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Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research.
Diks, A M; Bonroy, C; Teodosio, C; Groenland, R J; de Mooij, B; de Maertelaere, E; Neirynck, J; Philippé, J; Orfao, A; van Dongen, J J M; Berkowska, M A.
Afiliación
  • Diks AM; Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, the Netherlands.
  • Bonroy C; Department of Diagnostic Sciences, Ghent University, Ghent, Belgium; Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium.
  • Teodosio C; Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, the Netherlands.
  • Groenland RJ; Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, the Netherlands.
  • de Mooij B; Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, the Netherlands.
  • de Maertelaere E; Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium.
  • Neirynck J; Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium.
  • Philippé J; Department of Diagnostic Sciences, Ghent University, Ghent, Belgium; Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium.
  • Orfao A; Cancer Research Centre (IBMCC, USAL-CSIC; CIBERONC CB16/12/00400), Institute for Biomedical Research of Salamanca (IBSAL), Department of Medicine and Cytometry Service (NUCLEUS Research Support Platform), University of Salamanca (USAL), Salamanca, Spain.
  • van Dongen JJM; Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, the Netherlands. Electronic address: J.J.M.van_Dongen@lumc.nl.
  • Berkowska MA; Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, the Netherlands.
J Immunol Methods ; 475: 112616, 2019 12.
Article en En | MEDLINE | ID: mdl-31181213
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.
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Texto completo: 1 Bases de datos: MEDLINE Asunto principal: Manejo de Especímenes / Conservación de la Sangre / Inmunofenotipificación / Citometría de Flujo Tipo de estudio: Clinical_trials / Guideline Límite: Humans Idioma: En Revista: J Immunol Methods Año: 2019 Tipo del documento: Article País de afiliación: Países Bajos

Texto completo: 1 Bases de datos: MEDLINE Asunto principal: Manejo de Especímenes / Conservación de la Sangre / Inmunofenotipificación / Citometría de Flujo Tipo de estudio: Clinical_trials / Guideline Límite: Humans Idioma: En Revista: J Immunol Methods Año: 2019 Tipo del documento: Article País de afiliación: Países Bajos