ABSTRACT
BACKGROUND: Hemopoiesis and erythropoiesis have been studied mainly in immortalized cell lines and semisolid medium. But cell lines do not represent normal erythropoiesis, besides, in semisolid medium the cells are immobilized that it is difficult to do additional immunologic, biochemical, and molecular biologic experiments. In the present study we used a two-phase liquid culture system to isolate and quantify erythroid progenitors from peripheral blood and cord blood. METHODS: Peripheral and cord blood were obtained from three healthy donors and three full-term deliveries, respectively. Mononuclear cells were separated by density gradient centrifugation and were cultured in the first phase at a density of 5x106/mL in alpha- minimal essential medium ( -MEM). After 5~7 days of incubation at 37degrees C in an atmosphere of 5% CO2 with extra humidity, the nonadherent cells were harvested and recultured in the original volume of -MEM containing 10ng/mL stem cell factor and 1U/mL erythropoietin (EPO). Cellular morphology was observed by preparing cytocentrifuge slides stained with Wright Giemsa. On days 8, 10, 12, and 16 of the second phase, hemoglobin (Hb)- containing cells were counted on hemocytometer after staining with acid benzidine and glycophorin A-positive erythroid cells were scored by a flow cytometer. RESULTS: Pronormoblasts first started to appear on days 4~5 in the secondary culture. On day 10 basophilic normoblasts could be seen and on days 12~14 orthochromatic normoblasts were present. Both from peripheral and cord blood the maximum number of benzidine and glycophorin A-positive cells were achieved after 10 days and the total erythroid cell yield was approximately 1x106/mL. CONCLUSION: Using two-phase liquid culture, erythroid cell yield reached 1x106/mL both from peripheral and cord blood. In addition, this culture system permits the study of the effect of various culture conditions and components without terminating the culture, therefore it might provide us a useful experimental tool for studying pathogenesis and therapeutic modalities in genetic erythroid disorders as well as erythroid cell development.
Subject(s)
Humans , Atmosphere , Basophils , Cell Line , Centrifugation, Density Gradient , Erythroblasts , Erythroid Cells , Erythropoiesis , Erythropoietin , Fetal Blood , Glycophorins , Humidity , Stem Cell Factor , Tissue DonorsABSTRACT
BACKGROUND: Methicillin-resistant Staphylococcus aureus(MRSA) is an increasingly common cause of nosocomial infections worldwide. Epidemiologic investigation of MRSA outbreaks and identification of pathways of nosocomial MRSA spread require the ability to distinguish individual MRSA strains. We applied molecular tap ing of the methicillin-resistant determinant (mec) and coagulase typing in the investigation of a nosocomial MRSA infections. METHODS: We randomly selected 79 strains of mecA positive MRSA isolated from patients who visited Dong-A university Hospital from Dec. 1995 to Oct. 1996. Molecular typing of MRSA was performed by comparing the size of the mac-associated hypervariable region amplified by the polymerase chain reaction (PCR). Coagulase typing with type I-VIII antisera was also used for classification of MRSA based on its phenotype. Each isolates were classified by the combination of molecular analyses and coagulase type. RESULTS: The 79 MRSA isolates were grouped Into sin hypervariable legion (HVR) genotypes on the basis of the size of the PGR products. In coagulase typing, the most predominant type was II(46.8%) and type V was not found. Nine strains were not typable. The combination of HVR genotypes and coagulase types showed 23 different types in 79 MRSA Isolates. The strains which were repeatedly isolated from the same patients showed the same HYR genotypes and coagulate types. CONCLUSION: The combination of HVR genotypes and coagulase types is thought to be useful in epidemiolgical Investigation of nosocomial infections caused by MRSA ,because of its simplicity and reproducibility.