Development of a New Blood Typing Kit Using the Microfluidics Separation Technique / 대한혈액학회지

BACKGROUND: Blood typing is an essential test for transfusion. Generally, blood typing is performed using a slide test, tube test or microcolumn agglutination test. The aims of this study were to develop a new blood typing kit using micromachining, microfluidics and microseparation methods, and to evaluate the clinical usefulness of the new blood typing kit. METHODS: We designed and manufactured a blood typing microchip using polydimethylsiloxane (PDMS), which contained a microchannel (25~200 micrometer). The blood sample and antisera to be tested were dropped on the microwell for movement and mixing by capillary action. Once agglutination occurred, the microchannel acts as a filter and the blood type was determined by observation by the naked eye. To evaluate the newtyping kit, we tested sensitivity using artificially diluted blood and compared the results of the new typing method with the slide and tube methods using 70 samples. RESULTS: The new blood typing kit could differentiate a +4~+2 agglutination reaction, but could not detect a +1 agglutination reaction as observed by the naked eye. Among 70 samples, the results of ABO and Rh typing by the new typing method (n=66, > or = +2 agglutination reaction by the column agglutination method) were in accord with the results of the tube and slide methods, but couldnot detect agglutination in all 4 clinical samples, below a +1 agglutination reaction. CONCLUSION: The new blood typing kit is inadequate for routine use in the clinical laboratory due to low sensitivity, but with further improvement, it can be used economically, conveniently and objectively for blood typing without any special equipment. Moreover, the microfludics and separation method may be broadly applicable in other tests using the hemagglutination method.

Measurement of Smooth Muscle Cell Migration on the Micromachined Groove Topology

PURPOSE: The spreading, orientation, and chemotaxis with the gradient of a chemoattractant of smooth muscle cells (SMCs) were studied on the micro-grooved substrata by the light, fluorescence and scanning electron microscopy. METHOD: Vertical-walled grooves were produced in silicon wafers by the micromachining technique. All grooves were 4~20micrometer deep and 10~80 micrometer wide. SMCs were cultured on each microgroove and examined under stereo-microscope. RESULT: Cell clusters were markedly oriented by all the grooved substrata examined. Time-lapse images acquired from CCD (Charge Coupled Device) showed that the grooves directed the migration of SMCs. There was no prominent difference in the migration speed of SMCs according to the grooves. All the cytoskeletal fibers were reorganized in the same direction with grooves. Especially the alignments of microtubule and intermediate filaments were distinguished in the SMCs on the micro grooves. CONCLUSION: These results could be applied to the analysis of vascular restenosis and the development of artificial blood vessels.

Expression of Heat Shock Protein 70 in Vein Endothelial Cells Induced by Shear Stress

PURPOSE: Endothelial cells (ECs) are exposed to continuous shear stress due to the blood flow. Heat shock protein (hsp) 70 is a well-known stress-response protein. The objective of this study is to investigate the expression of hsp70 in human umbilical vein endothelial cells induced by shear stress. METHOD: The pattern of hsp70 was investigated by the in vitro laminar flow system and its image was analyzed by PV-WAVE software program. The image of heat shock-induced hsp70 expression was analyzed and compared with that of shear stress-induced hsp70 expression. RESULT: Under the condition of shear stress produced by flow, the ECs change their morphology and lineup along the flow direction. As to expression of flow stress-induced hsp70, it was accumulated in perinuclear region of endothelial cells, which is different from heat shock-induced hsp70 exression. CONCLUSION: ECs alignment along the direction of the flow shear stress and changes their phenotype are due to the remodeling of the actin microfilaments. Perinuclear expression of hsp70 may play an important roles in this shear stress induced remodeling response.

The Response of Human Umbilical Vein Endothelial Cells Under Complicated Flow Conditions

PURPOSE: Endothelial cells (ECs) are exposed to continuous shear stress from their birth and also respond to the hemodynamic environmental changes which may be localizing factor in vascular diseases, such as atherosclerosis. The hemodynamic shear stress is implicated in the pathogenesis of atherosclerosis, thrombosis, and also restenosis. The objective of this study is to investigate the morphological and molecular biological changes of vein ECs under complicated flow flield which could occur in the anastomosis site of the autogenous vein bypass graft. METHOD: We developed a laminar flow chamber for the normal vessel and a sudden expansion flow chamber to simulate the recirculation or the stagnation zone of vascular graft. RESULT: Normal flow shear stress transformed ECs from random oriented polygonal, cobblestone shape to elongated shape aligned along the flow direction. However the stagnation and flow separation zone could not show the morphologic change of ECs and could be the region of low shear stress prone for intimal hyperplasia and atherosclerosis initiation. CONCLUSION: It also represents that the ECs can sense the magnitude and the direction of the flow shear stress and change their phenotype through the remodeling of the actin microfilaments.

Morphological Changes of the Endothelial Cell under the Influence of the Hemodynamic Stresses

PURPOSE: The objective of this study is to investigate the effects of hemodynamics on morphological changes of human endothelial cells. METHODS: The changes under the laminar flow condition are investigated by the in-vitro experiment and computer simulation. Micrographs of the endothelial cells in the laminar flow chamber are taken as a function of the exposed time. Idealized geometric shapes of the cells whose shapes are changing with the exposed time due to the flow stresses are portrayed by the computer simulation. Drag force on the cell due to the pressure and shear stress is calculated for two constraining conditions, that is, the cell changes its shape keeping its initial volume or initial surface area. RESULTS AND CONCLUSION: The drag force of the cell which keeps constant volume is smaller than that of the cell which keeps constant surface area.

Morphological Changes of the Endothelial Cell under the Influence of the Hemodynamic Stresses

PURPOSE: The objective of this study is to investigate the effects of hemodynamics on morphological changes of human endothelial cells. METHODS: The changes under the laminar flow condition are investigated by the in-vitro experiment and computer simulation. Micrographs of the endothelial cells in the laminar flow chamber are taken as a function of the exposed time. Idealized geometric shapes of the cells whose shapes are changing with the exposed time due to the flow stresses are portrayed by the computer simulation. Drag force on the cell due to the pressure and shear stress is calculated for two constraining conditions, that is, the cell changes its shape keeping its initial volume or initial surface area. RESULTS AND CONCLUSION: The drag force of the cell which keeps constant volume is smaller than that of the cell which keeps constant surface area.

Chromosome Analysis in Clinical Samples by Chromosome Diagnostic System Using Fluorescence in Situ Hybridization / 대한불임학회지

Fluorescence in situ hybridization (FISH) techniques allow the enumeration of chromosome abnormalities and from a great potential for many clinical applications. In order to produce quantitative and reproducible results, expensive tools such as a cooled CCD camera and a computer software are required. We have developed a Chromosome Image Processing System (Chips) using FISH that allows the detection and mapping of the genetic aberrations. The aim of our study, therefore, is to evaluate the capabilities of our original system using a black-and-white video camera. As a model system, three repetitive DNA probes (D18Zl, DXZI, and DYZ3) were hybridized to variety different clinical samples such as human metaphase spreads and interphase nuclei obtained from uncultured peripheral blood lymphocytes, uncultured amniocytes, and germ cells. The visualization of the FISH signals was performed using our system for image acquisition and pseudocoloring. FISH images were obtained by combining images from each of probes and DAPI counterstain captured separately. Using our original system, the aberrations of single or multiple chromosomes in a single hybridization experiment using chromosomes and interphase nuclei from a variety of cell types, including lymphocytes, amniocytes, sperm, and biopsied blastomeres, were enabled to evaluate. There were no differences in the image quality in accordance with FISH method, fluorochrome types, or different clinical samples. Always bright signals were detected using our system. Our system also yielded constant results. Our Chips would permit a level of performance of FISH analysis on metaphase chromosomes and interphase nuclei with unparalleled capabilities. Thus, it would be useful for clinical purposes.