Harmonization of light scatter and fluorescence flow cytometry profiles obtained after staining peripheral blood leucocytes for cell surface‐only versus intracellular antigens with the Fix &amp; Perm™ reagent

Costa, Elaine Sobral da; Peres, Rodrigo Tosta; Almeida, Júlia; Lécrevisse, Quentin; Arroyo, María Elena; Teodósio, Cristina; Pedreira, Carlos Eduardo; Dongen, Jacques J. M. van; Órfão, Alberto

doi:10.1002/cyto.b.20486

Cited by 16 publications

(9 citation statements)

References 30 publications

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“…33,64 The EuroFlow Consortium works on full standardization of instrument setups, sample preparation, immunostaining procedures, fluorochromes and eight-color antibody panels, as well as bioinformatics-assisted expert independent automated analysis of the acquired data. [65][66][67] The major advantage of FCM is its rapidity, which allows result reporting within 1 day. This is specifically useful when MRD results are required quickly to guide therapy.…”

Section: Multiparameter Fcmmentioning

confidence: 99%

Standardized MRD quantification in European ALL trials: Proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18–20 September 2008

et al. 2009

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Assessment of minimal residual disease (MRD) has acquired a prominent position in European treatment protocols for patients with acute lymphoblastic leukemia (ALL), on the basis of its high prognostic value for predicting outcome and the possibilities for implementation of MRD diagnostics in treatment stratification. Therefore, there is an increasing need for standardization of methodologies and harmonization of terminology. For this purpose, a panel of representatives of all major European study groups on childhood and adult ALL and of international experts on PCR-and flow cytometry-based MRD assessment was built in the context of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008. The panel summarized the current state of MRD diagnostics in ALL and developed recommendations on the minimal technical requirements that should be fulfilled before implementation of MRD diagnostics into clinical trials. Finally, a common terminology for a standard description of MRD response and monitoring was established defining the terms 'complete MRD response', 'MRD persistence' and 'MRD reappearance'. The proposed MRD terminology may allow a refined and standardized assessment of response to treatment in adult and childhood ALL, and provides a sound basis for the comparison of MRD results between different treatment protocols.

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Section: Multiparameter Fcmmentioning

confidence: 99%

Standardized MRD quantification in European ALL trials: Proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18–20 September 2008

et al. 2009

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“…Such tools were developed by the EuroFlow group and they proved to be essential for the critical evaluation and (re)design of the antibody combinations. 31, 32, 33 Among the new tools, reference data files of normal and distinct disease entities were built and the panels evaluated through paired multidimensional statistical comparisons of such normal versus disease-specific data files, as well as of data files corresponding to different well-characterized leukemia/lymphoma entities (Figure 2). In this way we could objectively evaluate the overall performance of the proposed panels for answering the specific clinical questions.…”

Section: Introductionmentioning

confidence: 99%

“…Each tube should be treated with the appropriate sample preparation protocol for staining of surface markers or surface plus intracellular markers. The different impact of the pre-analytical steps on light scatter parameters and fluorochrome intensities of backbone markers is automatically recognized and adjusted by the software (harmonization process), 33 allowing merging and calculation of marker expression based on parameters measured under different preanalytical conditions. This approach has been validated in B-CLPD 33 as well as on a series of 9 BCP-ALL samples using the BCP-ALL panel (data not shown).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2012

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Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2–7 sequential design–evaluation–redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies.

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“…Therefore, variations in the FSC/SSC values or in the fluorescence levels of the backbone markers may occur because of the different sample preparation procedures (Table 9). In order to allow the calculation process when cells are treated with different staining protocols, a harmonization procedure was developed 56 and applied to those cell populations of interest, for all parameters measured in common in the different sample aliquots, which are prepared differently (Figure 11). Such harmonization process consists of the translation of a data matrix defined in a tube by a given set of parameters for a given cell population into a data matrix defined by the same parameters for the same cell population measured in another tube under different conditions (for example, surface versus surface plus intracellular stainings).…”

Section: Introductionmentioning

confidence: 99%

EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols

et al. 2012

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The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database.

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Harmonization of light scatter and fluorescence flow cytometry profiles obtained after staining peripheral blood leucocytes for cell surface‐only versus intracellular antigens with the Fix & Perm™ reagent

Cited by 16 publications

References 30 publications

Standardized MRD quantification in European ALL trials: Proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18–20 September 2008

Standardized MRD quantification in European ALL trials: Proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18–20 September 2008

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

EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols

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