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Numerous applications in biology and medicine requires efficient and reliable separation of cellular or sub-cellular components from blood for disease diagnosis, genetic analysis, drug screening, and therapeutics. However, analyzing undiluted whole human blood is challenging due to its complex composition of hematopoietic cellular populations, plasma and sub-cellular components such as nucleic acids, metabolites, and proteins. In addition, current state-of-art for blood fractionation relies on multi-step density-based centrifugation or flow cytometry which are manual, time-consuming and unable to process whole blood. In this dissertation, we aim to demonstrate a multi-functional, low-cost microfluidic acoustic streaming platform to enrich and characterize cellular components from whole blood and utilize blood’s sub-cellular components for simultaneous detection of multiple infections. First, we present Lateral Cavity Acoustic Transducers (LCATs) that enables (1) the sorting of undiluted donor whole blood into its cellular subsets (platelets, RBCs, and WBCs), (2) the enrichment and retrieval of breast cancer cells (MCF-7) spiked in donor whole blood at rare cell relevant concentrations (10/ml), and (3) on-chip immunofluorescent labeling for the detection of specific target cellular populations by their known marker expression patterns. Our approach thus demonstrates a compact system that integrates upstream sample processing with downstream separation/enrichment, to carry out multi-parametric cell analysis for blood-based diagnosis and liquid biopsy blood sampling. Second, we integrated a passive (spiral inertial microfluidic device) and an active device (LCAT) for high-throughput separation, enrichment and release of specific target cellular population (such as larger side-population (SP) of DU-145 cells from tissue biopsy or larger monocytic cell population from blood sample). After optimization with particles spiked in blood, this platform removed >90% of the smaller cells, such as RBCs in a blood sample or smaller cancer cells in a heterogeneous cell line, and provided 44,000x enrichment from remaining sample within 5 minutes of device operation. Third, since whole blood comprises not only cellular subsets, but also sub-cellular components, we utilized the antibodies in both blood serum and saliva for the multiplexed screening of HIV, HPV and HSV within 17.5 minutes. We integrated this acoustic microstreaming platform with a protein microarray and verified reactivity of patient antibodies with HIV, HPV and HSV antigens. This integrated microfluidic protein array platform is the basis of a potent strategy to delay progression of primary infection, reduce the risk of co-infections and prevent onward transmission of infections by point-of-care detection of multiple pathogens.

Dynamic and on-site analysis of serum from human blood is crucial, however, state-of-the-art blood-assay methods can only collect single or discrete data of physiological analytes; thus, the online reports of the dynamic fluctuation of key analytes remains a great challenge. Here, we propose a novel separation-sensing membrane by constructing a heterogeneous-nanostructured architecture, wherein a surface nanoporous layer continuously extracts serum, while the biosensing nanochannels underneath dynamically recognise biotargets, thereby achieving a continuous testing of vital clinical indices as blood is drawn. By precisely controlling the pore structure and nanoshape of biosensing crystals, this membrane achieved accurate and online glucose and lactate monitoring in patients with a variety of medical conditions within 1 min, which is one order of magnitude faster than state-of-the-art techniques. Moreover, various kinds of bio-recognisers can be introduced into this membrane to accurately detect glutamate, transaminase, and cancer biomarkers.

Antibodies are important biomarkers in clinical diagnostics in addition to being increasingly used for therapeutic purposes. Although numerous methods for their detection and quantification exist, they predominantly require benchtop instruments operated by specialists. To enable the detection of antibodies at point-of-care (POC), the development of simple and rapid assay methods independent of laboratory equipment is of high relevance. In this study, we demonstrate microfluidic thread-based analytical devices (μTADs) as a new platform for antibody detection by means of bioluminescence resonance energy-transfer (BRET) switching sensor proteins. The devices consist of vertically assembled layers including a blood separation membrane and a plastic film with a sewn-in cotton thread, onto which the BRET sensor proteins together with the substrate furimazine have been predeposited. In contrast to intensity-based signaling, the BRET mechanism enables time-independent, ratiometric readout of bioluminescence signals with a digital camera in a darkroom or a smartphone camera with a 3D-printed lens adapter. The device design allows spatially separated deposition of multiple bioluminescent proteins on a single sewn thread, enabling quantification of multiple antibodies in 5 μL of whole blood within 5 min. The bioluminescence response is independent of the applied sample volume within the range of 5-15 μL. Therefore, μTADs in combination with BRET-based sensor proteins represent user-friendly analytical tools for POC quantification of antibodies without any laboratory equipment in a finger prick (5 μL) of whole blood.

New processing techniques for manipulating blood and its components at a microfluidic scale are currently implemented. As for extracorporeal circulation, the in-line evaluation and monitoring of blood properties during these microfluidic techniques is a challenging task. Here, we show that the blood hematocrit can be measured non-invasively in a sub-millimeter medical tube using the non-Newtonian behavior of blood velocity profile. This hematocrit measurement is demonstrated on human blood with a simple Doppler ultrasound system. Results show a mean measurement error of 4.6 ± 1.3%Hct for hematocrit up to 52% and for 5 s-long ultrasonic signals. The simplicity and the measurement scale of the approach make it highly valuable for measuring hematocrit in new blood separation techniques. The approach may have an impact on in-vitro blood processing in general.

Background One of the elements that makes up blood safety is automation of processing whole blood into ingredients In 2017, Reveos was installed in Blood Bank in Lodz. By combining the centrifuge function and the press, Reveos provides full automation of the whole blood separation process, minimizing the risk of human error, thus ensuring accuracy and repeatability. The aim is to compare the results of the quality control of ingredients obtained after the blood separation using the Compomat G5, Optipress II and the Reveos system as well as to compare the level of compliance with the recommendations on the quality of blood components contained in the applicable regulations. Materials and methods 150 units were separated into ingredients using above mentioned systems showing choosen parameters. Results Parameters were determined for Compomat, Optipress i Reveos: plasma: volume 260±13ml, 262±14ml i 239±20ml, platelets: 16, 23 i 23x109/l. RBC: volume 286±13ml, 272±13ml i 295±17ml, hemoglobin 55, 51, 58g/unit., hematocrit 59, 58 i 52%, leukocytes 0,6x109/unit., 0,6x109/unit., 0,1x106/unit. Buffy coat volume: 56,6ml i 56,1ml for Compomat i Optipress and interim platelets volume 37,8ml. The platelet content in the above-mentioned components was 0.8, 0.7, 0.66x1011/unit, respectively. Conclusions The comparison of the quality of ingredients after the blood separation showed slight differences in the parameters determined. The main difference concerns the volume of the obtained plasma and red blood cells. Nevertheless, all the ingredients obtained meet rigorous quality control assumptions and separation methods can be successfully used in routine work. The Reveos system allows to obtain qualitatively good ingredients in a one-step process. This reduces staff interference in the whole process, which may result in minimizing errors during processing of the whole blood.

Developing simple, portable, rapid, and easy-to-use diagnostic technologies is essential for point-of-care (POC) blood molecular testing. Integrated microfluidic devices that include the functionalities of blood separation, microfluidic pumping, and molecular detection are desirable for POC testing; however, current technologies still rely on off-chip sample processing or require bulky equipment. We report a fully-integrated microfluidic diagnostic device, i.e., an integrated pneumatic microfluidic circuit (iPC), that can autonomously pump whole blood, continuously sort blood plasma, and readily enable blood plasma proteomic analysis. The iPC contains vacuum pillars as a vacuum source and waste reservoir, as well as microchannels connecting the pillars as a plasma separator or a flow stabilizer. We combined the iPC and a miniaturized fluorescence microscope to create a portable diagnostic platform that enables fluorescence-based biomarker detection. First, we performed systematic parametric studies to establish design rules for determining the transport and distribution of fluid streams in the iPC. We then demonstrated the capability of the iPC-based diagnostic platform by successfully separating blood plasma from microliter quantities of whole blood while simultaneously quantifying thrombin in blood samples using an aptamer beacon within 5 min of sample injection. Our platform holds potential as a rapid, field-deployable, essentially universal diagnostic tool in POC settings.

Separation of blood plasma or serum from blood is essential for accurate analysis. Conventional blood separation requires instrumentation, reagents, and large sample volumes, limiting this process to laboratory environments with trained personnel. Full implementation of effective blood separation and analysis on microliter sample volumes for point of care (POC) diagnostics has proven extremely challenging resulting in a growing market demand, with common challenges such as expensive device fabrication processes or devices being comprised of materials which are not easily disposable. We developed a membrane-based wicking microfluidic device which is made using a simple fabrication process. This device uses a unique 3D flow channel geometry, fabricated in a polycaprolactone-filled glass microfiber membrane, to efficiently separate microliter sample volumes of blood. Colorimetric assay chemistries were integrated to demonstrate utility of these devices in POC diagnostics. The devices are capable of separating both fresh and anticoagulant-treated blood at microscale sample volumes (

This work introduces the procedure of using non-immunoassay distance-based paper analytical devices (dPADs) to accurately measure any traces of the cardiac troponin I (TnI) in whole blood samples without the use of any external blood separation. This enables a rapid clinical diagnosis and the subsequent follow-up in regard to identifying acute myocardial infarction. These dPADs are designed and constructed to accommodate three parts: (1) a blood separation zone that is immobilized with a hemostatic agent, this no longer requires a blood separation membrane for the isolation of the plasma from the blood element, (2) a pretreatment zone, and (3) a detection zone coated with thymol blue. The quantitative TnI level in the whole blood was determined by measuring the blue color length found in the detection zone, which is proportional to the concentration, owing to the dry protein binding principle. Correspondingly, a mere single drop of human whole blood performs adequately within our proposed method. This reduces both the size of the collection process and the sample volumes needed in the respective medical fields. As we cover all of the optimization studies, our dPADs provide an evaluation of the linearity range from 0.025 to 2.5 ng/mL (R2 = 0.9989) of TnI, with a detection limit as low as 0.025 ng/mL by use of an observation just using the naked eye. To validate the clinical utilities of our proposed method, our dPADs were then applied for the detection of TnI in humans using the whole blood sample of 15 volunteers. A great amount of accuracy was required in this assay because there was no significant difference between both methods, with the confidence level being as high as 95%. This technique also showed that the recoveries ranged from 99.40 to 104.27%, with the highest relative standard deviation being at 3.77%. Thus, our proposed dPADs offer more benefits for a rapid TnI determination.

A microfluidic chip, which can separate and enrich leukocytes from whole blood, is proposed. The chip has 10 switchback curve channels, which are connected by straight channels. The straight channels are designed to permit the inertial migration effect and to concentrate the blood cells, while the curve channels allow the Dean flow to further classify the blood cells based on the cell sizes. Hydrodynamic suction is also utilized to remove smaller blood cells (e.g., red blood cell (RBC)) in the curve channels for higher separation purity. By employing the inertial migration, Dean flow force, and hydrodynamic suction in a continuous flow system, our chip successfully separates large white blood cells (WBCs) from the whole blood with the processing rates as high as 1 &times, 108 cells/sec at a high recovery rate at 93.2% and very few RBCs (~0.1%).

Interest in developing paper-based devices for point-of-care diagnostics in resource-limited settings has risen remarkably in recent decades. In this paper, we demonstrate what we refer to as “High Yield Passive Erythrocyte Removal” (HYPER) technology, which utilizes capillary forces in a unique cross-flow filtration for the separation of whole blood with performance comparable to centrifuges. As we will demonstrate, state-of-the-art passive blood separation methods implemented in paper-based systems exhibit rapid blood cell clogging on the filtration media or serum outlet and yield only about 10%−30% of the total serum present in the sample. Our innovation results from the inclusion of a differentiation pad, which exploits hydrodynamic effects to reduce the formation of a fouling layer on the blood filtration membrane resulting in more than 60% serum yield with undiluted whole blood as direct input. To demonstrate the effectiveness of the HYPER technology we implement it in a lateral flow system and demonstrate the accurate quantification of vitamin A and iron levels in whole blood samples in 15 minutes.

A disposable microfluidic chip for measuring the impedimetric ratio of blood cells was developed. The undiluted blood sample was separated into erythrocytes and leukocytes in the blood separation microchannel of the chip, and the erythrocytes and leukocytes were then driven into the measurement chambers, respectively. Inside each measurement chamber was an interdigitated electrode (IDE) array acting as a sensor for the impedimetric measurement of the blood cells. On the surface of the IDE array, ZnO nanowires were synthesized to promote the sensitivity of the impedimetric measurement. The experimental result showed that the measured impedance was proportional to the number of blood cells. The IDE array with ZnO nanowires was more sensitive than the IDE without nanowires. We also used freshly drawn venous blood samples from five healthy donors and ten informed and consenting patients for comparison. The impedimetric ratios of the five healthy donors were similar with low standard deviation, indicating that the impedimetric ratio would tend to be a reliable threshold as the indicator of erythrocytes to leukocytes. The impedimetric measurements of all patients were below the threshold. Using impedimetric measurement, the change in leukocytes could be easily monitored.

Background. In 2013, the World Health Organization reported a shortage of 17 million red blood cell units, a number that remains growing. Acts to relieve this shortage have primarily focused on allogeneic blood collection. Nevertheless, autologous transfusion can partially alleviate the current pressure and dependence on blood banking systems. To achieve this, current gold standard autotransfusion devices should be complemented with widely available, cost-efficient, and time-efficient devices. The novel HemoClear cell salvage device (HemoClear BV, Zwolle, Netherlands), a gravity-driven microfilter, potentially is widely employable. We evaluated its performance in the cardiac postoperative setting compared to the centrifugal XTRA™ autotransfusion device. Methods. In a split-unit study (n = 18), shed blood collected 18 hours after cardiothoracic surgery was divided into two equal volumes. One-half was processed by the XTRA™ device and the other with the HemoClear blood separation system. In this paired set-up, equal washing volumes were used for both methods. Washing effectivity and cellular recovery were determined by measuring of complete blood count, free hemoglobin, complement C3, complement C4, and D-dimer in both concentrate as filtrate. Also, processing times and volumes were evaluated. Results. The HemoClear and XTRA™ devices showed equal effectiveness in concentrating erythrocytes and leucocytes. Both methods reduced complement C3, complement C4, and D-dimer by ≥90%. The centrifugal device reduced solutes more significantly by up to 99%. Free hemoglobin load was reduced to 12.9% and 15.5% by the XTRA™ and HemoClear, respectively. Conclusion. The HemoClear device effectively produced washed concentrated red blood cells comparably to the conventional centrifugal XTRA™ autotransfusion device. Although the centrifugal XTRA™ device achieved a significantly higher reduction in contaminants, the HemoClear device achieved acceptable blood quality and seems promising in settings where gold standard cell savers are unaffordable or unpractical.

As the point of care diagnosis devices are becoming ever more popular, this paper suggest a miniaturized testing device from a drop of blood to diagnosis of disease for the global healthcare. The minimal requirements for the POC blood-testing device are blood microsampling, blood separation, immunoassay, and detection and communication of the signals. The microsampling of the blood can be achieved by specialized needle, which can be connected to the microchip or analytical devices. The sampled blood is then separated using either a filter (weir or pillar type), or by the phenomena unique to microfluidic system. The separated blood should then go through sandwich, homogeneous non-competitive, or competitive immunoassay, which can effectively diagnose diverse diseases. Lastly, the device should detect and translate the immune-signals to readable, and clinically significant signals. The development of such device will play a great role for improving healthcare technology.

The paper presents numerical analysis in three-dimensional (3D) of a hybrid micro-separator that uses magnetophoresis (MAP) and hydrodynamic forces for blood cells separation. The separation between red blood cells (RBC) and white blood cells (WBC) is done by manipulating the differences of their physical and magnetic susceptibilities. The MAP force is induced from ferromagnetic line fabricated on a glass slide while the hydrodynamic force is obtained by a ‘U’ shape microfluidic channel. The analysis was conducted using finite element software, COMSOL Multiphysics®. The particles trajectories, which represent the actual blood cells' movements on the micro-separator, were profiled and show successful separation between RBCs and WBCs by using the MAP and hydrodynamic forces. This study also provides insights of challenges associated with blood separation towards the realization of better diagnostic devices.

The aim of this work is to construct a computational fluid dynamics model capable of simulating the quasitransient process of apheresis. To this end a Lagrangian-Eulerian model has been developed which tracks the blood particles within a delineated two-dimensional flow domain. Within the Eulerian method, the fluid flow conservation equations within the separator are solved. Taking the calculated values of the flow field and using a Lagrangian method, the displacement of the blood particles is calculated. Thus, the local blood density within the separator at a given time step is known. Subsequently, the flow field in the separator is recalculated. This process continues until a quasisteady behavior is reached. The simulations show good agreement with experimental results. They shows a complete separation of plasma and red blood cells, as well as nearly complete separation of red blood cells and platelets. The white blood cells build clusters in the low concentrate cell bed.

Determining cell mechanical properties is increasingly recognized as a marker-free way to characterize and separate biological cells. This emerging realization has led to the development of a plethora of appropriate measurement techniques. Here, we use a fairly novel approach, deterministic lateral displacement (DLD), to separate blood cells based on their mechanical phenotype with high throughput. Human red blood cells were treated chemically to alter their membrane deformability and the effect of this alteration on the hydrodynamic behaviour of the cells in a DLD device was investigated. Cells of defined stiffness (glutaraldehyde cross-linked erythrocytes) were used to test the performance of the DLD device across a range of cell stiffness and applied shear rates. Optical stretching was used as an independent method for quantifying the variation in stiffness of the cells. Lateral displacement of cells flowing within the device, and their subsequent exit position from the device were shown to correlate with cell stiffness. Data showing how the isolation of leucocytes from whole blood varies with applied shear rate are also presented. The ability to sort leucocyte sub-populations (T-lymphocytes and neutrophils), based on a combination of cell size and deformability, demonstrates the potential for using DLD devices to perform continuous fractionation and/or enrichment of leucocyte sub-populations from whole blood.

To add to the traditional method of blood separation by centrifugation, a membrane method has in recent years been coming into wide clinical use in treating autoimmune diseases by plasma exchange. The membrane method which not only is easy but also needs simple equipment has been the focus of attention as a new means to collect blood components, especially plasma.This paper discusses the results of clinical evaluation of the safety and performance of Terumo Corporation's membrane type plasma separator Donor Set GDS-1.As a result, it was demonstrated that 174ml of plasma could be separated from approximately 408ml of blood in a little more than 10min, and that the N-N time from needle insertion to withdrawal was approximately 21min. The laboratory data of the donors were not remarkable after plasma separation, and no adverse effects plasma separation were observed. Accordingly, plasmapheresis by this system may be considered to be for practical purpose adequately safe. The activation of the coagulation, fibrinolysis and complement systems was so slight that biocompatibility was considered satisfactory.This system which separates plasma from the blood by gravity is high of clinical use for its safety and procedural simplicity.

Most clinical chemistry tests are performed on cell-free serum or plasma. Therefore micro assay devices for blood tests require integrated on-chip microfluidics for separation of plasma or serum from blood. This is achieved by a new blood separation technique based on a microchannel bend structure developed within the collaborative microtele-biochip (/spl mu/TBC) project co-funded by the German Ministry for education and research (BMBF). Different prototype polymer chips have been manufactured with an UV-LIGA process and hot embossing technology. The separation efficiency of these chips has been determined with samples of human whole blood as well as diluted blood samples. The results show different separation efficiencies up to 90% for blood cells and plasma depending on microchannel geometry as well as cell concentration. As compared to present microfluidic devices for the separation of blood cells like filters or filtration by diffusion the microchannel bend is an integrated on-chip blood separation method, which combines the advantages of rapid separation times and a simple geometry.

We propose a MEMS device which integrates a dielectrophoretic blood separation technique with a cytometer and pump for use in low cost blood analysis. Our technical analysis focuses on fluid flow, blood modeling for electrical systems and the dielectrophoresis and cytometry methods. Simulation results of the flow, separation and cytometry are presented.

A blood analysis chip including a power-free micro blood separator capable of separating cells-free plasma from whole blood using a specially designed sub-micron structure and electrochemical electrodes for glucose sensing is developed. Preliminary tests show the possibility of the presented chip.

The aim of this work is to construct a com- putational fluid dynamics model capable of simulating the transient non-Newtonian process of apheresis. A Lagrangian-Eulerian model has been developed which tracks the blood particles within a two-dimensional flow configuration. Within the Eulerian method, the fluid mass and momentum conservation equations within the separa- tor are solved using the density and the viscosity is calcu- lated from the blood particle concentrations. Subsequently, the displacement of the blood particles is calculated with a Lagrangian method. Hawksley's model for the density of supensions is used in the variable density calculation. The viscosity is calculated with two models based on Vand's rigid particle suspension viscosity concepts, followed by the flow field calculation in the separator. Simulations were performed for various inlet hematocrit values and separa- tor lengths. The simulations are in satisfactory agreement with experimental results reported in literature, indicating a complete separation of plasma and red blood cells (RBCs), as well as nearly complete separation of red blood cells and platelets. No hemolysis was observed in the sim- ulations because the shear rate remained under the criti- cal value of 150 N/m 2 . Ke y Words: Blood separation —Apheresis—Centrifugation—Particle flow—Computa- tional fluid dynamics—Variable viscosity.

We propose a blood separation microfluidic device suitable for point-of-care (POC) applications. By utilizing the high gas permeability of polydimethylsiloxane (PDMS) and phaseguide structures, a simple blood separation device is presented. The device consists of two main parts. A separation chamber with the phaseguide structures, where a sample inlet, a tape-sealed outlet, and a dead-end ring channel are connected, and pneumatic chambers, in which manually operating syringes are plugged. The separation chamber and pneumatic chambers are isolated by a thin PDMS wall. By manually pulling out the plunger of the syringe, a negative pressure is instantaneously generated inside the pneumatic chamber. Due to the gas diffusion from the separation chamber to the neighboring pneumatic chamber through the thin permeable PDMS wall, low pressure can be generated, and then the whole blood at the sample inlets starts to be drawn into the separation chamber and separated through the phaseguide structures. Reversely, after removing the tape at the outlet and manually pushing in the plunger of the syringe, a positive pressure will be created which will cause the air to diffuse back into the ring channel, and therefore allow the separated plasma to be recovered at the outlet on demand. In this paper, we focused on the study of the plasma separation and associated design parameters, such as the PDMS wall thickness, the air permeable overlap area between the separation and pneumatic chambers, and the geometry of the phaseguides. The device required only 2 μl of whole blood but yielding approximately 0.38 μl of separated plasma within 12 min. Without any of the requirements of sophisticated equipment or dilution techniques, we can not only separate the plasma from the whole blood for on-chip analysis but also can push out only the separated plasma to the outlet for off-chip analysis.

Diagnostics plays a critical role in human health, while blood analysis is one of the most common clinical diagnoses. Biomedical engineers have been developing technologies that can robotically perform accurate and rapid blood analysis for field application. The simplified fabrication processes and low cost materials become the tendency of point-of-care (POC) devices for blood analysis. Recently, lab-in-a-tube technique comes up as a simple and robust method for POC measurements of multiple analytes. This paper proposed a tubing system with out-of-plane valves using tygon tubing assembled on a circular disc (CD) for blood separation. Pure plasma could be achieved within minutes after extracting blood from human body. The out-of-plane valve showed excellent capability in preventing backflow and reverse mixing due to diffusion. Therefore, the lab-in-a-tube platform has promising applications for the automation of POC analysis, especially important for the rural areas without enough medical resources.

In this work, a new lab-on-a-chip for rapid analysis of low volume blood samples was designed, fabricated and demonstrated for integration of serum separation, hematocrit evaluation, and protein quantitation. Blood separation was achieved using microchannel flow-based separation. A novel method for evaluating hematocrit from microfluidic flow-separated blood samples was developed using gray scale analysis of a point-and-shoot digital photograph of separated blood in a micochannel. Protein quantitation was subsequently performed in a high surface area-to-volume ratio microfluidic chemiluminescent immunoassay using cell depleted serum produced by microfluidic flow-based separation of whole blood samples. All three steps were achieved in a single microchannel with separation of blood samples and hematocrit evaluation in less than 1 min, and protein quantitation in 5 min.