Influence of storage and buffer composition on the mechanical behavior of flowing red blood cells.

On-chip study of blood flow has emerged as a powerful tool to assess the contribution of each component of blood to its overall function. Blood has indeed many functions, from gas and nutrient transport to immune response and thermal regulation. Red blood cells play a central role therein, in particular through their specific mechanical properties, which directly influence pressure regulation, oxygen perfusion, or platelet and white cell segregation toward endothelial walls. As the bloom of in-vitro studies has led to the apparition of various storage and sample preparation protocols, we address the question of the robustness of the results involving cell mechanical behavior against this diversity. The effects of three conservation media (EDTA, citrate, and glucose-albumin-sodium-phosphate) and storage time on the red blood cell mechanical behavior are assessed under different flow conditions: cell deformability by ektacytometry, shape recovery of cells flowing out of a microfluidic constriction, and cell-flipping dynamics under shear flow. The impact of buffer solutions (phosphate-buffered saline and density-matched suspension using iodixanol/Optiprep) are also studied by investigating individual cell-flipping dynamics, relative viscosity of cell suspensions, and cell structuration under Poiseuille flow. Our results reveal that storing blood samples up to 7 days after withdrawal and suspending them in adequate density-matched buffer solutions has, in most experiments, a moderate effect on the overall mechanical response, with a possible rapid evolution in the first 3 days after sample collection.

Multi-laboratory validation of DNAxs including the statistical library DNAStatistX.

This study describes a multi-laboratory validation of DNAxs, a DNA eXpert System for the data management and probabilistic interpretation of DNA profiles [1], and its statistical library DNAStatistX to which, besides the organising laboratory, four laboratories participated. The software was modified to read multiple data formats and the study was performed prior to the release of the software to the forensic community. The first exercise explored all main functionalities of DNAxs with feedback on user-friendliness, installation and general performance. Next, every laboratory performed likelihood ratio (LR) calculations using their own dataset and a dataset provided by the organising laboratory. The organising laboratory performed LR calculations using all datasets. The datasets were generated with different STR typing kits or analysis systems and consisted of samples varying in DNA amounts, mixture ratios, number of contributors and drop-out level. Hypothesis sets had the correct, under- and over-assigned number of contributors and true and false donors as person of interest. When comparing the results between laboratories, the LRs were foremost within one unit on log10 scale. The few LR results that deviated more had differences for the parameters estimated by the optimizer within DNAStatistX. Some of these were indicated by failed iteration results, others by a failed model validation, since unrealistic hypotheses were included. When these results that do not meet the quality criteria were excluded, as is in accordance with interpretation guidelines, none of the analyses in the different laboratories yielded a different statement in the casework report. Nonetheless, changes in software parameters were sought that minimized differences in outcomes, which made the DNAStatistX module more robust. Overall, the software was found intuitive, user-friendly and valid for use in multiple laboratories.

Mechanical Signature of Red Blood Cells Flowing Out of a Microfluidic Constriction Is Impacted by Membrane Elasticity, Cell Surface-to-Volume Ratio and Diseases.

Despite the fact that Red Blood Cells (RBCs) have been intensively studied in the past 50 years to characterize mechanical phenotypes associated with both healthy and pathological states, only ektacytometry (i.e., laser diffractometry) is currently used by hematologists to screen for RBC membrane disorders. Therefore, the development of new diagnostic tools able to perform analysis at the scale of a single cell, over a statistically relevant population, would provide important complementary information. But these new diagnostic tools would have to be able to discriminate between different disorders causing a change in RBCs mechanical properties. We evaluated the mechanical response of artificially rigidified RBCs flowing through a microfluidic constriction. The geometry consists in a 50 µm wide channel with a succession of 14 tooth-like patterns, each composed of a 5 µm wide and 10 µm long constriction, associated with a 25 µm wide and 10 µm long enlargement. RBCs deformability was altered using two chemical treatments, known to affect RBCs membrane surface area and membrane deformability, lysolecithine (LPC) and diamide, respectively. Differences between samples were highlighted by the representation of the inverse of the shape recovery time (1/τ r ), versus the extension at the exit of the constriction, D out . The results demonstrate that our approach is able to provide a direct signature of RBCs membrane composition and architecture, as it allows discriminating the effect of changes in RBCs membrane surface area from changes in RBCs membrane deformability. Finally, in order to evaluate the potential of our microsystem to detect pathological cells, we have performed preliminary experiments on patients with Hereditary Spherocytosis (HS) or Sickle Cell Anemia (SCA).

Sequenced-based French population data from 169 unrelated individuals with Verogen's ForenSeq DNA signature prep kit.

Massively Parallel Sequencing (MPS) applied to forensic genetics allows the simultaneous analysis of hundreds of genetic markers and the access to full amplicon sequences which help to increase available allele diversity. Meanwhile, sequence variation within the repeat regions represents the majority of the allele diversity, flanking regions adjacent to the repeat core provide an additional degree of variation. The forensic genetics community needs access to population data, from relevant parts of the world that contain this new sequence diversity in order to perform statistical calculations. In this study, we report sequence-based Short Tandem Repeat (STR) and identity Single Nucleotide Polymorphism (iSNPs) allele data for 169 French individuals across 58 STRs and 92 SNPs included in the Verogen ForenSeq DNA Signature Prep kit. 42 STRs out of 58 showed an increased number of alleles due to sequence variation in the repeat motif and/or the flanking regions. D9S1122 showed the largest overall gain with an increase of observed heterozygosities of almost 25 %. The combined match probability combining 27 autosomal STRs and 91 identity SNPs was 1.11E-69. Sequence-based allele frequencies included in this publication will help forensic laboratories to increase the power of discrimination for identification, kinship analysis and mixture interpretation.

Impact of surface-area-to-volume ratio, internal viscosity and membrane viscoelasticity on red blood cell deformability measured in isotonic condition.

Osmotic gradient ektacytometry is the gold standard to assess red blood cell (RBC) deformability. It has been proposed that, when measured in isotonic condition, RBC deformability at low shear stress would depend on membrane elasticity while it would be influenced by internal viscosity when measured at high shear stress, but this hypothesis needs to be further addressed. Healthy RBCs were rigidified by treatment with lysolecithine (LPC), diamide or nystatine associated with hyperosmolar solutions (OSMO), which reduces membrane surface area, decreases membrane elasticity or promotes cell dehydration, respectively. Diamide treatment resulted in a decrease in isotonic RBC deformability at all shear stresses tested (i.e. from 0.3 to 30 Pa). LPC and OSMO treatments caused a decrease in isotonic RBC deformability above 3 Pa only. Isotonic RBC deformability from patients with hereditary spherocytosis or sickle cell disease was mainly decreased above 1.69 Pa. Our findings indicate that decreased isotonic RBC deformability at shear stresses above 3 Pa would be related to a reduction in the surface-area-to-volume ratio and/or to a loss of membrane elasticity and/or to an increase in internal viscosity while a decrease of RBC deformability below 3 Pa would reflect a loss of membrane elasticity.

Automation and developmental validation of the ForenSeq™ DNA Signature Preparation kit for high-throughput analysis in forensic laboratories.

Massively parallel sequencing (MPS) applications in forensic science highlight the advantages of this technique compared to capillary electrophoresis (CE). The multiplexing of a wide range of genetic markers and access to the full amplicon sequence, allowing the detection of isoalleles, make it a very promising tool which could be applied to the most challenging casework DNA samples. However, the complexity of the manual library preparation protocol, potential DNA contamination and sample tracking issues are the main reasons why forensic scientists still hesitate to implement MPS analytical workflows in their laboratory. Here, we present the automation of all library preparation steps for up to 96 samples using the Verogen's ForenSeq™ DNA Signature Preparation kit. This automated protocol, developed on a Hamilton ID STARlet robotic platform, is designed to allow the combined sequencing of rich and poor DNA samples thanks to a final step which adjusts normalized library pooling volume to guarantee a uniform depth of coverage across all samples. Our study includes tests of concordance, repeatability, reproducibility and sensitivity (1000 pg, 700 pg, 500 pg, 250 pg, 100 pg and 50 pg). Sequencing results obtained with the automated protocol were found to be concordant with previous validation studies using the manual protocol in terms of depth of coverage and allele coverage ratio. The results of this study will assist forensic laboratories seeking to acquire a plug and play solution to optimize the processing and analysis of casework samples with MPS.

Dielectrophoretic capture of low abundance cell population using thick electrodes.

Enrichment of rare cell populations such as Circulating Tumor Cells (CTCs) is a critical step before performing analysis. This paper presents a polymeric microfluidic device with integrated thick Carbon-PolyDimethylSiloxane composite (C-PDMS) electrodes designed to carry out dielectrophoretic (DEP) trapping of low abundance biological cells. Such conductive composite material presents advantages over metallic structures. Indeed, as it combines properties of both the matrix and doping particles, C-PDMS allows the easy and fast integration of conductive microstructures using a soft-lithography approach while preserving O2 plasma bonding properties of PDMS substrate and avoiding a cumbersome alignment procedure. Here, we first performed numerical simulations to demonstrate the advantage of such thick C-PDMS electrodes over a coplanar electrode configuration. It is well established that dielectrophoretic force ([Formula: see text]) decreases quickly as the distance from the electrode surface increases resulting in coplanar configuration to a low trapping efficiency at high flow rate. Here, we showed quantitatively that by using electrodes as thick as a microchannel height, it is possible to extend the DEP force influence in the whole volume of the channel compared to coplanar electrode configuration and maintaining high trapping efficiency while increasing the throughput. This model was then used to numerically optimize a thick C-PDMS electrode configuration in terms of trapping efficiency. Then, optimized microfluidic configurations were fabricated and tested at various flow rates for the trapping of MDA-MB-231 breast cancer cell line. We reached trapping efficiencies of 97% at 20 µl/h and 78.7% at 80 µl/h, for 100 µm thick electrodes. Finally, we applied our device to the separation and localized trapping of CTCs (MDA-MB-231) from a red blood cells sample (concentration ratio of 1:10).

Magnetophoretic manipulation in microsystem using carbonyl iron-polydimethylsiloxane microstructures.

This paper reports the use of a recent composite material, noted hereafter i-PDMS, made of carbonyl iron microparticles mixed in a PolyDiMethylSiloxane (PDMS) matrix, for magnetophoretic functions such as capture and separation of magnetic species. We demonstrated that this composite which combine the advantages of both components, can locally generate high gradients of magnetic field when placed between two permanent magnets. After evaluating the magnetic susceptibility of the material as a function of the doping ratio, we investigated the molding resolution offered by i-PDMS to obtain microstructures of various sizes and shapes. Then, we implemented 500 µm i-PDMS microstructures in a microfluidic channel and studied the influence of flow rate on the deviation and trapping of superparamagnetic beads flowing at the neighborhood of the composite material. We characterized the attraction of the magnetic composite by measuring the distance from the i-PDMS microstructure, at which the beads are either deviated or captured. Finally, we demonstrated the interest of i-PDMS to perform magnetophoretic functions in microsystems for biological applications by performing capture of magnetically labeled cells.

Gel phase vesicles buckle into specific shapes.

Osmotic deflation of giant vesicles in the rippled gel phase P(ß') gives rise to a large variety of novel faceted shapes. These shapes are also found from a numerical approach by using an elastic surface model. A shape diagram is proposed based on the model that accounts for the vesicle size and ratios of three mechanical constants: in-plane shear elasticity and compressibility (usually neglected) and out-of-plane bending of the membrane. The comparison between experimental and simulated vesicle morphologies reveals that they are governed by a typical elasticity length, of the order of 1   µm, and must be described with a large Poisson's ratio.

Coagulation dynamics of a blood sample by multiple scattering analysis.

We report a new technique to measure coagulation dynamics on whole-blood samples. The method relies on the analysis of the speckle figure resulting from a whole-blood sample mixed with coagulation reagent and introduced in a thin chamber illuminated with a coherent light. A dynamic study of the speckle reveals a typical behavior due to coagulation. We compare our measured coagulation times to a reference method obtained in a medical laboratory.

Ultrasound internal tattooing.

PURPOSE: The ability of remotely tagging tissues in a controlled and three-dimensional manner during preoperative imaging could greatly help surgeons to identify targets for resection. The authors' objective is to selectively and noninvasively deposit markers under image guidance for such internal tattooing. METHODS: This study describes the production of new ultrasound-inducible droplets carrying large payloads of fluorescent markers and the in vivo proof of concept of their remote and controlled deposition via focused ultrasound. The droplets are monodispersed multiple emulsions produced in a microfluidic system, consisting of aqueous fluorescein in perfluorocarbon in water. Their conversion (either by vaporization or cavitation) is performed remotely using a clinical ultrasonic imaging probe. RESULTS: When submitted to 5 MHz imaging pulses, the droplets vaporize in vitro at 1.4 MPa peak-negative pressure and eject their content. After several seconds, a brightly fluorescent spot (0.5 mm diameter) is observed at the focus of the transducer. Experiments in the chorioallantoique membrane of chicken eggs and chicken embryo demonstrate that the spot is stable and is easily seen by naked eye. CONCLUSIONS: These ultrasound-inducible multiple emulsions could be used to deliver large amounts of contrast agents, chemotherapy, and genetic materials in vivo using a conventional ultrasound scanner.

Drop production and tip-streaming phenomenon in a microfluidic flow-focusing device via an interfacial chemical reaction.

Microfluidic flow-focusing technology is used to investigate the effect on drop formation due to the production of a surfactant via an interfacial chemical reaction. The reactants are an aqueous solution of sodium hydroxide (NaOH) and a mixture of oleic acid (C(17)H(33)-COOH) and mineral oil, for the dispersed and continuous phase fluids, respectively, at concentration < or = 5 mM. In the absence of a chemical reaction, the drop shapes remain constant from just after breakup into droplets down at the flow-focusing nozzle until the drops exit the channel. In the presence of the chemical reaction, there is modification of the shape depending on the concentration of reactants. The drop speeds, O(10) mm/s, lengths, O(1-100) microm, and relative displacements, O(100-1000) microm, are measured for a variety of flow conditions with observable trends that correlate with the reaction rate, which we rationalize by using the Damkohler number to characterize drop production and transport in these types of flows.

Cellular-scale hydrodynamics.

Microfluidic tools are providing many new insights into the chemical, physical and physicochemical responses of cells. Both suspension-level and single-cell measurements have been studied. We review our studies of these kinds of problems for red blood cells with particular focus on the shapes of individual cells in confined geometries, the development and use of a 'differential manometer' for evaluating the mechanical response of individual cells or other objects flowing in confined geometries, and the cross-streamline drift of cells that pass through a constriction. In particular, we show how fluid mechanical effects on suspended cells can be studied systematically in small devices, and how these features can be exploited to develop methods for characterizing physicochemical responses and possibly for the diagnosis of cellular-scale changes to environmental factors.

Swinging of red blood cells under shear flow.

We reveal that under moderate shear stress (etagamma[over ] approximately 0.1 Pa) red blood cells present an oscillation of their inclination (swinging) superimposed to the long-observed steady tank treading (TT) motion. A model based on a fluid ellipsoid surrounded by a viscoelastic membrane initially unstrained (shape memory) predicts all observed features of the motion: an increase of both swinging amplitude and period (1/2 the TT period) upon decreasing etagamma[over ], a etagamma[over ]-triggered transition toward a narrow etagamma[over ] range intermittent regime of successive swinging and tumbling, and a pure tumbling at low etagamma[over ] values.

Geometrical focusing of cells in a microfluidic device: an approach to separate blood plasma.

It is well known that when a suspension of cells flows in small vessels (arterioles or venules), there exists a cell-free layer of a few microns adjacent to the vascular walls. Using an in vitro model, we show experimentally that for a fixed flow rate a geometrical constriction in the flow can artificially enhance the cell-free layer. Also, we show that rapid variation of the geometry coupled to the deformability of the cells can dramatically modify their spatial distribution in the channel. The effects of the constriction geometry, flow rate, suspending fluid viscosity, cell concentration, and cell deformability are studied and the results are interpreted in terms of a model of the hydrodynamic drift of an ellipsoidal cell in a shear flow. We propose a microfluidic application of this focusing effect for separation of the red blood cells from the suspending plasma.

High-speed microfluidic differential manometer for cellular-scale hydrodynamics.

We propose a broadly applicable high-speed microfluidic approach for measuring dynamical pressure-drop variations along a micrometer-sized channel and illustrate the potential of the technique by presenting measurements of the additional pressure drop produced at the scale of individual flowing cells. The influence of drug-modified mechanical properties of the cell membrane is shown. Finally, single hemolysis events during flow are recorded simultaneously with the critical pressure drop for the rupture of the membrane. This scale-independent measurement approach can be applied to any dynamical process or event that changes the hydrodynamic resistance of micro- or nanochannels.

Microfluidic flow focusing: drop size and scaling in pressure versus flow-rate-driven pumping.

We experimentally study the production of micrometer-sized droplets using microfluidic technology and a flow-focusing geometry. Two distinct methods of flow control are compared: (i) control of the flow rates of the two phases and (ii) control of the inlet pressures of the two phases. In each type of experiment, the drop size l, velocity U and production frequency f are measured and compared as either functions of the flow-rate ratio or the inlet pressure ratio. The minimum drop size in each experiment is on the order of the flow focusing contraction width a. The variation in drop size as the flow control parameters are varied is significantly different between the flow-rate and inlet pressure controlled experiments.