J Flores-Montero

Publications (5)28.03 Total impact

• Source

  Available from: Tomas Kalina

  Dataset: leu2012122a

  T Kalina, J Flores-Montero, V H J van der Velden, M Martin-Ayuso, S Böttcher, M Ritgen, J Almeida, L Lhermitte, V Asnafi, A Mendonça, [......], M Cullen, L Sedek, M B Vidriales, J J Pérez, J G Te Marvelde, E Mejstrikova, O Hrusak, T Szczepański, J J M van Dongen, A Orfao
• Source

  Available from: Tomas Kalina

  Article: EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols.

  T Kalina, J Flores-Montero, V H J van der Velden, M Martin-Ayuso, S Böttcher, M Ritgen, J Almeida, L Lhermitte, V Asnafi, A Mendonça, [......], M Cullen, L Sedek, M B Vidriales, J J Pérez, J G Te Marvelde, E Mejstrikova, O Hrusak, T Szczepański, J J M van Dongen, A Orfao
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  ABSTRACT: The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database.
  Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 09/2012; 26(9):1986-2010. · 8.30 Impact Factor
• Article: Flow cytometric immunobead assay for fast and easy detection of PML-RARA fusion proteins for the diagnosis of acute promyelocytic leukemia.

  E H A Dekking, V H J van der Velden, R Varro, H Wai, S Böttcher, M Kneba, E Sonneveld, A Koning, N Boeckx, N Van Poecke, [......], A Orfao, J Almeida, P Evans, M Cullen, A L Noordijk, P M Vermeulen, M T de Man, E P Dixon, W M Comans-Bitter, J J M van Dongen
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  ABSTRACT: The PML-RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML-RARA fusion protein arrests maturation of myeloid cells at the promyelocytic stage, leading to the accumulation of neoplastic promyelocytes. Complete remission can be obtained by treatment with all-trans-retinoic acid (ATRA) in combination with chemotherapy. Diagnosis of APL is based on the detection of t(15;17) by karyotyping, fluorescence in situ hybridization or PCR. These techniques are laborious and demand specialized laboratories. We developed a fast (performed within 4-5 h) and sensitive (detection of at least 10% malignant cells in normal background) flow cytometric immunobead assay for the detection of PML-RARA fusion proteins in cell lysates using a bead-bound anti-RARA capture antibody and a phycoerythrin-conjugated anti-PML detection antibody. Testing of 163 newly diagnosed patients (including 46 APL cases) with the PML-RARA immunobead assay showed full concordance with the PML-RARA PCR results. As the applied antibodies recognize outer domains of the fusion protein, the assay appeared to work independently of the PML gene break point region. Importantly, the assay can be used in parallel with routine immunophenotyping for fast and easy diagnosis of APL.
  Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 09/2012; 26(9):1976-85. · 8.30 Impact Factor
• Source

  Available from: Anton W Langerak

  Article: EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes.

  J J M van Dongen, L Lhermitte, S Böttcher, J Almeida, V H J van der Velden, J Flores-Montero, A Rawstron, V Asnafi, Q Lécrevisse, P Lucio, [......], M Brüggemann, L Sedek, M Cullen, A W Langerak, A Mendonça, E Macintyre, M Martin-Ayuso, O Hrusak, M B Vidriales, A Orfao
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  ABSTRACT: Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2-7 sequential design-evaluation-redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies.
  Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 05/2012; 26(9):1908-75. · 8.30 Impact Factor
• Source

  Available from: Paulo Jorge Lucio

  Article: Detection of fusion genes at the protein level in leukemia patients via the flow cytometric immunobead assay.

  E Dekking, V H J van der Velden, S Böttcher, M Brüggemann, E Sonneveld, A Koning-Goedheer, N Boeckx, P Lucio, L Sedek, T Szczepański, T Kalina, M Kovac, P Evans, P G Hoogeveen, J Flores-Montero, A Orfao, W M Comans-Bitter, F J T Staal, J J M van Dongen
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  ABSTRACT: Nowadays, the presence of specific genetic aberrations is progressively used for classification and treatment stratification, because acute leukemias with the same oncogenetic aberration generally form a clinically and diagnostically homogenous disease entity with comparable prognosis. Many oncogenetic aberrations in acute leukemias result in a fusion gene, which is transcribed into fusion transcripts and translated into fusion proteins, which are assumed to play a critical role in the oncogenetic process. Fusion gene aberrations are detected by karyotyping, FISH, or RT-PCR analysis. However, these molecular genetic techniques are laborious and time consuming, which is in contrast to flow cytometric techniques. Therefore we developed a flow cytometric immunobead assay for detection of fusion proteins in lysates of leukemia cell samples by use of a bead-bound catching antibody against one side of the fusion protein and fluorochrome-conjugated detection antibody. So far, we have been able to design such fusion protein immunobead assays for BCR-ABL, PML-RARA, TEL-AML1, E2A-PBX1, MLL-AF4, AML1-ETO and CBFB-MYH11. The immunobead assay for detection of fusion proteins can be performed within 3 to 4 hours in a routine diagnostic setting, without the need of special equipment other than a flow cytometer. The novel immunobead assay will enable fast and easy classification of acute leukemia patients that express fusion proteins. Such patients can be included at an early stage in the right treatment protocols, much faster than by use of current molecular techniques. The immunobead assay can be run in parallel to routine immunophenotyping and is particularly attractive for clinical settings without direct access to molecular diagnostics.
  Best practice & research. Clinical haematology 09/2010; 23(3):333-45. · 3.13 Impact Factor