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1.
Klin Onkol ; 25(1): 17-25, 2012.
Artículo en Checo | MEDLINE | ID: mdl-22348216

RESUMEN

BACKGROUNDS: Recently, the term biomarker has become, especially in connection with the term clinical proteomics, one of the most frequent terms in the field of biomedical research. The aim of this work was to select an appropriate pre-fractionation method of blood plasma prior to a subsequent proteomic analysis of low-abundant fraction of proteins by two dimensional gel electrophoresis (2-DE) and mass spectrometry to improve the resolution of 2-DE maps and protein identification. MATERIALS AND METHODS: First, we compared two prefractionation methods (MARS versus ProteoMiner) preceding 2-DE analysis using 10 blood plasma samples. Based on the results of the comparative experiments, low-abundant plasma protein fractions from 18 multiple myeloma patients treated with bortezomib were analyzed. Patients were divided into two groups: a group resistant to chemotherapy (9 patients--disease progression, stable disease) and a group with positive clinical response (9 patients--complete and partial remission). RESULTS AND CONCLUSION: Samples prefractioned by ProteoMiner method yielded 2-DE maps with a significantly increased number of detected protein spots, as compared to immunodepletion method MARS (Multiple Affinity Removal System). Between groups of chemoresistant and sensitive patients treated with bortezomib, 15 differently intense spots were revealed by image analysis. These spots were found to correspond to 10 proteins, as confirmed by mass spectrometry. Seven proteins had significantly lower protein level in the group of chemosensitive patients (serum amyloid P, fibrinogen--gamma chain, retinol-binding protein 4, complement factor C4-A, apolipoprotein E, carboxypeptidase N and complement factor H-related protein 1) and 3 proteins showed significantly higher levels of protein (or were only detected) in the group of chemosensitive patients (serum paraoxonase 1, alpha-1-antitrypsin and complement factor B).


Asunto(s)
Antineoplásicos/uso terapéutico , Proteínas Sanguíneas/análisis , Ácidos Borónicos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Proteómica , Pirazinas/uso terapéutico , Anciano , Biomarcadores/sangre , Bortezomib , Electroforesis en Gel Bidimensional , Femenino , Humanos , Masculino , Espectrometría de Masas , Mieloma Múltiple/sangre
2.
Klin Onkol ; 24(1): 35-40, 2011.
Artículo en Checo | MEDLINE | ID: mdl-21542274

RESUMEN

BACKGROUNDS: The aim of this paper is to present an algorithm for plasma cell separation from bone marrow samples of multiple myeloma patients. The main prerequisite for applying modern research methods in this disease is gaining pure cell populations. MATERIAL AND METHODS: Bone marrow samples were collected from outpatients or inpatients of the Internal Haematology and Oncology Clinic of the Faculty Hospital Brno, after they had signed an Informed Consent Form. The bone marrow was first depleted of red cells (by density gradient centrifugation or erythrolysis), plasma cells were labelled by monoclonal antibody against syndecan-1 (CD138) and separated either magnetically or by cell sorter. The purity of separated population was evaluated by flow cytometry or, alternatively, morfologically. RESULTS: We processed 28 bone marrow samples, in parallel, by magnetic or fluorescence-based separation, and we evaluated the purity of the separated fractions. Based on a statistical evaluation of resulting purities in the entire sample set as well as the individual groups divided according to the initial plasma cell content, a separation algorithm was proposed with a cut-off value of 5% of plasma cells in mononuclear fraction of bone marrow: samples with less than 5% of plasma cells are henceforth separated on cell sorter, samples with more than 5% are separated magnetically.The effectiveness of this algorithm was evaluated after the first year of its application on a dataset of 210 bone marrow samples: median purity of the separated plasma cells increased from 62.4% (0.4-99.6%) to 94.0% (23.9-100%). CONCLUSION: The introduction of a fluorescence-based separation markedly increased the effectiveness of plasma cell separation from bone marrow samples, mainly in samples with low plasma cell content where magnetic separation used thus far is not sufficient. This finding also opened a door for plasma cell separation of bone marrow samples from patients with monoclonal gammopathy of undetermined significance, where plasma cell count is typically below or just over one percent.


Asunto(s)
Algoritmos , Médula Ósea/patología , Separación Inmunomagnética , Mieloma Múltiple/patología , Células Plasmáticas/patología , Citometría de Flujo , Humanos
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