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1. Immunodeficiëntie

Author

de Vries, E., van Dongen, J. J. M., Driessen, G. J. A., Derksen-Lubsen, G., editor, Moll, H.A., editor, Oudesluys-Murphy, A.M., editor, Sprij, A.J., editor, Bolt-Wieringa, J.W., editor, van den Elzen, A.P.M., editor, Leeuwenburgh-Pronk, W.G., editor, Ropers, F.G., editor, and Verhoeven, J.J., editor

Published
2018
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3. Automated EuroFlow approach for standardized in-depth dissection of human circulating B-cells and plasma cells

Author

Delgado, Alejandro H., primary, Fluxa, Rafael, additional, Perez-Andres, Martin, additional, Diks, Annieck M., additional, van Gaans-van den Brink, Jacqueline A. M., additional, Barkoff, Alex-Mikael, additional, Blanco, Elena, additional, Torres-Valle, Alba, additional, Berkowska, Magdalena A., additional, Grigore, Georgiana, additional, van Dongen, J .J .M., additional, and Orfao, Alberto, additional

Published
2023
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5. Minimal residual disease assessment in B-cell precursor acute lymphoblastic leukemia by semi-automated identification of normal hematopoietic cells: A EuroFlow study

Author

Verbeek, M, Rodríguez, B, Sedek, L, Laqua, A, Buracchi, C, Buysse, M, Reiterová, M, Oliveira, E, Morf, D, Oude Alink, S, Barrena, S, Kohlscheen, S, Nierkens, S, Hofmans, M, Fernandez, P, de Costa, E, Mejstrikova, E, Szczepanski, T, Slota, L, Brüggemann, M, Gaipa, G, Grigore, G, van Dongen, J, Orfao, A, van der Velden, V, Verbeek M. W. C., Rodríguez B. S., Sedek L., Laqua A., Buracchi C., Buysse M., Reiterová M., Oliveira E., Morf D., Oude Alink S. R., Barrena S., Kohlscheen S., Nierkens S., Hofmans M., Fernandez P., de Costa E. S., Mejstrikova E., Szczepanski T., Slota L., Brüggemann M., Gaipa G., Grigore G., van Dongen J. J. M., Orfao A., van der Velden V. H. J., Verbeek, M, Rodríguez, B, Sedek, L, Laqua, A, Buracchi, C, Buysse, M, Reiterová, M, Oliveira, E, Morf, D, Oude Alink, S, Barrena, S, Kohlscheen, S, Nierkens, S, Hofmans, M, Fernandez, P, de Costa, E, Mejstrikova, E, Szczepanski, T, Slota, L, Brüggemann, M, Gaipa, G, Grigore, G, van Dongen, J, Orfao, A, van der Velden, V, Verbeek M. W. C., Rodríguez B. S., Sedek L., Laqua A., Buracchi C., Buysse M., Reiterová M., Oliveira E., Morf D., Oude Alink S. R., Barrena S., Kohlscheen S., Nierkens S., Hofmans M., Fernandez P., de Costa E. S., Mejstrikova E., Szczepanski T., Slota L., Brüggemann M., Gaipa G., Grigore G., van Dongen J. J. M., Orfao A., and van der Velden V. H. J.

Abstract

Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating & Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data.

Published
2023

8. Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1

Author

Pfeifer, H., Cazzaniga, G., van der Velden, V. H. J., Cayuela, J. M., Schäfer, B., Spinelli, O., Akiki, S., Avigad, S., Bendit, I., Borg, K., Cavé, H., Elia, L., Reshmi, S. C., Gerrard, G., Hayette, S., Hermanson, M., Juh, A., Jurcek, T., Chillón, M. C., Homburg, C., Martinelli, G., Kairisto, V., Lange, T., Lion, T., Mueller, M. C., Pane, F., Rai, L., Damm-Welk, C., Sacha, T., Schnittger, S., Touloumenidou, T., Valerhaugen, H., Vandenberghe, P., Zuna, J., Serve, H., Herrmann, E., Markovic, S., van Dongen, J. J. M., and Ottmann, O. G.

Published
2020
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9. Differentiation stage of myeloma plasma cells: biological and clinical significance

Author

Paiva, B, Puig, N, Cedena, M T, de Jong, B G, Ruiz, Y, Rapado, I, Martinez-Lopez, J, Cordon, L, Alignani, D, Delgado, J A, van Zelm, M C, Van Dongen, J J M, Pascual, M, Agirre, X, Prosper, F, Martín-Subero, J I, Vidriales, M-B, Gutierrez, N C, Hernandez, M T, Oriol, A, Echeveste, M A, Gonzalez, Y, Johnson, S K, Epstein, J, Barlogie, B, Morgan, G J, Orfao, A, Blade, J, Mateos, M V, Lahuerta, J J, and San-Miguel, J F

Published
2017
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10. Next generation flow for minimally-invasive blood characterization of MGUS and multiple myeloma at diagnosis based on circulating tumor plasma cells (CTPC)

Author

Sanoja-Flores, L., Flores-Montero, J., Garcés, J. J., Paiva, B., Puig, N., García-Mateo, A., García-Sánchez, O., Corral-Mateos, A., Burgos, L., Blanco, E., Hernández-Martín, J., Pontes, R., Díez-Campelo, M., Millacoy, P., Rodríguez-Otero, P., Prosper, F., Merino, J., Vidriales, M. B., García-Sanz, R., Romero, A., Palomera, L., Ríos-Tamayo, R., Pérez-Andrés, M., Blanco, J. F., González, M., van Dongen, J. J. M., Durie, B., Mateos, M. V., San-Miguel, J., Orfao, A., and on behalf of the EuroFlow consortium

Published
2018
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11. Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies — a EuroFlow study

Author

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

Abstract

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

Published
2022

12. Impact of Pre-Analytical and Analytical Variables Associated with Sample Preparation on Flow Cytometric Stainings Obtained with EuroFlow Panels

Author

Sedek, L, Flores-Montero, J, van der Sluijs, A, Kulis, J, Marvelde, J, Philippe, J, Bottcher, S, Bitter, M, Caetano, J, van der Velden, V, Sonneveld, E, Buracchi, C, Santos, A, Lima, M, Szczepanski, T, van Dongen, J, Orfao, A, Sedek L., Flores-Montero J., van der Sluijs A., Kulis J., Marvelde J. T., Philippe J., Bottcher S., Bitter M., Caetano J., van der Velden V. H. J., Sonneveld E., Buracchi C., Santos A. H., Lima M., Szczepanski T., van Dongen J. J. M., Orfao A., Sedek, L, Flores-Montero, J, van der Sluijs, A, Kulis, J, Marvelde, J, Philippe, J, Bottcher, S, Bitter, M, Caetano, J, van der Velden, V, Sonneveld, E, Buracchi, C, Santos, A, Lima, M, Szczepanski, T, van Dongen, J, Orfao, A, Sedek L., Flores-Montero J., van der Sluijs A., Kulis J., Marvelde J. T., Philippe J., Bottcher S., Bitter M., Caetano J., van der Velden V. H. J., Sonneveld E., Buracchi C., Santos A. H., Lima M., Szczepanski T., van Dongen J. J. M., and Orfao A.

Abstract

Objective interpretation of FC results may still be hampered by limited technical stan-dardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients’ samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA-vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B-and T-cells (but not on leukemic blasts, clonal B-and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein.

Published
2022

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

Author

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

Abstract

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

Published
2022

14. POS0185 BELIMUMAB DISRUPTS MEMORY B-CELL TRAFFICKING IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

Author

Arends, E. J., primary, Zlei, M., additional, Tipton, C. M., additional, Cotic, J., additional, Osmani, Z., additional, de Bie, F., additional, Kamerling, S., additional, van Maurik, A., additional, Dimelow, R., additional, Gregan, Y. I., additional, Fox, N. L., additional, Rabelink, T., additional, Roth, D., additional, Sanz, I., additional, van Dongen, J. J. M., additional, van Kooten, C., additional, and Teng, Y. K. O., additional

Published
2022
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16. Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1 (Leukemia, (2019), 33, 8, (1910-1922), 10.1038/s41375-019-0413-0)

Author

Pfeifer H., Cazzaniga G., van der Velden V. H. J., Cayuela J. M., Schafer B., Spinelli O., Akiki S., Avigad S., Bendit I., Borg K., Cave H., Elia L., Reshmi S. C., Gerrard G., Hayette S., Hermanson M., Juh A., Jurcek T., Chillon M. C., Homburg C., Martinelli G., Kairisto V., Lange T., Lion T., Mueller M. C., Pane F., Rai L., Damm-Welk C., Sacha T., Schnittger S., Touloumenidou T., Valerhaugen H., Vandenberghe P., Zuna J., Serve H., Herrmann E., Markovic S., van Dongen J. J. M., Ottmann O. G., Pfeifer, H., Cazzaniga, G., van der Velden, V. H. J., Cayuela, J. M., Schafer, B., Spinelli, O., Akiki, S., Avigad, S., Bendit, I., Borg, K., Cave, H., Elia, L., Reshmi, S. C., Gerrard, G., Hayette, S., Hermanson, M., Juh, A., Jurcek, T., Chillon, M. C., Homburg, C., Martinelli, G., Kairisto, V., Lange, T., Lion, T., Mueller, M. C., Pane, F., Rai, L., Damm-Welk, C., Sacha, T., Schnittger, S., Touloumenidou, T., Valerhaugen, H., Vandenberghe, P., Zuna, J., Serve, H., Herrmann, E., Markovic, S., van Dongen, J. J. M., and Ottmann, O. G.

Abstract

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Published
2020

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

Author

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

Abstract

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

Published
2021

20. Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph plus ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1

Author

Pfeifer, H., Cazzaniga, G., van der Velden, V. H. J., Cayuele, J. M., Schafer, B., Spinelli, O., Akiki, S., Avigad, S., Bendit, I, Borg, K., Cave, H., Elia, L., Reshmi, S. C., Gerrard, G., Hayette, S., Hermansson, Monica, Juh, A., Jurcek, T., Chillon, M. C., Homburg, C., Martinelli, G., Kairisto, V, Langen, T., Lion, T., Mueller, M. C., Pane, F., Rai, L., Damm-Welk, C., Sacha, T., Schnittger, S., Touloumenidou, T., Valerhaugen, H., Vandenberghe, P., Zuna, J., Server, H., Herrmann, E., Markovic, S., van Dongen, J. J. M., Ottmann, O. G., Pfeifer, H., Cazzaniga, G., van der Velden, V. H. J., Cayuele, J. M., Schafer, B., Spinelli, O., Akiki, S., Avigad, S., Bendit, I, Borg, K., Cave, H., Elia, L., Reshmi, S. C., Gerrard, G., Hayette, S., Hermansson, Monica, Juh, A., Jurcek, T., Chillon, M. C., Homburg, C., Martinelli, G., Kairisto, V, Langen, T., Lion, T., Mueller, M. C., Pane, F., Rai, L., Damm-Welk, C., Sacha, T., Schnittger, S., Touloumenidou, T., Valerhaugen, H., Vandenberghe, P., Zuna, J., Server, H., Herrmann, E., Markovic, S., van Dongen, J. J. M., and Ottmann, O. G.

Abstract

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10(-3) and 36/67 (53%) and 53/67 (79%) at 10(-4)BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

Published
2019
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21. Quality control and quantification in IG/TR next-generation sequencing marker identification: protocols and bioinformatic functionalities by EuroClonality-NGS

Author

Knecht, H, Reigl, T, Kotrova, M, Appelt, F, Stewart, P, Bystry, V, Krejci, A, Grioni, A, Pal, K, Stranska, K, Plevova, K, Rijntjes, J, Songia, S, Svaton, M, Fronkova, E, Bartram, J, Scheijen, B, Herrmann, D, Garcia-Sanz, R, Hancock, J, Moppett, J, van Dongen, J, Cazzaniga, G, Davi, F, Groenen, P, Hummel, M, Macintyre, E, Stamatopoulos, K, Trka, J, Langerak, A, Gonzalez, D, Pott, C, Bruggemann, M, Darzentas, N, Knecht H., Reigl T., Kotrova M., Appelt F., Stewart P., Bystry V., Krejci A., Grioni A., Pal K., Stranska K., Plevova K., Rijntjes J., Songia S., Svaton M., Fronkova E., Bartram J., Scheijen B., Herrmann D., Garcia-Sanz R., Hancock J., Moppett J., van Dongen J. J. M., Cazzaniga G., Davi F., Groenen P. J. T. A., Hummel M., Macintyre E. A., Stamatopoulos K., Trka J., Langerak A. W., Gonzalez D., Pott C., Bruggemann M., Darzentas N., Knecht, H, Reigl, T, Kotrova, M, Appelt, F, Stewart, P, Bystry, V, Krejci, A, Grioni, A, Pal, K, Stranska, K, Plevova, K, Rijntjes, J, Songia, S, Svaton, M, Fronkova, E, Bartram, J, Scheijen, B, Herrmann, D, Garcia-Sanz, R, Hancock, J, Moppett, J, van Dongen, J, Cazzaniga, G, Davi, F, Groenen, P, Hummel, M, Macintyre, E, Stamatopoulos, K, Trka, J, Langerak, A, Gonzalez, D, Pott, C, Bruggemann, M, Darzentas, N, Knecht H., Reigl T., Kotrova M., Appelt F., Stewart P., Bystry V., Krejci A., Grioni A., Pal K., Stranska K., Plevova K., Rijntjes J., Songia S., Svaton M., Fronkova E., Bartram J., Scheijen B., Herrmann D., Garcia-Sanz R., Hancock J., Moppett J., van Dongen J. J. M., Cazzaniga G., Davi F., Groenen P. J. T. A., Hummel M., Macintyre E. A., Stamatopoulos K., Trka J., Langerak A. W., Gonzalez D., Pott C., Bruggemann M., and Darzentas N.

Abstract

Assessment of clonality, marker identification and measurement of minimal residual disease (MRD) of immunoglobulin (IG) and T cell receptor (TR) gene rearrangements in lymphoid neoplasms using next-generation sequencing (NGS) is currently under intensive development for use in clinical diagnostics. So far, however, there is a lack of suitable quality control (QC) options with regard to standardisation and quality metrics to ensure robust clinical application of such approaches. The EuroClonality-NGS Working Group has therefore established two types of QCs to accompany the NGS-based IG/TR assays. First, a central polytarget QC (cPT-QC) is used to monitor the primer performance of each of the EuroClonality multiplex NGS assays; second, a standardised human cell line-based DNA control is spiked into each patient DNA sample to work as a central in-tube QC and calibrator for MRD quantification (cIT-QC). Having integrated those two reference standards in the ARResT/Interrogate bioinformatic platform, EuroClonality-NGS provides a complete protocol for standardised IG/TR gene rearrangement analysis by NGS with high reproducibility, accuracy and precision for valid marker identification and quantification in diagnostics of lymphoid malignancies.

Published
2019

22. Standardized next-generation sequencing of immunoglobulin and T-cell receptor gene recombinations for MRD marker identification in acute lymphoblastic leukaemia; a EuroClonality-NGS validation study

Author

Bruggemann, M, Kotrova, M, Knecht, H, Bartram, J, Boudjogrha, M, Bystry, V, Fazio, G, Fronkova, E, Giraud, M, Grioni, A, Hancock, J, Herrmann, D, Jimenez, C, Krejci, A, Moppett, J, Reigl, T, Salson, M, Scheijen, B, Schwarz, M, Songia, S, Svaton, M, van Dongen, J, Villarese, P, Wakeman, S, Wright, G, Cazzaniga, G, Davi, F, Garcia-Sanz, R, Gonzalez, D, Groenen, P, Hummel, M, Macintyre, E, Stamatopoulos, K, Pott, C, Trka, J, Darzentas, N, Langerak, A, Bruggemann M., Kotrova M., Knecht H., Bartram J., Boudjogrha M., Bystry V., Fazio G., Fronkova E., Giraud M., Grioni A., Hancock J., Herrmann D., Jimenez C., Krejci A., Moppett J., Reigl T., Salson M., Scheijen B., Schwarz M., Songia S., Svaton M., van Dongen J. J. M., Villarese P., Wakeman S., Wright G., Cazzaniga G., Davi F., Garcia-Sanz R., Gonzalez D., Groenen P. J. T. A., Hummel M., Macintyre E. A., Stamatopoulos K., Pott C., Trka J., Darzentas N., Langerak A. W., Bruggemann, M, Kotrova, M, Knecht, H, Bartram, J, Boudjogrha, M, Bystry, V, Fazio, G, Fronkova, E, Giraud, M, Grioni, A, Hancock, J, Herrmann, D, Jimenez, C, Krejci, A, Moppett, J, Reigl, T, Salson, M, Scheijen, B, Schwarz, M, Songia, S, Svaton, M, van Dongen, J, Villarese, P, Wakeman, S, Wright, G, Cazzaniga, G, Davi, F, Garcia-Sanz, R, Gonzalez, D, Groenen, P, Hummel, M, Macintyre, E, Stamatopoulos, K, Pott, C, Trka, J, Darzentas, N, Langerak, A, Bruggemann M., Kotrova M., Knecht H., Bartram J., Boudjogrha M., Bystry V., Fazio G., Fronkova E., Giraud M., Grioni A., Hancock J., Herrmann D., Jimenez C., Krejci A., Moppett J., Reigl T., Salson M., Scheijen B., Schwarz M., Songia S., Svaton M., van Dongen J. J. M., Villarese P., Wakeman S., Wright G., Cazzaniga G., Davi F., Garcia-Sanz R., Gonzalez D., Groenen P. J. T. A., Hummel M., Macintyre E. A., Stamatopoulos K., Pott C., Trka J., Darzentas N., and Langerak A. W.

Abstract

Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) was spiked into each sample as a library-specific quality control and calibrator. NGS identified 259 (average 5.2/sample, range 0–14) clonal sequences vs. Sanger-sequencing 248 (average 5.0/sample, range 0–14). NGS primers covered possible IG/TR rearrangement types more completely compared with local multiplex PCR sets and enabled sequencing of bi-allelic rearrangements and weak PCR products. The cPT-QC showed high reproducibility across all laboratories. These validated and reproducible quality-controlled EuroClonality-NGS assays can be used for standardized NGS-based identification of IG/TR markers in lymphoid malignancies.

Published
2019

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

Author

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

Abstract

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

Published
2017

24. Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma

Author

Flores-Montero, J, primary, Sanoja-Flores, L, additional, Paiva, B, additional, Puig, N, additional, García-Sánchez, O, additional, Böttcher, S, additional, van der Velden, V H J, additional, Pérez-Morán, J-J, additional, Vidriales, M-B, additional, García-Sanz, R, additional, Jimenez, C, additional, González, M, additional, Martínez-López, J, additional, Corral-Mateos, A, additional, Grigore, G-E, additional, Fluxá, R, additional, Pontes, R, additional, Caetano, J, additional, Sedek, L, additional, del Cañizo, M-C, additional, Bladé, J, additional, Lahuerta, J-J, additional, Aguilar, C, additional, Bárez, A, additional, García-Mateo, A, additional, Labrador, J, additional, Leoz, P, additional, Aguilera-Sanz, C, additional, San-Miguel, J, additional, Mateos, M-V, additional, Durie, B, additional, van Dongen, J J M, additional, and Orfao, A, additional

Published
2017
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25. Germline activating TYK2 mutations in pediatric patients with two primary acute lymphoblastic leukemia occurrences

Author

Waanders, E, primary, Scheijen, B, additional, Jongmans, M C J, additional, Venselaar, H, additional, van Reijmersdal, S V, additional, van Dijk, A H A, additional, Pastorczak, A, additional, Weren, R D A, additional, van der Schoot, C E, additional, van de Vorst, M, additional, Sonneveld, E, additional, Hoogerbrugge, N, additional, van der Velden, V H J, additional, Gruhn, B, additional, Hoogerbrugge, P M, additional, van Dongen, J J M, additional, Geurts van Kessel, A, additional, van Leeuwen, F N, additional, and Kuiper, R P, additional

Published
2016
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26. IgE‐expressing memory B cells and plasmablasts are increased in blood of children with asthma, food allergy, and atopic dermatitis.

Author

Heeringa, J. J., Rijvers, L., Arends, N. J., Driessen, G. J., Pasmans, S. G., van Dongen, J. J. M., de Jongste, J. C., and van Zelm, M. C.

Subjects
IMMUNOGLOBULIN E, B cells, MEMORY, ASTHMA in children, FOOD allergy in children, ATOPIC dermatitis, BLOOD
Abstract

Abstract: Despite the critical role of soluble IgE in the pathology of IgE‐mediated allergic disease, little is known about abnormalities in the memory B cells and plasma cells that produce IgE in allergic patients. We here applied a flow cytometric approach to cross‐sectionally study blood IgE+ memory B cells and plasmablasts in 149 children with atopic dermatitis, food allergy, and/or asthma and correlated these to helper T(h)2 cells and eosinophils. Children with allergic disease had increased numbers of IgE+CD27‐ and IgE+CD27+ memory B cells and IgE+ plasmablasts, as well as increased numbers of eosinophils and Th2 cells. IgE+ plasmablast numbers correlated positively with Th2 cell numbers. These findings open new possibilities for diagnosis and monitoring of treatment in patients with allergic diseases. [ABSTRACT FROM AUTHOR]

Published
2018
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27. Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia

Author

Lhermitte, L, Mejstrikova, E, van der Sluijs-Gelling, A J, Grigore, G E, Sedek, L, Bras, A E, Gaipa, G, Sobral da Costa, E, Novakova, M, Sonneveld, E, Buracchi, C, de Sá Bacelar, T, te Marvelde, J G, Trinquand, A, Asnafi, V, Szczepanski, T, Matarraz, S, Lopez, A, Vidriales, B, Bulsa, J, Hrusak, O, Kalina, T, Lecrevisse, Q, Martin Ayuso, M, Brüggemann, M, Verde, J, Fernandez, P, Burgos, L, Paiva, B, Pedreira, C E, van Dongen, J J M, Orfao, A, and van der Velden, V H J

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
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28. Germline activating TYK2mutations in pediatric patients with two primary acute lymphoblastic leukemia occurrences

Author

Waanders, E, Scheijen, B, Jongmans, M C J, Venselaar, H, van Reijmersdal, S V, van Dijk, A H A, Pastorczak, A, Weren, R D A, van der Schoot, C E, van de Vorst, M, Sonneveld, E, Hoogerbrugge, N, van der Velden, V H J, Gruhn, B, Hoogerbrugge, P M, van Dongen, J J M, Geurts van Kessel, A, van Leeuwen, F N, and Kuiper, R P

Abstract

The contribution of genetic predisposing factors to the development of pediatric acute lymphoblastic leukemia (ALL), the most frequently diagnosed cancer in childhood, has not been fully elucidated. Children presenting with multiple de novoleukemias are more likely to suffer from genetic predisposition. Here, we selected five of these patients and analyzed the mutational spectrum of normal and malignant tissues. In two patients, we identified germline mutations in TYK2, a member of the JAK tyrosine kinase family. These mutations were located in two adjacent codons of the pseudokinase domain (p.Pro760Leu and p.Gly761Val). In silicomodeling revealed that both mutations affect the conformation of this autoregulatory domain. Consistent with this notion, both germline mutations promote TYK2 autophosphorylation and activate downstream STAT family members, which could be blocked with the JAK kinase inhibitor I. These data indicate that germline activating TYK2 mutations predispose to the development of ALL.

Published
2017
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29. Basal Ca2+ signaling is particularly increased in mutated chronic lymphocytic leukemia.

Author

Muggen, A F, Pillai, S Y, Kil, L P, van Zelm, M C, van Dongen, J J M, Hendriks, R W, and Langerak, A W

Subjects
CHRONIC lymphocytic leukemia, ANTIGEN receptors, CARCINOGENESIS, IMMUNOGLOBULINS, EPITOPES
Abstract

On the basis of somatic hypermutation status of their B-cell antigen receptor (BCR) genes, chronic lymphocytic leukemia (CLL) patients can be divided into unmutated CLL (U-CLL) or mutated CLL (M-CLL). Approximately 30% of CLL patients express a stereotypic BCR, which may indicate that specific antigenic stimulation is driving CLL pathogenesis. Recently, it was reported that BCRs from CLL cells are capable of antigen-independent, cell-autonomous signaling, through recognition of an internal framework 2 (FR2) BCR epitope. We hypothesized that the level of cell-autonomous signaling may differ between CLL subgroups. Therefore, we analyzed Ca2+ signaling in a series of primary stereotypic or heterogeneous U-CLL and M-CLL (n=68) and healthy controls (n=14). We confirmed that basal Ca2+ signaling in CLL cells is higher than in normal B cells. Interestingly, we found that basal signaling was particularly increased in M-CLL. The degree of basal signaling did not correlate with membrane immunoglobulin levels, HCDR3 characteristics or FR2/FR3 sequence. We conclude that the level of basal Ca2+ signaling is not uniformly enhanced in CLL B cells, but is associated with CLL immunoglobulin heavy chain V mutational status, reflecting a distinct cellular origin and possibly a different anergic state induced by repetitive or continuous antigen binding in vivo. [ABSTRACT FROM AUTHOR]

Published
2015
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30. Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research.

Author

Diks AM, Bonroy C, Teodosio C, Groenland RJ, de Mooij B, de Maertelaere E, Neirynck J, Philippé J, Orfao A, van Dongen JJM, and Berkowska MA

Subjects
Blood Preservation standards, Flow Cytometry standards, Humans, Immunophenotyping standards, Multicenter Studies as Topic, Reproducibility of Results, Specimen Handling standards, Blood Preservation methods, Flow Cytometry methods, Immunophenotyping methods, Specimen Handling methods
Abstract

Obtaining reliable and reproducible high quality data in multicenter clinical research settings requires design of optimal standard operating procedures. While the need for standardization in sample processing and data analysis is well-recognized, the impact of sample handling in the pre-analytical phase remains underestimated. We evaluated the impact of sample storage time (≈transport time) and temperature, type of anticoagulant, and limited blood volume on reproducibility of flow cytometric studies. EDTA and Na-Heparin samples processed with the EuroFlow bulk lysis protocol, stained and stored at 4 °C showed fairly stable expression of cell surface markers and distribution of the major leukocyte populations for up to 72 h. Additional sample fixation (1% PFA, Fix & Perm) did not have any beneficial effects. Blood samples stored for <24 h at room temperature before processing and staining seemed suitable for reliable immunophenotyping, although losses in absolute cell numbers were observed. The major losses were observed in myeloid cells and monocytes, while lymphocytes seemed less affected. Expression of cell surface markers and population distribution were more stable in Na-Heparin blood than in EDTA blood. However, storage of Na-Heparin samples was associated with faster decrease in leukocyte counts over time. Whole blood fixation strategies (Cyto-Chex, TransFix) improved long-term population distribution, but were detrimental for expression of cellular markers. The main conclusions from this study on healthy donor blood samples were successfully confirmed in EDTA clinical (patient) blood samples with different time delays until processing. Finally, we recognized the need for adjustments in bulk lysis in case of insufficient blood volumes. Despite clear overall conclusions, individual markers and cell populations had different preferred conditions. Therefore, specific guidelines for sample handling should always be adjusted to the clinical application and the main target leukocyte population., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Published
2019
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31. From big flow cytometry datasets to smart diagnostic strategies: The EuroFlow approach.

Author

Pedreira CE, Costa ESD, Lecrevise Q, Grigore G, Fluxa R, Verde J, Hernandez J, van Dongen JJM, and Orfao A

Subjects
Humans, Big Data, Datasets as Topic, Flow Cytometry methods, Immunophenotyping methods
Abstract

The rise in the analytical speed of mutiparameter flow cytometers made possible by the introduction of digital instruments, has brought up the possibility to manage progressively higher number of parameters simultaneously on significantly greater numbers of individual cells. This has led to an exponential increase in the complexity and volume of flow cytometry data generated about cells present in individual samples evaluated in a single measurement. This increase demands for new developments in flow cytometry data analysis, graphical representation, and visualization and interpretation tools to address the new big data challenges, i.e. processing data files of ≥10-25 parameters per cell in samples with >5-10 million cells (= up to 250 million data points per cell sample) obtained in a few minutes. Here, we present a comprehensive review of some of the tools developed by the EuroFlow consortium for processing flow cytometric big data files in diagnostic laboratories, particularly focused on automated EuroFlow approaches for: i) identification of all cell populations coexisting in a sample (automated gating); ii) smart classification of aberrant cell populations in routine diagnostics; iii) automated reporting; together with iv) new tools developed to visualize n-dimensional data in 2-dimensional plots to support expert-guided automated data analysis. The concept of using reference data bases implemented into software programs, in combination with multivariate statistical analysis pioneered by EuroFlow, provides an innovative, highly efficient and fast approach for diagnostic screening, classification and monitoring of patients with distinct hematological and immune disorders, as well as other diseases., (Copyright © 2019. Published by Elsevier B.V.)

Published
2019
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