RESUMO
Cell and gene therapy is a fast-growing field for cancer therapeutics requiring reliable instrumentation and technologies. Key parameters essential for satisfying Chemistry Manufacturing and Controls criteria standards are routinely performed using flow cytometry. Recently, image cytometry was developed for cell characterization and cell-based assays but had not yet demonstrated sufficient sensitivity for surface marker detection. We developed the Cellaca® PLX image cytometry system and the respective methodologies required for immunophenotyping, GFP and RFP transfection/transduction efficiencies, and cell health analyses for routine cell characterization. All samples tested were compared directly to results from the CytoFLEX flow cytometer. PBMCs were stained with T-cell surface markers for immunophenotyping, and results show highly comparable CD3, CD4, and CD8 populations (within 5 %). GFP- or RFP-expressing cell lines were analyzed for transfection/transduction efficiencies, and the percentage positive cells and respective viabilities were equivalent on both systems. Staurosporine-treated Jurkat cells were stained for apoptotic markers, where annexin V and caspase-3 positive cells were within 5 % comparing both instruments. The proposed system may provide a complementary tool for performing routine cell-based experiments with improved efficiency and sensitivity compared to prior image cytometers, which may be significantly valuable to the cell and gene therapy field.
Assuntos
Apoptose , Humanos , Imunofenotipagem , Transfecção , Linhagem Celular , Células Jurkat , Citometria de Fluxo/métodosRESUMO
RESUMEN Objetivos. Determinar y correlacionar la densidad mineral ósea (DMO) y los marcadores bioquímicos del metabolismo óseo de adultos de 30 a 40 años de Lima, Perú, en el año 2018. Métodos. Estudio descriptivo, transversal, realizado a 84 mujeres y 53 varones, de 30 a 40 años de edad, residentes en Lima, Perú, sin manifestaciones ni tratamiento de enfermedades metabólicas óseas primarias ni secundarias. Se registró información del estado actual, antecedentes personales fisiológicos, patológicos, familiares y examen clínico. Se midió la DMO por absorciometría dual de rayos X en columna, cadera y antebrazo; en sangre, parathormona, osteocalcina, 25-hidroxi-vitamina-D, hormona luteinizante, estradiol en las mujeres, testosterona en los varones, calcio sérico, fósforo sérico, fosfatasa alcalina, proteínas totales y fracciones, creatinina; en orina N-telopéptido de enlaces de colágeno tipo I, calcio y fósforo, por métodos convencionales. Resultados. La DMO se efectuó a 62 mujeres y 40 varones; 2 mujeres y 1 varón tuvieron osteoporosis, y 3 mujeres y 2 varones osteopenia, retirados para la evaluación estadística; 58 mujeres y 35 varones tuvieron una DMO normal. Los varones tuvieron mayor DMO y concentraciones mayores de calcio, fosfatasa alcalina y creatinina; 14 mujeres y 2 varones tuvieron cifras bajas de 25-OH-vitaminaD. Hubo correlación positiva entre DMO y el IMC. Conclusiones. La DMO normal de personas de 30 a 40 años de Lima fue lineal y hubo una relación positiva con el IMC; hubo osteoporosis en 2 mujeres y 1 varón, osteopenia en 3 mujeres y 2 varones, cifras bajas de 25-hidroxi-vitamina-D en 16 personas.
ABSTRACT Objectives. To determine and correlate the bone mineral density (BMD) and the biochemical markers of bone metabolism in adults 30 to 40 years old from Lima, Perú, in year 2018. Methods. A total of 84 women and 53 men, 30 to 40 years old from Lima, Perú, were submmited to a descriptive, transversal study. They did not have symptoms nor treatment for bone metabolic diseases. Information about their present health state, personal and familiar physiological and pathological history and physical examinatión were registered. BMD was measured by dual energy-X-ray absorptiometry in spine, hip and forearm. Parathormone, osteocalcine, 25-hidroxi-vitamine-D, luteinizing hormone, estradiol in women, testosterone in men, calcium, phosphorus, alkaline fosfatase, total and fractionated proteins, creatinine were measured in blood; in urine N-telopéptide, calcium and phosphorus by conventional methods. Results. BMD was measured in 62 women and 40 males; 2 women and 1 man had osteoporosis and 3 women and 2 men osteopenia, who were taken out of statistical evaluation; 58 women and 35 men showed a normal BMD. Male subjects had a greater BMD, higher calcium, alkaline fosfatase and creatine blood concentrations than the female ones; 14 women y 2 men had low 25-OH-vitaminaD concentrations. A positive correlation between the BMD and BMI was found. Conclusions. The normal BMD of persons 30 to 40 years old showed a linear figure and there was a positive correlate with the BMI; premature osteoporosis in 2 women and 1 men as well as osteopenia in 3 women y 2 men was found, low concentrations of 25-hidroxi-vitamine-D was detected in 16 persons.