EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols

Abstract

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.

Multiparametric Whole Blood Dissection: A one‐shot comprehensive picture of the human hematopoietic system

“…Over the past three decades, flow cytometry has been widely applied for immune‐phenotyping in clinical and research settings 1, 2, 29, 30, 47, 65, 66, 67, 68, 69, 70. A 17‐multiparamentric assay has been developed in the past for analyzing in details PB mature cells 68.…”

Analysis of the immune system of multiple myeloma patients achieving long-term disease control by multidimensional flow cytometry

“…17,[19][20][21] In all cases, data were acquired in a FACSCanto II flow cytometer (BDB) using the FACSDiva software (version 6.1, BDB) for ≥ 10 6 leukocytes/tube using the EuroFlow instrument setup data acquisition standard operating procedures. 22 INFINI-CYT software (Cytognos SL, Salamanca, Spain) was used for the data analysis.…”

Determinación de la clonalidad en tejidos humanos

“…El consorcio Euroflow, conformado en parte por los mismos laboratorios del consorcio BIOMED-2, propone un panel de 24 anticuerpos, que permiten determinar el 70% de los dominios del TCRβ (21). Según Lima y colaboradores (22), este análisis citométri-co es una buena herramienta de tamización para la clonalidad de lesiones linfoides de linaje T en especial en el caso de la leucemia de células grandes granulares.…”

“…Sin embargo, los primers consensus que propone el consorcio reconocen secuencias conservadas entre los fragmentos y permiten detectar la mayor parte de combinaciones posibles. El protocolo de amplificación permite la utilización de cualquier termociclador (7,21).…”