Measurement of RBC agglutination with microscopic cell image analysis in a microchannel chip.

Since Landsteiner's discovery of ABO blood groups, RBC agglutination has been one of the most important immunohematologic techniques for ABO and RhD blood groupings. The conventional RBC agglutination grading system for RhD blood typings relies on macroscopic reading, followed by the assignment of a grade ranging from (-) to (4+) to the degree of red blood cells clumping. However, with the new scoring method introduced in this report, microscopically captured cell images of agglutinated RBCs, placed in a microchannel chip, are used for analysis. Indeed, the cell images' pixel number first allows the differentiation of agglutinated and non-agglutinated red blood cells. Finally, the ratio of agglutinated RBCs per total RBC counts (CRAT) from 90 captured images is then calculated. During the trial, it was observed that the agglutinated group's CRAT was significantly higher (3.77-0.003) than that of the normal control (0). Based on these facts, it was established that the microchannel method was more suitable for the discrimination between agglutinated RBCs and non-agglutinated RhD negative, and thus more reliable for the grading of RBCs agglutination than the conventional method.

Analytical performance of a new one-step quantitative prostate-specific antigen assay, the FREND™ PSA Plus.

BACKGROUND: We evaluated the analytical performance of a new one-step rapid quantitative sandwich immunoassay for total prostate-specific antigen (tPSA), the FREND™ PSA Plus (FREND PSA) (NanoEnTek Inc., Seoul, Korea). METHODS: The imprecision, linearity, hook effect, detection limit (LoD), and interference were evaluated and trueness verification and matrix validation were performed. For method comparison, 79 patient specimens were analyzed with FREND PSA and two comparative tPSA assays (Architect® total PSA and cobas® total PSA assay). RESULTS: Total CVs of the imprecision for low (0.208 ng/mL), medium (4.051 ng/mL), and high PSA levels (5.469 ng/mL) were 15.9%, 6.4%, and 9.1%, respectively. Linearity was observed from 1.01 to 19.15 ng/mL and the hook phenomenon was absent up to 171.48 ng/mL. The LoD was 0.094 ng/mL. The regression equations between FREND (y) and Architect or cobas were as follows: y=0.0133+1.054x (r=0.973), y=-0.2144+1.066x (r=0.977), respectively. Differences between FREND PSA and the comparative methods at a medical decision level of 4.0 ng/mL were less than the optimum specification bias (9.3%). The percentage biases from the trueness verification and interference test were less than the desirable specifications for bias (18.7%). The plasma tPSA level measured with lithium heparin or K2EDTA was comparable to that in the serum. CONCLUSIONS: The FREND PSA provided reliable analytical performance and test results in comparison to two widely used tPSA assays. It is a simple and rapid test for tPSA and can be applied in point-of-care testing.

Ongoing development of image cytometers.

Image cytometry is a method for quantitative cellular analysis using images generally captured on slides or microfabricated chips. The flowless nature of data acquisition in image cytometry allows the use of value components, such as light-emitting diode excitation sources or low-cost charge-coupled device detectors. Unlike flow cytometry, the stationary cellular samples can be exposed to lower-intensity light and utilize less sensitive detectors with higher exposure times. Images are acquired and data is processed using recognition software to identify, count and analyze cells. Current image cytometers cannot replicate the quality of the data from flow cytometers or fluorescence microscopes with full functionality and performance components. Yet, the production of inexpensive image cytometers for use in small laboratories and clinics has made a compelling argument. The addition of fluorescence detection to the new generation of image cytometers has opened the field to a broader range of applications. This article will review the technical aspects and application of image cytometers, the recent progress in the field and available commercial devices.

Simultaneous counting of two subsets of leukocytes using fluorescent silica nanoparticles in a sheathless microchip flow cytometer.

A portable flow cytometer has been recognized as an important tool for many clinical applications such as HIV/AIDS screening in developing countries and regions with limited medical facilities and resources. Conventional flow cytometers typically require multiple detectors for simultaneous identification of multiple subsets of immune cell. To minimize the number of detectors toward portable flow cytometry or to analyze multi-parametric cellular information with minimum number of detectors in conventional flow cytometers, we propose a versatile multiplexed cell-counting method using functional silica nanoparticles (SiNPs). FITC-doped SiNPs, which are 100 times brighter than the FITC molecules itself, were used as new intensity-based fluorescent dye complexes to simultaneously measure two subsets of leukocytes using a single detector. CD45(+)CD4(+) cells tagged with these FITC-doped SiNPs were 50 times brighter than CD45(+)CD4(-) cells tagged only with FITC. To make the overall system compact, a disposable microchip flow cytometer that does not require sheath flow was developed. Combining these dye-doped SiNPs based detection schemes and the sheathless microchip flow cytometer scheme, we successfully identified and counted two subsets of leukocytes simultaneously (R(2) = 0.876). These approaches can be the building blocks for a truly portable and disposable flow cytometer for various clinical cytometry applications.

Absolute CD4+ cell count using a plastic microchip and a microscopic cell counter.

We have designed and evaluated the performance of a simple, rapid, and affordable method for counting CD4(+) T-cells with the use of plastic microchips. This new system is an adaptation of a "no-lyse, no-wash," volumetric single platform assay, and absolute CD4(+) counts are determined with the use of a microscopic scanning cell counter. To assess the CD4(+) count test precision and linearity of the system, measured CD4(+) counts were compared with two other reference assays (single and dual platform flow cytometry) with the use of 123 clinical samples including samples obtained from 35 HIV-infected patients, and artificially diluted samples. A correlation between the results from the use of the new method and from the use of the two other reference assays was r = 0.98 for the clinical samples. A dilution test of the new method demonstrated a linearity of r >or= 0.99, with coefficients of variation

A novel electroporation method using a capillary and wire-type electrode.

Electroporation is widely used to achieve gene transfection. A common problem in electroporation is that it has a lower viability than any other transfection method. In this study, we developed a novel electroporation device using a capillary tip and a pipette that was effective on a wide range of mammalian cells, including cell lines, primary cells, and stem cells. The capillary electroporation system considerably reduced cell death during electroporation because of its wire-type electrode, which has a small surface area. The experimental results also indicated that the cell viability was dependent on the change in pH induced by electrolysis during electroporation. Additionally, the use of a long and narrow capillary tube combined with simple pipetting shortened the overall time of the electroporation process by up to 15 min, even under different conditions with 24 samples. These results were supported by comparison with a conventional electroporation system. The transfection rate and the cell viability were enhanced by the use of the capillary system, which had a high transfection rate of more than 80% in general cell lines such as HeLa and COS-7, and more than 50% in hard-to-transfect cells such as stem or primary cells. The viability was approximately 70-80% in all cell types used in this study.

An impulsive, electropulsation-driven backflow in microchannels during electroporation.

We describe for the first time an impulsive, electropulsation-driven backflow in microchannels for on-chip cell electroporation.

A multi-channel electroporation microchip for gene transfection in mammalian cells.

We developed a multi-channel electroporation microchip made of polydimethylsiloxane (PDMS) and glass for gene transfer in mammalian cells. This chip produces multiple electric field gradients in a single microchip by varying the lengths of the microchannels from 2 to 4 cm. Electric fields of 0.65, 0.57, 0.49, 0.41, and 0.33 kV/cm were simultaneously produced in a single chip when the voltage of 1.3 kV was applied. We transferred enhanced green fluorescent protein genes (pEGFP) into HEK-293 and CHO cells, which were cultured within the microchannels. The feasibility of our device was demonstrated because it was able to produce five different transfection rates and survival rates at different electric fields produced in a single microchip. This system is expected to optimize the experimental conditions in gene transfection research more easily and faster than conventional electroporation methods.

On-chip erythrocyte deformability test under optical pressure.

This paper presents a novel method for an on-chip erythrocyte deformability test under optical pressure, especially to enhance the level of sensitivity with respect to the detection of cancerous diseases. To demonstrate the performance and sensitivity of the combined method, we introduce the concept of transit velocity, a modified elongation index, and shape recovery time of individual erythrocytes in a strictly confined region (2 microm deep, 4 microm wide, and 100 microm long). Finally, we investigate a synergy or convergence effect due to the combination of these parameters for in situ detection of cancerous diseases under optical pressure.

Expansion channel for microchip flow cytometers.

This paper presents a novel way of designing a flow focusing channel for microchip flow cytometers. With this method we increased throughput and sensitivity of particle detection at the same time. Generally, to increase the detection throughput of a flow cytometer, the speed of the flow inside the focusing channel needs to be increased, hence reducing the time of exposure to laser beam. With the shorter exposure time, both the fluorescence and scatter signal from the target particles become dimmer. To increase the sensitivity of signal detection, however, the speed of the flow should be decreased so as to decrease throughput of detection. To overcome this dilemmatic problem, we integrated an expansion channel inside a focusing channel. Signals from particles in an expansion channel were about 10 times brighter than those in a normal channel. With this enhanced sensitivity, we could also speed up the inlet flow, which in turn increases the overall throughput of detection.

Microchip capillary gel electrophoresis using programmed field strength gradients for the ultra-fast analysis of genetically modified organisms in soybeans.

We have developed a novel method for the ultra-fast analysis of genetically modified organisms (GMOs) in soybeans by microchip capillary gel electrophoresis (MCGE) using programmed field strength gradients (PFSG) in a conventional glass double-T microchip. Under the programmed electric field strength and 0.3% poly(ethylene oxide) sieving matrix, the GMO in soybeans was analyzed within only 11 s of the microchip. The MCGE-PFSG method was a program that changes the electric field strength during GMO analysis, and was also applied to the ultra-fast analysis of PCR products. Compared to MCGE using a conventional and constantly applied electric field, the MCGE-PFSG analysis generated faster results without the loss of resolving power and reproducibility for specific DNA fragments (100- and 250-bp DNA) of GM-soybeans. The MCGE-PFSG technique may prove to be a new tool in the GMO analysis due to its speed, simplicity, and high efficiency.

Perinuclear translocation of hsp 27 in shear stress exposed human endothelial cells.

Expression of hsp 27 in human umbilical vein endothelial cells exposed to a shear stress was investigated. Using immunostaining, it was concluded that shear stress results in perinuclear translocation of hsp 27. Polymerization of actin microfilaments plays an important role in this process.

Electrotransfection of mammalian cells using microchannel-type electroporation chip.

Transfection of DNA molecules into mammalian cells with electric pulsations, which is so-called electroporation, is a powerful and widely used method that can be directly applied to gene therapy. However, very little is known about the basic mechanisms of DNA transfer and cell response to the electric pulse. We developed a microelectroporation chip with poly(dimethylsiloxane) (PDMS) to investigate the mechanism of electroporation as a first step of DNA transfer and to introduce the benefits of miniaturization into the genetic manipulation. The microelectroporation chip has a microchannel with a height of 20 microm and a length of 2 cm. Owing to the transparency of PDMS, we could in situ observe the uptake process of propidium iodide (PI) into SK-OV-3 cells, which shows promise in visualization of gene delivery in living cells. We also noticed the geometric effect on the degree of electroporation in microchannels with diverse channel width. This experimental result shows that the geometry can be another parameter to be considered for the electroporation when it is performed in microchannels with an exponential decaying pulse generator. Cell culturing is possible within the microelectroporation chip, and we also successfully transfected SK-OV-3 cells with enhanced green fluorescent protein genes, which demonstrates the feasibility of the microelectroporation chip in genetic manipulation.

Effects of peak anomalies with the hydrophilic or hydrophobic properties of reservoirs during serial injection on a capillary electrophoresis microchip.

Several anomalies, e.g., in peak shape, migration time, and baseline drift, all due to pressure-driven backflow, were previously reported to occur during serial injection on capillary electrophoresis (CE) chips. Since these anomalies were worse for polydimethylsiloxane (PDMS) microchips than for glass microchips, reproducible data on PDMS microchips were difficult to obtain. In this paper, we found that these problems were affected by the hydrophilic or hydrophobic properties of the reservoirs on the microchip and demonstrated that these anomalies were reduced by converting the hydrophobic properties of the reservoirs on the PDMS microchip into hydrophilic ones. Thus, compared with hydrophobic reservoirs, hydrophilic reservoirs were suitable for the formation of a stable plug. Several chip designs were suggested to reduce these pressure-driven backflows.