Diamagnetic capture mode magnetophoretic microseparator for blood cells

This paper presents the characterization of a continuous diamagnetic capture (DMC) mode magnetophoretic microseparator for separating red and white blood cells from diluted whole blood based on their native magnetic properties. The DMC microseparator separated the blood cells using a high-gradient magnetic separation (HGMS) method without the use of additives such as magnetic beads. The microseparator was fabricated using microfabrication technology, enabling the integration of microscale magnetic flux concentrators in an aqueous microenvironment. Experimental results show that the DMC microseparator can continuously separate out 89.7% of red blood cells (RBCs) from diluted whole blood within 5 min using an external magnetic flux of 0.2 T from a permanent magnet. Monitoring white blood cells (WBCs) probed with a fluorescence dye show that 72.7% of WBCs were separated out within 10 min in the DMC microseparator using a 0.2 T external applied magnetic flux. Consequently, the DMC microseparator may facilitate the separation of WBCs from whole blood in applications such as a genetic sample preparation and blood borne disease detection. [1574].     

Integrated microfluidic system for electrochemical sensing of glycosylated hemoglobin

This article reports a new miniature electrochemical detection system integrating a sample pretreatment device for fast detection of glycosylated hemoglobin (HbA_1C), which is a common indicator for diabetes mellitus. In this system, circular micropumps, normally closed microvalves, dielectrophoretic (DEP) electrodes, and electrochemical sensing electrode are integrated to perform several crucial processes. These processes include separation of red blood cells (RBCs), sample/reagent transportation, mixing, cell lysis, and electrochemical sensing. For the HbA_1C measurement, the RBCs are separated and are collected from whole human blood by using a positive DEP force generated by the DEP electrodes. The collected RBCs are then lysed to release HbA_1C for the subsequent electrochemical detection processes. Experimental data show that the RBCs are successfully separated and are collected using the developed system with a RBCs capture rate of 84.2%. The subsequent detection of HbA_1C is automatically completed by utilizing electrochemical sensing electrode. The microfluidic system only consumes a sample volume of 200 μl. The entire process is automatically performed within a short period of time (10 min). The development of this integrated microfluidic system may be promising for the clinical monitoring of diabetes mellitus.     

Arrays of high-aspect ratio microchannels for high-throughput isolation of circulating tumor cells (CTCs)

Microsystem-based technologies are providing new opportunities in the area of in vitro diagnostics due to their ability to provide process automation enabling point-of-care operation. As an example, microsystems used for the isolation and analysis of circulating tumor cells (CTCs) from complex, heterogeneous samples in an automated fashion with improved recoveries and selectivity are providing new opportunities for this important biomarker. Unfortunately, many of the existing microfluidic systems lack the throughput capabilities and/or are too expensive to manufacture to warrant their widespread use in clinical testing scenarios. Here, we describe a disposable, all-polymer, microfluidic system for the high-throughput (HT) isolation of CTCs directly from whole blood inputs. The device employs an array of high aspect ratio (HAR), parallel, sinusoidal microchannels (25 × 150 μm; W × D; AR = 6.0) with walls covalently decorated with anti-EpCAM antibodies to provide affinity-based isolation of CTCs. Channel width, which is similar to an average CTC diameter (10–20 μm), plays a critical role in maximizing the probability of cell/wall interactions and allows for achieving high CTC recovery. The extended channel depth allows for increased throughput at the optimized flow velocity (2 mm/s in a microchannel); maximizes cell recovery, and prevents clogging of the microfluidic channels during blood processing. Fluidic addressing of the microchannel array with a minimal device footprint is provided by large cross-sectional area feed and exit channels poised orthogonal to the network of the sinusoidal capillary channels (so-called Z-geometry). Computational modeling was used to confirm uniform addressing of the channels in the isolation bed. Devices with various numbers of parallel microchannels ranging from 50 to 320 have been successfully constructed. Cyclic olefin copolymer (COC) was chosen as the substrate material due to its superior properties during UV-activation of the HAR microchannels surfaces prior to antibody attachment. Operation of the HT-CTC device has been validated by isolation of CTCs directly from blood secured from patients with metastatic prostate cancer. High CTC sample purities (low number of contaminating white blood cells) allowed for direct lysis and molecular profiling of isolated CTCs.     

The synergistic effect of periodic immunomagnetics and microfluidics on universally capturing circulating tumor cells

Efficient capture data on circulating tumor cells (CTCs) determines early-stage cancer diagnosis and contributes to timely clinical treatment. We present an enhanced method of capturing CTCs accomplished by a microfluidic device integrated with magnetic field to activate the kinetic motion of in-device magnetic beads. The device, consisting of a microfluidic chamber and two electrode chips applied with pulsatile alternating current in both, is designed based on simulations on the periodicity characteristic of magnetic beads as well as the effect of heat dissipation on cell culture medium manifest. Using MEMS technologies, the prototype is fabricated and assembled. The cell capture experiments based on active magnetic beads are achieved in separation of rare cancer cells (MCF7 cells) with low concentration. The capture rate is estimated up to 88 %, with great potential of dramatically improving detection efficiency in disease diagnostics.     

Exploiting aggregate channel features for urine sediment detection

Urine sediment examination refers to the use of microscopes to examine various tangible components in urine sediment, e.g. red blood cells (RBCs), white blood cells (WBCs), tube, and crystal, etc., having a very important role in infectious diseases and circulatory diseases diagnosis. The traditional method about urine sediment analysis depends on the observation of medical staffs. So the workload is particularly large and inefficient, and relevant staff need to own some experience. Recently, the automation of urine sediment analysis can be realized. However, due to the complexity of the urine sediment microscopic image, the accuracy and efficiency of the automatic recognition for the tangible components are still very low. To solve this problem, we investigate channel features to urine sediment detection which include diverse feature types like color channel features and gradient magnitude, etc. We propose aggregate channel features plus (ACF+) detector which is based on aggregate channel features (ACF) for urine sediment detection. We adopt improved Adaboost classifier. The input image does not require any preprocessing and the specific ingredients such as RBCs can be detected directly with a high precision and efficiency. On the testing set, our proposed ACF+ detector suppresses several competitive baselines e.g. Support Vector Machine (SVM) combined with Histogram of Oriented Gradient (HOG), vanilla ACF, and ACDS. In terms of speed, it runs 3FPS on 2592?×?2048 images.

     

Design of microfluidic channels for magnetic separation of malaria-infected red blood cells

This study is motivated by the development of a blood cell filtration device for removal of malaria-infected, parasitized red blood cells (pRBCs). The blood was modeled as a multi-component fluid using the computational fluid dynamics discrete element method (CFD-DEM), wherein plasma was treated as a Newtonian fluid and the red blood cells (RBCs) were modeled as soft-sphere solid particles which move under the influence of drag, collisions with other RBCs, and a magnetic force. The CFD-DEM model was first validated by a comparison with experimental data from Han and Frazier (Lab Chip 6:265–273, 2006) involving a microfluidic magnetophoretic separator for paramagnetic deoxygenated blood cells. The computational model was then applied to a parametric study of a parallel-plate separator having hematocrit of 40 % with 10 % of the RBCs as pRBCs. Specifically, we investigated the hypothesis of introducing an upstream constriction to the channel to divert the magnetic cells within the near-wall layer where the magnetic force is greatest. Simulations compared the efficacy of various geometries upon the stratification efficiency of the pRBCs. For a channel with nominal height of 100 µm, the addition of an upstream constriction of 80 % improved the proportion of pRBCs retained adjacent to the magnetic wall (separation efficiency) by almost twofold, from 26 to 49 %. Further addition of a downstream diffuser reduced remixing and hence improved separation efficiency to 72 %. The constriction introduced a greater pressure drop (from 17 to 495 Pa), which should be considered when scaling up this design for a clinical-sized system. Overall, the advantages of this design include its ability to accommodate physiological hematocrit and high throughput, which is critical for clinical implementation as a blood-filtration system.     

On the thin-film-dominated passing pressure of cancer cell squeezing through a microfluidic CTC chip

Detection of circulating tumor cells (CTCs) shows strong promise for early cancer diagnosis, and cell-deformation-based microfluidic CTC chips have been playing an important role. For the design and optimization of high-throughput CTC chips, the dynamic pressure drop in the microfluidic chip during the CTC passing process is a key parameter related to the device sensitivity and filtering performance and has to be given very serious consideration. Although insights have been provided by previous researches, there is still a lack of understanding of the fundamental physics and complex interplay between viscous tumor cell and the flow inside the microfluidic filtering channel. In this paper, the process of the viscous cell squeezing through a microchannel is modeled by solving the governing equations of microscopic multiphase flows, with the tumor cell modeled by a droplet model and the immiscible cell–blood interface tracked by the volume-of-fluid method. Detailed dynamics regarding the filtering process is discussed, including the cell deformation, flow characteristics, passing pressure characteristics as well as the relationship between the pressure drop across the device and the thin film formed in the filtration channel. Current simulation shows a good agreement with analytic results, and an analytical formula is proposed to predict the passing pressure in the microchannel. Our study provides insights into the fluid physics of a viscous cell passing through a constricted microchannel, and the proposed formula can be readily applied to the design and optimization of cell-deformation-based microchannels for CTC detection.     

Optimization of red blood cell inventory: a blood-type compatibility-preference and emergency model

Transfusion of red blood cells (RBCs) is essential for different medical conditions such as surgery, anemia, and cancer. Blood is a precious living composition consisting of eight types of RBCs that can be transfused by either exact or compatible substitutes. As stored blood gets older, it begins to deteriorate; therefore, excessive blood supply at hospitals results in spoilage, while limited supply results in shortage. This research aims at finding the optimal order-up-to-level quantities of blood, which minimize its shortage and outdated units. Stochastic demand, blood aging, emergency orders, and compatibility preference are considered in this study. A customized simulated annealing algorithm is built for optimization. Several simulation experiments are conducted to test the merits of the model in a selected hospital. Not only does the model demonstrate improved results in the mean inventory/age, shortage/spoilage, and substitution percentage but it also serves as a test bed for better understanding of RBC inventory behavior under different compatibility policies.     

无透镜微流控成像流动细胞检测与计数系统

在未来面向个人化的生物医疗诊断中,实时的细胞检测与计数具有重要需求.现有的细胞检测和计数系统例如流式细胞仪和血细胞计数器不适用于小型化流动细胞实时检测和计数.通过将CMOS图像传感器芯片和微流控芯片结合,提出了一种用于流动细胞检测和计数的无透镜微流控成像系统,与用于计数静态细胞的其它无透镜微流控成像系统不同,该系统可以通过基于时域差分的运动检测算法检测和计数微流体通道中连续流动的细胞样本.测试结果表明:该系统可以对微流控通道中流动的人体骨髓基质细胞实现自动检测和计数,并具有-6.53％的低统计错误率.该系统提供了面向未来即时应用的细胞检测和计数解决方案.     

一种基于CCD的全自动循环肿瘤细胞检测系统

文章旨在介绍一种基于CCD的全自动循环肿瘤细胞检测系统。该系统主要采用多路阀控制实验进程,通过对血液中过量表达EpCAM的细胞进行分离富集,然后经过免疫化学染色,通过CCD摄像头来将采集到的多重荧光成像的图像传递给LabView进行处理,根据图像表征的不同来确定是否为循环肿瘤细胞以及检测个数并对其进行相关图像特性的分析。     

Detection of Cellular Parameters Using a Microfluidic Chip-Based System

Lab-on-a-chip technology achieves a reduction of sample and reagent volume and automates complex laboratory processes. Here, we present the implementation of cell assays on a microfluidic platform using disposable microfluidic chips. The applications are based on the controlled movement of cells by pressure-driven flow inside networks of microfluidic channels. Cells are hydrodynamically focused and pass the fluorescence detector in single file. Initial applications are the determination of protein expression and apoptosis parameters. The microfluidic system allows unattended measurement of six samples per chip. Results obtained with the microfluidic chips showed good correlation with data obtained using a standard flow cytometer.     

Miniaturized flow cytometer with 3D hydrodynamic particle focusing and integrated optical elements applying silicon photodiodes

In this study, the design, realization and measurement results of a novel optofluidic system capable of performing absorbance-based flow cytometric analysis is presented. This miniaturized laboratory platform, fabricated using SU-8 on a silicon substrate, comprises integrated polymer-based waveguides for light guiding and a biconcave cylindrical lens for incident light focusing. The optical structures are detached from the microfluidic sample channel resulting in a significant increase in optical sensitivity. This allows the application of standard solid-state laser and standard silicon-based photodiodes operated by lock-in-amplification resulting in a highly practical and effective detection system. The easy-to-fabricate single-layer microfluidic structure enables independently adjustable 3D hydrodynamic sample focusing to an arbitrary position in the channel. To confirm the fluid dynamics and raytracing simulations and to characterize the system, different sets of microparticles and T-lymphocyte cells (Jurkat cell line) for vital staining were investigated by detecting the extinction (axial light loss) signal. The analytical classification via signal peak height/width demonstrates the high sensitivity and sample discrimination capability of this compact low-cost/low-power microflow cytometer.     

Microfluidic device for continuous magnetophoretic separation of white blood cells

This paper presents a microfluidic device for magnetophoretic separation of red blood cells from blood under continuous flow. The separation method consists of continuous flow of a blood sample (diluted in PBS) through a microfluidic channel which presents on the bottom “dots” of ferromagnetic layer. By applying a magnetic field perpendicular on the flowing direction, the ferromagnetic “dots” generate a gradient of magnetic field which amplifies the magnetic force. As a result, the red blood cells are captured on the bottom of the microfluidic channel while the rest of the blood is collected at the outlet. Experimental results show that an average of 95% of red blood cells is trapped in the device.     

Machine aided malaria parasitemia detection in Giemsa-stained thin blood smears

Malaria parasitemia is the quantitative measurement of the parasites in the blood to grade the degree of infection. Light microscopy is the most well-known method used to examine the blood for parasitemia quantification. The visual quantification of malaria parasitemia is laborious, time-consuming and subjective. Although automating the process is a good solution, the available techniques are unable to evaluate the same cases such as anemia and hemoglobinopathies due to deviation from normal RBCs’ morphology. The main aim of this research is to examine the microscopic images of stained thin blood smears using a variety of computer vision techniques, grading malaria parasitemia on independent factors (RBC’s morphology). The proposed methodology is based on inductive approach, color segmentation of malaria parasites through adaptive algorithm of Gaussian mixture model (GMM). The quantification accuracy of RBCs is improved, splitting the occlusions of RBCs with distance transform and local maxima. Further, the classification of infected and non-infected RBCs has been made to properly grade parasitemia. The training and evaluation have been carried out on image dataset with respect to ground truth data, determining the degree of infection with the sensitivity of 98 % and specificity of 97 %. The accuracy and efficiency of the proposed scheme in the context of being automatic were proved experimentally, surpassing other state-of-the-art schemes. In addition, this research addressed the process with independent factors (RBCs’ morphology). Eventually, this can be considered as low-cost solutions for malaria parasitemia quantification in massive examinations.

     

MayGAN: Mayfly Optimization with Generative Adversarial Network-Based Deep Learning Method to Classify Leukemia Form Blood Smear Images

Leukemia, often called blood cancer, is a disease that primarily affects white blood cells (WBCs), which harms a person’s tissues and plasma. This condition may be fatal when if it is not diagnosed and recognized at an early stage. The physical technique and lab procedures for Leukaemia identification are considered time-consuming. It is crucial to use a quick and unexpected way to identify different forms of Leukaemia. Timely screening of the morphologies of immature cells is essential for reducing the severity of the disease and reducing the number of people who require treatment. Various deep-learning (DL) model-based segmentation and categorization techniques have already been introduced, although they still have certain drawbacks. In order to enhance feature extraction and classification in such a practical way, Mayfly optimization with Generative Adversarial Network (MayGAN) is introduced in this research. Furthermore, Generative Adversarial System (GAS) is integrated with Principal Component Analysis (PCA) in the feature-extracted model to classify the type of blood cancer in the data. The semantic technique and morphological procedures using geometric features are used to segment the cells that makeup Leukaemia. Acute lymphocytic Leukaemia (ALL), acute myelogenous Leukaemia (AML), chronic lymphocytic Leukaemia (CLL), chronic myelogenous Leukaemia (CML), and aberrant White Blood Cancers (WBCs) are all successfully classified by the proposed MayGAN model. The proposed MayGAN identifies the abnormal activity in the WBC, considering the geometric features. Compared with the state-of-the-art methods, the proposed MayGAN achieves 99.8% accuracy, 98.5% precision, 99.7% recall, 97.4% F1-score, and 98.5% Dice similarity coefficient (DSC).     

Fabrication and integration of microscale permanent magnets for particle separation in microfluidics

Microfluidic magnetophoresis is an effective technique to separate magnetically labeled bioconjugates in lab-on-a-chip applications. However, it is challenging and expensive to fabricate and integrate microscale permanent magnets into microfluidic devices with conventional methods that use thin-film deposition and lithography. Here, we propose and demonstrate a simple and low-cost technique to fabricate microscale permanent magnetic microstructures and integrate them into microfluidic devices. In this method, microstructure channels were fabricated next to a microfluidic channel and were injected with a liquid mixture of neodymium (NdFeB) powders and polydimethylsiloxane (PDMS). After the mixture was cured, the resulted solid NdFeB–PDMS microstructure was permanently magnetized to form microscale magnets. The microscale magnets generate strong magnetic forces capable of separating magnetic particles in microfluidic channels. Systematic experiments and numerical simulations were conducted to study the geometric effects of the microscale magnets. It was found that rectangular microscale magnets generate larger \(({\mathbf {H}}\cdot \nabla ) {\mathbf {H}}\) which is proportional to magnetic force and have a wider range of influence than the semicircle or triangle magnets. For multiple connected rectangular microscale magnet, additional geometric parameters, including separation distance, height and width of the individual elements, further influence the particle separation and were characterized experimentally. With an optimal size combination, complete separation of yeast cells and magnetic microparticles of similar sizes (\(4\;\upmu \hbox {m}\)) was demonstrated with the multi-rectangular magnet microfluidic device.     

A microfabricated module for isolating cervical carcinoma cells from peripheral blood utilizing dielectrophoresis in stepping electric fields

The ability to isolate rare cells, such as circulating tumor cells (CTC), circulating fetal cells, and stem cells, is important for medical diagnostics and characterization. The present study develops a microfabricated module that can effectively isolate cervical carcinoma cells (HeLa) from a peripheral blood sample. Circular microelectrodes that generate a stepping electric field by switching the electric field between adjacent electrode pairs by relays are designed herein. Positive dielectrophoretic cells are guided by the movement of the high-electric-field region. The magnitude of the dielectrophoresis (DEP) force acting on HeLa cells is about sevenfold that on red blood cells (RBCs) under a given electric field distribution in a sucrose medium, making it possible to separate HeLa cells from normal blood cells. Both HeLa cells and RBCs are pushed to the outermost electrodes when an outward stepping electric field (16?V peak-to-peak; 1?MHz) is applied. When an inward stepping electric field (10?V peak-to-peak; 1?MHz) is applied, the movement of HeLa cells toward the center electrodes is faster than that of RBCs. As a result, HeLa cells are concentrated onto the central microelectrode and isolated from the blood sample. Experimental results demonstrate the feasibility of isolating HeLa cells from blood samples.     

Rapid prototyping of PMMA microfluidic chips utilizing a CO<Subscript>2</Subscript> laser

A commercially available CO₂ laser scriber is used to perform the direct-writing ablation of polymethyl-methacrylate (PMMA) substrates for microfluidic applications. The microfluidic designs are created using commercial layout software and are converted into the command signals required to drive the laser scriber in such a way as to reproduce the desired microchannel configuration on the surface of a PMMA substrate. The aspect ratio and surface quality of the ablated microchannels are examined using scanning electron microscopy and atomic force microscopy surface measurement techniques. The results show that a smooth channel wall can be obtained without the need for a post-machining annealing operation by performing the scribing process with the CO₂ laser beam in an unfocused condition. The practicality of the proposed approach is demonstrated by fabricating two microfluidic chips, namely a cytometer, and an integrating microfluidic chip for methanol detection, respectively. The results confirm that the proposed unfocused ablation technique represents a viable solution for the rapid and economic fabrication of a wide variety of PMMA-based microfluidic chips.     

An integrated microfluidic system for counting of CD4+/CD8+ T lymphocytes

This study reports on an integrated microfluidic system capable of counting CD4⁺/CD8⁺ T lymphocytes from a whole blood sample, which may be further applied for the rapid screening of the human immunodeficiency virus (HIV) infection. This system is composed of a sample incubation module for fluorescence-labeling of the target cells and a micro-fabricated flow cytometry module for cell counting. First, a pneumatically driven, vortex-type micro-mixer has been adopted for the fluorescence-labeling of CD4⁺/CD8⁺ T lymphocytes from whole blood. After the labeling process, different laser-excited fluorescent signals are detected and are used for counting of CD4⁺/CD8⁺ T lymphocytes as they pass through the detection region of the microflow cytometer. A concentration of 963 cells/μl is counted for cultured CD4⁺ T lymphocytes with a reference concentration of 1000 cells/μl. The ratio of CD4⁺/CD8⁺ T lymphocytes is then calculated. Experimental results show that the results from the microsystem are in agreement with the ones from large-scale flow cytometers. In addition, the entire diagnostic procedure, including the sample incubation and the cell counting, can be automatically performed within 35 min. Therefore, this may become a powerful tool for further biomedical applications, especially for fast screening of HIV infection.     

Histogram-Based Decision Support System for Extraction and Classification of Leukemia in Blood Smear Images

An abnormality that develops in white blood cells is called leukemia. The diagnosis of leukemia is made possible by microscopic investigation of the smear in the periphery. Prior training is necessary to complete the morphological examination of the blood smear for leukemia diagnosis. This paper proposes a Histogram Threshold Segmentation Classifier (HTsC) for a decision support system. The proposed HTsC is evaluated based on the color and brightness variation in the dataset of blood smear images. Arithmetic operations are used to crop the nucleus based on automated approximation. White Blood Cell (WBC) segmentation is calculated using the active contour model to determine the contrast between image regions using the color transfer approach. Through entropy-adaptive mask generation, WBCs accurately detect the circularity region for identification of the nucleus. The proposed HTsC addressed the cytoplasm region based on variations in size and shape concerning addition and rotation operations. Variation in WBC imaging characteristics depends on the cytoplasmic and nuclear regions. The computation of the variation between image features in the cytoplasm and nuclei regions of the WBCs is used to classify blood smear images. The classification of the blood smear is performed with conventional machine-learning techniques integrated with the features of the deep-learning regression classifier. The designed HTsC classifier comprises the binary classifier with the classification of the lymphocytes, monocytes, neutrophils, eosinophils, and abnormalities in the WBCs. The proposed HTsC identifies the abnormal activity in the WBC, considering the color and shape features. It exhibits a higher classification accuracy value of 99.6% when combined with the other classifiers. The comparative analysis expressed that the proposed HTsC model exhibits an overall accuracy value of 98%, which is approximately 3%–12% higher than the conventional technique.