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2. Multiparameter flow cytometry in the evaluation of myelodysplasia: Analytical issues: Recommendations from the European LeukemiaNet/International Myelodysplastic Syndrome Flow Cytometry Working Group.

Author

Porwit, A., Béné, M.C., Duetz, C., Matarraz, S., Oelschlaegel, U., Westers, Theresia M., Wagner-Ballon, O., Kordasti, S., Valent, P., Preijers, F.W., Alhan, C., Bellos, F., Bettelheim, P., Burbury, K., Chapuis, N., Cremers, E., Porta, M.G. Della, Dunlop, A., Eidenschink-Brodersen, L., Font, P., Fontenay, M., Hobo, W., Ireland, R., Johansson, U., Loken, M.R., Ogata, K., Orfao, A., Psarra, K., Saft, L., Subira, D., Marvelde, J. Te, Wells, D.A., Velden, V.H. van der, Kern, W., Loosdrecht, A.A. van de, Porwit, A., Béné, M.C., Duetz, C., Matarraz, S., Oelschlaegel, U., Westers, Theresia M., Wagner-Ballon, O., Kordasti, S., Valent, P., Preijers, F.W., Alhan, C., Bellos, F., Bettelheim, P., Burbury, K., Chapuis, N., Cremers, E., Porta, M.G. Della, Dunlop, A., Eidenschink-Brodersen, L., Font, P., Fontenay, M., Hobo, W., Ireland, R., Johansson, U., Loken, M.R., Ogata, K., Orfao, A., Psarra, K., Saft, L., Subira, D., Marvelde, J. Te, Wells, D.A., Velden, V.H. van der, Kern, W., and Loosdrecht, A.A. van de

Abstract

01 januari 2023, Item does not contain fulltext, Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34(+) CD19(-) ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS.

Published
2023

4. Flow cytometric analysis of myelodysplasia: Pre-analytical and technical issues-Recommendations from the European LeukemiaNet

Author

van der Velden, VHJ, Preijers, F, Johansson, U, Westers, TM, Dunlop, A, Porwit, A, Bene, MC, Valent, P, te Marvelde, J, Wagner-Ballon, O, Oelschlaegel, U, Saft, L, Kordasti, S, Ireland, R, Cremers, E, Alhan, C, Duetz, C, Hobo, W, Chapuis, N, Fontenay, M, Bettelheim, P, Eidenshink-Brodersen, L, Font, P, Loken, MR, Matarraz, S, Ogata, K, Orfao, A, Psarra, K, Subira, D, Wells, DA, Della Porta, MG, Burbury, K, Bellos, F, Weiss, E, Kern, W, van de Loosdrecht, A, van der Velden, VHJ, Preijers, F, Johansson, U, Westers, TM, Dunlop, A, Porwit, A, Bene, MC, Valent, P, te Marvelde, J, Wagner-Ballon, O, Oelschlaegel, U, Saft, L, Kordasti, S, Ireland, R, Cremers, E, Alhan, C, Duetz, C, Hobo, W, Chapuis, N, Fontenay, M, Bettelheim, P, Eidenshink-Brodersen, L, Font, P, Loken, MR, Matarraz, S, Ogata, K, Orfao, A, Psarra, K, Subira, D, Wells, DA, Della Porta, MG, Burbury, K, Bellos, F, Weiss, E, Kern, W, and van de Loosdrecht, A

Abstract

BACKGROUND: Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre-analytical procedures. METHODS: Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group. RESULTS: We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies. CONCLUSIONS: These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice.

Published
2021

5. Clinical application of flow cytometry in patients with unexplained cytopenia and suspected myelodysplastic syndrome: A report of the European LeukemiaNet International MDS-Flow Cytometry Working Group

Author

Loosdrecht, AA, Kern, W, Porwit, A, Valent, P, Kordasti, S, Cremers, E, Alhan, C, Duetz, C, Dunlop, A, Hobo, W, Preijers, F, Wagner-Ballon, O, Chapuis, N, Fontenay, M, Bettelheim, P, Eidenschink-Brodersen, L, Font, P, Johansson, U, Loken, MR, Marvelde, JG, Matarraz, S, Ogata, K, Oelschlaegel, U, Orfao, A, Psarra, K, Subira, D, Wells, DA, Bene, MC, Della Porta, MG, Burbury, K, Bellos, F, van der Velden, VHJ, Westers, TM, Saft, L, Ireland, R, Loosdrecht, AA, Kern, W, Porwit, A, Valent, P, Kordasti, S, Cremers, E, Alhan, C, Duetz, C, Dunlop, A, Hobo, W, Preijers, F, Wagner-Ballon, O, Chapuis, N, Fontenay, M, Bettelheim, P, Eidenschink-Brodersen, L, Font, P, Johansson, U, Loken, MR, Marvelde, JG, Matarraz, S, Ogata, K, Oelschlaegel, U, Orfao, A, Psarra, K, Subira, D, Wells, DA, Bene, MC, Della Porta, MG, Burbury, K, Bellos, F, van der Velden, VHJ, Westers, TM, Saft, L, and Ireland, R

Abstract

This article discusses the rationale for inclusion of flow cytometry (FCM) in the diagnostic investigation and evaluation of cytopenias of uncertain origin and suspected myelodysplastic syndromes (MDS) by the European LeukemiaNet international MDS Flow Working Group (ELN iMDS Flow WG). The WHO 2016 classification recognizes that FCM contributes to the diagnosis of MDS and may be useful for prognostication, prediction, and evaluation of response to therapy and follow-up of MDS patients.

Published
2021

11. Diagnostic screening of paroxysmal nocturnal hemoglobinuria: Prospective multicentric evaluation of the current medical indications

Author

Morado, M, Freire Sandes, A, Colado, E, Subira, D, Isusi, P, Soledad Noya M, Belén Vidriales M, Sempere, A, Ángel Díaz J, Minguela, A, Alvarez, B, Serrano, C, Caballero, T, Rey, M, Pérez Corral A, Cristina Fernández Jiménez M, Magro, E, Lemes, A, Benavente, C, Bañas H, Merino, J, Castejon, C, Gutierrez, O, Rabasa, P, Vescosi Gonçalves M, Perez-Andres, M, Orfao, A, and PNH working group of the Iberian Society of Cytometry (SIC)

Subjects
standardization, Male, Erythrocytes, hematology, flow cytometry, Hemoglobinuria, Paroxysmal, Anemia, Aplastic, myelodysplastic syndrome, hemic and lymphatic diseases, Myelodysplastic Syndromes, Prevalence, Humans, Female, fluorescence cytometry, Prospective Studies, Retrospective Studies
Abstract

BackgroundAlthough consensus guidelines have been proposed in 2010 for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometry (FCM), so far no study has investigated the efficiency of such medical indications in multicentric vs. reference laboratory settings. MethodsHere we evaluate the efficiency of consensus medical indications for PNH testing in 3,938 peripheral blood samples submitted to FCM testing in 24 laboratories in Spain and one reference center in Brazil. ResultsOverall, diagnostic screening based on consensus medical indications was highly efficient (14% of PNH+ samples) both in the multicenter setting in Spain (10%) and the reference laboratory in Brazil (16%). The highest frequency of PNH+ cases was observed among patients screened because of bone marrow (BM) failure syndrome (33%), particularly among those with aplastic anemia (AA; 45%) and to a less extent also a myelodysplastic syndrome (MDS; 10%). Among the other individuals studied, the most efficient medical indications for PNH screening included: hemolytic anemia (19%), hemoglobinuria (48%) and unexplained cytopenias (9%). In contrast, only a minor fraction of the patients who had been submitted for PNH testing because of unexplained thrombosis in the absence of cytopenia, were positive (0.4%). ConclusionsIn summary, our results demonstrate that the current medical indications for PNH screening by FCM are highly efficient, although improved screening algorithms are needed for patients presenting with thrombosis and normal blood cell counts. (c) 2016 International Clinical Cytometry Society

Published
2016

13. TRAF1 as a marker of exhaustion during chronic hepatitis C

Author

Moreno, E., primary, Subira, D., additional, Miquel, J., additional, de Villalobos, E.S., additional, Praetorius, A.G., additional, Madejon, A., additional, Olveira, A., additional, Garcia-Samaniego, J., additional, Garcia, A., additional, Parra, T., additional, Romero, M., additional, and Larrubia, J.-R., additional

Published
2017
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16. Flow cytometric immunophenotyping of cerebrospinal fluid

Author

Kraan, Jaco, Gratama, Jan willem, Haioun, C, Orfao, A, Plonquet, A, Porwit, A, Quijano, S, Stetler-Stevenson, MA, Subira, D, Wilson, W, Robinson, J.P., Darzynkiewicz, Z., Hoffman, R., Nolan, J.P., Orfao, A., Rabinovitch, P.S., Watkins, S., and Medical Oncology

Published
2008

20. Rationale for the clinical application of flow cytometry in patients with myelodysplastic syndromes: position paper of an International Consortium and the European LeukemiaNet Working Group

Author

Loosdrecht, A.A. van de, Ireland, R., Kern, W., Della Porta, M.G., Alhan, C., Balleisen, J.S., Bettelheim, P., Bowen, D.T., Burbury, K., Eidenschink, L., Cazzola, M., Chu, S.S., Cullen, M., Cutler, J.A., Drager, A.M., Feuillard, J., Fenaux, P., Font, P., Germing, U., Haase, D., Hellstrom-Lindberg, E., Johansson, U., Kordasti, S., Loken, M.R., Malcovati, L., Marvelde, J.G. Te, Matarraz, S., Milne, T., Moshaver, B., Mufti, G.J., Nikolova, V., Ogata, K., Oelschlaegel, U., Orfao, A., Ossenkoppele, G.J., Porwit, A., Platzbecker, U., Preijers, F.W.M.B., Psarra, K., Richards, S.J., Subira, D., Seymour, J.F., Tindell, V., Vallespi, T., Valent, P., Velden, V.H. van der, Wells, D.A., Witte, T.J.M. de, Zettl, F., Bene, M.C., Westers, T.M., Loosdrecht, A.A. van de, Ireland, R., Kern, W., Della Porta, M.G., Alhan, C., Balleisen, J.S., Bettelheim, P., Bowen, D.T., Burbury, K., Eidenschink, L., Cazzola, M., Chu, S.S., Cullen, M., Cutler, J.A., Drager, A.M., Feuillard, J., Fenaux, P., Font, P., Germing, U., Haase, D., Hellstrom-Lindberg, E., Johansson, U., Kordasti, S., Loken, M.R., Malcovati, L., Marvelde, J.G. Te, Matarraz, S., Milne, T., Moshaver, B., Mufti, G.J., Nikolova, V., Ogata, K., Oelschlaegel, U., Orfao, A., Ossenkoppele, G.J., Porwit, A., Platzbecker, U., Preijers, F.W.M.B., Psarra, K., Richards, S.J., Subira, D., Seymour, J.F., Tindell, V., Vallespi, T., Valent, P., Velden, V.H. van der, Wells, D.A., Witte, T.J.M. de, Zettl, F., Bene, M.C., and Westers, T.M.

Abstract

Item does not contain fulltext, An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

Published
2013

21. Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group.

Author

Westers, T.M., Ireland, R., Kern, W., Alhan, C., Balleisen, J.S., Bettelheim, P., Burbury, K., Cullen, M., Cutler, J.A., Porta, M.G. Della, Drager, A.M., Feuillard, J., Font, P., Germing, U., Haase, D., Johansson, U., Kordasti, S., Loken, M.R., Malcovati, L., Marvelde, J.G. Te, Matarraz, S., Milne, T., Moshaver, B., Mufti, G.J., Ogata, K., Orfao, A., Porwit, A., Psarra, K., Richards, S.J., Subira, D., Tindell, V., Vallespi, T., Valent, P., Velden, V.H. van der, Witte, T.J.M. de, Wells, D.A., Zettl, F., Bene, M.C., Loosdrecht, A.A. van de, Westers, T.M., Ireland, R., Kern, W., Alhan, C., Balleisen, J.S., Bettelheim, P., Burbury, K., Cullen, M., Cutler, J.A., Porta, M.G. Della, Drager, A.M., Feuillard, J., Font, P., Germing, U., Haase, D., Johansson, U., Kordasti, S., Loken, M.R., Malcovati, L., Marvelde, J.G. Te, Matarraz, S., Milne, T., Moshaver, B., Mufti, G.J., Ogata, K., Orfao, A., Porwit, A., Psarra, K., Richards, S.J., Subira, D., Tindell, V., Vallespi, T., Valent, P., Velden, V.H. van der, Witte, T.J.M. de, Wells, D.A., Zettl, F., Bene, M.C., and Loosdrecht, A.A. van de

Abstract

1 juli 2012, Item does not contain fulltext, Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as 'normal', 'suggestive of', or 'diagnostic of' MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

Published
2012

23. Standardization of flow cytometry in myelodysplastic syndromes: report from the first European LeukemiaNet working conference on flow cytometry in myelodysplastic syndromes.

Author

Loosdrecht, A.A. van de, Alhan, C., Bene, M.C., Porta, M.G. Della, Drager, A.M., Feuillard, J., Font, P., Germing, U., Haase, D., Homburg, C.H., Ireland, R., Jansen, J.H., Kern, W., Malcovati, L., Marvelde, J.G. Te, Mufti, G.J., Ogata, K., Orfao, A., Ossenkoppele, G.J., Porwit, A., Preijers, F.W.M.B., Richards, S.J., Schuurhuis, G.J., Subira, D., Valent, P., Velden, V.H. van der, Vyas, P., Westra, A.H., Witte, T.J.M. de, Wells, D.A., Loken, M.R., Westers, T.M., Loosdrecht, A.A. van de, Alhan, C., Bene, M.C., Porta, M.G. Della, Drager, A.M., Feuillard, J., Font, P., Germing, U., Haase, D., Homburg, C.H., Ireland, R., Jansen, J.H., Kern, W., Malcovati, L., Marvelde, J.G. Te, Mufti, G.J., Ogata, K., Orfao, A., Ossenkoppele, G.J., Porwit, A., Preijers, F.W.M.B., Richards, S.J., Schuurhuis, G.J., Subira, D., Valent, P., Velden, V.H. van der, Vyas, P., Westra, A.H., Witte, T.J.M. de, Wells, D.A., Loken, M.R., and Westers, T.M.

Abstract

Contains fulltext : 81483.pdf (publisher's version ) (Open Access), The myelodysplastic syndromes are a group of clonal hematopoietic stem cell diseases characterized by cytopenia(s), dysplasia in one or more cell lineages and increased risk of evolution to acute myeloid leukemia (AML). Recent advances in immunophenotyping of hematopoietic progenitor and maturing cells in dysplastic bone marrow point to a useful role for multiparameter flow cytometry (FCM) in the diagnosis and prognostication of myelodysplastic syndromes. In March 2008, representatives from 18 European institutes participated in a European LeukemiaNet (ELN) workshop held in Amsterdam as a first step towards standardization of FCM in myelodysplastic syndromes. Consensus was reached regarding standard methods for cell sampling, handling and processing. The group also defined minimal combinations of antibodies to analyze aberrant immunophenotypes and thus dysplasia. Examples are altered numbers of CD34(+) precursors, aberrant expression of markers on myeloblasts, maturing myeloid cells, monocytes or erythroid precursors and the expression of lineage infidelity markers. When applied in practice, aberrant FCM patterns correlate well with morphology, the subclassification of myelodysplastic syndromes, and prognostic scoring systems. However, the group also concluded that despite strong evidence for an impact of FCM in myelodysplastic syndromes, further (prospective) validation of markers and immunophenotypic patterns are required against control patient groups as well as further standardization in multi-center studies. Standardization of FCM in myelodysplastic syndromes may thus contribute to improved diagnosis and prognostication of myelodysplastic syndromes in the future.

Published
2009

24. Standardization of flow cytometry in myelodysplastic syndromes: report from the first European LeukemiaNet working conference on flow cytometry in myelodysplastic syndromes

Author

van de Loosdrecht, AA, Alhan, C, Bene, MC, Della Porta, MG, Drager, AM, Feuillard, J, Font, P, Germing, U, Haase, D, Homburg, CH, Ireland, R, Jansen, JH (Joop), Kern, W, Malcovati, L, te Marvelde, Jeroen, Mufti, GJ, Ogata, K, Orfao, A, Ossenkoppele, GJ, Porwit, A, Preijers, FW, Richards, SJ, Schuurhuis, GJ, Subira, D, Valent, P, van der Velden, Vincent, Vyas, P, Westra, AH, de Witte, TM, Wells, DA, Loken, MR, Westers, TM, van de Loosdrecht, AA, Alhan, C, Bene, MC, Della Porta, MG, Drager, AM, Feuillard, J, Font, P, Germing, U, Haase, D, Homburg, CH, Ireland, R, Jansen, JH (Joop), Kern, W, Malcovati, L, te Marvelde, Jeroen, Mufti, GJ, Ogata, K, Orfao, A, Ossenkoppele, GJ, Porwit, A, Preijers, FW, Richards, SJ, Schuurhuis, GJ, Subira, D, Valent, P, van der Velden, Vincent, Vyas, P, Westra, AH, de Witte, TM, Wells, DA, Loken, MR, and Westers, TM

Abstract

The myelodysplastic syndromes are a group of clonal hematopoietic stem cell diseases characterized by cytopenia(s), dysplasia in one or more cell lineages and increased risk of evolution to acute myeloid leukemia (AML). Recent advances in immunophenotyping of hematopoietic progenitor and maturing cells in dysplastic bone marrow point to a useful role for multiparameter Row cytometry (FCM) in the diagnosis and prognostication of myelodysplastic syndromes. In March 2008, representatives from 18 European institutes participated in a European LeukemiaNet (ELN) workshop held in Amsterdam as a first step towards standardization of FCM in myelodysplastic syndromes. Consensus was reached regarding standard methods for cell sampling, handling and processing. The group also defined minimal combinations of antibodies to analyze aberrant immunophenotypes and thus dysplasia. Examples are altered numbers of CD34(+) precursors, aberrant expression of markers on myeloblasts, maturing myeloid cells, monocytes or erythroid precursors and the expression of lineage infidelity markers. When applied in practice, aberrant FCM patterns correlate well with morphology, the subclassification of myelodysplastic syndromes, and prognostic scoring systems. However, the group also concluded that despite strong evidence for an impact of FCM in myelodysplastic syndromes, further (prospective) validation of markers and immunophenotypic patterns are required against control patient groups as well as further standardization in multi-center studies. Standardization of FCM in myelodysplastic syndromes may thus contribute to improved diagnosis and prognostication of myelodysplastic syndromes in the future.

Published
2009

25. V44 Left laparoscopic radical nephrectomy: Step-by-step

Author

Sotelo, Noguera R., primary, Carmona, O., additional, De Andrade, R., additional, Santinelli, F., additional, Subira, D., additional, Ignacio, C., additional, Fernández, G., additional, Garza, R., additional, Castro, J., additional, Birkhauser, F., additional, Cisneros, R., additional, Canes, D., additional, and Clayman, R., additional

Published
2013
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28. Standardization of flow cytometry in myelodysplastic syndromes: report from the first European LeukemiaNet working conference on flow cytometry in myelodysplastic syndromes

Author

van de Loosdrecht, A. A., primary, Alhan, C., additional, Bene, M. C., additional, Della Porta, M. G., additional, Drager, A. M., additional, Feuillard, J., additional, Font, P., additional, Germing, U., additional, Haase, D., additional, Homburg, C. H., additional, Ireland, R., additional, Jansen, J. H., additional, Kern, W., additional, Malcovati, L., additional, te Marvelde, J. G., additional, Mufti, G. J., additional, Ogata, K., additional, Orfao, A., additional, Ossenkoppele, G. J., additional, Porwit, A., additional, Preijers, F. W., additional, Richards, S. J., additional, Schuurhuis, G. J., additional, Subira, D., additional, Valent, P., additional, van der Velden, V. H.J., additional, Vyas, P., additional, Westra, A. H., additional, de Witte, T. M., additional, Wells, D. A., additional, Loken, M. R., additional, and Westers, T. M., additional

Published
2009
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29. P088 Challenging diagnosis of refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS): detection of myeloid dysplasia by flow cytometry (FC)

Author

Font, P., primary, Subira, D., additional, Loscertales, J., additional, Serrano, C., additional, Castanón, S., additional, Gonzalo, R., additional, Blas, C., additional, Lopez-Pascual, J., additional, Martí, E., additional, Alaez, C., additional, Escudero, A., additional, and Fernandez-Ranada, J.M., additional

Published
2007
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36. 137 Standardization of flow cytometry in myelodysplastic syndromes: A report from an international consortium and the European LeukemiaNet Working Group

Author

Westers, T.M., Ireland, R., Kern, W., Alhan, C., Balleisen, J.S., Béné, M.C., Bettelheim, P., Burbury, K., Cullen, M., Cutler, J.A., Porta, M.G. Della, Drager, A.M., Feuillard, J., Font, P., Germing, U., Haase, D., Johansson, U., Kordasti, S., Loken, M.R., Malcovati, L., te Marvelde, J.G., Matarraz, S., Milne, T., Moshaver, B., Mufti, G.J., Ogata, K., Orfao, A., Porwit, A., Psarra, K., Richards, S.J., Subirá, D., Tindell, V., Vallespi, T., Valent, P., van der Velden, V.H., de Witte, T.M., Wells, D.A., Zettl, F., and van de Loosdrecht, A.A.

Published
2011
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37. JAK2 as a Molecular Marker in Myeloproliferative Diseases

Author

Mata, R., Subira, D., Garcia-Raso, A., and Llamas, P.

Abstract

The molecular pathogenesis of the myeloproliferative disorders (MPD) is poorly understood, except for chronic myeloid leukemia (CML). Recently, several groups have discovered a novel recurrent unique acquired clonal mutation in a tyrosine-kinase JAK2 in patients with Philadelphia-negative MPD and other myeloid disorders. It consists in a guanineto- thymine change encoding a valine to phenylalanine at codon 617 (JAK2 V617F). JAK2 and the other members of the Janus kinase family are tyrosine kinases that function as intermediates between membrane receptors and intracellular signalling molecules. The mutation occurs within the enzymatically inactive JH2 pseudo-kinase domain that regulates the active JH1 kinase domain. The JAK2 activation leads to constitutive JAK and STAT (activators of transcription) hyperactivation with induction of growth factor hypersensitivity and cell transformation. Some authors have found a higher risk of vascular thrombosis and higher platelet activation when the mutation is present. Therefore, the JAK2 mutation offers a molecular target for new drugs investigation in a similar way to bcr/abl rearrangement in CML. For all these reasons, several studies related to JAK2 have arisen in the last year. In this report, we will review the literature and discuss its possible clinical and prognostic significance.

Published
2007

40. Role of flow cytometry immunophenotyping in the diagnosis of leptomeningeal carcinomatosis

Author

Subira, D. (Dolores)

Subjects
Flow cytometry, Immunophenotype, Meningeal carcinomatosis
Abstract

PURPOSE: To explore the contribution of flow cytometry immunophenotyping (FCI) in detecting leptomeningeal disease in patients with solid tumors. EXPERIMENTAL DESIGN: Cerebrospinal fluid (CSF) samples from 78 patients who received a diagnosis of epithelial-cell solid tumors and had clinical data suggestive of leptomeningeal carcinomatosis (LC) were studied. A novel FCI protocol was used to identify cells expressing the epithelial cell antigen EpCAM and their DNA content. Accompanying inflammatory cells were also described. FCI results (positive or negative for malignancy) were compared with those from CSF cytology and with the diagnosis established by the clinicians: patients with LC (n = 49), without LC (n = 26), and undetermined (n = 3). RESULTS: FCI described a wide range of EpCAM-positive cells with a hyperdiploid DNA content in the CSF of patients with LC. Compared with cytology, FCI showed higher sensitivity (75.5 vs 65.3) and negative predictive value (67.6 vs 60.5), and similar specificity (96.1 vs 100) and positive predictive value (97.4 vs 100). Concordance between cytology and FCI was high (Kp = 0.83), although misdiagnosis of LC did not show differences between evaluating the CSF with 1 or 2 techniques (P = .06). Receiver-operator characteristic curve analyses showed that lymphocytes and monocytes had a different distribution between patients with and without LC. CONCLUSION: FCI seems to be a promising new tool for improving the diagnostic examination of patients with suspicion of LC. Detection of epithelial cells with a higher DNA content is highly specific of LC, but evaluation of the nonepithelial cell compartment of the CSF might also be useful for supporting this diagnosis.

Published
2012

42. Moderated Posters 1.

Author

Minervini, A., Suks, M., Rees, R., Goorney, S., Bettocchi, C., Ralph, D.J., Pryor, J.P., Lledo-Garcia, E., Moncada-Iribarren, I., Jara-Rascon, J., Gonzalez-Chamorro, F., Hernandez-Fernandez, C., Llorente-Abarca, C., Mooreville, M., Martin-Morales, A., Jara, J., Bonilla, R., Subira, D., and Wilson, S.K.

Subjects
IMPOTENCE, PENILE prostheses
Abstract

Presents various studies related to impotence. Long term results of penile prostheses insertion for the treatment of erectile dysfunction; Analysis of sildenafil citrate as a treatment for cold glans syndrome after implantation of penile prostheses; Insertion of penile prostheses in fibrotic corpora.

Published
2001
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43. ELN iMDS flow working group validation of the monocyte assay for chronic myelomonocytic leukemia diagnosis by flow cytometry.

Author

Wagner-Ballon O, Bettelheim P, Lauf J, Bellos F, Della Porta M, Travaglino E, Subira D, Lopez IN, Tarfi S, Westers TM, Johansson U, Psarra K, Karathanos S, Matarraz S, Colado E, Gupta M, Ireland R, Kern W, and Van De Loosdrecht AA

Subjects
Humans, Monocytes, Flow Cytometry methods, Immunophenotyping, Leukemia, Myelomonocytic, Chronic diagnosis, Myeloproliferative Disorders
Abstract

Background: It was proposed that peripheral blood (PB) monocyte profiles evaluated by flow cytometry, called "monocyte assay," could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay requires a large multicenter validation and the assessment of its feasibility on bone marrow (BM) samples, as some centers may not have access to PB., Methods: PB and/or BM samples from patients displaying monocytosis were assessed with the "monocyte assay" by 10 ELN iMDS Flow working group centers with harmonized protocols. The corresponding files were reanalyzed in a blind fashion and the cMo percentages obtained by both analyses were compared. Confirmed diagnoses were collected when available., Results: The comparison between cMo percentages from 267 PB files showed a good global significant correlation (r = 0.88) with no bias. Confirmed diagnoses, available for 212 patients, achieved a 94% sensitivity and an 84% specificity. Hence, 95/101 CMML patients displayed cMo ≥94% while cMo <94% was observed in 83/99 patients with reactive monocytosis and in 10/12 patients with myeloproliferative neoplasms (MPN) with monocytosis. The established Receiver Operator Curve again provided a 94% cut-off value of cMo. The 117 BM files reanalysis led to an 87% sensitivity and an 80% specificity, with excellent correlation between the 43 paired samples to PB., Conclusions: This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples., (© 2021 International Clinical Cytometry Society.)

Published
2023
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44. Multiparameter flow cytometry in the evaluation of myelodysplasia: Analytical issues: Recommendations from the European LeukemiaNet/International Myelodysplastic Syndrome Flow Cytometry Working Group.

Author

Porwit A, Béné MC, Duetz C, Matarraz S, Oelschlaegel U, Westers TM, Wagner-Ballon O, Kordasti S, Valent P, Preijers F, Alhan C, Bellos F, Bettelheim P, Burbury K, Chapuis N, Cremers E, Della Porta MG, Dunlop A, Eidenschink-Brodersen L, Font P, Fontenay M, Hobo W, Ireland R, Johansson U, Loken MR, Ogata K, Orfao A, Psarra K, Saft L, Subira D, Te Marvelde J, Wells DA, van der Velden VHJ, Kern W, and van de Loosdrecht AA

Subjects
Humans, Flow Cytometry methods, Antigens, CD34, Granulocytes pathology, Monocytes pathology, Immunophenotyping, Myelodysplastic Syndromes diagnosis, Myelodysplastic Syndromes pathology
Abstract

Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34 + CD19 - ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS., (© 2022 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Published
2023
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45. Flow cytometric immunophenotyping of cerebrospinal fluid.

Author

Kraan J, Gratama JW, Haioun C, Orfao A, Plonquet A, Porwit A, Quijano S, Stetler-Stevenson M, Subira D, and Wilson W

Subjects
Animals, Antigens, Surface analysis, Humans, Immunophenotyping methods, Leukemia complications, Leukemia pathology, Lymphoma complications, Lymphoma pathology, Meningeal Neoplasms diagnosis, Cerebrospinal Fluid cytology, Flow Cytometry methods
Abstract

Leptomeningeal disease is an important adverse complication occurring in patients with B and T cell lymphomas and acute leukemias of lymphoid and myeloid origin. Recent reports suggest that multiparameter flow cytometry immunophenotypic assessment of spinal fluid samples could improve the efficiency of detection of CNS involvement, due to its high specificity and greater sensitivity. However, spinal fluid samples are frequently paucicellular with a rapidly decreasing cell viability. Staining of spinal fluid therefore requires dedicated sample storage/transport, staining, and preparation protocols. The Basic Protocol in this unit outlines a consensus multiparameter (3- to 8-color) flow cytometry immunophenotypic protocol for the evaluation of CNS involvement of cerebrospinal fluid (CSF) samples by neoplastic cells. A Support Protocol describing the simultaneous assessment of surface and cytoplasmic antigens is also provided. Finally, in the Alternate Protocol, we describe a method to calculate absolute numbers of both normal and pathological cell subpopulations by adding counting beads to the assay.

Published
2008
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