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2. Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study

Author

Lhermitte, Ludovic, Barreau, Sylvain, Morf, Daniela, Fernandez, Paula, Grigore, Georgiana, Barrena, Susana, de Bie, Maaike, Flores-Montero, Juan, Brüggemann, Monika, Mejstrikova, Ester, Nierkens, Stefan, Burgos, Leire, Caetano, Joana, Gaipa, Giuseppe, Buracchi, Chiara, da Costa, Elaine Sobral, Sedek, Lukasz, Szczepański, Tomasz, Aanei, Carmen-Mariana, van der Sluijs-Gelling, Alita, Delgado, Alejandro Hernández, Fluxa, Rafael, Lecrevisse, Quentin, Pedreira, Carlos E., van Dongen, Jacques J.M., Orfao, Alberto, van der Velden, Vincent H.J., van Dongen, J. J.M., Bitter, W.M., Lubbers, B.R., Teodosio, C.I., Zlei, M., van der Sluijs-Gelling, A.J., de Bie, F., de Bruin-Versteeg, S., van der Burg, M., Schilham, M.W., van der Velden, V. H.J., Langerak, A.W., te Marvelde, J., Bras, A.E., Schilperoord-Vermeulen, J., Jugooa, R., Heezen, K.C., Orfao, A., Almeida, J., Vidriales, M.B., Flores-Montero, J., Pérez-Andrés, M., Matarraz, S., Martín, L., Lecrevisse, Q., Pérez-Morán, J.J., Puig, N., Almeida, A. Medina, Gomes da Silva, M., Faria, T., Brüggemann, M., Ritgen, M., Szczepanowski, M., Kohlscheen, S., Laqua, A., Harbst, E., Finke, J., Asnafi, V., Lhermitte, L., Duroyon, E., Trka, J., Hrusak, O., Kalina, T., Mejstrikova, E., Novakova, M., Thurner, D., Kanderova, V., Szczepanski, T., Sędek, L., Bulsa, J., Slota, L., Kulis, J., Pedreira, C.E., da Costa, E. Sobral, Nierkens, S., de Jong, A., de Koning, A., Lima, M., Santos, A.H., Böttcher, S., Lange, S., Engelmann, R., Paape, D., Machka, C., Gaipa, G., Burracchi, C., Bugarin, C., Lopez-Granados, E., del Pino Molina, L., Campos-Guyotat, L., Aanei, C., Miguel, J. F. San, Paiva, B., Burgos, L., Villamor-Casas, N., Magnano, L., Philippé, J., Bonroy, C., Denys, B., Willems, A., Breughe, P., de Wolf, J., Sousa, A.E., Silva, S.L., Fernandez, P., and Morf, D.

Published
2021
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6. Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies — a EuroFlow study

Author

Verbeek, M, Buracchi, C, Laqua, A, Nierkens, S, Sedek, L, Flores-Montero, J, Hofmans, M, Sobral de Costa, E, Novakova, M, Mejstrikova, E, Barrena, S, Kohlscheen, S, Szczepanowski, M, Kulis, J, Oliveira, E, Jugooa, R, de Jong, A, Szczepanski, T, Philippe, J, van Dongen, J, Orfao, A, Bruggemann, M, Gaipa, G, van der Velden, V, Verbeek M. W. C., Buracchi C., Laqua A., Nierkens S., Sedek L., Flores-Montero J., Hofmans M., Sobral de Costa E., Novakova M., Mejstrikova E., Barrena S., Kohlscheen S., Szczepanowski M., Kulis J., Oliveira E., Jugooa R., de Jong A. X., Szczepanski T., Philippe J., van Dongen J. J. M., Orfao A., Bruggemann M., Gaipa G., van der Velden V. H. J., Verbeek, M, Buracchi, C, Laqua, A, Nierkens, S, Sedek, L, Flores-Montero, J, Hofmans, M, Sobral de Costa, E, Novakova, M, Mejstrikova, E, Barrena, S, Kohlscheen, S, Szczepanowski, M, Kulis, J, Oliveira, E, Jugooa, R, de Jong, A, Szczepanski, T, Philippe, J, van Dongen, J, Orfao, A, Bruggemann, M, Gaipa, G, van der Velden, V, Verbeek M. W. C., Buracchi C., Laqua A., Nierkens S., Sedek L., Flores-Montero J., Hofmans M., Sobral de Costa E., Novakova M., Mejstrikova E., Barrena S., Kohlscheen S., Szczepanowski M., Kulis J., Oliveira E., Jugooa R., de Jong A. X., Szczepanski T., Philippe J., van Dongen J. J. M., Orfao A., Bruggemann M., Gaipa G., and van der Velden V. H. J.

Abstract

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.

Published
2022

7. Phenotypic profiling of CD34+ cells by advanced flow cytometry improves diagnosis of Juvenile Myelomonocytic Leukemia

Author

Bugarin, C, Antolini, L, Buracchi, C, Matarraz, S, Coliva, T, Van der Velden, V, Szczepanski, T, Da Costa, E, Van der Sluijs, A, Novakova, M, Mejstrikova, E, Nierkens, S, De Mello, F, Fernandez, P, Aanei, C, Sędek, Ł, Strocchio, L, Masetti, R, Sainati, L, Philippé, J, Valsecchi, M, Locatelli, F, Van Dongen, J, Biondi, A, Orfao, A, Gaipa, G, Bugarin, Cristina, Antolini, Laura, Buracchi, Chiara, Matarraz, Sergio, Coliva, Tiziana Angela, Van der Velden, Vincent H, Szczepanski, Tomasz, Da Costa, Elaine Sobral, Van der Sluijs, Alita, Novakova, Michaela, Mejstrikova, Ester, Nierkens, Stefan, De Mello, Fabiana Vieira, Fernandez, Paula, Aanei, Carmen, Sędek, Łukasz, Strocchio, Luisa, Masetti, Riccardo, Sainati, Laura, Philippé, Jan, Valsecchi, Maria Grazia, Locatelli, Franco, Van Dongen, Jacques J M, Biondi, Andrea, Orfao, Alberto, Gaipa, Giuseppe, Bugarin, C, Antolini, L, Buracchi, C, Matarraz, S, Coliva, T, Van der Velden, V, Szczepanski, T, Da Costa, E, Van der Sluijs, A, Novakova, M, Mejstrikova, E, Nierkens, S, De Mello, F, Fernandez, P, Aanei, C, Sędek, Ł, Strocchio, L, Masetti, R, Sainati, L, Philippé, J, Valsecchi, M, Locatelli, F, Van Dongen, J, Biondi, A, Orfao, A, Gaipa, G, Bugarin, Cristina, Antolini, Laura, Buracchi, Chiara, Matarraz, Sergio, Coliva, Tiziana Angela, Van der Velden, Vincent H, Szczepanski, Tomasz, Da Costa, Elaine Sobral, Van der Sluijs, Alita, Novakova, Michaela, Mejstrikova, Ester, Nierkens, Stefan, De Mello, Fabiana Vieira, Fernandez, Paula, Aanei, Carmen, Sędek, Łukasz, Strocchio, Luisa, Masetti, Riccardo, Sainati, Laura, Philippé, Jan, Valsecchi, Maria Grazia, Locatelli, Franco, Van Dongen, Jacques J M, Biondi, Andrea, Orfao, Alberto, and Gaipa, Giuseppe

Abstract

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well-established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs vs controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML vs patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single 8-color antibody combination.

Published
2023

10. Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study

Author

Lhermitte, L, Barreau, S, Morf, D, Fernandez, P, Grigore, G, Barrena, S, de Bie, M, Flores-Montero, J, Bruggemann, M, Mejstrikova, E, Nierkens, S, Burgos, L, Caetano, J, Gaipa, G, Buracchi, C, da Costa, E, Sedek, L, Szczepanski, T, Aanei, C, van der Sluijs-Gelling, A, Delgado, A, Fluxa, R, Lecrevisse, Q, Pedreira, C, van Dongen, J, Orfao, A, van der Velden, V, Bitter, W, Lubbers, B, Teodosio, C, Zlei, M, de Bie, F, de Bruin-Versteeg, S, van der Burg, M, Schilham, M, Langerak, A, te Marvelde, J, Bras, A, Schilperoord-Vermeulen, J, Jugooa, R, Heezen, K, Almeida, J, Vidriales, M, Perez-Andres, M, Matarraz, S, Martin, L, Perez-Moran, J, Puig, N, Almeida, A, Gomes da Silva, M, Faria, T, Ritgen, M, Szczepanowski, M, Kohlscheen, S, Laqua, A, Harbst, E, Finke, J, Asnafi, V, Duroyon, E, Trka, J, Hrusak, O, Kalina, T, Novakova, M, Thurner, D, Kanderova, V, Bulsa, J, Slota, L, Kulis, J, de Jong, A, de Koning, A, Lima, M, Santos, A, Bottcher, S, Lange, S, Engelmann, R, Paape, D, Machka, C, Burracchi, C, Bugarin, C, Lopez-Granados, E, del Pino Molina, L, Campos-Guyotat, L, Miguel, J, Paiva, B, Villamor-Casas, N, Magnano, L, Philippe, J, Bonroy, C, Denys, B, Willems, A, Breughe, P, de Wolf, J, Sousa, A, Silva, S, Lhermitte L., Barreau S., Morf D., Fernandez P., Grigore G., Barrena S., de Bie M., Flores-Montero J., Bruggemann M., Mejstrikova E., Nierkens S., Burgos L., Caetano J., Gaipa G., Buracchi C., da Costa E. S., Sedek L., Szczepanski T., Aanei C. -M., van der Sluijs-Gelling A., Delgado A. H., Fluxa R., Lecrevisse Q., Pedreira C. E., van Dongen J. J. M., Orfao A., van der Velden V. H. J., Bitter W. M., Lubbers B. R., Teodosio C. I., Zlei M., van der Sluijs-Gelling A. J., de Bie F., de Bruin-Versteeg S., van der Burg M., Schilham M. W., Langerak A. W., te Marvelde J., Bras A. E., Schilperoord-Vermeulen J., Jugooa R., Heezen K. C., Almeida J., Vidriales M. B., Perez-Andres M., Matarraz S., Martin L., Perez-Moran J. J., Puig N., Almeida A. M., Gomes da Silva M., Faria T., Ritgen M., Szczepanowski M., Kohlscheen S., Laqua A., Harbst E., Finke J., Asnafi V., Duroyon E., Trka J., Hrusak O., Kalina T., Novakova M., Thurner D., Kanderova V., Bulsa J., Slota L., Kulis J., de Jong A., de Koning A., Lima M., Santos A. H., Bottcher S., Lange S., Engelmann R., Paape D., Machka C., Burracchi C., Bugarin C., Lopez-Granados E., del Pino Molina L., Campos-Guyotat L., Aanei C., Miguel J. F. S., Paiva B., Villamor-Casas N., Magnano L., Philippe J., Bonroy C., Denys B., Willems A., Breughe P., de Wolf J., Sousa A. E., Silva S. L., Lhermitte, L, Barreau, S, Morf, D, Fernandez, P, Grigore, G, Barrena, S, de Bie, M, Flores-Montero, J, Bruggemann, M, Mejstrikova, E, Nierkens, S, Burgos, L, Caetano, J, Gaipa, G, Buracchi, C, da Costa, E, Sedek, L, Szczepanski, T, Aanei, C, van der Sluijs-Gelling, A, Delgado, A, Fluxa, R, Lecrevisse, Q, Pedreira, C, van Dongen, J, Orfao, A, van der Velden, V, Bitter, W, Lubbers, B, Teodosio, C, Zlei, M, de Bie, F, de Bruin-Versteeg, S, van der Burg, M, Schilham, M, Langerak, A, te Marvelde, J, Bras, A, Schilperoord-Vermeulen, J, Jugooa, R, Heezen, K, Almeida, J, Vidriales, M, Perez-Andres, M, Matarraz, S, Martin, L, Perez-Moran, J, Puig, N, Almeida, A, Gomes da Silva, M, Faria, T, Ritgen, M, Szczepanowski, M, Kohlscheen, S, Laqua, A, Harbst, E, Finke, J, Asnafi, V, Duroyon, E, Trka, J, Hrusak, O, Kalina, T, Novakova, M, Thurner, D, Kanderova, V, Bulsa, J, Slota, L, Kulis, J, de Jong, A, de Koning, A, Lima, M, Santos, A, Bottcher, S, Lange, S, Engelmann, R, Paape, D, Machka, C, Burracchi, C, Bugarin, C, Lopez-Granados, E, del Pino Molina, L, Campos-Guyotat, L, Miguel, J, Paiva, B, Villamor-Casas, N, Magnano, L, Philippe, J, Bonroy, C, Denys, B, Willems, A, Breughe, P, de Wolf, J, Sousa, A, Silva, S, Lhermitte L., Barreau S., Morf D., Fernandez P., Grigore G., Barrena S., de Bie M., Flores-Montero J., Bruggemann M., Mejstrikova E., Nierkens S., Burgos L., Caetano J., Gaipa G., Buracchi C., da Costa E. S., Sedek L., Szczepanski T., Aanei C. -M., van der Sluijs-Gelling A., Delgado A. H., Fluxa R., Lecrevisse Q., Pedreira C. E., van Dongen J. J. M., Orfao A., van der Velden V. H. J., Bitter W. M., Lubbers B. R., Teodosio C. I., Zlei M., van der Sluijs-Gelling A. J., de Bie F., de Bruin-Versteeg S., van der Burg M., Schilham M. W., Langerak A. W., te Marvelde J., Bras A. E., Schilperoord-Vermeulen J., Jugooa R., Heezen K. C., Almeida J., Vidriales M. B., Perez-Andres M., Matarraz S., Martin L., Perez-Moran J. J., Puig N., Almeida A. M., Gomes da Silva M., Faria T., Ritgen M., Szczepanowski M., Kohlscheen S., Laqua A., Harbst E., Finke J., Asnafi V., Duroyon E., Trka J., Hrusak O., Kalina T., Novakova M., Thurner D., Kanderova V., Bulsa J., Slota L., Kulis J., de Jong A., de Koning A., Lima M., Santos A. H., Bottcher S., Lange S., Engelmann R., Paape D., Machka C., Burracchi C., Bugarin C., Lopez-Granados E., del Pino Molina L., Campos-Guyotat L., Aanei C., Miguel J. F. S., Paiva B., Villamor-Casas N., Magnano L., Philippe J., Bonroy C., Denys B., Willems A., Breughe P., de Wolf J., Sousa A. E., and Silva S. L.

Abstract

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r2 > 0.95 for all cell types in PB and r2 > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a “sample-in and result-out” approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal p

Published
2021

20. Blood monitoring of circulating tumor plasma cells by next generation flow in multiple myeloma after therapy

Author

Sanoja-Flores, L., Flores-Montero, J., Puig, N., Contreras-Sanfeliciano, T., Pontes, R., Corral-Mateos, A., Garcia-Sanchez, O., Diez-Campelo, M., Magalhaes, R.J.P. de, Garcia-Martin, L., Alonso-Alonso, J.M., Garcia-Mateo, A., Aguilar-Franco, C., Labrador, J., Barez-Garcia, A., Maiolino, A., Paiva, B., San Miguel, J., Costa, E.S. da, Gonzalez, M., Mateos, M.V., Durie, B., Dongen, J.J.M. van, Orfao, A., Bitter, W.M., Lubbers, B.R., Meij, A.S.M. van der, Teodosio, C.I., Zlei, M., Sluijs-Gelling, A.J. van der, Burg, M. van der, Velden, V.H.J. van der, Langerak, A.W., Marvelde, J. te, Schilperoord-Vermeulen, J., Blijkerk, A., Heezen, K.C., Almeida, J., Vidriales, M.B., Perez-Andres, J.F.M.M., Matarraz, S., Blanco, E., Martin, L., Lecrevisse, Q., Perez-Moran, J.J., Almeida, A.M., Silva, M.G. da, Faria, T., Bruggemann, M., Ritgen, M., Szczepanowski, M., Kohlscheen, S., Steinert, A., Harbst, E., Finke, J., Asnafi, V., Lhermitte, L., Duroyon, E., Trka, J., Hrusak, O., Kalina, T., Mejstrikova, E., Novakova, M., Thurner, D., Kanderova, V., Szczepanski, T., Sedek, L., Bulsa, J., Slota, L., Kulis, J., Pedreira, C.E., Nierkens, S., Jong, A. de, Koning, A. de, Lima, M., Santos, A.H., Bottcher, S., Lange, S., Engelmann, R., Paape, D., Machka, C., Gaipa, G., Burracchi, C., Bugarin, C., Lopez-Granados, E., Molina, L.D., Vlkova, M., Nechvatalova, J., Roussel, M., Campos-Guyotat, L., Aanei, C., Burgos, L., Villamor-Casas, N., Magnano, L., Philippe, J., Bonroy, C., Denys, B., Willems, A., Breughe, P., Wolf, J. de, Sousa, A.E., Silva, S.L., Fernandez, P., Morf, D., EuroFlow Consortium, and Immunology

Subjects
Pathology, medicine.medical_specialty, medicine.diagnostic_test, business.industry, Immunology, Plasma Cells, Cell Biology, Hematology, medicine.disease, Flow Cytometry, Neoplastic Cells, Circulating, Prognosis, Biochemistry, Flow cytometry, Immunophenotyping, Text mining, Flow (mathematics), medicine, Humans, business, Multiple Myeloma, Letter to Blood, Multiple myeloma
Published
2019

23. Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia

Author

Lhermitte, L., Mejstrikova, E., Sluijs-Gelling, A.J. van der, Grigore, G.E., Sedek, L., Bras, A.E., Gaipa, G., Costa, E.S. da, Novakova, M., Sonneveld, E., Buracchi, C., Bacelar, T.D., Marvelde, J.G.T., Trinquand, A., Asnafi, V., Szczepanski, T., Matarraz, S., Lopez, A., Vidriales, B., Bulsa, J., Hrusak, O., Kalina, T., Lecrevisse, Q., Ayuso, M.M., Bruggemann, M., Verde, J., Fernandez, P., Burgos, L., Paiva, B., Pedreira, C.E., Dongen, J.J.M. van, Orfao, A., Velden, V.H.J. van der, EuroFlow Consortium, Instituto de Salud Carlos III, Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brasil), Ministerio de Sanidad y Consumo (España), Polish Academy of Sciences, Fundações de Amparo à Pesquisa (Brasil), Ministerio de Economía y Competitividad (España), Immunology, Lhermitte, L, Mejstrikova, E, Van Der Sluijs-Gelling, A, Grigore, G, Sedek, L, Bras, A, Gaipa, G, Sobral Da Costa, E, Novakova, M, Sonneveld, E, Buracchi, C, De Sa Bacelar, T, Te Marvelde, J, Trinquand, A, Asnafi, V, Szczepanski, T, Matarraz, S, Lopez, A, Vidriales, B, Bulsa, J, Hrusak, O, Kalina, T, Lecrevisse, Q, Martin Ayuso, M, Bruggemann, M, Verde, J, Fernandez, P, Burgos, L, Paiva, B, Pedreira, C, Van Dongen, J, Orfao, A, and Van Der Velden, V

Subjects
Adult, Male, 0301 basic medicine, Cancer Research, Myeloid, Adolescent, T-cell acute lymphoblastic leukemia (T-ALL), Immunophenotyping, Young Adult, 03 medical and health sciences, Text mining, Acute leukemia (AL), EuroFlow, medicine, Humans, AL orientation tube (ALOT), Child, Aged, Aged, 80 and over, Acute leukemia, Leukemia, business.industry, Orientation (computer vision), Infant, Newborn, Infant, Myeloid leukemia, Pattern recognition, Hematology, Middle Aged, Precursor Cell Lymphoblastic Leukemia-Lymphoma, medicine.disease, Leukemia, Myeloid, Acute, Statistical classification, 030104 developmental biology, medicine.anatomical_structure, Oncology, Child, Preschool, Acute Disease, B-cell precursor (BCP), Female, Original Article, Artificial intelligence, business
Abstract

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications., The research was performed within the EuroFlow Consortium, which started with an EU-FP6 grant (LSHB-CT-2006-018708) and obtained sustainability by protecting and licensing intellectual property, thereby obtaining royalties, which are exclusively being used for supporting the EuroFlow research program (chairmen: JJMvD and AO). LS, JB and TS were supported by STRATEGMED3/304586/5/NCBR/2017 PersonALL grant of the Polish National Center for Research and Development. ESC acknowledges FAPERJ, Rio de Janeiro, Brazil (E26/110.105/2014; E26/102.191/2013) and Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPQ of Brazil (400194/2014-7). AO, SM and AL acknowledge the Instituto de Salud Carlos III (MINECO, Madrid, Spain) for the DTS15/00119, and CIBERONC-FEDER-CB16/12/00400 grants. EM, OH, TK and MN were supported by Ministry of Health grant number 15-28525A and NPU LO1604. The research for this manuscript was in part performed within the framework of the Erasmus Postgraduate School Molecular Medicine.

Published
2018

24. Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia

Author

Theunissen, P, Mejstrikova, E, Sedek, L, Van Der Sluijs-Gelling, A, Gaipa, G, Bartels, M, Sobral da Costa, E, Kotrova, M, Novakova, M, Sonneveld, E, Buracchi, C, Bonaccorso, P, Oliveira, E, Te Marvelde, J, Szczepanski, T, Lhermitte, L, Hrusak, O, Lecrevisse, Q, Grigore, G, Fronkova, E, Trka, J, Bruggemann, M, Orfao, A, Van Dongen, J, Van Der Velden, V, Theunissen P., Mejstrikova E., Sedek L., Van Der Sluijs-Gelling A. J., Gaipa G., Bartels M., Sobral da Costa E., Kotrova M., Novakova M., Sonneveld E., Buracchi C., Bonaccorso P., Oliveira E., Te Marvelde J. G., Szczepanski T., Lhermitte L., Hrusak O., Lecrevisse Q., Grigore G. E., Fronkova E., Trka J., Bruggemann M., Orfao A., Van Dongen J. J. M., Van Der Velden V. H. J., Theunissen, P, Mejstrikova, E, Sedek, L, Van Der Sluijs-Gelling, A, Gaipa, G, Bartels, M, Sobral da Costa, E, Kotrova, M, Novakova, M, Sonneveld, E, Buracchi, C, Bonaccorso, P, Oliveira, E, Te Marvelde, J, Szczepanski, T, Lhermitte, L, Hrusak, O, Lecrevisse, Q, Grigore, G, Fronkova, E, Trka, J, Bruggemann, M, Orfao, A, Van Dongen, J, Van Der Velden, V, Theunissen P., Mejstrikova E., Sedek L., Van Der Sluijs-Gelling A. J., Gaipa G., Bartels M., Sobral da Costa E., Kotrova M., Novakova M., Sonneveld E., Buracchi C., Bonaccorso P., Oliveira E., Te Marvelde J. G., Szczepanski T., Lhermitte L., Hrusak O., Lecrevisse Q., Grigore G. E., Fronkova E., Trka J., Bruggemann M., Orfao A., Van Dongen J. J. M., and Van Der Velden V. H. J.

Abstract

A fully-standardized EuroFlow 8–color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of £1025, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)–based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR–based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (£1025), if sufficient cells (>4 3 106, preferably more) are evaluated.

Published
2017

26. Differential expression of CD73, CD86 and CD304 in normal vs. leukemic B-cell precursors and their utility as stable minimal residual disease markers in childhood B-cell precursor acute lymphoblastic leukemia

Author

Sedek, L, Theunissen, P, Sobral da Costa, E, van der Sluijs-Gelling, A, Mejstrikova, E, Gaipa, G, Sonsala, A, Twardoch, M, Oliveira, E, Novakova, M, Buracchi, C, van Dongen, J, Orfao, A, van der Velden, V, Szczepanski, T, van Dongen, JJM, van der Velden, VHJ, Sedek, L, Theunissen, P, Sobral da Costa, E, van der Sluijs-Gelling, A, Mejstrikova, E, Gaipa, G, Sonsala, A, Twardoch, M, Oliveira, E, Novakova, M, Buracchi, C, van Dongen, J, Orfao, A, van der Velden, V, Szczepanski, T, van Dongen, JJM, and van der Velden, VHJ

Abstract

Background: Optimal discrimination between leukemic blasts and normal B-cell precursors (BCP) is critical for treatment monitoring in BCP acute lymphoblastic leukemia (ALL); thus identification of markers differentially expressed on normal BCP and leukemic blasts is required. Methods: Multicenter analysis of CD73, CD86 and CD304 expression levels was performed in 282 pediatric BCP-ALL patients vs. normal bone marrow BCP, using normalized median fluorescence intensity (nMFI) values. Results: CD73 was expressed at abnormally higher levels (vs. pooled normal BCP) at diagnosis in 71/108 BCP-ALL patients (66%), whereas CD304 and CD86 in 119/202 (59%) and 58/100 (58%) patients, respectively. Expression of CD304 was detected at similar percentages in common-ALL and pre-B-ALL, while found at significantly lower frequencies in pro-B-ALL. A significant association (p = 0.009) was found between CD304 expression and the presence of the ETV6-RUNX1 fusion gene. In contrast, CD304 showed an inverse association with MLL gene rearrangements (p = 0.01). The expression levels of CD73, CD86 and CD304 at day 15 after starting therapy (MRD15) were stable or higher than at diagnosis in 35/37 (95%), 40/56 (71%) and 19/41 (46%) cases investigated, respectively. This was also associated with an increased mean nMFI at MRD15 vs. diagnosis of +24 and +3 nMFI units for CD73 and CD86, respectively. In addition, gain of expression of CD73 and CD86 at MRD15 for cases that were originally negative for these markers at diagnosis was observed in 16% and 18% of cases, respectively. Of note, CD304 remained aberrantly positive in 63% of patients, despite its levels of expression decreased at follow-up in 54% of cases. Conclusions: Here we show that CD73, CD86 and CD304 are aberrantly (over)expressed in a substantial percentage of BCP-ALL patients and that their expression profile remains relatively stable early after starting therapy, supporting their potential contribution to improved MRD analysis by fl

Published
2019

27. Strategic Environmental Assessment for Nuclear Power Programmes: Guidelines

Author

Athar, G.R., Aulavuo, T., Bond, A., Bonilla, M., Chrobak, G., Croal, P., Tomšić, Željko, Fischer, T.B., Haladyj, A., Jalal, A.I., Kallenbach-Herbert, B., Kremlis, G., Magné, B., Monken- Fernandes, H., Montaño, M., Mwaura, F., Novakova, M., Parvez, A., Phaneuf, M., Platzer- Schneider, U., Smutny, M., Sternath, F., Subki, H., Szkudlarek, Ł., Van Heek, A., and Welsch, M.

Subjects
ComputerApplications_COMPUTERSINOTHERSYSTEMS, Nuclear Power Programmes, Strategic Environmental Assessment, decision-making, stakeholder engagement, public participation
Abstract

This publication provides practical guidance for performing strategic environmental assessments (SEAs) for nuclear power programmes. It incorporates the latest knowledge and draws on best practices in conducting SEAs. Based on inputs from SEA experts from across the world, it lays down an effective SEA process that contributes to: strengthening decision making for nuclear power programmes ; achieving environmentally sound and sustainable development ; and improving good governance and building public trust and confidence in decision-making. Importantly, SEA for nuclear power programmes can ensure effective communication with the public and other stakeholders. Consequently, significant emphasis is placed on stakeholder engagement and public participation. Further, appropriate tools for assessment and quality review are presented for all stages of the SEA process.

Published
2018

29. PF237 THE PROGNOSTIC IMPACT OF LYMPHOCYTE REPERTOIRE RECOVERY AFTER INDUCTION TREATMENT OF CHILDHOOD AML

Author

Svaton, M., primary, Novakova, M., additional, Reznickova, L., additional, Darzentas, N., additional, Stuchly, J., additional, Svenkrtova, A., additional, Zaliova, M., additional, Zuna, J., additional, Mejstrikova, E., additional, Kotrova, M., additional, Reigl, T., additional, Hrusak, O., additional, Stary, J., additional, Trka, J., additional, and Fronkova, E., additional

Published
2019
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30. PF176 MONOCYTIC SWITCH AND DISCREPANCY BETWEEN FLOW CYTOMETRIC AND MOLECULAR MINIMAL RESIDUAL DISEASE ARE FREQUENT IN DUX4 REARRANGED AND PAX5-P80R MUTATED B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

Author

Novakova, M., primary, Vakrmanova, B., additional, Slamova, L., additional, Musilova, A., additional, Bruggemann, M., additional, Ritgen, M., additional, Kolenovska, A., additional, Svec, P., additional, Fronkova, E., additional, Kalina, T., additional, Trka, J., additional, Stary, J., additional, Vaskova, M., additional, Winkowska, L., additional, Zaliova, M., additional, Fiser, K., additional, Hrusak, O., additional, and Mejstrikova, E., additional

Published
2019
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32. Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia

Author

Lhermitte, L, Mejstrikova, E, Van Der Sluijs-Gelling, A, Grigore, G, Sedek, L, Bras, A, Gaipa, G, Sobral Da Costa, E, Novakova, M, Sonneveld, E, Buracchi, C, De Sa Bacelar, T, Te Marvelde, J, Trinquand, A, Asnafi, V, Szczepanski, T, Matarraz, S, Lopez, A, Vidriales, B, Bulsa, J, Hrusak, O, Kalina, T, Lecrevisse, Q, Martin Ayuso, M, Bruggemann, M, Verde, J, Fernandez, P, Burgos, L, Paiva, B, Pedreira, C, Van Dongen, J, Orfao, A, Van Der Velden, V, Lhermitte, L, Mejstrikova, E, Van Der Sluijs-Gelling, A, Grigore, G, Sedek, L, Bras, A, Gaipa, G, Sobral Da Costa, E, Novakova, M, Sonneveld, E, Buracchi, C, De Sa Bacelar, T, Te Marvelde, J, Trinquand, A, Asnafi, V, Szczepanski, T, Matarraz, S, Lopez, A, Vidriales, B, Bulsa, J, Hrusak, O, Kalina, T, Lecrevisse, Q, Martin Ayuso, M, Bruggemann, M, Verde, J, Fernandez, P, Burgos, L, Paiva, B, Pedreira, C, Van Dongen, J, Orfao, A, and Van Der Velden, V

Abstract

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

Published
2018

34. Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia

Author

Theunissen, Prisca, Mejstrikova, E, Sedek, L, van der Sluijs-Gelling, AJ, Gaipa, G, Bartels, M, Costa, ES, Kotrova, M, Novakova, M, Sonneveld, E, Buracchi, C, Bonaccorso, P, Oliveira, E, te Marvelde, Jeroen, Szczepanski, T, Lhermitte, L, Hrusak, O, Lecrevisse, Q, Grigore, GE, Fronkova, E, Trka, J, Bruggemann, M, Orfao, A, Dongen, Jacques, van der Velden, Vincent, Theunissen, Prisca, Mejstrikova, E, Sedek, L, van der Sluijs-Gelling, AJ, Gaipa, G, Bartels, M, Costa, ES, Kotrova, M, Novakova, M, Sonneveld, E, Buracchi, C, Bonaccorso, P, Oliveira, E, te Marvelde, Jeroen, Szczepanski, T, Lhermitte, L, Hrusak, O, Lecrevisse, Q, Grigore, GE, Fronkova, E, Trka, J, Bruggemann, M, Orfao, A, Dongen, Jacques, and van der Velden, Vincent

Published
2017

37. Loss of B cells and their precursors is the most constant feature of GATA-2 deficiency in childhood myelodysplastic syndrome

Author

Novakova, M, Zaliova, M, Sukova, M, Wlodarski, M, Janda, A, Fronkova, E, Campr, V, Lejhancova, K, Zapletal, O, Pospisilova, D, Cerna, Z, Kuhn, T, Svec, P, Pelkova, V, Zemanova, Z, Kerndrup, G, Van den Heuvel - Eibrink, Marry, van der Velden, Vincent, Niemeyer, C, Kalina, T, Trka, J, Stary, J, Hrusak, O, Mejstrikova, E, and Immunology

Subjects
B-Lymphocytes, Adolescent, Precursor Cells, B-Lymphoid, Anemia, Aplastic, Infant, Bone Marrow Cells, Article, Immunophenotyping, Diagnosis, Differential, GATA2 Transcription Factor, Young Adult, Phenotype, ROC Curve, Bone Marrow, T-Lymphocyte Subsets, hemic and lymphatic diseases, Child, Preschool, Lymphopenia, Myelodysplastic Syndromes, Mutation, Humans, Myeloid Cells, Lymphocyte Count, Child, Biomarkers
Abstract

GATA-2 deficiency was recently described as common cause of overlapping syndromes of immunodeficiency, lymphedema, familiar myelodysplastic syndrome or acute myeloid leukemia. The aim of our study was to analyze bone marrow and peripheral blood samples of children with myelodysplastic syndrome or aplastic anemia to define prevalence of the GATA2 mutation and to assess whether mutations in GATA-2 transcription factor exhibit specific immunophenotypic features. The prevalence of a GATA2 mutation in a consecutively diagnosed cohort of children was 14% in advanced forms of myelodysplastic syndrome (refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and myelodysplasia-related acute myeloid leukemia), 17% in refractory cytopenia of childhood, and 0% in aplastic anemia. In GATA-2-deficient cases, we found the most profound B-cell lymphopenia, including its progenitors in blood and bone marrow, which correlated with significantly diminished intronRSS-Kde recombination excision circles in comparison to other myelodysplastic syndrome/aplastic anemia cases. The other typical features of GATA-2 deficiency (monocytopenia and natural killer cell lymphopenia) were less discriminative. In conclusion, we suggest screening for GATA2 mutations in pediatric myelodysplastic syndrome, preferentially in patients with impaired B-cell homeostasis in bone marrow and peripheral blood (low number of progenitors, intronRSS-Kde recombination excision circles and naïve cells).

Published
2016

38. Standardized Next-Generation Flow Cytometry for Highly Sensitive Mrd Measurements in B-Cell Acute Lymphoblastic Leukemia

Author

Theunissen, Prisca, Mejstrikova, E, Sedek, L, van der Sluijs, A, Gaipa, G, Bartels, M, Sobral de Costa, E, Kotrova, M, Novakova, M, Sonneveld, E, Baracchi, C, Bonaccorso, P, Oliviera, E, te Marvelde, Jeroen, Szczepanski, Tomek, Lhermitte, L, Hrusak, O, Lecrevisse, Q, Grigore, GE, Fronkova, E, Trka, J, Bruggemann, M, Orfao, A, Dongen, Jacques, van der Velden, Vincent, van Dongen, JJM, Immunology, and Plastic and Reconstructive Surgery and Hand Surgery

Published
2016

40. Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds

Author

Kalina, T, Flores-Montero, J, Lecrevisse, Q, Pedreira, CE, van der Velden, Vincent, Novakova, M, Mejstrikova, E, Hrusak, O, Bottcher, S, Karsch, D, Sedek, L, Trinquand, A, Boeckx, N, Caetano, J, Asnafi, V, Lucio, P, Lima, M, Santos, AH, Bonaccorso, P, van der Sluijs-Gelling, AJ, Langerak, Ton, Martin-Ayuso, M, Szczepanski, T, Dongen, Jacques, Orfao, A, and Immunology

Subjects
Europe, Quality Control, Leukemia, Lymphoma, Reference Values, Reference Standards, Flow Cytometry, Healthy Volunteers, Lymphocyte Subsets, Immunophenotyping
Abstract

Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for laboratories that use standardized EuroFlow protocols. It may be used to complement, but not replace, established proficiency tests. © 2014 International Society for Advancement of Cytometry.

Published
2014

41. Bone marrow immunophenotyping by flow cytometry in refractory cytopenia of childhood

Author

Aalbers, A. M., Van Den Heuvel-Eibrink, M. M., Baumann, I., Dworzak, M., Hasle, H., Locatelli, Franco, De Moerloose, B., Schmugge, M., Mejstrikova, E., Novakova, M., Zecca, M., Michel Zwaan, C., Te Marvelde, J. G., Langerak, A. W., Van Dongen, J. J., Pieters, R., Niemeyer, C. M., Van Der Velden, V. H., Locatelli F. (ORCID:0000-0002-7976-3654), Aalbers, A. M., Van Den Heuvel-Eibrink, M. M., Baumann, I., Dworzak, M., Hasle, H., Locatelli, Franco, De Moerloose, B., Schmugge, M., Mejstrikova, E., Novakova, M., Zecca, M., Michel Zwaan, C., Te Marvelde, J. G., Langerak, A. W., Van Dongen, J. J., Pieters, R., Niemeyer, C. M., Van Der Velden, V. H., and Locatelli F. (ORCID:0000-0002-7976-3654)

Abstract

Refractory cytopenia of childhood is the most common type of childhood myelodysplastic syndrome. Because the majority of children with refractory cytopenia have a normal karyotype and a hypocellular bone marrow, differentiating refractory cytopenia from the immune-mediated bone marrow failure syndrome (very) severe aplastic anemia can be challenging. Flow cytometric immunophenotyping of bone marrow has been shown to be a valuable diagnostic tool in differentiating myelodysplastic syndrome from non-clonal cytopenias in adults. Here, we performed the first comprehensive flow cytometric analysis of immature myeloid, lymphoid cells and erythroid cells, and granulocytes, monocytes, and lymphoid cells in bone marrow obtained from a large prospective cohort of 81 children with refractory cytopenia. Children with refractory cyotopenia had a strongly reduced myeloid compartment, but not as severe as children with aplastic anemia. Furthermore, the number of flow cytometric abnormalities was significantly higher in children with refractory cytopenia than in healthy controls and in children with aplastic anemia, but lower than in advanced myelodysplastic syndrome. We conclude that flow cytometric immunophenotyping could be a relevant addition to histopathology in the diagnosis of refractory cytopenia of childhood. (The multi-center studies EWOG-MDS RC06 and EWOG-MDS 2006 are registered at clinicaltrials.gov identifiers 00499070 and00662090, respectively).

Published
2015

42. Bone marrow immunophenotyping by flow cytometry in refractory cytopenia of childhood

Author

Aalbers, A. M., primary, van den Heuvel-Eibrink, M. M., additional, Baumann, I., additional, Dworzak, M., additional, Hasle, H., additional, Locatelli, F., additional, De Moerloose, B., additional, Schmugge, M., additional, Mejstrikova, E., additional, Novakova, M., additional, Zecca, M., additional, Zwaan, C. M., additional, te Marvelde, J. G., additional, Langerak, A. W., additional, van Dongen, J. J. M., additional, Pieters, R., additional, Niemeyer, C. M., additional, and van der Velden, V. H. J., additional

Published
2014
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48. Phytoremediation of polychlorinated biphenyls

Author

Macková, M., Dowling, D.N., Macek, T., Barriault, D., Francova, K., Sylvestre, M., Möder, Monika, Vrchotova, B., Lovecka, P., Najmanová, J., Demnerova, K., Novakova, M., Rezek, J., Macková, M., Dowling, D.N., Macek, T., Barriault, D., Francova, K., Sylvestre, M., Möder, Monika, Vrchotova, B., Lovecka, P., Najmanová, J., Demnerova, K., Novakova, M., and Rezek, J.

Published
2006

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