-required pre-knowledge of biological aspects of diseases. Likewise, limitations can be faced with the machine learning approach due to insufficient training data, confounding effects, reproducibility and accessibility, and the potential slow-performing algorithm when dealing with large data sets ,75. Furtp-value threshold of 5 × 10−8 to account for multiple testing correction. Different from sequencing-based studies, a GWAS typically has a larger sample size due to the lower cost of microarray genotyping, but it is better powered to examine common variant associations than those for variants with lower frequencies due to poor imputation quality of rare variants, and a lack of ability for common variants to tag rare variants through LD.A GWAS aims to identify associations between  SNPs and a disease or trait of interest. SNP genotypes are usually obtained using a genotyping microarray for a set of pre-determined variants. The genotype information for each bi-allelic SNP is stored as the count of a reference allele, which can be coded as 0, 1, or 2. It is also a common practice to impute relatively common but ungenotyped SNPs based on a population haplotype reference panel . A GWAS Despite the simplicity, GWAS have identified tens of thousands of associations for numerous diseases and traits . In partGWAS also has some inherent limitations. One major challenge in population-based GWAS is the unadjusted confounding due to population stratification where different ancestry groups differ in both variant allele frequencies and the trait under study. In addition, recent evidence suggests that parental genotypes can be a major confounder for genetic associations identified in GWAS . A persoSince the proportion of complex trait variance explained by the additive genetic components in GWAS is often smaller than heritability estimated from twin studies, gene–gene interactions have been hypothesized to partially account for this discrepancy ,88. HoweA key goal in human genetic research is to identify individuals at higher disease risks for early screening and intervention. Thanks to the widely accessible summary-level data from GWAS, PRS models that can be trained directly using GWAS summary statistics have quickly gained popularity in recent years. In a nutshell, a PRS is a weighted (by variant effect sizes) sum of risk allele counts across a (possibly large) number of SNPs. It quantifies the genetic predisposition of disease risk for an individual and thus can be used to stratify individuals into high and low risk groups .Methodological challenges in computing PRS reside in estimating the highly polygenic yet typically weak SNP effects for most complex traits and accounting for extensive LD in the human genome. Recently, penalized regression models that re-estimate SNP effects from GWAS summary statistics while explicitly modeling LD have been shown to effectively improve the predictive performance of PRS ,102,103,However, challenges remain before clinical use of PRS becomes a reality. Currently, the vast majority of published GWAS have been conducted on the non-Hispanic white population . PRS traA primary objective of human genetic studies is to uncover novel genetic etiology to disease and elucidate pathomechanistic features to develop meaningful therapies for patients. Among the most-promulgated forms of novel therapies stemming from human genetic studies is gene therapy, which seeks to alter the biological properties of living cells by modifying or modulating the gene function and expression in cells . Being pWhile gene therapy techniques can target both somatic and germline cells, ethical concerns about introducing heritable changes to humans have prevented the U.S. Food and Drug Administration (FDA) from approving any therapies targeting germline cells. Different strategies for different types of diseases have been developed in past decades: (a) inserting a functional copy of a gene to restore the biological function disrupted by a deficient copy ; (b) proGenerally, drug development is divided into five steps: discovery, preclinical research, clinical research, FDA review, and post-market monitoring. This process is lengthy and expensive, taking up to 12–15 years with costs of more than USD 1 billion and increasing every year. At the same time, conventional drug development has slowed exponentially, with the number of new drugs brought to market per billion USD spent on research and development decreasing ten-fold since 1980 and fifty-fold since 1960 . Thus, rClinicalTrials.gov numbers, NCT03282656) showed a promising outcome, whereby the patient had prompt hematopoietic reconstitution after treatment [In a poignant example of this ‘base pairs-to-bedside’ approach to drug development, until 2017 sickle cell disease (SCD), one of the most common inherited blood disorders, had seen no therapeutic innovation to meet unmet clinical needs in over 20 years. Thanks to the progress of disease association analysis and advanced genetic engineering, more-specific drugs  have become available in the past 3 years ,116,117.reatment . There areatment , amyotroreatment , autosomreatment , hemophireatment ,124, retreatment ,128,129,reatment , and cysreatment  , genomic technology , and the type of genetic variations under investigation . We also discussed the current best practices of genomic study in human disorders—gene therapy—and summarized currently available treatments for diseases .As demonstrated in many studies, genetic variations alter patient responses to clinical treatments ,143,144.Many in the field speculate that, as WES/WGS becomes increasingly more common and affordable, increased understanding of variant–phenotype relationships and novel integrative genomic and pharmacogenomic therapeutic approaches tailored to patient-specific genetic information may revolutionize clinical care by increasing treatment specificity ,147. Qua


Machine learning (ML) provides incredible opportunities to answer some of the most important and difficult questions in a wide range of applications. However, ML systems often face a major challenge when applied in the real world: the conditions under which the system was deployed can differ from those under which it was developed. Recent examples have shown that ML methods are highly susceptible to minor changes in image orientation, minute amounts of adversarial corruptions, or bias in the data. Susceptibility of ML methods to test-time shift is a major hurdle in a universal acceptance of these solutions in several high-regret applications. To overcome this challenge, in this research topic “Safe and Trustworthy Machine Learning”, a wide range of solutions are contributed as potentially viable solutions to address trust, safety and security issues faced by ML methods.Song, et al., considered the problem of dataset shift detection for safety-critical graph applications. The authors proposed a practical two-sample test approach for shift detection in large-scale graph structured data.Anirudh, et al., considered the problem of post-hoc interpretability tasks, such as, prediction explanation, noisy label detection, adversarial example detection. The authors introduced MARGIN, a simple yet general approach, that exploits ideas rooted in graph signal analysis to determine the most influential nodes in a graph to solve the aforementioned tasks.Majumdar, et al., considered the problem of mitigation of bias arising due to unbalanced representation of sub-groups in the training data. The authors proposed a bias mitigation algorithm to generate Subgroup Invariant Perturbation (SIP) which when added the input dataset reduces the bias in model predictions.Huang, et al., showed that seq2seq models, successful in natural language correction, is also applicable in programming language correction. Their results show that seq2seq models can provide suggestions to potential errors and have a decent correct rate in code auto-correction task.Qayyum, et al., conducted a systematic evaluation of literature of cloud-hosted ML/DL models along both the important dimensions -- attacks and defenses -- related to their security. The authors identified the limitations and pitfalls of the analyzed papers and highlight open research issues that require further investigation.Berghoff, et al., presented a comprehensive list of threats and possible mitigations of IT security of connectionist artificial intelligence (AI) applications. AI-specific vulnerabilities such as adversarial attacks and poisoning attacks as well as their AI-specific root causes are discussed in detail. The article concluded that single protective measures are not sufficient but rather multiple measures on different levels must be combined to achieve a minimum level of IT security for AI applications.Kusters, et al., analyzed key challenges to interdisciplinary AI research, and deliver three broad conclusions: 1) future development of AI should not only impact other scientific domains but should also take inspiration and benefit from other fields of science, 2) AI research must be accompanied by decision explainability, dataset bias transparency as well as development of evaluation methodologies and creation of regulatory agencies to ensure responsibility, and 3) AI education should receive more attention, efforts and innovation from the educational and scientific communities.The papers included in this research topic “Safe and Trustworthy Machine Learning” discussed some promising solutions, highlighted open research issues, and offered visionary perspectives regarding trust, safety and security issues faced by machine learning. We hope that challenges and potential solutions presented here will help researchers better understand the current limitations of machine learning methods and motivate future work in the direction of developing trustworthy, safe, and robust machine learning methods, and their applications to high-regret application areas.


Exercise-based, multidisciplinary programs have been associated with improved clinical outcomes post myocardial infarction and is now recommended for patients with cancer at elevated risk for cardiovascular complications. Imaging studies have documented numerous beneficial effects of exercise on cardiac structure and function, vascular function and more recently on the cardiovascular risk profile. In this contemporary review, we will discuss the effects of exercise training on imaging-derived cardiovascular outcomes. For cardiac imaging via echocardiography or magnetic resonance, we will review the effects of exercise on left ventricular function and remodeling in patients with established or at risk for cardiac disease , and the potential utility of exercise stress to assess cardiac reserve. Exercise training also has salient effects on vascular function and health including the attenuation of age-associated arterial stiffness and thickening as assessed by Doppler ultrasound. Finally, we will review recent data on the relationship between exercise training and regional adipose tissue deposition, an emerging marker of cardiovascular risk. Imaging provides comprehensive and accurate quantification of cardiac, vascular and cardiometabolic health, and may allow refinement of risk stratification in select patient populations. Future studies are needed to evaluate the clinical utility of novel imaging metrics following exercise training.Exercise is a commonly prescribed therapy for patients with established cardiovascular disease or those at high risk for  Regular physical exercise provides many benefits to the cardiovascular system and overall health at all stages of life. As such, aerobic exercise  and more recently resistance exercise , have been an integral component of clinical guidelines, and the cornerstone of cardiac rehabilitation for patients with or at risk for cardiovascular disease including coronary artery disease (CAD), heart failure, and cancer –4.The most common form of exercise for primary and secondary prevention of cardiovascular disease is aerobic training, which improves oxygen delivery (i.e. cardiac output) during physical effort, and resistance training, which increases skeletal muscle mass and strength. Patients with established cardiovascular disease are commonly referred to cardiac rehabilitation, which typically involves a 6–24-week program of supervised, moderate-intensity  continuous aerobic exercise, supplemental resistance training and other health interventions. High-intensity interval training  is a time efficient aerobic exercise alternative for lower risk patients and appears to offer similar health benefits  are the primary imaging modalities used to evaluate the cardiovascular effects of exercise based on their accuracy, versatility, and safety profile for repeat testing. In assessing the heart, these modalities are principally used to provide information on ventricular size, mass, and function as well as hemodynamic and flow quantification. Cardiac MRI also allows the evaluation of myocardial tissue characterization, including macro- and microscopic fibrosis using quantitative mapping sequences and contrast enhanced imaging . In asseIn this review article, we will review the potential benefits of exercise on left ventricular (LV) function and remodeling, vascular function and structure, and body composition in adult patients with or at risk for cardiovascular disease and discuss the application and utility of imaging to characterize these changes.Before examining the role of exercise training in patients with cardiovascular disease, it is important to understand its effects in healthy individuals. Public health recommendations for the general population are to include 150 min per week of moderate-intensity aerobic exercise or 75 min if the intensity is classified as vigorous . SustainSoluble suppression of tumorigenicity 2 (sST2) is an emerging prognostic biomarker that has been associated with myocardial fibrosis, inflammation, and function and is an independent predictor of long-term mortality . High-inn = 468 healthy individuals) found similar LV hypertrophy adaptations to endurance training among men and women, but that LV end-diastolic, end-systolic and stroke volumes increased more in men  (LVEF < 40%), exercise training has been shown to reverse LV remodeling, with favorable effects on ejection fraction, stroke volume, and end-diastolic and end-systolic diameters –62.2) by ~2 ml/kg/min] (2) after 12 months of training  is an important clinical consideration for patients receiving anthracycline and/or trastuzumab-based adjuvant treatment. Early recognition and management of CTRCD is important as it has the potential to delay cancer treatment and directly impact cardiovascular morbidity and mortality. Cardiac imaging plays a critical role in the early detection and monitoring of CTRCD. This adverse complication is most commonly assessed by measuring LVEF by echocardiography, multi-gated blood pool imaging or cardiac MRI . Left ve2  of exercise training in patients with breast cancer receiving trastuzumab and/or anthracyclines showed no consistent benefit in the prevention of LV dilatation or worsening systolic function as assessed by echocardiography or cardiac MRI . However2 .Cardiac reserve function is defined as the difference in cardiac function from rest to peak exercise captured by echocardiography or cardiac MRI. Its potential importance stems from its ability to detect stress-induced myocardial dysfunction and evaluate exercise-related changes to chronotropy, inotropy, lusitropy, and vasodilation in patients with normal resting parameters .In patients with HFpEF, a number of metrics have been studied with exercise imaging including LVEF, LV global longitudinal strain, E/E' ratio, mitral annular systolic velocity, and tricuspid regurgitation maximal velocity –93. RestExercise stress imaging with cardiac MRI offers the advance of enhanced accuracy and reproducible quantification of cardiac volumes and biventricular function relative to other imaging modalities , 95. TheAge, cardiometabolic disease and toxic exposures  each affect the arterial wall matrix, reduce elasticity and thus increase the potential for arterial stiffening and impaired blood flow . EndotheAerobic exercise training has been linked to reduced arterial stiffness in patients with CAD. A systematic review of 5 studies of arterial stiffness in patients with CAD found that aerobic training was associated with lower PWV measures on Doppler ultrasound . One stu2)  . In a cr2) . However2) . While v2) .2 max + 4.3 ml/kg/min) and PWV (−0.9 m/s) in 51 patients  reduced arterial PWV . Howeverpatients . A recenpatients   in patients with established cardiovascular disease or with cardiovascular risk factors.Late gadolinium enhancement sequences on cardiac MRI are frequently utilized to identify and quantify myocardial scar (replacement fibrosis) arising from acute ischemic injury . Follow- disease , 123. Fo6 months . Similar6 months . Increasotherapy , 127. Kiications . A recenfraction . To our It is now well-established that the location of fat deposition is much more closely linked to cardiovascular disease risk than the total quantity of fat . For insImaging modalities used to quantify body composition in clinical practice and research include computed tomography, dual energy X-ray absorptiometry and MRI . MRI is Exercise training and caloric restriction  are the primary interventions used to improve body composition. A meta-analysis comparing the effects of exercise training to caloric restriction in patients with obesity found that both interventions independently reduce visceral fat with a potential trend toward greater reduction with exercise . In patiOur group is conducting a randomized-controlled trial of a multi-modal intervention including combined aerobic and resistance exercise training and dietary counseling  on LV function in patients receiving cardiotoxic breast cancer treatment and includes quantification of ectopic fat as a secondary outcome measure . In a reExercise training is a cornerstone treatment for patients with cardiovascular disease due its demonstrated impact on risk profile and clinical outcomes. Ultrasound or MR imaging for quantification of changes to the cardiac and extra-cardiac phenotype of patients, provide valuable whole-body information about the cardiovascular risk profile and have an excellent safety profile to allow for repeat testing.The choice of imaging modality to evaluate changes in cardiovascular structure and function is an important consideration. In general, echocardiography is the most readily available modality and offers a comprehensive assessment of ventricular volumes, mass and function. It can also more easily be used to evaluate cardiac function in real time during exercise on a semi-recumbent bicycle. Cardiac MRI is considered the gold standard imaging test for ventricular volumes, mass and function due to high reproducibility. As mentioned previously, it also allows assessment of cardiac and extra cardiac tissue characterization which has increasingly been linked to prognosis in patients with or at risk for cardiovascular disease. Therefore, cardiac MRI should likely be used to study the effects of exercise training in higher risk patient groups when available.There is strong evidence supporting a beneficial effect of exercise training, particularly >6 months duration, on cardiac function and remodeling in patients with CAD or heart failure . Imaging1 mapping sequences also provide incremental prognostic information however, the effect of exercise training on this parameter has not well-studied. MRI and/or ultrasound is well-suited for multiparametric evaluation of exercise training interventions and is easily translatable to the clinical setting to help guide patient management. Incorporation of MRI-derived lung water quantification could also help to identify cardiogenic causes of exercise intolerance and evaluate exercise training in patients with subclinical pulmonary edema (Ectopic fat deposition, particularly, visceral and intermuscular fat are important indicators of cardiovascular risk that can be reduced by exercise training in numerous patient populations. MRI-acquired myocardial tissue characterization using Try edema , 143. Exry edema , 145.Targeted nutrition interventions can also positively impact cardiovascular health and should be further explored in combination with exercise interventions . DietaryPreclinical models of cardiovascular disease have been used extensively to study the cardioprotective effects of exercise training. Aerobic training has been shown to attenuate cardiac injury in animal models of myocardial infarction , ischemiWA and DIP proposed the review subject matter and drafted the manuscript. All authors made critical revisions to the manuscript, contributed to the article, and approved the submitted version.MH was supported by the Faculty of Nursing Research Chair in aging and quality of life at the University of Alberta, EP was supported by a Tier 2 Canada Research Chair, and CP was supported by a Campus Alberta Innovates Program Chair in Nutrition, Food, and Health as well as a Canadian Institutes of Health Research new investigator award.DIP reports consultant fees from Alnylam and Pfizer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Globally, antimicrobial resistance is one of the most important public health challenges in which the clinical microbiology laboratory plays a critical role by providing guidance for antimicrobial treatment. Despite the recognition of its importance, there is still a real need for the standardized training of clinical microbiologists and harmonization of diagnostic procedures. This is particularly true for veterinary clinical microbiology, where additional challenges exist when microbiologists are trying to fulfill a professional role very similar to that of their colleagues working in human microbiology laboratories. The specific points that need addressing to improve the outputs of veterinary microbiology laboratories discussed here include (i) harmonization of methodologies used by veterinary laboratories for antimicrobial susceptibility testing (AST); (ii) specific guidelines for interpretation and reporting of AST results for animal pathogens; (iii) guidelines for detection of antimicrobial resistance mechanisms in animal isolates; (iv) standardization of diagnostic procedures for animal clinical specimens; and (v) the need to train more veterinary clinical microbiology specialists. However, there is now a plan to address these issues, led by the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), which is bringing together experts in veterinary microbiology, pharmacology, epidemiology, and antimicrobial stewardship from Europe and wider afield. ENOVAT is aiming to work with project partners toward standardization and harmonization of laboratory methodologies and optimization of veterinary antimicrobial treatment. Ultimately, the project may provide a mechanism for standardization and harmonization of veterinary clinical microbiology methodologies that could then be used as a template for implementation at a wider international level. Antimicrobial resistance (AMR) is a global multifactorial issue that endangers the ability to treat bacterial infections and hinders the implementation of important medical advances  in both human and veterinary medicine. The emergence of AMR has highlighted the key role that clinical microbiology laboratories play in driving antimicrobial stewardship and appropriate antimicrobial use .The underuse or suboptimal use of microbiological culture and antimicrobial susceptibility testing (AST) and overreliance on empirical antimicrobial therapy can exacerbate AMR in both human and veterinary settings; therefore, to overcome these obstacles, a closer partnership between diagnostic laboratories and clinicians is required for successful antimicrobial stewardship , 2. In ahttps://www.iso.org/standard/70464.html), their implementation is dependent on local factors. Furthermore, currently there is no worldwide consensus for the use of a common methodology in veterinary laboratories. When performing culture and AST, veterinary laboratories generally follow methodologies developed for processing human clinical isolates. In that regard, laboratories adhere to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) or the American Clinical and Laboratory Standards Institute (CLSI) guidelines, or, less commonly, guidelines issued by various national committees. This approach serves the immediate needs of clinicians, and the data can be useful for detecting shifts in local antimicrobial susceptibility patterns. However, the use of multiple standards is a major limitation when comparing susceptibility data between laboratories or countries, thereby compromising global AMR surveillance in animal pathogens. Hence, the early detection of emergent resistant pathogens or meaningful comparison of resistance rates within or between countries is hampered, as shown in a study comparing antimicrobial susceptibility data in canine urinary tract infection isolates from across Europe  and EUCAST (VetCAST), there is still a shortage of animal-, infection-, and pathogen-specific clinical breakpoints (CBPs) for antimicrobial drugs used in veterinary medicine. Both subcommittees are actively developing more clinical breakpoints for veterinary antimicrobial agents; however, this is a slow process due to the complexity of the tasks for various pathogen-antimicrobial combinations in different infections and animal hosts. In the meantime, the lack of specific interpretative criteria for animal pathogens represents a great difficulty for laboratory staff. Thus, developing best practice guidelines for interpreting and reporting AST results for animal pathogens for which CBPs are not yet available must be regarded as a priority for the veterinary profession.https://www.eumonitor.eu/9353000/1/j9vvik7m1c3gyxp/vk0vn25n5e9o), AMR surveillance in companion animals, primarily cats, dogs, and horses, has not been included. Veterinary laboratories, which actively perform AMR surveillance, often follow either the CLSI (https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Resistance_mechanisms/EUCAST_detection_of_resistance_mechanisms_170711.pdf) for specific detection of resistance mechanisms; however, these are not entirely applicable for veterinary clinical isolates. For instance, consensus on detection methods for methicillin resistance in important animal pathogens, such as methicillin-resistant Staphylococcus pseudintermedius (MRSP) or S. schleiferi (MRSS), is still lacking  is https://www.gov.uk/guidance/uk-standards-for-microbiology-investigations-smi-quality-and-consistency-in-clinical-laboratories), should be created through a similar consultation process involving all partners and organizations active in this field. Ideally, these laboratory procedures should be standardized at a European level and made available to all veterinary microbiology laboratories. In addition, a new framework for Microbiology Investigation Criteria for Reporting Objectively (MICRO), to ensure accurate and comparable microbiology laboratory results are produced among human laboratories, was recently published and could be adopted by veterinary laboratories , ENOVAT is aiming to use online surveys to critically review the current methodologies and interpretive criteria used by veterinary microbiology diagnostic laboratories and identify gaps and challenges of microbiological diagnostic procedures. The survey outcome will provide an invaluable data source that can be used to draw a roadmap outlining how ENOVAT can work with project partners toward standardization and harmonization of veterinary microbiology methodologies.Although the points highlighted here are long-held goals, there is now a plan for action that is being led by the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT). ENOVAT is an EU COST Action project bringing together experts in veterinary microbiology, pharmacology, epidemiology, and antimicrobial stewardship throughout Europe and wider afield via collaborations with Near Neighbor Countries and International Partner Countries. Among other important objectives  technologies, loop-mediated isothermal assays (LAMP), mass spectrometry, and next-generation sequencing (NGS)   associated with clinical syndromes. These molecular tools include point-of-care tests (POCTs), gene-based resistance detection platforms, single or multiplex PCR assays, immune-chromatographic tests, peptide nucleic acid fluorescent ng (NGS) , 23. POCng (NGS) . This stng (NGS) . Multiplng (NGS) . These mng (NGS) . Not lasng (NGS) . The rolng (NGS) . Furtherng (NGS) . Howeverng (NGS) . The issng (NGS) .https://www.acvm.us/about-acvm/). In Europe, the formation of the European College of Veterinary Microbiology (ECVM) became a reality in 2016 (https://ebvs.eu/colleges/ECVM). In addition, the Study Group of Veterinary Microbiology (ESGVM), established within the European Society for Clinical Microbiology and Infectious Diseases, also promotes the need for training and specialization in veterinary microbiology in Europe (https://www.escmid.org/research_projects/study_groups/study_groups_o_z/veterinary_microbiology/). Furthermore, the European Association for Veterinary Diagnosticians  provides a platform for networking and communication among veterinary laboratories.As technical advances continue to emerge in clinical microbiology, careful integration of what is technically possible with what is clinically relevant will require regular appropriate training of staff to keep pace with the developments in the field , 27. ThiUltimately, the increasing threat from AMR and zoonotic emerging infectious diseases underlies the need to improve and integrate veterinary microbiology services with public health services worldwide to provide the backbone of a global One Health approach. Ensuring that veterinary microbiology laboratories have the technical facilities and the expertise of veterinary microbiology specialists provides the necessary infrastructure to change and adapt to new challenges, such as the one represented by the SARS-COV-2 pandemic. This major public health issue has created unprecedented pressure on global health services and provided an opportunity for veterinary microbiology services to rise to the challenge and show their adaptability by joining the global effort of controlling the pandemic through PCR testing when it was most needed .Within the ENOVAT project, we are developing united complementary approaches in the veterinary microbiology profession to help achieve the long-held goals of harmonization of AST methods and standardization of diagnostic procedures across veterinary microbiology laboratories in Europe and beyond. We are also lobbying for more training of clinical veterinary microbiologists to enable the rollout of high-quality diagnostic and treatment protocols for animals. This would ensure the implementation of common strategies and a level playing field across all laboratories, which will positively reduce the AMR burden and ultimately improve animal and public health. The outcomes may well bring benefits to veterinary diagnosticians worldwide.


Retroperitoneal fibrosis (RPF) is an uncommon disease characterised by the presence of fibroinflammatory reaction which starts around the infrarenal portion of the abdominal aorta in the retroperitoneum and frequently entrap the ureter causing obstructive uropathy. Approximately, two thirds of the cases are idiopathic, where aetiopathogenesis is not known. Ergotamine-induced RPF, although rare, is considered under secondary group. The fibrogenic process here is thought to be due to serotonergic activity. We report a case of RPF in a young female with obstructive uropathy who had history of long-term ergotamine intake for migraine. Histopathological evaluation revealed different evolving stages of necrotising vasculitis. In addition, the patient has responded to withdrawal of offending drug along with immunosuppressive therapy. We believe, apart from serotonergic activity, ergotamine can lead to RPF through a vasculitic process which has not been reported earlier. This condition has an idiopathic origin in majority of the cases ; however, this may arise secondary to malignancy, infection, drugs, chemoradiotherapy, and surgery, or very rarely as a part of Erdheim-Chester disease.2 In recent times, the pathophysiology of RPF is better understood, as some of these cases are speculated to be a part of clinically heterogenous immunoglobulin G4 (IgG4) related disease (IgG4-RD).3 Therefore, differentiating idiopathic from the secondary RPF is crucial due to completely different therapeutic approach. Although non-contrast enhanced computerised tomogram scan (NCCT) and magnetic resonance imaging (MRI) are useful imaging modalities which help in mapping the extent and severity of fibrosis, these are not useful in differentiating benign versus malignant forms of RPF. Moreover, CT with contrast media is contraindicated in RPF with renal complications.4 18F-fluorodeoxyglucose (FDG-PET) or PET/CT scan helps in diagnosis as well as post therapy monitoring by evaluating the disease activity and its extent.5 Histopathological evaluation of a surgical open biopsy specimen is considered as ‘gold standard’ for diagnosis, characterising the true nature of the lesion with atypical presentation such as localised mass-like lesion, confirming IgG4-RD, and monitoring response to immunosuppressive therapy.6 In this report, we describe a young adult female with histopathologically proven RPF with a history of prolonged ergotamine therapy for migraine and associated vasculitis, which, to the best of our knowledge, has not been described in world literature. Furthermore, the proposed pathophysiologic mechanism of RPF is also briefly highlighted.Retroperitoneal fibrosis (RPF) is a rare fibroinflammatory disorder which is characterised by deposition of fibrous tissue around the abdominal aorta and iliac arteries with a propensity for contiguous involvement of surrounding structures within the retroperitoneal space and frequently causing ureteral obstruction and renal failure.thpost-operative day, she developed vomiting and was re-evaluated with NCCT of abdomen which showed diffuse omental thickening in sub hepatic, infraumbilical, left iliac fossa, and pelvic region with mild thickening of peritoneal layer with mild to moderate ascites; and involvement of bilateral ureters causing mild bilateral hydronephrosis along with mild thickening of ascending colon and prepyloric region . Ascitic fluid cytological analysis showed lymphocyte rich effusion without any evidence of malignant cells based on which she was advised a course of antitubercular therapy (ATT). After 3 months of ATT, there was no improvement and vomiting were persisting. Cartridge-based nucleic acid amplification test (CBNAAT) for tuberculosis was found to be negative; and her serum CA-125 was found to be elevated  based on which a clinical suspicion of malignancy was raised, and she was referred to our institute for surgical oncology consultation. Upper gastrointestinal endoscopy and colonoscopy did not show any abnormality.In December 2017, a 41-year-old female presented with dull aching diffuse pain abdomen of one-year duration. Ultra-sonography (USG) of abdomen and pelvis revealed an ovarian cyst, for which she underwent hysterectomy and bilateral salpingo-oophorectomy at an outside hospital. However, she remained symptomatic after the surgery. On the 7rd and 4th part of duodenum causing restricted mobility. In addition, bilateral ureters were also seen to be encased and medially displaced due to the fibrosing bands. Obstructions were relieved by gastro-jejunostomy and jejuno-jejunostomy; and by putting bilateral ureteric double J (DJ) stents. Biopsies taken from omental and peritoneal tissue confirmed the diagnosis of RPF (see below). She was referred to rheumatologist for further work up; and was found to be negative for anti-neutrophil cytoplasmic antibody (ANCA), antinuclear antibody (ANA), anti-ds-DNA, rheumatoid factor, HLA-B27. Her serum IgG4 level was within normal reference limits. Her microbiological testing for viral serology  was negative.In view of the diagnostic dilemma, she was subjected to exploratory laparotomy of abdomen. Per operatively, omentum was found adhered to pelvis and anterior abdominal wall on multiple bands. Retroperitoneum was hard and fixed with presence of hypervascular fibrosing bands which were seen encroaching small and large bowel mesentery as well as encasing 3Table 1.On further enquiry, it was found that she was a known case of migraine for which she had been on Vasograin tablet (caffeine + ergotamine + paracetamol + prochlorperazine) for last 20 years. So, a final diagnosis of RPF secondary to prolonged ergotamine use was made. Vasograin was withdrawn and she was started on tablet prednisolone (40mg/day) and mycophenolate mofetil (MMF) (2gm/day). After twelve weeks, MMF was discontinued as the patient developed recurrent vomiting and diarrhoea which was attributed to MMF; prednisolone was tapered slowly to 5 mg alternate days over 6 months and tablet tamoxifen (20mg) was added as an alternative steroid sparing agent with regular imageological follow up. A repeat MRI scan performed 2 ½ years after initiation of therapy showed no significant retroperitoneal soft tissue thickening; normal course of the ureters, and significant reduction in hydronephrosis, more in right side. FDG-PET scan showed mild diffuse thickening around small bowel and peri-pyloric region but without any abnormal metabolic activity or FDG avid lesions. She is presently having a stable disease with alternate day prednisolone and tamoxifen. Her latest follow up abdominal USG (February 2020) showed mild bilateral hydronephrosis with no ureteric DJ stent in place. The sequential laboratory parameters and management details are presented in . Besides these, patchy areas of vascular congestion and lymphoplasmacytic inflammatory infiltrate were seen mostly around small veins (perivenulitis) with evidence of infiltration of their walls (phlebitis)  . Foci of old haemorrhage with collection of haemosiderophages were also noted.Multiple sections taken from the omentum and peritoneum showed fibrous septae of variable thickness extending from periphery to centre replacing and encircling lobules of adipocytes. The fibrotic process, noted more commonly in peritoneal biopsy, ranged from active fibroblastic proliferation with inflammation to inactive diffuse collagenisation resembling old scar  mediated antigenic presentation to macrophages and subsequent adventitial lymphoplasmacytic inflammation and periadventitial fibrosis.13 However, occurrence of RPF among children and in individuals without any evidence of atherosclerosis challenged this theory. Moreover, vascular segments which are commonly spared by atherosclerotic process are also involved in RPF pathogenesis. Furthermore, this theory did not support the systemic nature of the disease as has been evidenced by presence of constitutional symptoms; raised serum inflammatory biomarkers and concomitance of other autoimmune conditions such as Hashimoto thyroiditis, Graves disease, or ANCA-associated vasculitis.15The pathophysiologic mechanism of RPF is complex and is poorly understood. Idiopathic retroperitoneal fibrosis (IRPF) is postulated to be a part of chronic periaortitis (CP) that also includes inflammatory abdominal aortic aneurysms (IAAAs) and perianeurysmal retroperitoneal fibrosis (PRF).*03, an allele linked to many autoimmune conditions such as type 1 diabetes mellitus, myasthenia gravis, and autoimmune thyroiditis.14 A review of 608 consecutive patients with gastrointestinal (GI) vasculitis from Mayo Clinic, only 5 had evidence of CP at the time of onset of GI symptoms, and none of these were positive for autoimmune markers such as ANA, ANCA, anti-ds-DNA, cryoglobulin, or had any evidence infections such as syphilis, HIV, or Hepatitis B and C.16 The presence of necrotising vasculitis and lymphocytic phlebitis; as well as favourable response to immunosuppressive therapy in our case also points towards an immune-mediated vasculitis reaction. Ergotamine related/induced vasculitis was speculated as the possible underlying mechanism in our case though the same was not mentioned as a cause in 2012 Chapel Hill Consensus Conference classification of drug induced vasculitis. 17Martorana et al. in their case control study have highlighted the strong association between IRPF and HLA-DRB118 A previous report described a distinctive restrictive type pleuropulmonary pattern of fibrosis following long-term therapy with ergot derivatives , as opposed to typical interstitial pattern observed in other drugs. None of the cases were associated with either clinical or biological evidence of a systemic illness such as rheumatoid arthritis, ankylosing spondylitis, mixed connective tissue disease, lupus erythematosus, or inflammatory bowel disease, and vasculitic mechanism was not described in any of those cases. The outcome was favourable following the withdrawal of ergot derivatives.19Drug induced RPF is a rarely reported complication in subjects following the usage of ergot alkaloids .To conclude, histopathological demonstration of the entire process of retroperitoneal fibrosis and vasculitis at different stages of evolution with 20-year history of ergotamine ingestion in our case possibly points to a cause and effect relationship. We believe, prolonged ergotamine exposure might have acted like a heptane in this situation to give rise to a small vessel vasculitis culminating in a fibrotic process. The response to immune suppression therapy further substantiates our hypothesis.


The selection of circulating tumor cells (CTCs) directly from blood as a real-time liquid biopsy has received increasing attention over the past ten years, and further analysis of these cells may greatly aid in both research and clinical applications. CTC analysis could advance understandings of metastatic cascade, tumor evolution, and patient heterogeneity, as well as drug resistance. Until now, the rarity and heterogeneity of CTCs have been technical challenges to their wider use in clinical studies, but microfluidic-based isolation technologies have emerged as promising tools to address these limitations. This review provides a detailed overview of latest and leading microfluidic devices implemented for CTC isolation. In particular, this study details must-have device performances and highlights the tradeoff between recovery and purity. Finally, the review gives a report of CTC potential clinical applications that can be conducted after CTC isolation. Widespread microfluidic devices, which aim to support liquid-biopsy-based applications, will represent a paradigm shift for cancer clinical care in the near future. Cancer is a leading health issue, accounting for nearly 1 in 6 deaths worldwide. By 2040, the disease burden is expected to reach 30.2 million new cancer cases and 16.3 million cancer deaths, according to the Global Cancer Observatory (GCO). Cancer burden can be reduced through early detection and appropriate treatment. In particular, early diagnosis could prevent the formation of metastasis, a multistep process responsible for cancer spread and high morbidity rates [In the early stage of the disease, the small size of the primary tumor, as well as the lack of symptoms, are stumbling blocks for early screening. To date, tissue biopsies are core components of cancer patient management to diagnose, assess disease stage, and prescribe appropriate therapeutic regimens. However, biopsies are not only invasive and risky but they may also not fully reflect intratumor heterogeneity; although, the latter has been shown to reduce therapy effectiveness . For allThe study of CTCs may overcome these limitations and become complementary to tissue biopsies. CTCs are released into the bloodstream from primary and metastatic tumors; in addition, besides playing an important role in cancer metastasis , they of9 red blood cells (RBCs) and 107 white blood cells (WBCs) per mL). Moreover, their isolation is challenging, due to th following characteristics: (i) a morphology similarity with some WBCs, reducing size-based sorting effectiveness; (ii) a phenotypic heterogeneity, which makes the use of biomarkers more complex and limits the effectiveness of biomarker-based separation. The latter challenge results from the epithelial-to-mesenchymal transition (EMT) that CTCs can undergo [Nevertheless, until now, isolating CTCs has been a technical challenge, limiting their wider use in research and clinical studies. The main issue is the rarity of CTCs (1–1000 CTCs per mL) among a high background of blood cells  is the only device approved by the US Food and Drug Administration (FDA) for CTC detection and enumeration for clinical use. The device uses ferrofluid particles, coated with antibodies, targeting the epithelial cell adhesion molecule (EpCAM) for the enrichment of CTCs from the patient’s blood. Isolated cells are subsequently immunostained with fluorescently labeled antibodies and then counted using automated cell image capture and analysis. Since its introduction in 2004, the CellSearch™ system has been used as a diagnostic and prognostic tool in patients with metastatic breast, colorectal, and prostate cancer . AlthougOver the past decade, microfluidic devices have emerged as promising tools to address these challenges . Microfl®  [® FX1  [Several microfluidic technologies awaiting FDA clearance have been commercialized for CTC isolation, such as Parsortixrey, UK) , ClearCengapore) , and VTXngapore) . These tIn the following sections, relevant microfluidic-based CTC isolation devices will be reported, categorized by the separation method adopted , which will be compared by examining the figures of merit, detailed in Box 1Recovery: The fraction of injected CTCs that are collected downstream.Purity: The fraction of CTCs among the collected cells in the output. Sample purity determines the panel of downstream analyses that can be performed.Throughput: The applied flow rate for blood sample processing.Viability: The assay’s ability to preserve the viability of recovered CTCs.Clinical yield or clinical recovery: The number of isolated CTCs obtained from patient samples with a defined cancer type.Clinical sensitivity: The minimum number of detected CTCs in patient samples.Clinical relevance: The biological and therapeutic information that can be obtained beyond the enumeration of the isolated CTCs.Numerous studies  have highlighted the physical and biomechanical properties of CTCs, enabling their distinction from other blood cells . Indeed,Microfiltration techniques have been implemented in microfluidics for CTC isolation ,23,24,25The first approach consists in integrating an array of micrometric-sized posts that act as filters to capture CTCs. To this end, several microstructure post shapes were imagined: from microellipse  to microStrategies were investigated by other researchers to tackle these issues. Park et al. developed a deformability-based device to enrich viable CTCs directly from whole blood by integrating funnel-shaped constrictions with openings smaller than the diameter of the cell . The devThe second filtration approach consists of microporous membranes, which leads to higher throughput (>3 mL/h). Hosokawa et al. developed a microfluidic device equipped with a nickel-based microcavity array (MCA) filter to enrich CTCs from blood samples B. The fiRecently, another MCA structure was imagined by Yin et al., since vertical entrances (presented above) keep blood cells from entering and escaping the microcavities and thus decrease the efficiency and purity of separation . They inFinally, the last filtration approach consists of narrowing the dimensions of the fluidic path, through which cells flow to entrap CTCs. Hvichia et al., developed a semi-automated separation system, the Parsortix™ D, which Further label-free separation methods using hydrodynamic forces were developed. Compared with microfiltration techniques, hydrodynamic isolation exerts low fluidic stress on cancer cells as they do not pass through physical obstacles. Besides, samples are processed at high flow rates to ensure the generation of relevant hydrodynamic forces, which leads to high-throughput sorting, while cancer cells can be retrieved for subsequent analysis. Some of the most promising hydrodynamic isolation strategies can be classified into size-dependent deterministic flow pathways in pillar arrays , inertial migration of cells in a multi-flow straight microchannel, inertial focusing in spiral microfluidic channels , and microfluidic vortices generated in micro-reservoirs aside the channel.The deterministic lateral displacement (DLD) utilizes an array of posts within microchannels, where each post is laterally shifted at a set distance from the previous post. By optimizing the gap distance and post size, one can determine a critical size. Cells smaller than the critical size flow between the post gaps, while larger cells constantly collide with posts and are forced to move laterally following the post arrangement, achieving continuous CTC sorting. When DLD was firstly reported, a circular shape of post was used, with a gap of 10 µm ,53. HoweAnother hydrodynamic size separation technology utilizes inertial focusing to create size-dependent equilibrium positions within the channel. This phenomenon relies on the balance of lift forces arising from the curvature of the velocity profile (the shear gradient lift) and the interaction between the cell and the channel wall  for Reynold number of the order of 1 or greater . It resu® (Biolidics) and has recently been recognized through its ISO certifications  Class I Medical Device registered). Other channel geometries are reported in inertial microfluidics for CTC separation and can generate secondary flows that create additional hydrodynamic effects beyond shear gradient and wall effect lift forces for improved separation. In a spiral microchannel, the channel curvature introduces two symmetrical counter-rotating flows, called Dean vortices, within the transverse plane of the channel. This Dean flow fractionation (DFF) separation method causes large cells (CTCs) to move toward the inner wall, because of the balance of inertial lift force and Dean drag force, while small cells (RBCs and WBCs) flow toward the outer wall. Cells with different size can thus be collected in two separate outlets. Spiral microchannels can provide inertial focusing but in a much smaller footprint . The groRecently, Lin et al. created the Labyrinth device to address the challenge of focusing on smaller cells, such as WBCs, which remain unfocused in most DFF technologies. It was achieved by incorporating numerous sharp corners placed across the flow pattern to enhance Dean forces for the migration of smaller cells to their equilibrium positions . It resuFinally, similarly to DFF, another inertial-based device was reported using contraction–expansion arrays, whose cross-sections periodically widen and narrow to differentiate the focusing positions of particles depending on their sizes. When cells flow through a series of expansion–contraction reservoirs within a microchannel, they experience multiple microvortices because of the shear gradient lift force in expansion reservoirs. Cells larger than a critical size are collected in the center of the vortices; therefore, CTCs can be separated from other blood cells using this method. The Vortex technology was developed by the group of Di Carlo and has been well described for CTC enrichment over the years ,72,73,74Inertial-based sorting methods therefore have numerous advantages: high throughput, high recovery, and CTC retrieval for subsequent analysis. However, the main drawback of this method is the risk of CTC loss during the process, leading to a false negative result. Reducing the cut-off size can help to minimize this loss with the tradeoff of the increased contamination of white blood cells, reducing purity.Hence, despite the straightforward and label-free separation methods based on CTC size, the non-specificity of the size criteria limits their efficacies. Indeed, the separation methods were optimized on cancer cell lines, but studies have shown the morphological heterogeneity of CTCs found in patient bloods, from round to oval shapes, and with diameters varying from 4 to 30 µm . BesidesBesides size-based and deformability-based separation methods, dielectrophoresis (DEP) utilizes the electrophysical properties of CTCs to isolate them under a nonuniform electric field. When applying an AC voltage across two electrodes of different sizes, the non-uniform distribution of the charges generates a net DEP force will move the cell either towards the higher electric field gradient region , or in the opposite direction, towards the lower electric field gradient region (negative DEP). At a given electric field frequency  and depending on the electrical conductivity of the cells and its suspending medium, cell may experience either positive DEP (higher cell conductivity) or negative DEP (higher medium conductivity). Generally, viable cells express negative DEP at low frequencies and positive DEP at high frequencies. In particular, cells with different membrane surface area exhibit distinct DEP frequency responses. As previously mentioned, CTCs are larger, but they also present a 60% greater surface area than a WBC of the same size , which gTwo DEP-based devices were commercialized ten years ago for CTC isolation, the DEPArray™  for single CTC DEP trapping , and theOther DEP strategies were reported for CTC isolation, including DEP field flow fractionation (DEP-FFF) and optically induced dielectrophoresis (ODEP), but they suffer from relatively low throughput in the range of 0.01−1.0 mL/h ,86,87. RThus, DEP-based separation methods provide high recovery rates, but their implementation can be challenging. Indeed, they require specific electrode geometries and controlled microfabrication. In addition, DEP separation systems rely on cells polarization differences; therefore, any cell exhibiting a damaged membrane may influence isolation efficiencies. The use of specific buffers, such as sucrose, can induce osmotic stress and cause leakage of cytosolic ions over time . FinallySome advantages and limitations of each presented separation method can be found in More recently, acoustophoresis has evolved as a promising field for CTC sorting ,90,91,92The isolation of CTCs from other blood cells can also be achieved by exploiting biological properties of CTCs, such as their surface marker expression. These methods rely on the high specificity of the bonding between antibodies and expressed antigens in targeted cells. CTC isolation can be performed either by positive selection, where CTCs are collected as the target cell population, or negative selection, with WBCs as targeted cells. Biological-based separation methods can be categorized into either surface affinity approach through microchannel functionalization or the immunomagnetic approach using functionalized magnetic particles .HBCTC-Chip), integrating herringbone grooves on the roof of the anti-EpCAM-coated microchannel [HBCTC-Chip allowed for higher sample throughput and increased CTC capture efficiency and purity. A capture efficiency of 92% on spiked cancer cells was achieved at 1 mL/h, with a 5% better purity. Clinical use of HBCTC-Chip was further established and enabled the determination of CTC signaling pathways by RNA sequencing [The very first geometrically patterned microfluidic device with antibody-coated surfaces is the CTC-Chip, reported by Toner’s group in 2007 . The devochannel . These squencing , identifquencing , and invquencing . HBCTC-Chip with a gold nanoparticle coating and utilized a thiolated ligand-exchange reaction to isolate and release CTCs from whole blood [HBCTC-Chip (35% decrease). For non-epithelial cancer lines, a cocktail of antibodies had to be used within the chip to increase capture efficiency, from 16% to >90%. In addition, the chip successfully released 90% of the captured cells that were further cultured for 5 days with a preserved viability (78–87%). The use of nanostructured substrates, such as silicon nanopillars (NanoVelcro Chip)  Figure B or graple blood . Indeed,Similarly, Tseng’s group reported the tuning of their NanoVelcro Chip with a phenylboronic acid (PBA)-grafted PEDOT nanosubstrate to release captured CTCs upon exposure to a glycan molecule  , which hThe main advantage of this method, based on antibody–antigen reaction within functionalized microfluidic systems, is the high sensitivity for a given cellular type with a preserved viability. Nonetheless, lower throughput is achieved compared with physical-based separation approaches. In addition, special attention should be paid to enable the collection of captured CTCs. The main drawback of this method remains that, most of the time, a unique antigen is targeted , therefore limiting the recovery of heterogeneous CTCs.The immunomagnetic separation relies on the conjugation of magnetic particles to cells via antigen–antibody recognition in order to confer them magnetic properties. When subjected to a non-uniform magnetic field, magnetically labelled cells can be manipulated within the microchannel for sorting applications ,107,108.The immunomagnetic separation can either target CTCs (positive selection) or WBCs (negative selection). The benefit of the negative selection over the positive one is the ability to collect all CTCs regardless their surface marker expression. Nonetheless, given the high concentration of WBCs in blood, their depletion is more challenging. Various strategies were implemented for the immunomagnetic separation of CTCs in a microfluidic device and will be further detailed. 3) located at the bottom of the microfluidic channel. CTCs in blood were labelled with EpCAM-functionalized Fe3O4 magnetic nanoparticles and captured by the magnetic field as the blood flows through the microchannel [Magnetophoresis, which, as introduced earlier, is the motion of an object in a non-uniform magnetic field, and coupled with microfluidic technology, can be used for sorting applications. Researchers have carried out hard work on the optimization of magnetic field gradient sources to reach high magnetic forces, therefore maximizing sorting efficiencies. For example, Hoshino’s group and Kelley’s group demonstrated the advantage of downscaling the size of the magnetic source. Historically, Hoshino et al. implemented a CTC sorting device using an array of three NdFeB block magnets  microstructures were integrated into the microfluidic channel which act like microtraps  Figure B. Arraye2 . They weSimilarly, Kelley’s group developed a more complex device, integrating X-shaped microstructures as capture spots . These cFurthermore, other various immunomagnetic-based approaches have been implemented for CTC-positive selection. Viovy’s group reported the Ephesia technology, which consists of self-assembled anti-EpCAM functionalized magnetic beads forming columnar arrays along the microchannel height and acting as a trap for target cells A. They fHBGO chip for ex vivo capture of intravenously infused CTCs in canine [Magnetic columns must be tightly anchored to the bottom layer of the chip to stand firm against hydrodynamic flow during the whole capture and analysis. They later proposed a capillary assembly technique , using an canine .Nevertheless, the approaches cited above do not take into account the surface marker expression heterogeneity in CTCs. Special designs were imagined to track this heterogeneity in immunomagnetic separation-based systems for CTC sorting. Kwak et al. reported the fabrication of a spiral-shaped channel capable of capturing magnetically labeled CTCs by magnetophoresis regarding their EpCAM expression level . This waOther immunomagnetic-based strategies relying on WBC depletion (negative selection) were also studied. Such tumor–antigen independent immunomagnetic separation methods were investigated to overcome marker expression variability among CTCs. By specifically removing WBCs, typically using anti-CD45 antibodies, CTCs can further be collected for downstream analysis. These approaches offer an opportunity to isolate CTCs regardless of their phenotype and ensure that CTC viability is maintained. Hyun et al. fabricated a two-stage microfluidic chip (μ-MixMACS chip) for negative selection of CTCs ,127. The6). Recently, Mishra et al., reported an ultra-high-throughput magnetic sorting chip, the LPCTC-iChip, which processed very large blood volumes (65 mL) for negative selection of CTCs [Large volumes of blood have to be processed to ensure the collection of a sufficient number of CTCs, which might cause clogging due to the large number of WBCs per 1 mL of blood .Integrated microfluidic systems, as reported with combined magnetophoresis with other size-based separation methods, for example, have emerged as next-generation CTC isolation systems, since they can offer the following: (i) effective recovery of CTCs and CTCs clusters simultaneously; (ii) ultra-pure samples with minimal contamination of normal blood cells; (iii) high-throughput sorting with preserved viability. The widespread use and development of microfluidic systems, aimed at supporting liquid-biopsy-based applications, will represent a paradigm shift for cancer clinical care. In particular, the detection, enumeration, and characterization of CTCs will play a significant role in clinical applications involving early detection of aggressive cancers, selection of therapies, identification of drug resistance, and discovery of novel therapeutic targets. Tremendous research efforts have already been made within the past decade to provide robust CTC separation methods and the next generation of devices will certainly provide a complete workflow integrating both isolation and characterization  steps into a single chip.