A Novel Approach to Hematology Testing at the Point of Care.

BACKGROUND: The need for rapid point-of-care (POC) diagnostics is now becoming more evident due to the increasing need for timely results and improvement in healthcare service. With the recent COVID-19 pandemic outbreak, POC has become critical in managing the spread of disease. Applicable diagnostics should be readily deployable, easy to use, portable, and accurate so that they fit mobile laboratories, pop-up treatment centers, field hospitals, secluded wards within hospitals, or remote regions, and can be operated by staff with minimal training. Complete blood count (CBC), however, has not been available at the POC in a simple-to-use device until recently. The HemoScreen, which was recently cleared by the FDA for POC use, is a miniature, easy-to-use instrument that uses disposable cartridges and may fill this gap. CONTENT: The HemoScreen's analysis method, in contrast to standard laboratory analyzers, is based on machine vision (image-based analysis) and artificial intelligence (AI). We discuss the different methods currently used and compare their results to the vision-based one. The HemoScreen is found to correlate well to laser and impedance-based methods while emphasis is given to mean cell volume (MCV), mean cell hemoglobin (MCH), and platelets (PLT) that demonstrate better correlation when the vision-based method is compared to itself due to the essential differences between the underlying technologies. SUMMARY: The HemoScreen analyzer demonstrates lab equivalent performance, tested at different clinical settings and sample characteristics, and might outperform standard techniques in the presence of certain interferences. This new approach to hematology testing has great potential to improve quality of care in a variety of settings.

Advanced microfluidic droplet manipulation based on piezoelectric actuation.

As droplet-based microfluidic devices evolve, the demand for simple-to-fabricate droplet manipulation modules increases. Of these modules, droplet sorting has drawn much attention due to its ability not only to enrich, but also to selectively isolate droplet subpopulations of interest. In this paper, we present an innovative piezoelectric-driven droplet sorter that is simple to fabricate, reproducible and robust, which provides extensive control over spatio-temporal droplet pattern. This degree of control is demonstrated by sorting droplets of alternating volumes and by grouping defined number of droplets into traveling clusters. The ability to automatically sort droplets is demonstrated by computerized detection and sorting of droplets based on their color. The sorter performance was investigated and found to work on a wide range of sorting parameters. The sorter is created by a single step fabrication process and does not rely on complex electronics or optics. These advantages simplify the adoption of droplet-based microfluidic technology by the scientific community and provide an ideal platform for single cell assays.

A microfluidic traps system supporting prolonged culture of human embryonic stem cells aggregates.

The unlimited proliferative and differentiative capacities of embryonic stem cells (ESCs) are tightly regulated by their microenvironment. Local concentrations of soluble factors, cell-cell interactions and extracellular matrix signaling are just a few variables that influence ESC fate. A common method employed to induce ESC differentiation involves the formation of cell aggregates called embryoid bodies (EBs), which recapitulate early stages of embryonic development. EBs are normally formed in suspension cultures, producing heterogeneously shaped and sized aggregates. The present study demonstrates the usage of a microfluidic traps system which supports prolonged EB culturing. The traps are uniquely designed to facilitate cell capture and aggregation while offering efficient gas/nutrients exchange. A finite element simulation is presented with emphasis on several aspects critical to appropriate design of such bioreactors for ESC culture. Finally, human ESC, mouse Nestin-GFP ESC and OCT4-EGFP ESCs were cultured using this technique and demonstrated extended viability for more than 5 days. In addition, EBs developed and maintained a polarized differentiation pattern, possibly as a result of the nutrient gradients imposed by the traps bioreactor. The novel microbioreactor presented here can enhance future embryogenesis research by offering tight control of culturing conditions.

A microfluidic droplet generator based on a piezoelectric actuator.

Droplet based microfluidic systems have been shown to be most valuable in biology and chemistry research. However droplet modulation and manipulation requires still further improvement in order to make this technology feasible particularly for biological applications. On demand generation of droplets and droplet synchronization, which is crucial for coalescence, remain largely unanswered. The present study describes a simple and robust droplet generator based on a piezoelectric actuator which is integrated into a microfluidic device. The droplet generator is able to independently control the droplet size, rate of formation and distance between droplets. Moreover, the droplet uniformity is especially high, deviating from the mean value by less than 0.3%. The cross flow and T-junction configurations are tested and show no significant differences, yet the inlet to main channel ratio is found to be important. As this ratio increases, droplets tend to be generated in bursts instead of individually. The physical mechanisms involved are discussed, providing insight into optimized design of such systems.

Periodic "flow-stop" perfusion microchannel bioreactors for mammalian and human embryonic stem cell long-term culture.

The present study examines the use of automated periodic "flow-stop" perfusion systems for long-term culture of mammalian cells in a microchannel bioreactor. The method is used to culture Human Foreskin Fibroblasts (HFF) and Human Umbilical Vein Endothelial Cells (HUVEC) for long periods of time (>7 d) in a microchannel (height 100 mum). Design parameters, mass transport and shear stress issues are theoretically examined via numerical simulations. Cell growth and morphology are experimentally monitored and an enhanced growth rate was measured compared to constant perfusion micro-reactors and to traditional culture in Petri dishes. Moreover, we demonstrate the use of the method to co-culture undifferentiated colonies of human Embryonic Stem Cells (hESC) on HFF feeder cells in microchannels. The successful hESC-HFF co-culture in the microbioreactor is achieved due to two vital characteristics of the developed method-short temporal exposure to flow followed by long static incubation periods. The short pulsed exposure to shear enables shear sensitive cells (e.g., hESC) to withstand the medium renewal flow. The long static incubation period may enable secreted factors (e.g., feeder cells secreted factors) to accumulate locally. Thus the developed method may be suitable for long-term culture of sensitive multi-cellular complexes in microsystems.

Design of well and groove microchannel bioreactors for cell culture.

Microfluidic bioreactors have been shown valuable for various cellular applications. The use of micro-wells/grooves bioreactors, in which micro-topographical features are used to protect sensitive cells from the detrimental effects of fluidic shear stress, is a promising approach to culture sensitive cells in these perfusion microsystems. However, such devices exhibit substantially different fluid dynamics and mass transport characteristics compared to conventional planar microchannel reactors. In order to properly design and optimize these systems, fluid and mass transport issues playing a key role in microscale bioreactors should be adequately addressed. The present work is a parametric study of micro-groove/micro-well microchannel bioreactors. Operation conditions and design parameters were theoretically examined via a numerical model. The complex flow pattern obtained at grooves of various depths was studied and the shear protection factor compared to planar microchannels was evaluated. 3D flow simulations were preformed in order to examine the shear protection factor in micro-wells, which were found to have similar attributes as the grooves. The oxygen mass transport problem, which is coupled to the fluid mechanics problem, was solved for various groove geometries and for several cell types, assuming a defined shear stress limitation. It is shown that by optimizing the groove depth, the groove bioreactor may be used to effectively maximize the number of cells cultured within it or to minimize the oxygen gradient existing in such devices. Moreover, for sensitive cells having a high oxygen demand (e.g., hepatocytes) or low endurance to shear (e.g., human embryonic stem cells), results show that the use of grooves is an enabling technology, since under the same physical conditions the cells cannot be cultured for long periods of time in a planar microchannel. In addition to the theoretical model findings, the culture of human foreskin fibroblasts in groove (30 microm depth) and well bioreactors (35 microm depth) was experimentally examined at various flow rates of medium perfusion and compared to cell culture in regular flat microchannels. It was shown that the wells and the grooves enable a one order of magnitude increase in the maximum perfusion rate compared to planar microchannels. Altogether, the study demonstrates that the proper design and use of microgroove/well bioreactors may be highly beneficial for cell culture assays.

A parametric study of human fibroblasts culture in a microchannel bioreactor.

The culture of cells in a microbioreactor can be highly beneficial for cell biology studies and tissue engineering applications. The present work provides new insights into the relationship between cell growth, cell morphology, perfusion rate, and design parameters in microchannel bioreactors. We demonstrate the long-term culture of mammalian (human foreskin fibroblasts, HFF) cells in a microbioreactor under constant perfusion in a straightforward simple manner. A perfusion system was used to culture human cells for more than two weeks in a plain microchannel (130 microm x 1 mm x 2 cm). At static conditions and at high flow rates (>0.3 ml h(-1)), the cells did not grow in the microchannel for more than a few days. For low flow rates (<0.2 ml h(-1)), the cells grew well and a confluent layer was obtained. We show that the culture of cells in microchannels under perfusion, even at low rates, affects cell growth kinetics as well as cell morphology. The oxygen level in the microchannel was evaluated using a mass transport model and the maximum cell density measured in the microchannel at steady state. The maximum shear stress, which corresponds to the maximum flow rate used for long term culture, was 20 mPa, which is significantly lower than the shear stress cells may endure under physiological conditions. The effect of channel size and cell type on long term cell culture were also examined and were found to be significant. The presented results demonstrate the importance of understanding the relationship between design parameters and cell behavior in microscale culture system, which vary from physiological and traditional culture conditions.

Theoretical model and experimental study of red blood cell (RBC) deformation in microchannels.

The motion and deformation of red blood cells (RBCs) flowing in a microchannel were studied using a theoretical model and a novel automated rheoscope. The theoretical model was developed to predict the cells deformation under shear as a function of the cells geometry and mechanical properties. Fluid dynamics and membrane mechanics are incorporated, calculating the traction and deformation in an iterative manner. The model was utilized to evaluate the effect of different biophysical parameters, such as: inner cell viscosity, membrane shear modulus and surface to volume ratio on deformation measurements. The experimental system enables the measurement of individual RBCs velocity and their deformation at defined planes within the microchannel. Good agreement was observed between the simulation results, the rheoscope measurements and published ektacytometry results. The theoretical model results imply that such deformability measuring techniques are weakly influenced by changes in the inner viscosity of the cell or the ambient fluid viscosity. However, these measurements are highly sensitive to RBC shear modulus. The shear modulus, estimated by the model and the rheoscope measurements, falls between the values obtained by micropipette aspiration and laser trapping. The study demonstrates the integration of a theoretical model with a microfabricated device in order to achieve a better understanding of RBC mechanics and their measurement using microfluidic shear assays. The system and the model have the potential of serving as quantitative clinical tools for diagnosing deformability disorders in RBCs.

An automated cell analysis sensing system based on a microfabricated rheoscope for the study of red blood cells physiology.

An automated rheoscope has been developed, utilizing a microfabricated glass flow cell, high speed camera and advanced image-processing software. RBCs suspended in a high viscosity medium were filmed flowing through a microchannel. Under these conditions, RBCs exhibit different orientations and deformations according to their location in the velocity profile. The rheoscope system produces valuable data such as velocity profile of RBCs, spatial distribution within a microchannel and deformation index (DI) curves. The variation of DI across the channel height, due to change in shear stress, was measured carrying implications for diffractometry methods. These curves of DI were taken at a constant flow rate and cover most of the relevant shear stress spectrum. This is an improvement of the existing techniques for deformability measurements and may serve as a diagnostic tool for certain blood disorders. The DI curves were compared to measurements of the flowing RBCs velocity profile. In addition, we found that RBCs flowing in a microchannel are mostly gathered in the center of the flow and maintain a characteristic spatial distribution. The spatial distribution in this region changes slightly with increasing flow rate. Hence, the system described, provides means for examining the behavior of individual RBCs, and may serve as a microfabricated diagnostic device for deformability measurement.

The HemoScreen, a novel haematology analyser for the point of care.

BACKGROUND AND AIMS: A haematology analyser, based on a new technology, is presented herein. The analyser that provides a complete blood count (CBC) and five-part differential accepts disposable cartridges containing all required reagents, making it maintenance-free and ideal for point-of-care (POC) settings. The test reproducibility and imperviousness to analytical errors are attributed to the imaging-based analysis employed. Imaging enables cell-morphology-based differentiation, which is analogous to the gold standard microscopic analysis. This article presents the HemoScreen new technology and evaluates its performance through a small-scale study conducted in its designated clinical settings. METHODS: Thirty anticoagulated whole blood samples were analysed on the HemoScreen and Sysmex XE-2100. Linear regression was performed for the methods comparison. Two samples with 15 replicates were processed for imprecision. Ease of use of the device was also considered. RESULTS: The HemoScreen demonstrated acceptable imprecision and good agreement with the Sysmex XE-2100. The white blood cells (WBCs), red blood cells (RBCs), haemoglobin (HGB), haematocrit (HCT), platelets (PLT), neutrophils, lymphocytes and eosinophils have coefficients of correlation (r) >0.97. For mean cell volume (MCV), mean cell HGB (MCH) and RBC distribution width (RDW), r values ranged from 0.92 to 0.96. For mean cell HGB concentration (MCHC) and monocytes r=0.82 was demonstrated. User-friendliness and suitability of the device for operation in the designated POC settings was also confirmed. CONCLUSIONS: The HemoScreen employs innovative technologies of viscoelastic focusing and microfluidics within a disposable cartridge for an image-based blood cell analysis. By providing accurate and repeatable CBC and five-part differential results within minutes and maintaining the simplicity of operation, the HemoScreen could have far-reaching implications for use at POC. Further extended evaluation is in progress.

Coalescence-assisted generation of single nanoliter droplets with predefined composition.

We demonstrate the generation of highly accurate nanoliter droplets with a predefined composition. This composition control over a single droplet is achieved by merging two droplets with known concentrations and defined volumes. A forced coalescence is accomplished by synchronizing two piezoelectric-based active droplet generators. A microscope-mounted CCD camera is used to record, quantify and monitor the process to assure its high fidelity. The device is disposable, surfactant free, simple to operate and does not require microelectrode fabrication. It delivers a single on-demand droplet with adjustable high resolution mixing ratios up to 9 at a volume range of 1-10 nanoliters. The presented platform offers, for the first time, a means to perform droplet-based high-throughput screening in the nanoliter range.

Experimental and theoretical study of selective protein deposition using focused micro laminar flows.

The present work describes an experimental method and design tools which enable the precise localization of an analyte, a few microns in width, both temporally and spatially using laminar flows and thus improves previous methods in hydrodynamic focusing. The technique is used to adsorb proteins to selected regions within a microfluidic device without any contamination of the surroundings and may serve in applications requiring selective conveying of other reagents. The regions not coated by proteins are modified with poly(ethylene glycol; PEG), known to efficiently resist protein and cell adhesion. Human endothelial and fibroblast cells are later introduced into the device selectively attaching to the protein coated regions and cultured for a few days. A simulation of the convection-diffusion characteristics of the system is presented and compared to the known T-sensor. The results reveal that, by proper design, reagents concentration may be kept nearly constant along the flow direction. This phenomenon is demonstrated here by achieving particularly precise patterning of cells but may be utilized for numerous other applications as well.

Correlation between erythrocytes deformability and size: a study using a microchannel based cell analyzer.

The deformability of erythrocytes is of great importance for oxygen delivery in the microcirculation [Lipowsky, H.H., 2005. Microvascular rheology and hemodynamics. Microcirculation 12, 5-15]. Aging of erythrocytes is associated with a reduction in deformability and also in size. The present work describes an automated cell analyzer which utilizes a glass microchannel and advanced image processing software. Erythrocytes suspended in a high viscosity medium are filmed flowing through the microchannel. Under these conditions, the cells assume different orientations and undergo varying deformations according to their location in the velocity profile. The cell analyzer enables the measurement of individual erythrocyte velocity, deformability and volume at varying depths within the microchannel. The volume of the cells is calculated based on the experimental data and a fluid mechanics model. The results obtained show that, on average, the deformability of the cells increases with increase in their size. Additionally, the behavior of RBCs in a microchannel is investigated, showing promising diagnostic possibilities.