Statistical methods for building better biomarkers of chronic kidney disease.

The last two decades have witnessed an explosion in research focused on the development and assessment of novel biomarkers for improved prognosis of diseases. As a result, best practice standards guiding biomarker research have undergone extensive development. Currently, there is great interest in the promise of biomarkers to enhance research efforts and clinical practice in the setting of chronic kidney disease, acute kidney injury, and glomerular disease. However, some have questioned whether biomarkers currently add value to the clinical practice of nephrology. The current state of the art pertaining to statistical analyses regarding the use of such measures is critical. In December 2014, the National Institute of Diabetes and Digestive and Kidney Diseases convened a meeting, "Toward Building Better Biomarker Statistical Methodology," with the goals of summarizing the current best practice recommendations and articulating new directions for methodological research. This report summarizes its conclusions and describes areas that need attention. Suggestions are made regarding metrics that should be commonly reported. We outline the methodological issues related to traditional metrics and considerations in prognostic modeling, including discrimination and case mix, calibration, validation, and cost-benefit analysis. We highlight the approach to improved risk communication and the value of graphical displays. Finally, we address some "new frontiers" in prognostic biomarker research, including the competing risk framework, the use of longitudinal biomarkers, and analyses in distributed research networks.

(Re)Building a Kidney.

(Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for in vitro engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons in situ to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of in vitro and in vivo differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.

Hydrodynamic stretching of single cells for large population mechanical phenotyping.

Cell state is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as the ability to mechanically deform under a load, are advantageous in that they do not require costly labeling or sample preparation. However, current techniques that assay cell mechanical properties have had limited adoption in clinical and cell biology research applications. Here, we demonstrate an automated microfluidic technology capable of probing single-cell deformability at approximately 2,000 cells/s. The method uses inertial focusing to uniformly deliver cells to a stretching extensional flow where cells are deformed at high strain rates, imaged with a high-speed camera, and computationally analyzed to extract quantitative parameters. This approach allows us to analyze cells at throughputs orders of magnitude faster than previously reported biophysical flow cytometers and single-cell mechanics tools, while creating easily observable larger strains and limiting user time commitment and bias through automation. Using this approach we rapidly assay the deformability of native populations of leukocytes and malignant cells in pleural effusions and accurately predict disease state in patients with cancer and immune activation with a sensitivity of 91% and a specificity of 86%. As a tool for biological research, we show the deformability we measure is an early biomarker for pluripotent stem cell differentiation and is likely linked to nuclear structural changes. Microfluidic deformability cytometry brings the statistical accuracy of traditional flow cytometric techniques to label-free biophysical biomarkers, enabling applications in clinical diagnostics, stem cell characterization, and single-cell biophysics.

High-throughput single-microparticle imaging flow analyzer.

Optical microscopy is one of the most widely used diagnostic methods in scientific, industrial, and biomedical applications. However, while useful for detailed examination of a small number (< 10,000) of microscopic entities, conventional optical microscopy is incapable of statistically relevant screening of large populations (> 100,000,000) with high precision due to its low throughput and limited digital memory size. We present an automated flow-through single-particle optical microscope that overcomes this limitation by performing sensitive blur-free image acquisition and nonstop real-time image-recording and classification of microparticles during high-speed flow. This is made possible by integrating ultrafast optical imaging technology, self-focusing microfluidic technology, optoelectronic communication technology, and information technology. To show the system's utility, we demonstrate high-throughput image-based screening of budding yeast and rare breast cancer cells in blood with an unprecedented throughput of 100,000 particles/s and a record false positive rate of one in a million.

Mediating millisecond reaction time around particles and cells.

Precise spatiotemporal control of how particles and cells interact with reagents is critical for numerous laboratory and industrial processes. Novel tools for exerting this control at shorter time scales will enable development of new chemical processes and biomedical assays. Previously, we have developed a generalized approach to manipulate cells and particles across fluid streams termed rapid inertial solution exchange (RInSE), which utilizes inertial lift forces at finite Reynolds number and high Peclet number to transfer particles from an initial solution to another within a millisecond. Here, we apply these principles toward developing a continuous flow microfluidic platform that enables transient chemical treatments of cells and particles (on the order of 1 ms). We also demonstrate how the reactant stream can be employed as a diffusion barrier, preventing adverse reactions between coflowing solutions. In order to demonstrate the utility of the method, we applied it to various operations in molecular biology and automated cell staining including cell permeabilization, fluorescent staining, and molecular delivery to viable cells. We expect this method will enable previously unexplored studies of the dynamics of molecular events, improve uniformity of reactions carried on the surface of beads, and increase uniformity in cell-based assays through automation.

Three dimensional, sheathless, and high-throughput microparticle inertial focusing through geometry-induced secondary flows.

A novel inertial focusing platform creates a single-stream microparticle train in a single-focal plane without sheath fluids and external forces, all in a high-throughput manner. The proposed design consists of a low-aspect-ratio straight channel interspersed with a series of constrictions in height arranged orthogonally, making use of inertial focusing and geometry-induced secondary flows. Focusing efficiency as high as 99.77% is demonstrated with throughput as high as 36 000 particles s(-1) for a variety of different sized particles and cells.

Inertial manipulation and transfer of microparticles across laminar fluid streams.

A general strategy for controlling particle movement across streams would enable new capabilities in single-cell analysis, solid-phase reaction control, and biophysics research. Transferring cells across streams is difficult to achieve in a well-controlled manner, since it requires precise control of fluid flow along with external force fields or precisely manufactured mechanical structures. Herein a strategy is introduced for particle transfer based on passive inertial lift forces and shifts in the distribution of these forces for channels with shifting aspect ratios. Uniquely, use of the dominant wall-effect lift parallel to the particle rotation direction is explored and utilized to achieve controllable cross-stream motion. In this way, particles are positioned to migrate across laminar streams and enter a new solution without significant disturbance of the interface at rates exceeding 1000 particles per second and sub-millisecond transfer times. The capabilities of rapid inertial solution exchange (RInSE) for preparation of hematological samples and other cellular assays are demonstrated. Lastly, improvements to inline flow cytometry after RInSE of excess fluorescent dye and focusing for downstream analysis are characterized. The described approach is simply applied to manipulating cells and particles and quickly exposing them to or removing them from a reacting solution, with broader applications in control and analysis of low affinity interactions on cells or particles.

Label-free cell separation and sorting in microfluidic systems.

Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible.

Particle focusing mechanisms in curving confined flows.

Particles in finite-inertia confined channel flows are known to segregate and focus to equilibrium positions whose number corresponds with the fold of symmetry of the channel's cross section. The addition of curvature into channels presumably modifies these equilibrium inertial focusing positions, because of the secondary flow induced in curved channels. Here, we identify the critical interaction of the secondary flow field with inertial lift forces to create complex sets of particle focusing positions that vary with the channel Reynolds number (Re(C)) and the inertial force ratio, which is a new dimensionless parameter that is based on the ratio of inertial lift to drag forces from the secondary flow. We use these results to identify microfluidic channel geometries to focus particles at rates an order of magnitude higher than previously shown (channel Reynolds number, Re(C) = 270) and develop design criteria for the focusing of potentially arbitrary-sized particles. In addition, our results indicate that channel curvature can lead to microfluidic designs with reduced fluidic resistance, useful for lower power inertial focusing or separation. These results will enable design of practical particle/cell separation, filtration, and focusing systems for critical applications in biomedicine and environmental cleanup.

High-Throughput Processing to Preserve Viable Cells: A Precision Medicine Initiative Cohort Program Workshop.

Conventionally, biobanks supporting clinical research studies have preserved serum, plasma, urine, saliva, a variety of tissue types, and stool. With the emergence of increasingly sophisticated technologies for analyzing single cells, there is growing interest in preserving viable blood cells for future functional studies. The new All of Us Research Program (formerly the Precision Medicine Initiative Cohort Program) biobank plans to house samples from a million or more individuals as part of a cohort with rich phenotypic data and longitudinal follow-up ( www.nih.gov/research-training/allofus-research-program ). Storage of viable cells for future single-cell analysis offers the promise of new biology, discovery of novel biomarkers, and advances toward the goal of precision medicine. A workshop was held in the summer of 2016 to evaluate the case for preservation of viable mononuclear blood cells and its feasibility within the collection plan for the biobank.

Multiparameter mechanical and morphometric screening of cells.

We introduce a label-free method to rapidly phenotype and classify cells purely based on physical properties. We extract 15 biophysical parameters from cells as they deform in a microfluidic stretching flow field via high-speed microscopy and apply machine-learning approaches to discriminate different cell types and states. When employing the full 15 dimensional dataset, the technique robustly classifies individual cells based on their pluripotency, with accuracy above 95%. Rheological and morphological properties of cells while deforming were critical for this classification. We also show the application of this method in accurate classifying cells based on their viability, drug screening and detecting populations of malignant cells in mixed samples. We show that some of the extracted parameters are not linearly independent, and in fact we reach maximum classification accuracy by using only a subset of parameters. However, the informative subsets could vary depending on cell types in the sample. This work shows the utility of an assay purely based on intrinsic biophysical properties of cells to identify changes in cell state. In addition to a label-free alternative to flow cytometry in certain applications, this work, also can provide novel intracellular metrics that would not be feasible with labeled approaches (i.e. flow cytometry).

Rapid inertial solution exchange for enrichment and flow cytometric detection of microvesicles.

Exosomes, nanosized membrane-bound vesicles released by cells, play roles in cell signaling, immunology, virology, and oncology. Their study, however, has been hampered by difficulty in isolation and quantification due to their size and the complexity of biological samples. Conventional approaches to improved isolation require specialized equipment and extensive sample preparation time. Therefore, isolation and detection methods of exosomes will benefit biological and clinical studies. Here, we report a microfluidic platform for inline exosome isolation and fluorescent detection using inertial manipulation of antibody-coated exosome capture beads from biological fluids.

Continuous-flow cytomorphological staining and analysis.

Cells suspended in bodily fluids are routinely analyzed by cytopathologists as a means of diagnosing malignancies and other diseases. The physical and morphological properties of these suspended cells are evaluated in making diagnostic decisions, which often requires manual concentration, staining, and washing procedures to extract information about intracellular architecture. The need to manually prepare slides for analysis by a cytopathologist is a labor-intensive process, which is ripe for additional automation to reduce costs but also to potentially provide more repeatable and improved accuracy in diagnoses. We have developed a microfluidic system to perform several steps in the preparation of samples for cytopathology that (i) automates colorimetric staining on-chip, and (ii) images cells in flow, as well as provides (iii) additional quantitative analyses of captured images to aid cytopathologists. A flow-through approach provides benefits by allowing staining and imaging to be performed in a continuous, integrated manner, which also overcomes previous challenges with in-suspension colorimetric staining. We envision such a tool may reduce costs and aid cytopathologists in identifying rare or characteristic cells of interest by providing isolated images along with quantitative metrics on single cells from various rotational angles, allowing efficient determination of disease etiology.

Size-selective collection of circulating tumor cells using Vortex technology.

A blood-based, low cost alternative to radiation intensive CT and PET imaging is critically needed for cancer prognosis and management of its treatment. "Liquid biopsies" of circulating tumor cells (CTCs) from a relatively non-invasive blood draw are particularly ideal, as they can be repeated regularly to provide up to date molecular information about the cancer, which would also open up key opportunities for personalized therapies. Beyond solely diagnostic applications, CTCs are also a subject of interest for drug development and cancer research. In this paper, we adapt a technology previously introduced, combining the use of micro-scale vortices and inertial focusing, specifically for the high-purity extraction of CTCs from blood samples. First, we systematically varied parameters including channel dimensions and flow rates to arrive at an optimal device for maximum trapping efficiency and purity. Second, we validated the final device for capture of cancer cell lines in blood, considering several factors, including the effect of blood dilution, red blood cell lysis and cell deformability, while demonstrating cell viability and independence on EpCAM expression. Finally, as a proof-of-concept, CTCs were successfully extracted and enumerated from the blood of patients with breast (N = 4, 25-51 CTCs per 7.5 mL) and lung cancer (N = 8, 23-317 CTCs per 7.5 mL). Importantly, samples were highly pure with limited leukocyte contamination (purity 57-94%). This Vortex approach offers significant advantages over existing technologies, especially in terms of processing time (20 min for 7.5 mL of whole blood), sample concentration (collecting cells in a small volume down to 300 µL), applicability to various cancer types, cell integrity and purity. We anticipate that its simplicity will aid widespread adoption by clinicians and biologists who desire to not only enumerate CTCs, but also uncover new CTC biology, such as unique gene mutations, vesicle secretion and roles in metastatic processes.

Pinched-flow hydrodynamic stretching of single-cells.

Reorganization of cytoskeletal networks, condensation and decondensation of chromatin, and other whole cell structural changes often accompany changes in cell state and can reflect underlying disease processes. As such, the observable mechanical properties, or mechanophenotype, which is closely linked to intracellular architecture, can be a useful label-free biomarker of disease. In order to make use of this biomarker, a tool to measure cell mechanical properties should accurately characterize clinical specimens that consist of heterogeneous cell populations or contain small diseased subpopulations. Because of the heterogeneity and potential for rare populations in clinical samples, single-cell, high-throughput assays are ideally suited. Hydrodynamic stretching has recently emerged as a powerful method for carrying out mechanical phenotyping. Importantly, this method operates independently of molecular probes, reducing cost and sample preparation time, and yields information-rich signatures of cell populations through significant image analysis automation, promoting more widespread adoption. In this work, we present an alternative mode of hydrodynamic stretching where inertially-focused cells are squeezed in flow by perpendicular high-speed pinch flows that are extracted from the single inputted cell suspension. The pinched-flow stretching method reveals expected differences in cell deformability in two model systems. Furthermore, hydraulic circuit design is used to tune stretching forces and carry out multiple stretching modes (pinched-flow and extensional) in the same microfluidic channel with a single fluid input. The ability to create a self-sheathing flow from a single input solution should have general utility for other cytometry systems and the pinched-flow design enables an order of magnitude higher throughput (65,000 cells s(-1)) compared to our previously reported deformability cytometry method, which will be especially useful for identification of rare cell populations in clinical body fluids in the future.

Quantitative diagnosis of malignant pleural effusions by single-cell mechanophenotyping.

Biophysical characteristics of cells are attractive as potential diagnostic markers for cancer. Transformation of cell state or phenotype and the accompanying epigenetic, nuclear, and cytoplasmic modifications lead to measureable changes in cellular architecture. We recently introduced a technique called deformability cytometry (DC) that enables rapid mechanophenotyping of single cells in suspension at rates of 1000 cells/s-a throughput that is comparable to traditional flow cytometry. We applied this technique to diagnose malignant pleural effusions, in which disseminated tumor cells can be difficult to accurately identify by traditional cytology. An algorithmic diagnostic scoring system was developed on the basis of quantitative features of two-dimensional distributions of single-cell mechanophenotypes from 119 samples. The DC scoring system classified 63% of the samples into two high-confidence regimes with 100% positive predictive value or 100% negative predictive value, and achieved an area under the curve of 0.86. This performance is suitable for a prescreening role to focus cytopathologist analysis time on a smaller fraction of difficult samples. Diagnosis of samples that present a challenge to cytology was also improved. Samples labeled as "atypical cells," which require additional time and follow-up, were classified in high-confidence regimes in 8 of 15 cases. Further, 10 of 17 cytology-negative samples corresponding to patients with concurrent cancer were correctly classified as malignant or negative, in agreement with 6-month outcomes. This study lays the groundwork for broader validation of label-free quantitative biophysical markers for clinical diagnoses of cancer and inflammation, which could help to reduce laboratory workload and improve clinical decision-making.

Introduction: why analyze single cells?

Powerful methods in molecular biology are abundant; however, in many fields including hematology, stem cell biology, tissue engineering, and cancer biology, data from tools and assays that analyze the average signals from many cells may not yield the desired result because the cells of interest may be in the minority-their behavior masked by the majority-or because the dynamics of the populations of interest are offset in time. Accurate characterization of samples with high cellular heterogeneity may only be achieved by analyzing single cells. In this chapter, we discuss the rationale for performing analyses on individual cells in more depth, cover the fields of study in which single-cell behavior is yielding new insights into biological and clinical questions, and speculate on how single-cell analysis will be critical in the future.

Sequential array cytometry: multi-parameter imaging with a single fluorescent channel.

Heterogeneity within the human population and within diseased tissues necessitates a personalized medicine approach to diagnostics and the treatment of diseases. Functional assays at the single-cell level can contribute to uncovering heterogeneity and ultimately assist in improved treatment decisions based on the presence of outlier cells. We aim to develop a platform for high-throughput, single-cell-based assays using well-characterized hydrodynamic cell isolation arrays which allow for precise cell and fluid handling. Here, we demonstrate the ability to extract spatial and temporal information about several intracellular components using a single fluorescent channel, eliminating the problem of overlapping fluorescence emission spectra. Integrated with imaging technologies such as wide field-of-view lens-free fluorescent imaging, fiber-optic array scanning technology, and microlens arrays, use of a single fluorescent channel will reduce the cost of reagents and optical components. Specifically, we sequentially stain hydrodynamically trapped cells with three biochemical labels all sharing the same fluorescence excitation and emission spectrum. These markers allow us to analyze the amount of DNA, and compare nucleus-to-cytoplasm ratio, as well as glycosylation of surface proteins. By imaging cells in real-time we enable measurements of temporal localization of cellular components and intracellular reaction kinetics, the latter is used as a measurement of multi-drug resistance. Demonstrating the efficacy of this single-cell analysis platform is the first step in designing and implementing more complete assays, aimed toward improving diagnosis and personalized treatments to complex diseases.

Strategies for implementing hardware-assisted high-throughput cellular image analysis.

Recent advances in imaging technology for biomedicine, including high-speed microscopy, automated microscopy, and imaging flow cytometry are poised to have a large impact on clinical diagnostics, drug discovery, and biological research. Enhanced acquisition speed, resolution, and automation of sample handling are enabling researchers to probe biological phenomena at an increasing rate and achieve intuitive image-based results. However, the rich image sets produced by these tools are massive, possessing potentially millions of frames with tremendous depth and complexity. As a result, the tools introduce immense computational requirements, and, more importantly, the fact that image analysis operates at a much lower speed than image acquisition limits its ability to play a role in critical tasks in biomedicine such as real-time decision making. In this work, we present our strategy for high-throughput image analysis on a graphical processing unit platform. We scrutinized our original algorithm for detecting, tracking, and analyzing cell morphology in high-speed images and identified inefficiencies in image filtering and potential shortcut routines in the morphological analysis stage. Using our "grid method" for image enhancements resulted in an 8.54× reduction in total run time, whereas origin centering allowed using a look up table for coordinate transformation, which reduced the total run time by 55.64×. Optimization of parallelization and implementation of specialized image processing hardware will ultimately enable real-time analysis of high-throughput image streams and bring wider adoption of assays based on new imaging technologies.

Nomarski serial time-encoded amplified microscopy for high-speed contrast-enhanced imaging of transparent media.

High-speed high-contrast imaging modalities that enable image acquisition of transparent media without the need for chemical staining are essential tools for a broad range of applications; from semiconductor process monitoring to blood screening. Here we introduce a method for contrast-enhanced imaging of unstained transparent objects that is capable of high-throughput imaging. This method combines the Nomarski phase contrast capability with the ultrahigh frame rate and shutter speed of serial time-encoded amplified microscopy. As a proof of concept, we show imaging of a transparent test structure and white blood cells in flow at a shutter speed of 33 ps and a frame rate of 36.1 MHz using a single-pixel photo-detector. This method is expected to be a valuable tool for high-throughput screening of unstained cells.