The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

Santos, Ana Paula dos; Bertho, Álvaro Luiz; Martins, Reinaldo de Menezes; Marcovistz, Rugimar

doi:10.1590/s0074-02762007000100020

Cited by 7 publications

(4 citation statements)

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“…In one published study evaluating time delay effects on lymphocyte populations, the authors found more cellular debris after 48 h with both whole blood lysis and density gradient methods, and loss of T lymphocyte subsets with density gradient separation. The investigators attributed T cell subset changes to red blood cell (RBC) and cellular debris contamination (6). Another of the studies standardizing PBMC collection concluded that time between collection and isolation was the variable that contributed most significantly to diminished T cell function.…”

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confidence: 99%

Stability of T cell phenotype and functional assays following heparinized umbilical cord blood collection

et al. 2012

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Umbilical cord blood has been used for a wide variety of immunologic investigations including assessments of developmental perturbations by antenatal exposures. Recent advances in multiparameter flow cytometry have allowed finer characterization of lymphocyte phenotype and function, revealing important differences between the fetal and adult immune systems. The degree of variability between human subjects confounds the ability to draw firm conclusions. Artifacts resulting from processing techniques exacerbate this variability. The unpredictable nature of deliveries, especially of premature infants, makes it difficult to control variables such as timing of umbilical cord mononuclear cell (UCMC) isolation and method of collection. Additionally, in multicenter studies dependent on central processing, delays are inevitable. However, little available literature describes systematic testing of the degree to which processing variations affect UCMC phenotype and function. Using multiparameter flow cytometry, we tested the effect of collection technique and length of time prior to UCMC isolation on T cell phenotype and function, with the goal of creating a standardized operating procedure for a multicenter investigation. The study also provides a benchmark data set including extensive surface and functional phenotyping of umbilical cord T cells. UCMC isolation delay of up to 24 h produced similar T cell phenotype and function as tested by in vitro SEB stimulation. There were few statistically significant differences between time points based on data medians. We conclude that, for the purpose of immunologic investigations, a 24-h time delay from sample collection to mononuclear cell isolation does not introduce a significant degree of variation in T cell phenotype and function when adhering to strict standard operating procedures.

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confidence: 99%

Stability of T cell phenotype and functional assays following heparinized umbilical cord blood collection

et al. 2012

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“…Despite a preliminary stability test having confirmed that delayed staining and acquisition of WB samples may generate artefacts in some population frequency evaluation [12], we set up WB staining 24 hours after blood withdrawn and sample acquisition on the following day. This procedure allowed simulating a realistic sample management in a multicentre study [42,43] and also fulfilled specific needs of some involved laboratories. Several reports used blood stabilisers in order to delay sample staining and acquisition, with different outcomes depending on analysed markers [44].…”

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confidence: 99%

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring

Macchia

Sorsa²,

Ruspantini³

et al. 2020

Journal of Immunology Research

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Background. Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intraand interoperator variabilities in a multicentre project. Methods. The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. Results. Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of crosscentre variability for naïve/memory subset frequencies particularly in the whole blood setting. Conclusion. Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

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“… The new seed lot is safe. dos Santos et al (2007) Study of lymphocyte subpopulations according to the time of processing of blood samples. Flow cytometry by the lysis method may be done at times 0, 24 or 48 h after blood collection.…”

Section: Resultsmentioning

confidence: 99%

Historical review of clinical vaccine studies at Oswaldo Cruz Institute and Oswaldo Cruz Foundation - technological development issues

Martins

Possas

Homma

2015

Mem. Inst. Oswaldo Cruz

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This paper presents, from the perspective of technological development and production, the results of an investigation examining 61 clinical studies with vaccines conducted in Brazil between 1938-2013, with the participation of the Oswaldo Cruz Institute (IOC) and the Oswaldo Cruz Foundation (Fiocruz). These studies have been identified and reviewed according to criteria, such as the kind of vaccine (viral, bacterial, parasitic), their rationale, design and methodological strategies. The results indicate that IOC and Fiocruz have accumulated along this time significant knowledge and experience for the performance of studies in all clinical phases and are prepared for the development of new vaccines products and processes. We recommend national policy strategies to overcome existing regulatory and financing constraints.

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The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

Cited by 7 publications

References 10 publications

Stability of T cell phenotype and functional assays following heparinized umbilical cord blood collection

Stability of T cell phenotype and functional assays following heparinized umbilical cord blood collection

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring

Historical review of clinical vaccine studies at Oswaldo Cruz Institute and Oswaldo Cruz Foundation - technological development issues

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