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

Abstract

01 januari 2023, Item does not contain fulltext, 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
2023

3. Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies in myelodysplastic syndromes by the ELN iMDS flow working group.

Author

Kern, W., Westers, Theresia M., Bellos, F., Bene, M.C., Bettelheim, P., Brodersen, L.E., Burbury, K., Chu, S.C., Cullen, M., Porta, M.D., Dunlop, A.S., Johansson, U., Matarraz, S., Oelschlaegel, U., Ogata, K., Porwit, A., Preijers, F.W., Psarra, K., Saft, L., Subirá, D., Weiß, E., Velden, V.H. van der, Loosdrecht, A. van de, Kern, W., Westers, Theresia M., Bellos, F., Bene, M.C., Bettelheim, P., Brodersen, L.E., Burbury, K., Chu, S.C., Cullen, M., Porta, M.D., Dunlop, A.S., Johansson, U., Matarraz, S., Oelschlaegel, U., Ogata, K., Porwit, A., Preijers, F.W., Psarra, K., Saft, L., Subirá, D., Weiß, E., Velden, V.H. van der, and Loosdrecht, A. van de

Abstract

01 januari 2023, Item does not contain fulltext, BACKGROUND: Myelodysplastic syndromes (MDS) represent a diagnostic challenge. This prospective multicenter study was conducted to evaluate pre-defined flow cytometric markers in the diagnostic work-up of MDS and chronic myelomonocytic leukemia (CMML). METHODS: Thousand six hundred and eighty-two patients with suspected MDS/CMML were analyzed by both cytomorphology according to WHO 2016 criteria and flow cytometry according to ELN recommendations. Flow cytometric readout was categorized 'non-MDS' (i.e. no signs of MDS/CMML and limited signs of MDS/CMML) and 'in agreement with MDS' (i.e., in agreement with MDS/CMML). RESULTS: Flow cytometric readout categorized 60% of patients in agreement with MDS, 28% showed limited signs of MDS and 12% had no signs of MDS. In 81% of cases flow cytometric readouts and cytomorphologic diagnosis correlated. For high-risk MDS, the level of concordance was 92%. A total of 17 immunophenotypic aberrancies were found independently related to MDS/CMML in ≥1 of the subgroups of low-risk MDS, high-risk MDS, CMML. A cut-off of ≥3 of these aberrancies resulted in 80% agreement with cytomorphology (20% cases concordantly negative, 60% positive). Moreover, >3% myeloid progenitor cells were significantly associated with MDS (286/293 such cases, 98%). CONCLUSION: Data from this prospective multicenter study led to recognition of 17 immunophenotypic markers allowing to identify cases 'in agreement with MDS'. Moreover, data emphasizes the clinical utility of immunophenotyping in MDS diagnostics, given the high concordance between cytomorphology and the flow cytometric readout. Results from the current study challenge the application of the cytomorphologically defined cut-off of 5% blasts for flow cytometry and rather suggest a 3% cut-off for the latter.

Published
2023

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

Author

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

Abstract

01 januari 2023, Item does not contain fulltext, 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
2023

5. 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

6. 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

7. 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

9. P1647: CONTRIBUTION OF NEXT GENERATION FLOW (NGF) CYTOMETRY IN PRIMARY IMMUNE THROMBOCYTOPENIA (ITP): UTILITY FOR THE DIFFERENTIAL DIAGNOSIS WITH MYELODYSPLASTIC SYNDROMES

Author

Gonzalez-Lopez, T. J., primary, Matarraz, S., additional, Caparros, I., additional, Lopez-Fernandez, M. F., additional, Mingot-Castellano, M. E., additional, Jimenez Martin, A., additional, Fernandez-Fuertes, F., additional, Sánchez Gallego, J. I., additional, Leoz, P., additional, Cuevas Ruiz, B., additional, Cuevas Ruiz, M. V., additional, Martin Marín, C., additional, Olazabal Herrero, J., additional, de Vicente Cámara, P., additional, and Orfao, A., additional

Published
2022
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10. Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes—proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS

Author

Porwit, A, van de Loosdrecht, A A, Bettelheim, P, Brodersen, L Eidenschink, Burbury, K, Cremers, E, Della Porta, M G, Ireland, R, Johansson, U, Matarraz, S, Ogata, K, Orfao, A, Preijers, F, Psarra, K, Subirá, D, Valent, P, van der Velden, V H J, Wells, D, Westers, T M, Kern, W, and Béné, M C

Published
2014
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11. Immunophenotypic alterations of bone marrow myeloid cell compartments in multiple myeloma patients predict for myelodysplasia-associated cytogenetic alterations

Author

Matarraz, S, Paiva, B, Díez-Campelo, M, Bárrena, S, Jara-Acevedo, M, Gutiérrez, M L, Sayagués, J M, Sánchez, M-L, Bárcena, P, Garrastazul, M P, Berruezo, M J, Duran, J M, Cerveró, C, García-Erce, J A, Florensa, L, Méndez, G D, Gutierrez, O, del Cañizo, M C, van Dongen, J J M, San Miguel, J F, and Orfao, A

Published
2014
Full Text
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13. 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

14. 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

15. 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, Della Porta, M G, Dräger, 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 J, de Witte, T M, Wells, D A, Zettl, F, Béné, M C, and van de Loosdrecht, A A

Published
2012
Full Text
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17. Biological and clinical significance of dysplastic hematopoiesis in patients with newly-diagnosed multiple myeloma

Author

Maia C, Puig N, Cedena M, Goicoechea I, Valdes-Mas R, Vazquez I, Chillon M, Aguirre P, Sarvide S, Gracia-Aznarez F, Alkorta-Aranburu G, Calasanz M, Garcia-Sanz R, Gonzalez M, Gutierrez N, Martinez-Lopez J, Perez J, Merino J, Moreno C, Burgos L, Alignani D, Botta C, Prosper F, Matarraz S, Orfao A, Oriol A, Teruel A, de Paz R, de Arriba F, Hernandez Garcia M, Palomera L, Martinez R, Rosinol L, Mateos M, Lahuerta J, Blade J, San Miguel J, and Paiva B

Subjects
hemic and lymphatic diseases
Abstract

Risk of developing myelodysplastic syndromes (MDS) is significantly increased in both multiple myeloma (MM) and MGUS, suggesting that is therapy independent. However, the incidence and sequelae of dysplastic hematopoiesis at diagnosis are unknown. Here, we used multidimensional flow cytometry (MFC) to prospectively screen for presence of MDS-associated phenotypic alterations (MDS-PA) in the bone marrow of 285 MM patients enrolled in the PETHEMA/GEM2012MENOS65 trial (NCT01916252), and investigated the clinical significance of monocytic MDS-PA in a larger series of 1,252 patients enrolled in four PETHEMA/GEM protocols. At diagnosis, 33/285 (11.6%) cases displayed MDS-PA. Bulk- and single-cell targeted sequencing of MDS recurrently mutated genes in CD34+ progenitors (and dysplastic lineages) from 67 patients unveiled clonal hematopoiesis in 13/26 (50%) cases with MDS-PA versus 9/41 (22%) without MDS-PA; TET2 and NRAS were the most frequently mutated genes. Dynamics of MDS-PA at diagnosis and after autologous transplant were evaluated in 86/285 patients, and showed that in most cases (69/86, 80%) MDS-PA either persisted or remained absent in patients with or without MDS-PA at diagnosis, respectively. Noteworthy, MDS-associated mutations unfrequently emerged after high-dose therapy. Based on MFC profiling, we found that patients with MDS-PA have altered hematopoiesis and Treg distribution in the tumor microenvironment. Importantly, presence of monocytic MDS-PA at diagnosis anticipated greater risk of hematological toxicity and was independently associated with inferior progression-free (HR:1.5, P=.02) and overall survival (HR:1.7, P=.01). This study unveils the biological and clinical significance of dysplastic hematopoiesis in newly-diagnosed MM, which can be screened with moderate sensitivity using cost-effective MFC. Copyright © 2020 American Society of Hematology.

Published
2020

18. Biological and clinical signi fi cance of dysplastic hematopoiesis in patients with newly diagnosed multiple myeloma

Author

Maia, C, Puig, N, Cedena, MT, Goicoechea, I, Valdes-Mas, R, Vazquez, I, Chillon, MC, Aguirre, P, Sarvide, S, Gracia-Aznarez, FJ, Alkorta, G, Calasanz, MJ, Garcia-Sanz, R, Gonzalez, M, Gutierrez, NC, Martinez-Lopez, J, Perez, JJ, Merino, J, Moreno, C, Burgos, L, Alignani, D, Botta, C, Prosper, F, Matarraz, S, Orfao, A, Oriol, A, Teruel, AI, de Paz, RD, de Arriba, F, Hernandez, MT, Palomera, L, Martinez, R, Rosinol, L, Mateos, MV, Lahuerta, JJ, Blade, J, San Miguel, JF, and Paiva, B

Abstract

Risk of developing myelodysplastic syndrome (MDS) is significantly increased in both multiple myeloma (MM) and monoclonal gammopathy of undetermined significance, suggesting that it is therapy independent. However, the incidence and sequelae of dys- plastic hematopoiesis at diagnosis are unknown. Here, we used multidimensional flow cytometry (MFC) to prospectively screen for the presence of MDS-associated phenotypic alterations (MDS-PA) in the bone marrow of 285 patients with MM enrolled in the PETHEMA/GEM2012MENOS65 trial (#NCT01916252). We investigated the clinical significance of monocytic MDS-PA in a larger series of 1252 patients enrolled in 4 PETHEMA/GEM protocols. At diagnosis, 33 (11.6%) of 285 cases displayed MDS-PA. Bulk and single-cell-targeted sequencing of MDS recurrently mutated genes in CD34(+) progenitors (and dysplastic lineages) from 67 patients revealed clonal hematopoiesis in 13 (50%) of 26 cases with MDS-PA vs 9 (22%) of 41 without MDS-PA; TET2 and NRAS were the most frequently mutated genes. Dynamics of MDS-PA at diagnosis and after autologous transplant were evaluated in 86 of 285 patients and showed that in most cases (69 of 86 [80%]), MDS-PA either persisted or remained absent in patients with or without MDS-PA at diagnosis, respectively. Noteworthy, MDS-associated mutations infrequently emerged after high-dose therapy. Based on MFC profiling, patients with MDS-PA have altered hematopoiesis and T regulatory cell distribution in the tumor microenvironment. Importantly, the presence of monocytic MDS-PA at diagnosis anticipated greater risk of hematologic toxicity and was independently associated with inferior progression-free survival (hazard ratio, 1.5; P = .02) and overall survival (hazard ratio, 1.7; P = .01). This study reveals the biological and clinical significance of dysplastic hematopoiesis in newly diagnosed MM, which can be screened with moderate sensitivity using cost-effective MFC. (Blood. 2020;135(26):2375-2387)

Published
2020

19. 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

20. 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

21. Distribution of subsets of blood monocytic cells throughout life

Author

Damasceno, D., Teodosio, C., Bossche, W.B.L. van den, Perez-Andres, M., Arriba-Mendez, S., Munoz-Bellvis, L., Romero, A., Blanco, J.F., Remesal, A., Puig, N., Matarraz, S., Vicente-Villardon, J.L., Dongen, J.J.M. van, Almeida, J., Orfao, A., TiMaScan Study Grp, and Immunology

Subjects
Adult, Male, Adolescent, Immunology, education, Spleen, Biology, Monocytes, Flow cytometry, Immunophenotyping, 03 medical and health sciences, Young Adult, 0302 clinical medicine, Antigen, Antigens, CD, Bone Marrow, medicine, Immunology and Allergy, Distribution (pharmacology), Humans, Child, 030304 developmental biology, Aged, Aged, 80 and over, 0303 health sciences, medicine.diagnostic_test, Infant, Newborn, Antibodies, Monoclonal, Infant, Middle Aged, Flow Cytometry, medicine.anatomical_structure, Child, Preschool, Monoclonal, biology.protein, Female, Bone marrow, Lymph Nodes, Antibody, 030215 immunology
Abstract

TiMaScan Study Group.

Published
2019

22. Distribution of subsets of blood monocytic cells throughout life

Author

Damasceno, D, Teodosio, C, Bossche, Wouter, Perez-Andres, M, Arriba-Mendez, S, Munoz-Bellvis, L, Romero, A, Blanco, JF, Remesal, A, Puig, N, Matarraz, S, Vicente-Villardon, JL, van Dongen, JJM, Almeida, J, Orfao, A, Damasceno, D, Teodosio, C, Bossche, Wouter, Perez-Andres, M, Arriba-Mendez, S, Munoz-Bellvis, L, Romero, A, Blanco, JF, Remesal, A, Puig, N, Matarraz, S, Vicente-Villardon, JL, van Dongen, JJM, Almeida, J, and Orfao, A

Published
2019

24. 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

25. Immunophenotypic analysis of erythroid dysplasia in myelodysplastic syndromes. A report from the IMDSFlow working group

Author

Westers, T.M., Cremers, Eline M. P., Oelschlaegel, U., Johansson, U., Bettelheim, P., Matarraz, S., Preijers, F.W.M.B., Kern, W., Loosdrecht, A.A. van de, Westers, T.M., Cremers, Eline M. P., Oelschlaegel, U., Johansson, U., Bettelheim, P., Matarraz, S., Preijers, F.W.M.B., Kern, W., and Loosdrecht, A.A. van de

Abstract

Contains fulltext : 189908.pdf (publisher's version ) (Open Access)

Published
2017

26. Immunophenotypic analysis of erythroid dysplasia in myelodysplastic syndromes. A report from the IMDSFlow working group

Author

Westers, T.M. (Theresia), Cremers, E.M.P. (Eline), Oelschlaegel, U. (Uta), Johansson, U. (Ulrika), Bettelheim, P. (Peter), Matarraz, S. (S.), Orfao, A. (Alberto), Moshaver, B. (Bijan), Brodersen, L.E. (Lisa Eidenschink), Loken, M.R. (Michael), Wells, D.A. (Denise), Subirá, D. (Dolores), Cullen, C., Marvelde, J.G. (Jeroen) te, Velden, V.H.J. (Vincent) van der, Preijers, F.W.M.B. (Frank), Chu, S.-C. (Sung-Chao), Feuillard, J. (Jean), Guérin, E. (Estelle), Psarra, K. (Katherina), Porwit, A. (Anna), Saft, L. (Leonie), Ireland, R. (Robin), Milne, T. (Timothy), Béné, M.C., Witte, B.I. (Birgit), Della Porta, M.G. (Matteo), Kern, W. (Wolfgang), Loosdrecht, A.A. (Arjan) van de, Westers, T.M. (Theresia), Cremers, E.M.P. (Eline), Oelschlaegel, U. (Uta), Johansson, U. (Ulrika), Bettelheim, P. (Peter), Matarraz, S. (S.), Orfao, A. (Alberto), Moshaver, B. (Bijan), Brodersen, L.E. (Lisa Eidenschink), Loken, M.R. (Michael), Wells, D.A. (Denise), Subirá, D. (Dolores), Cullen, C., Marvelde, J.G. (Jeroen) te, Velden, V.H.J. (Vincent) van der, Preijers, F.W.M.B. (Frank), Chu, S.-C. (Sung-Chao), Feuillard, J. (Jean), Guérin, E. (Estelle), Psarra, K. (Katherina), Porwit, A. (Anna), Saft, L. (Leonie), Ireland, R. (Robin), Milne, T. (Timothy), Béné, M.C., Witte, B.I. (Birgit), Della Porta, M.G. (Matteo), Kern, W. (Wolfgang), and Loosdrecht, A.A. (Arjan) van de

Abstract

Current recommendations for diagnosing myelodysplastic syndromes endorse flow cytometry as an informative tool. Most flow cytometry protocols focus on the analysis of progenitor cells and the evaluation of the maturing myelomonocytic lineage. However, one of the most frequently observed features of myelodysplastic syndromes is anemia, which may be associated with dyserythropoiesis. Therefore, analysis of changes in flow cytometry features of nucleated erythroid cells may complement current flow cytometry tools. The multicenter study within the IMDSFlow Working Group, reported herein, focused on defining flow cytometry parameters that enable discrimination of dyserythropoiesis associated with myelodysplastic syndromes from non-clonal cytopenias. Data from a learning cohort were compared between myelodysplasia and controls, and results were validated in a separate cohort. The learning cohort comprised 245 myelodysplasia cases, 290 pathological, and 142 normal controls; the validation cohort comprised 129 myelodysplasia cases, 153 pathological, and 49 normal controls. Multivariate logistic regression analysis performed in the learning cohort revealed that analysis of expression of CD36 and CD71 (expressed as coefficient of variation), in combination with CD71 fluorescence intensity and the percentage of CD117+ erythroid progenitors provided the best discrimination between myelodysplastic syndromes and non-clonal cytopenias (specificity 90%; 95% confidence interval: 84–94%). The high specificity of this marker set was confirmed in the validation cohort (92%; 95% confidence interval: 86–97%). This erythroid flow cytometry marker combination may improve the evaluation of cytopenic cases with suspected myelodysplasia, particularly when combined with flow cytometry assessment of the myelomonocytic lineage.

Published
2017
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28. Ex vivo identification and characterization of a population of CD13high CD105+ CD45- mesenchymal stem cells in human bone marrow

Author

Muñiz, C. (Carmen), Teodosio, C. (Cristina), Mayado, A. (Andrea), Amaral, A.T. (Ana Teresa), Matarraz, S. (S.), Bárcena, P. (Paloma), Sánchez, M.-L. (M.), Alvarez-Twose, I. (Iván), Díez-Campelo, M. (M.), García-Montero, A.C. (Andrés), Blanco, J.F. (Juan F.), Del Cañizo, M.C. (M.), Del Pino Montes, J. (Javier), Orfao, A. (Alberto), Muñiz, C. (Carmen), Teodosio, C. (Cristina), Mayado, A. (Andrea), Amaral, A.T. (Ana Teresa), Matarraz, S. (S.), Bárcena, P. (Paloma), Sánchez, M.-L. (M.), Alvarez-Twose, I. (Iván), Díez-Campelo, M. (M.), García-Montero, A.C. (Andrés), Blanco, J.F. (Juan F.), Del Cañizo, M.C. (M.), Del Pino Montes, J. (Javier), and Orfao, A. (Alberto)

Abstract

Introduction: Mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and multilineage differentiation. Their multipotential capacity and immunomodulatory properties have led to an increasing interest in their biological properties and therapeutic applications. Currently, the definition of MSCs relies on a combination of phenotypic, morphological and functional characteristics which are typically evaluated upon in vitro expansion, a process that may ultimately lead to modulation of the immunophenotypic, functional and/or genetic features of these cells. Therefore, at present there is great interest in providing markers and phenotypes for direct in vivo and ex vivo identification and isolation of MSCs. Methods: Multiparameter flow cytometry immunophenotypic studies were performed on 65 bone marrow (BM) samples for characterization of CD13high CD105+ CD45- cells. Isolation and expansion of these cells was performed in a subset of samples in parallel to the expansion of MSCs from mononuclear cells following currently established procedures. The protein expres

Published
2015
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29. Ex vivo identification and characterization of a population of CD13(high) CD105(+) CD45(-) mesenchymal stem cells in human bone marrow

Author

Muniz, C, Goncalves Grunho Teodosio, Cristina, Mayado, A, Amaral, AT, Matarraz, S, Barcena, P, Sanchez, ML, Alvarez-Twose, I, Diez-Campelo, M, Garcia-Montero, AC, Blanco, JF, del Canizo, MC, Montes, JDP, Orfao, A, Muniz, C, Goncalves Grunho Teodosio, Cristina, Mayado, A, Amaral, AT, Matarraz, S, Barcena, P, Sanchez, ML, Alvarez-Twose, I, Diez-Campelo, M, Garcia-Montero, AC, Blanco, JF, del Canizo, MC, Montes, JDP, and Orfao, A

Abstract

Introduction: Mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and multilineage differentiation. Their multipotential capacity and immunomodulatory properties have led to an increasing interest in their biological properties and therapeutic applications. Currently, the definition of MSCs relies on a combination of phenotypic, morphological and functional characteristics which are typically evaluated upon in vitro expansion, a process that may ultimately lead to modulation of the immunophenotypic, functional and/or genetic features of these cells. Therefore, at present there is great interest in providing markers and phenotypes for direct in vivo and ex vivo identification and isolation of MSCs. Methods: Multiparameter flow cytometry immunophenotypic studies were performed on 65 bone marrow (BM) samples for characterization of CD13(high) CD105(+) CD45(-) cells. Isolation and expansion of these cells was performed in a subset of samples in parallel to the expansion of MSCs from mononuclear cells following currently established procedures. The protein expression profile of these cells was further assessed on (paired) primary and in vitro expanded BM MSCs, and their adipogenic, chondrogenic and osteogenic differentiation potential was also determined. Results: Our results show that the CD13(high) CD105(+) CD45(-) immunophenotype defines a minor subset of cells that are systematically present ex vivo in normal/reactive BM (n = 65) and that display immunophenotypic features, plastic adherence ability, and osteogenic, adipogenic and chondrogenic differentiation capacities fully compatible with those of MSCs. In addition, we also show that in vitro expansion of these cells modulates their immunophenotypic characteristics, including changes in the expression of markers currently used for the definition of MSCs, such as CD105, CD146 and HLA-DR. Conclusions: BM MSCs can be identified ex vivo in normal/reactive BM, based on a robust CD13(high) CD105(+

Published
2015

30. Increased IL6 plasma levels in indolent systemic mastocytosis patients are associated with high risk of disease progression

Author

Mayado, A, primary, Teodosio, C, additional, Garcia-Montero, A C, additional, Matito, A, additional, Rodriguez-Caballero, A, additional, Morgado, J M, additional, Muñiz, C, additional, Jara-Acevedo, M, additional, Álvarez-Twose, I, additional, Sanchez-Muñoz, L, additional, Matarraz, S, additional, Caldas, C, additional, Muñoz-González, J I, additional, Escribano, L, additional, and Orfao, A, additional

Published
2015
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31. Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes-proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS

Author

Porwit, A., Loosdrecht, A.A. van de, Bettelheim, P., Brodersen, L.E., Burbury, K., Cremers, E., Porta, M.G. Della, Ireland, R., Johansson, U., Matarraz, S., Ogata, K., Orfao, A., Preijers, F.W., Psarra, K., Subirá, D., Valent, P., Velden, V.H. van der, Wells, D., Westers, T.M., Kern, W., Béné, M.C., Porwit, A., Loosdrecht, A.A. van de, Bettelheim, P., Brodersen, L.E., Burbury, K., Cremers, E., Porta, M.G. Della, Ireland, R., Johansson, U., Matarraz, S., Ogata, K., Orfao, A., Preijers, F.W., Psarra, K., Subirá, D., Valent, P., Velden, V.H. van der, Wells, D., Westers, T.M., Kern, W., and Béné, M.C.

Abstract

Item does not contain fulltext

Published
2014

33. 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

34. 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

37. 137 Standardization of flow cytometry in myelodysplastic syndromes: A report from an international consortium and the European LeukemiaNet Working Group

Author

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

Published
2011
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38. CD117 expression in gammopathies is associated with an altered maturation of the myeloid and lymphoid hematopoietic cell compartments and favorable disease features

Author

Schmidt-Hieber, M., primary, Perez-Andres, M., additional, Paiva, B., additional, Flores-Montero, J., additional, Perez, J. J., additional, Gutierrez, N. C., additional, Vidriales, M.-B., additional, Matarraz, S., additional, San Miguel, J. F., additional, and Orfao, A., additional

Published
2010
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39. The immunophenotype of different immature, myeloid and B-cell lineage-committed CD34+ hematopoietic cells allows discrimination between normal/reactive and myelodysplastic syndrome precursors.

Author

Matarraz, S., López, A., Barrena, S., Fernandez, C., Jensen, E., Flores, J., Bárcena, P., Rasillo, A., Sayagues, J. M., Sánchez, M. L., Hernandez-Campo, P., Rivas, J. M. Hernandez, Salvador, C., Fernandez-Mosteirín, N., Giralt, M., Perdiguer, L., and Orfao, A.

Subjects
*MYELOID leukemia, *MYELODYSPLASTIC syndromes, *HEMATOPOIETIC stem cell transplantation, *BONE marrow diseases, *DYSPLASIA
Abstract

Occurrence of phenotypic abnormalities in CD34+ hematopoietic progenitor and precursor cells (HPC) and their major B-cell and nonlymphoid compartments has been frequently reported in myelodysplastic syndromes (MDS). Here, we analyze for the first time the numerical and phenotypic abnormalities of different maturation-associated subsets of bone marrow (BM) CD34+ HPC from 50 newly diagnosed MDS patients in comparison to normal/reactive BM (n=29). Our results confirm the existence of heterogeneously altered phenotypes among CD34+ HPC from MDS and indicate that such variability depends both on the relative distribution of the different subsets of CD34+ HPC committed into the different myeloid and B-lymphoid compartments, and their immunophenotype (for example, higher reactivity for CD117 and CD13 and lower expression of CyMPO, CD64 and CD65 on CD34+ immature and neutrophil precursors), a clear association existing between the accumulation of CD34+ HPC and that of immature CD34+ HPC. Interestingly, expansion of erythroid- and neutrophil-lineage CD34+ cells is detected in low-grade MDS at the expense of CD34+ plasmacytoid dendritic cell and B-cell precursors, while expansion of immature CD34+ precursors occurs in high-grade MDS. On the basis of the number and severity of the phenotypic abnormalities detected, a scoring system is proposed that efficiently discriminates between normal/reactive and MDS CD34+ HPC, the mean score significantly increasing from low- to high-grade MDS.Leukemia (2008) 22, 1175–1183; doi:10.1038/leu.2008.49; published online 13 March 2008 [ABSTRACT FROM AUTHOR]

Published
2008
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40. Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study

Author

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

Subjects
0301 basic medicine, Pathology, medicine.medical_specialty, Standardization, Computer science, Leukaemia, Laboratory techniques and procedures, Article, Immunophenotyping, Pathology and Forensic Medicine, 03 medical and health sciences, 0302 clinical medicine, EuroFlow, Leukocytes, medicine, Humans, Leukaemia, Flow cytometry, Future, Acute leukemia, Orientation (computer vision), business.industry, Laboratory techniques and procedures, Pattern recognition, Flow Cytometry, Peripheral blood, Leukemia, Myeloid, Acute, Identification (information), 030104 developmental biology, Área de Biomedicina, T cell subset, Artificial intelligence, business, Algorithms, 030215 immunology
Abstract

© The Author(s) 2020., 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 ( 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 procedures., The EuroFlow Consortium received support from the FP6- 2004-LIFESCIHEALTH-5 program of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The Prague team received support from the grant number NV18-03-00343. The Salamanca team received support from the Instituto de Salud Carlos III (ISCIII) (PI16/00787-FEDER) and from Agencia Estatal de Investigación (RTC-2016-4865-1-FEDER), Ministerio de Economía y Competitividad, Madrid, Spain. AHD is supported from the program DI-17-09591 from Agencia Estatal de Investigación, Ministerio de Ciencia, Innovación y Universidades, Madrid, Spain. SB is supported from the program PTQ16-08364 from Agencia Estatal de Investigación, Ministerio de Ciencia, Innovación y Universidades, Madrid, Spain. Medical University of Silesia in Katowice team was supported by the Strategmed III PersonALL grant [No. 304586/5/NCBR/2017] from the Polish National Center of Research and Development. The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA).

Published
2021

41. Biological and clinical significance of dysplastic hematopoiesis in patients with newly diagnosed multiple myeloma

Author

Norma C. Gutiérrez, Felipe Prosper, Gorka Alkorta, Rafael Martínez, Felipe de Arriba, José de Jesús Pérez, Francisco Javier Gracia-Aznárez, Ibai Goicoechea, Diego Alignani, Leire Burgos, Noemi Puig, Maria-Teresa Cedena, Juan José Lahuerta, Bruno Paiva, Marcos González, Albert Oriol, Maria-Jose Calasanz, Maria-Victoria Mateos, Raquel de Paz, Alberto Orfao, Rafael Valdés-Mas, Catarina Maia, Jesús F. San Miguel, Juana Merino, Joan Bladé, Joaquin Martinez-Lopez, Sergio Matarraz, Cirino Botta, Laura Rosiñol, M C Chillón, Ana-Isabel Teruel, Miguel T. Hernandez, Luis Palomera, Iria Vázquez, Ramón García-Sanz, Sarai Sarvide, Cristina Moreno, Paula Martínez de Aguirre, Maia C., Puig N., Cedena M.-T., Goicoechea I., Valdes-Mas R., Vazquez I., Chillon M.-C., Aguirre P., Sarvide S., Gracia-Aznarez F.J., Alkorta G., Calasanz M.-J., Garcia-Sanz R., Gonzalez M., Gutierrez N.C., Martinez-Lopez J., Perez J.J., Merino J., Moreno C., Burgos L., Alignani D., Botta C., Prosper F., Matarraz S., Orfao A., Oriol A., Teruel A.-I., de Paz R., de Arriba F., Hernandez M.T., Palomera L., Martinez R., Rosinol L., Mateos M.-V., Lahuerta J.-J., Blade J., San Miguel J.F., and Paiva B.

Subjects
Male, Oncology, Physics::Instrumentation and Detectors, medicine.medical_treatment, Kaplan-Meier Estimate, Hematopoietic stem cell transplantation, Biochemistry, hemic and lymphatic diseases, Antineoplastic Combined Chemotherapy Protocols, Tumor Microenvironment, Prospective Studies, Computer Science::Operating Systems, In Situ Hybridization, Fluorescence, Multiple myeloma, Randomized Controlled Trials as Topic, Computer Science::Cryptography and Security, Hazard ratio, Hematopoietic Stem Cell Transplantation, High-Throughput Nucleotide Sequencing, Hematology, Middle Aged, Flow Cytometry, Prognosis, Combined Modality Therapy, Progression-Free Survival, medicine.anatomical_structure, Female, Clonal Hematopoiesis, Multiple Myeloma, medicine.medical_specialty, Immunology, Transplantation, Autologous, Internal medicine, medicine, Humans, Clinical significance, Progression-free survival, Computer Science::Distributed, Parallel, and Cluster Computing, Aged, Antineoplastic Combined Chemotherapy Protocol, business.industry, Cell Biology, medicine.disease, Transplantation, Clinical Trials, Phase III as Topic, Myelodysplastic Syndromes, Mutation, Bone marrow, business, Monoclonal gammopathy of undetermined significance
Abstract

On behalf of the PETHEMA/GEM Cooperative Group., Risk of developing myelodysplastic syndrome (MDS) is significantly increased in both multiple myeloma (MM) and monoclonal gammopathy of undetermined significance, suggesting that it is therapy independent. However, the incidence and sequelae of dysplastic hematopoiesis at diagnosis are unknown. Here, we used multidimensional flow cytometry (MFC) to prospectively screen for the presence of MDS-associated phenotypic alterations (MDS-PA) in the bone marrow of 285 patients with MM enrolled in the PETHEMA/GEM2012MENOS65 trial (#NCT01916252). We investigated the clinical significance of monocytic MDS-PA in a larger series of 1252 patients enrolled in 4 PETHEMA/GEM protocols. At diagnosis, 33 (11.6%) of 285 cases displayed MDS-PA. Bulk and single-cell–targeted sequencing of MDS recurrently mutated genes in CD34+ progenitors (and dysplastic lineages) from 67 patients revealed clonal hematopoiesis in 13 (50%) of 26 cases with MDS-PA vs 9 (22%) of 41 without MDS-PA; TET2 and NRAS were the most frequently mutated genes. Dynamics of MDS-PA at diagnosis and after autologous transplant were evaluated in 86 of 285 patients and showed that in most cases (69 of 86 [80%]), MDS-PA either persisted or remained absent in patients with or without MDS-PA at diagnosis, respectively. Noteworthy, MDS-associated mutations infrequently emerged after high-dose therapy. Based on MFC profiling, patients with MDS-PA have altered hematopoiesis and T regulatory cell distribution in the tumor microenvironment. Importantly, the presence of monocytic MDS-PA at diagnosis anticipated greater risk of hematologic toxicity and was independently associated with inferior progression-free survival (hazard ratio, 1.5; P = .02) and overall survival (hazard ratio, 1.7; P = .01). This study reveals the biological and clinical significance of dysplastic hematopoiesis in newly diagnosed MM, which can be screened with moderate sensitivity using cost-effective MFC.

Published
2020

42. 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 TA, Van der Velden VH, Szczepanski T, Da Costa ES, Van der Sluijs A, Novakova M, Mejstrikova E, Nierkens S, De Mello FV, Fernandez P, Aanei C, Sędek Ł, Strocchio L, Masetti R, Sainati L, Philippé J, Valsecchi MG, Locatelli F, Van Dongen JJM, Biondi A, Orfao A, and Gaipa G

Subjects
Child, Humans, Flow Cytometry, Antigens, CD34 genetics, Monocytes pathology, Leukemia, Myelomonocytic, Juvenile diagnosis, Leukemia, Myelomonocytic, Juvenile genetics
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-cell 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 versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus 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 eight-color antibody combination.

Published
2024
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43. Baseline immunophenotypic profile of bone marrow leukemia cells in acute myeloid leukemia with nucleophosmin-1 gene mutation: a EuroFlow study.

Author

Matarraz S, Leoz P, Yeguas-Bermejo A, van der Velden V, Bras AE, Sánchez Gallego JI, Lecrevisse Q, Ayala-Bueno R, Teodosio C, Criado I, González-González M, Flores-Montero J, Avendaño A, Vidriales MB, Chillón MC, González T, García-Sanz R, Prieto Conde MI, Villamor N, Magnano L, Colado E, Fernández P, Sonneveld E, Philippé J, Reiterová M, Caballero Berrocal JC, Diaz-Gálvez FJ, Ramos F, Dávila Valls J, Manjón Sánchez R, Solano Tovar J, Calvo X, García Alonso L, Arenillas L, Alonso S, Fonseca A, Quirós Caso C, van Dongen JJM, and Orfao A

Subjects
Humans, Bone Marrow, Mutation, Nucleophosmin, Leukemia, Myeloid, Acute genetics
Published
2023
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44. 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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45. Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies in myelodysplastic syndromes by the ELN iMDS flow working group.

Author

Kern W, Westers TM, Bellos F, Bene MC, Bettelheim P, Brodersen LE, Burbury K, Chu SC, Cullen M, Porta MD, Dunlop AS, Johansson U, Matarraz S, Oelschlaegel U, Ogata K, Porwit A, Preijers F, Psarra K, Saft L, Subirá D, Weiß E, van der Velden VHJ, and van de Loosdrecht A

Subjects
Humans, Flow Cytometry methods, Leukocytes, Immunophenotyping, Myelodysplastic Syndromes diagnosis, Leukemia, Myelomonocytic, Chronic diagnosis
Abstract

Background: Myelodysplastic syndromes (MDS) represent a diagnostic challenge. This prospective multicenter study was conducted to evaluate pre-defined flow cytometric markers in the diagnostic work-up of MDS and chronic myelomonocytic leukemia (CMML)., Methods: Thousand six hundred and eighty-two patients with suspected MDS/CMML were analyzed by both cytomorphology according to WHO 2016 criteria and flow cytometry according to ELN recommendations. Flow cytometric readout was categorized 'non-MDS' (i.e. no signs of MDS/CMML and limited signs of MDS/CMML) and 'in agreement with MDS' (i.e., in agreement with MDS/CMML)., Results: Flow cytometric readout categorized 60% of patients in agreement with MDS, 28% showed limited signs of MDS and 12% had no signs of MDS. In 81% of cases flow cytometric readouts and cytomorphologic diagnosis correlated. For high-risk MDS, the level of concordance was 92%. A total of 17 immunophenotypic aberrancies were found independently related to MDS/CMML in ≥1 of the subgroups of low-risk MDS, high-risk MDS, CMML. A cut-off of ≥3 of these aberrancies resulted in 80% agreement with cytomorphology (20% cases concordantly negative, 60% positive). Moreover, >3% myeloid progenitor cells were significantly associated with MDS (286/293 such cases, 98%)., Conclusion: Data from this prospective multicenter study led to recognition of 17 immunophenotypic markers allowing to identify cases 'in agreement with MDS'. Moreover, data emphasizes the clinical utility of immunophenotyping in MDS diagnostics, given the high concordance between cytomorphology and the flow cytometric readout. Results from the current study challenge the application of the cytomorphologically defined cut-off of 5% blasts for flow cytometry and rather suggest a 3% cut-off for the latter., (© 2022 International Clinical Cytometry Society.)

Published
2023
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46. 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

van de 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, Te Marvelde JG, Matarraz S, Ogata K, Oelschlaegel U, Orfao A, Psarra K, Subirá D, Wells DA, Béné MC, Della Porta MG, Burbury K, Bellos F, van der Velden VHJ, Westers TM, Saft L, and Ireland R

Subjects
Humans, Flow Cytometry, Myelodysplastic Syndromes diagnosis
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., (© 2021 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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47. 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, Béné 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, Subirá D, Wells DA, Della Porta MG, Burbury K, Bellos F, Weiß E, Kern W, and van de Loosdrecht A

Subjects
Humans, Flow Cytometry methods, Reference Standards, Biological Assay, Fluorescent Dyes, Myelodysplastic Syndromes diagnosis
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., (© 2021 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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48. 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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49. Quality Assessment of a Large Multi-Center Flow Cytometric Dataset of Acute Myeloid Leukemia Patients-A EuroFlow Study.

Author

Bras AE, Matarraz S, Nierkens S, Fernández P, Philippé J, Aanei CM, de Mello FV, Burgos L, van der Sluijs-Gelling AJ, Grigore GE, van Dongen JJM, Orfao A, van der Velden VHJ, and On Behalf Of The EuroFlow Consortium

Abstract

Flowcytometric analysis allows for detailed identification and characterization of large numbers of cells in blood, bone marrow, and other body fluids and tissue samples and therefore contributes to the diagnostics of hematological malignancies. Novel data analysis tools allow for multidimensional analysis and comparison of patient samples with reference databases of normal, reactive, and/or leukemia/lymphoma patient samples. Building such reference databases requires strict quality assessment (QA) procedures. Here, we compiled a dataset and developed a QA methodology of the EuroFlow Acute Myeloid Leukemia (AML) database, based on the eight-color EuroFlow AML panel consisting of six different antibody combinations, including four backbone markers. In total, 1142 AML cases and 42 normal bone marrow samples were included in this analysis. QA was performed on 803 AML cases using multidimensional analysis of backbone markers, as well as tube-specific markers, and data were compared using classical analysis employing median and peak expression values. Validation of the QA procedure was performed by re-analysis of >300 cases and by running an independent cohort of 339 AML cases. Initial evaluation of the final cohort confirmed specific immunophenotypic patterns in AML subgroups; the dataset therefore can reliably be used for more detailed exploration of the immunophenotypic variability of AML. Our data show the potential pitfalls and provide possible solutions for constructing large flowcytometric databases. In addition, the provided approach may facilitate the building of other databases and thereby support the development of novel tools for (semi)automated QA and subsequent data analysis.

Published
2022
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50. Immunophenotypic Analysis of Acute Megakaryoblastic Leukemia: A EuroFlow Study.

Author

Brouwer N, Matarraz S, Nierkens S, Hofmans M, Nováková M, da Costa ES, Fernandez P, Bras AE, de Mello FV, Mejstrikova E, Philippé J, Grigore GE, Pedreira CE, van Dongen JJM, Orfao A, van der Velden VHJ, and On Behalf Of The EuroFlow Consortium

Abstract

Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML). We evaluated the immunophenotypic profile of 72 AMKL and 114 non-AMKL AML patients using the EuroFlow AML panel. Univariate and multivariate/multidimensional analyses were performed to identify most relevant markers contributing to the diagnosis of AMKL. AMKL patients were subdivided into transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), AML-not otherwise specified with megakaryocytic differentiation (NOS-AMKL), and AMKL-other patients (AML patients with other WHO classification but with flowcytometric features of megakaryocytic differentiation). Flowcytometric analysis showed good discrimination between AMKL and non-AMKL patients based on differential expression of, in particular, CD42a.CD61, CD41, CD42b, HLADR, CD15 and CD13. Combining CD42a.CD61 (positive) and CD13 (negative) resulted in a sensitivity of 71% and a specificity of 99%. Within AMKL patients, TAM and ML-DS patients showed higher frequencies of immature CD34+/CD117+ leukemic cells as compared to NOS-AMKL and AMKL-Other patients. In addition, ML-DS patients showed a significantly higher expression of CD33, CD11b, CD38 and CD7 as compared to the other three subgroups, allowing for good distinction of these patients. Overall, our data show that the EuroFlow AML panel allows for straightforward diagnosis of AMKL and that ML-DS is associated with a unique immunophenotypic profile.

Published
2022
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