The CytoBead assay-a novel kind of multiparametric autoantibody analysis in the diagnostics of systemic autoimmune diseases [Der CytoBead-Assay-Eine neue Möglichkeit der multiparametrischen Autoantikörperanalytik bei systemischen Autoimmunerkrankungen]
Sowa M.,TU Brandenburg |
Grossmann K.,GA Generic Assays GmbH |
Scholz J.,GA Generic Assays GmbH |
Rober N.,Medizinische Fakultat der Technischen University |
And 5 more authors.
LaboratoriumsMedizin | Year: 2014
If there is a suspicion of a systemic autoimmune disease, a two-step assessment of autoantibodies (AAb) is recommended for the serological diagnosis routine. First, AAb will be determined using sensitive, cell-based indirect immunofluorescence. Then, a positive result must be confirmed with a more specific test due to the possibility of false-positive results. This gradual approach is necessary because there is currently no assay technique that fulfills the requirements for a one-stage procedure for sensitivity and specificity. For effective AAb analysis, simultaneous determination of several AAb with multiparametric confirmatory assays significantly shortens serological diagnosis, compared with conventional monoparametric testing. Yet, currently available multiparametric AAb detection techniques do not offer the combination of screening and confirmatory testing. Thus, a new approach based on digital fluorescence was developed by applying a novel CytoBead technology that is presented here. The aim was to combine the recommended stepwise approach consisting of sensitive screening and confirmation of specific diagnosis in a reaction environment and beyond the possibility of adaptation to the serological diagnosis of several autoimmune diseases. Using standard microscopic glass slides and the combination of native cellular or tissue substrates with autoantigen-loaded fluorescent microparticles (beads) in a reaction environment, along with the possibility of manual and automatic evaluation by IIF and the quantitative measurement of fluorescent signals, the disadvantages of currently existing test systems could be overcome. This novel concept is applicable for the determination of various multiparametric AAb, e.g., the determination of antinuclear antibodies and the corresponding AAb in molecular cytoplasmic and nuclear autoantigenic structures. Further, this becomes the basis for the simultaneous multiparametric AAb determination for the serology of celiac disease or ANCA-associated vasculitides. Source
Wermke M.,Universitatsklinikum Carl Gustav Carus der Technischen University |
Wermke M.,Medizinische Fakultat der Technischen University |
Camgoz A.,Medizinische Fakultat der Technischen University |
Paszkowski-Rogacz M.,Medizinische Fakultat der Technischen University |
And 11 more authors.
Blood | Year: 2015
Acute myeloid leukemia (AML) is characterized by a marked genetic heterogeneity, which complicates the development of novel therapeutics. The delineation of pathways essential within an individual patient's mutational background might overcome this limitation and facilitate personalized treatment. We report the results of a large-scale lentiviral lossof-function RNA interference (RNAi) screen in primary leukemic cells. Stringent validation identified 6 genes (BNIPL1, ROCK1, RPS13, STK3, SNX27, WDHD1) whose knockdown impaired growth and viability of the cells. Dependence on these genes was not caused by mutation or overexpression, and although some of the candidates seemed to be rather patient specific, others were essential in cells isolated from other AML patients. In addition to the phenotype observed after ROCK1 knockdown, treatment with the approved ROCK inhibitor fasudil resulted in increased apoptosis and decreased viability of primary AML cells. In contrast to observations in some other malignancies, ROCK1 inhibition did not foster growth of immature malignant progenitors but was toxic to this cell fraction in feeder coculture and xenotransplant experiments, indicating a distinct effect of ROCK1 inhibition on leukemic progenitors. We conclude that large-scale RNAi screens in primary patient-derived cells are feasible and can complement other methods for personalized cancer therapies, such as expression and mutation profiling. © 2015 by The American Society of Hematology. Source