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Microfluidic lab-on-a-chip technologies enable the analysis and manipulation of small fluid volumes and particles at small scales and the control of fluid flow and transport processes at the microscale, leading to the development of new methods to address a broad range of scientific and medical challenges. Microfluidic and lab-on-a-chip technologies have made a noteworthy impact in basic, preclinical, and clinical research, especially in hematology and vascular biology due to the inherent ability of microfluidics to mimic physiologic flow conditions in blood vessels and capillaries. With the potential to significantly impact translational research and clinical diagnostics, technical issues and incentive mismatches have stymied microfluidics from fulfilling this promise. We describe how accessibility, usability, and manufacturability of microfluidic technologies should be improved and how a shift in mindset and incentives within the field is also needed to address these issues. In this report, we discuss the state of the microfluidic field regarding current limitations and propose future directions and new approaches for the field to advance microfluidic technologies closer to translation and clinical use. While our report focuses on using blood as the prototypical biofluid sample, the proposed ideas and research directions can be extrapolated to other areas of hematology, oncology, biology, and medicine.

The significant biological and functional differences between small and large platelets suggested by recent studies could have profound implications for transfusion medicine. However, investigating the relationship between platelet size and function is challenging because separating platelets by size without affecting their properties is difficult. A standard approach is centrifugation, but it inevitably leads to premature activation and aggregation of separated platelets. This paper describes the development and validation of a microfluidic device based on controlled incremental filtration (CIF) for separating platelets by size without the cell damage and usability limitations associated with centrifugation. Platelet samples derived from whole blood were used to evaluate the dependence of the CIF device separation performance on design parameters and flow rate, and to compare the properties of PLT fractions generated by the CIF device with those produced using a centrifugation protocol in a split-sample study. This was accomplished by quantifying the platelet size distribution, mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet activation before and after processing for all input and output samples. The 'large platelet' fractions produced by the CIF device and the centrifugation protocol were essentially equivalent (no significant difference in MPV and P-LCR). Platelets in the 'small platelet' fraction produced by the CIF device were significantly smaller than those produced by centrifugation (lower MPV and P-LCR). This was because the CIF 'small platelet' fraction was contaminated by much fewer large platelets (∼2-times lower recovery of >12 fL platelets) and retained the smallest platelets that were discarded by the centrifugation protocol. There was no significant difference in platelet activation between the two methods. However, centrifugation required a substantial amount of additional anticoagulant to prevent platelet aggregation during pelleting. Unlike centrifugation, the CIF device offered continuous, flow-through, single-step processing that did not cause platelet aggregation. Such a capability has the potential to accelerate the basic studies of the relationship between platelet size and function, and ultimately improve transfusion practice, particularly in the pediatric setting, where the need for low-volume, high-quality platelet transfusions is most urgent.

The isolation of a specific lymphocyte subset from blood is the required first step in the manufacturing of many novel cellular immunotherapies. Microfluidic size-based separation methods are poised to significantly simplify this process because they require neither centrifugation nor magnetic or fluorescent labeling to operate. Lymphocytes can be separated from red blood cells (RBCs) and platelets as well as monocytes and granulocytes because their size differs from each of these cell types. However, further separation of a specific lymphocyte subset from other unwanted lymphocytes using size-based methods is impossible because all lymphocytes have approximately the same size and can only be distinguished by surface markers. This paper describes a new approach that made it possible for a size-based separation method to isolate a desired subset of lymphocytes by making unwanted lymphocytes as well as other blood cells artificially larger. The separation was enabled by selectively binding multiple RBCs to each unwanted cell to create 'rosettes' with an effective size significantly larger than the diameter of a typical lymphocyte. The desired lymphocytes remained unaffected by rosetting and were separated from the rosettes by passing the mixture through a microfluidic size-based separation device based on controlled incremental filtration (CIF). This new rosette-enabled size-based (RESIZE) separation approach demonstrated recovery of 80-90% for all lymphocyte subsets tested (CD3 + , CD4 + , CD56 + ) which was ∼2.5-fold higher than that for the standard immunodensity method (RBC rosetting followed by density gradient centrifugation). The purity of separation was >90% for CD3 + cells but declined with increasing cell rarity. Unlike the immunodensity approach, RESIZE required neither centrifugation nor cell washing after the separation and was ∼2.5-fold faster when processing the same sample volume. The results of this study suggest that integration of the RESIZE approach for high-yield isolation of lymphocyte subsets from blood could significantly streamline the manufacturing workflow and thus have a potentially transformative impact on the cost and availability of novel cellular immunotherapies.

The number of microfluidic strategies aimed at separating particles or cells of a specific size within a continuous flow system continues to grow. The wide array of biomedical and other applications that would benefit from successful development of such technology has motivated the extensive research in this area over the past 15 years. However, despite promising advancements in microfabrication capabilities, a versatile approach that is suitable for a large range of particle sizes and high levels of enrichment, with a volumetric throughput sufficient for large-scale applications, has yet to emerge. Here we describe a straightforward method that enables the rapid design of microfluidic devices that are capable of enriching/removing particles within a complex aqueous mixture, with an unprecedented range of potential cutoff diameter (below 1 μm to above 100 μm) and an easily scalable degree of enrichment/filtration (up to 10-fold and well beyond). A simplified model of a new approach to crossflow filtration - controlled incremental filtration - was developed and validated for its ability to generate microfluidic devices that efficiently separate particles on the order of 1-10 μm, with throughputs of tens of μL min(-1), without the use of a pump. Precise control of the amount of fluid incrementally diverted at each filtration "gap" of the device allows for the gap size (~20 μm) to be much larger than the particles of interest, while the simplicity of the model allows for many thousands of these filtration points to be readily incorporated into a desired device design. This new approach should enable truly high-throughput microfluidic particle-separation devices to be generated, even by users only minimally experienced in fluid mechanics and microfabrication techniques.

This communication describes a very simple, rapid and inexpensive point-of-care diagnostic test for sickle cell disease (SCD) that can conclusively differentiate between blood samples from normal healthy individuals, sickle cell trait carriers and SCD patients using the characteristic blood stain patterns produced by each sample on paper.

Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.

Traffic of leukocytes in microvascular networks (particularly through arteriolar bifurcations and venular convergences) affects the dynamics of capillary blood flow, initiation of leukocyte adhesion during inflammation, and localization and development of atherosclerotic plaques in vivo. Recently, a growing research effort has been focused on fabricating microvascular networks comprising artificial vessels with more realistic, rounded cross-sections. This paper investigated the impact of the cross-sectional geometry of microchannels on the traffic of leukocytes flowing with human whole blood through a non-symmetrical bifurcation that consisted of a 50 μm mother channel bifurcating into 30 μm and 50 μm daughter branches. Two versions of the same bifurcation comprising microchannels with rectangular and rounded cross-sections were fabricated using conventional multi-layer photolithography to produce rectangular microchannles that were then rounded in situ using a recently developed method of liquid PDMS/air bubble injection. For microchannels with rounded cross-sections, about two-thirds of marginated leukocytes traveling along a path in the top plane of the bifurcation entered the smallest 30 μm daughter branch. This distribution was reversed in microchannels with rectangular cross-sections--the majority of leukocytes traveling along a similar path continued to follow the 50 μm microchannels after the bifurcation. This dramatic difference in the distribution of leukocyte traffic among the branches of the bifurcation can be explained by preferential margination of leukocytes towards the corners of the 50 μm mother microchannels with rectangular cross-sections, and by the additional hindrance to leukocyte entry created by the sharp transition from the 50 μm mother microchannel to the 30 μm daughter branch at the intersection. The results of this study suggest that the trajectories of marginated leukocytes passing through non-symmetrical bifurcations are significantly affected by the cross-sectional geometry of microchannels and emphasize the importance of using microfludic systems with geometrical configurations closely matching physiological configurations when modeling the dynamics of whole blood flow in the microcirculation.

Recent evidence implicating leukocytes in angiogenesis raises the question of whether leukocytes and other cells circulating with the blood in microvascular networks can home to capillary sprouts intraluminally. This study describes an investigation of leukocyte trafficking in sprouting capillaries fabricated using soft lithography. The leukocytes passing with whole blood through existing capillaries were able to enter microfabricated capillary sprouts of variable length and sprouting angle due to the mechanical interaction with red blood cells (RBCs) at the sprouting bifurcation, in spite of the complete absence of blood flow through the blind-ended sprouts or any chemoattractants. The RBCs formed "comet tails" (the densely packed cellular trains forming behind leukocytes as they move through narrow capillaries) and effectively pushed leukocytes into the microfabricated sprouts while bypassing them at the sprouting bifurcation. Individual sprouts filled with several leukocytes, as wells as RBCs and platelets, were observed. The results of this study suggest that (i) blood cells are likely present in capillary sprouts throughout their development, (ii) leukocytes and other circulating cells may use this mechanism to home to capillary sprouts intraluminally for direct engraftment, and (iii) tissues may use this phenomenon as another mechanism for local recruitment of leukocytes from the blood stream.

This article describes the fabrication of a microfluidic device for the liquid culture of many individual nematode worms (Caenorhabditis elegans) in separate chambers. Each chamber houses a single worm from the fourth larval stage until death, and enables examination of a population of individual worms for their entire adult lifespans. Adjacent to the chambers, the device includes microfluidic worm clamps, which enable periodic, temporary immobilization of each worm. The device made it possible to track changes in body size and locomotion in individual worms throughout their lifespans. This ability to perform longitudinal measurements within the device enabled the identification of age-related phenotypic changes that correlate with lifespan in C. elegans.

This paper describes a microfluidic instrument that produces drops of supercooled water suspended in a moving stream of liquid fluorocarbon, and measures the temperatures at which ice nucleates in the drops. A microfluidic chip containing a monodisperse drop generator and a straight channel with 38 embedded resistance thermometers was placed in contact with a seven-zone temperature-control plate and imaged under a microscope with a high-speed camera. This instrument can record the freezing temperatures of tens of thousands of drops within minutes, with an accuracy of 0.4 degrees C. The ice-nucleation temperatures in approximately 80-microm drops were reported for the freezing of 37 061 drops of pure water, and of 8898 drops of water seeded with silver iodide. Nucleation of ice in pure water was homogenous and occurred at temperatures between -36 and -37.8 degrees C, while water containing silver iodide froze between -10 and -19 degrees C. The instrument recorded the largest sets of individual freezing temperatures (37 061), had the fastest data acquisition rate (75 measurements/s), and the best optical (3 microm) and temporal (70 micros) resolutions among instruments designed to study nucleation of ice. The dendritic growth of ice in 150-microm drops of supercooled water at -35 degrees C was observed and imaged at a rate of 16 000 frames/s.

This paper describes a method for prefabricating screw, pneumatic, and solenoid valves and embedding them in microfluidic devices. This method of prefabrication and embedding is simple, requires no advanced fabrication, and is compatible with soft lithography. Because prefabrication allows many identical valves to be made at one time, the performance across different valves made in the same manner is reproducible. In addition, the performance of a single valve is reproducible over many cycles of opening and closing: an embedded solenoid valve opened and closed a microfluidic channel more than 100,000 times with no apparent deterioration in its function. It was possible to combine all three types of prefabricated valves in a single microfluidic device to control chemical gradients in a microfluidic channel temporally and spatially.

This paper demonstrates that a hand-powered egg beater can be modified to serve as a centrifuge for separating plasma from human whole blood. Immunoassays used to diagnose infectious diseases often require plasma from whole blood, and obtaining plasma typically requires electrically-powered centrifuges, which are not widely available in resource-limited settings. Human whole blood was loaded into polyethylene (PE) tubing, and the tubing was attached to the paddle of an egg beater. Spinning the paddle pelleted the blood cells to the distal end of the PE tubing; the plasma remained as the supernatant. A cholesterol assay (run on patterned paper) demonstrated the suitability of this plasma for use in diagnostic assays. The physics of the system was also analyzed as a guide for the selection of other rotating systems for use in centrifugation. Egg beaters, polyethylene tubing, and paper are readily available devices and supplies that can facilitate the use of point-of-care diagnostics at sites far from centralized laboratory facilities.

This paper describes the fabrication of a composite agar/PDMS device for enriching short cells in a population of motile Escherichia coli. The device incorporated ratcheting microchannels, which directed the motion of swimming cells of E. coli through the device, and three sorting junctions, which isolated successively shorter populations of bacteria. The ratcheting microchannels guided cells through the device with an average rate of displacement of (32 +/- 9) microm s(-1). Within the device, the average length of the cells decreased from 3.8 microm (Coefficient of Variation, CV: 21%) at the entrance, to 3.4 microm (CV: 16%) after the first sorting junction, to 3.2 mum (CV: 19%) after the second sorting junction, to 3.0 mum (CV: 19%) after the third sorting junction.

This paper derives the difference in pressure between the beginning and the end of a rectangular microchannel through which a flowing liquid (water, with or without surfactant, and mixtures of water and glycerol) carries bubbles that contact all four walls of the channel. It uses an indirect method to derive the pressure in the channel. The pressure drop depends predominantly on the number of bubbles in the channel at both low and high concentrations of surfactant. At intermediate concentrations of surfactant, if the channel contains bubbles (of the same or different lengths), the total, aggregated length of the bubbles in the channel is the dominant contributor to the pressure drop. The difference between these two cases stems from increased flow of liquid through the "gutters"-the regions of the system bounded by the curved body of the bubble and the corners of the channel-in the presence of intermediate concentrations of surfactant. This paper presents a systematic and quantitative investigation of the influence of surfactants on the flow of fluids in microchannels containing bubbles. It derives the contributions to the overall pressure drop from three regions of the channel: (i) the slugs of liquid between the bubbles (and separated from the bubbles), in which liquid flows as though no bubbles were present; (ii) the gutters along the corners of the microchannels; and (iii) the curved caps at the ends of the bubble.

This paper describes the fabrication of a microfluidic device for rapid immobilization of large numbers of live C. elegans for performing morphological analysis, microsurgery, and fluorescence imaging in a high-throughput manner. The device consists of two principal elements: (i) an array of 128 wedge-shaped microchannels, or clamps, which physically immobilize worms, and (ii) a branching network of distribution channels, which deliver worms to the array. The flow of liquid through the device (driven by a constant pressure difference between the inlet and the outlet) automatically distributes individual worms into each clamp. It was possible to immobilize more than 100 worms in less than 15 min. The immobilization process was not damaging to the worms: following removal from the array of clamps, worms lived typical lifespans and reproduced normally. The ability to monitor large numbers of immobilized worms easily and in parallel will enable researchers to investigate physiology and behavior in large populations of C. elegans.

This paper describes a model of the motion of superparamagnetic beads in a microfluidic channel under the influence of a weak magnetic field produced by an electric current passing through a coplanar metal wire. The model based on the conventional expression for the magnetic force experienced by a superparamagnetic bead (suspended in a biologically relevant medium) and the parameters provided by the manufacturer failed to match the experimental data. To fit the data to the model, it was necessary to modify the conventional expression for the force to account for the non-zero initial magnetization of the beads, and to use the initial magnetization and the magnetic susceptibility of the beads as adjustable parameters. The best-fit value of susceptibility deviated significantly from the value provided by the manufacturer, but was in good agreement with the value computed using the magnetization curves measured independently for the beads from the same vial as those used in the experiment. The results of this study will be useful to researchers who need an accurate prediction of the behavior of superparamagnetic beads in aqueous suspensions under the influence of weak magnetic fields. The derivation of the force on a magnetic bead due to a magnetic field also identifies the correct treatment to use for this interaction, and resolves discrepancies present throughout the literature.

The ability of red blood cells (RBCs, erythrocytes) to deform and pass through capillaries is essential for continual flow of blood in the microvasculature, which ensures an adequate supply of oxygen and nutrients, prompt removal of metabolic waste products, transport of drugs and hormones, and traffic of circulating cells to and from all living tissues. This paper presents a novel tool for evaluating the impact of impaired deformability of RBCs on the flow of blood in the microvasculature by directly measuring perfusion of a test microchannel network with dimensions and topology similar to the real microcirculation. The measurement of microchannel network perfusion is compared with RBC filtration -- a conventional assay of RBC deformability. In contrast to RBC filterability, network perfusion depends linearly on RBC deformability modulated by graded exposure to glutaraldehyde, showing a higher sensitivity to small changes of deformability. The direct measurement of microchannel network perfusion represents a new concept for the field of blood rheology and should prove beneficial for basic science and clinical applications.