Method Validation for Automated Isolation of Viable Peripheral Blood Mononuclear Cells

Hamot, Gaël; Ammerlaan, Wim; Mathay, Conny; Kofanova, Olga; Betsou, Fay

doi:10.1089/bio.2014.0054

Cited by 22 publications

(19 citation statements)

References 39 publications

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“…A, B: freshly isolated PBMC (n = 6). C, D: cryopreserved PBMC (n = 3) Isolation by cell separation tubes requires less skill than isolation with Ficoll gradient, and is compatible with automation [40]. CPT, Accuspin, and Ficoll isolation methods have shown similar performance on PBMC recovery and viability [17].…”

Section: Pbmc Isolationmentioning

confidence: 99%

Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications

et al. 2019

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Purpose of Review Peripheral blood mononuclear cells (PBMCs) are used in a wide variety of preclinical assays. Preanalytical variations can have a devastating impact on the results. In this review, we list critical preanalytical factors for PBMC-based assays to develop awareness and orientation to the types of sample preparation and storage that one may consider employing. Recent Findings Critical factors during blood collection are the blood collection tube and anticoagulant, possible stabilizer used, and the pre-isolation blood storage temperature and time. During PBMC isolation, critical factors are the isolation method, density gradient or magnetic sorting, use of barrier, possible RBC lysis, and centrifuge type. During cryopreservation, attention is needed for the cryomedium type and temperature, freezing device and program, cell concentration, and the long-term storage temperature. During the thawing process, the thawing procedure/device used and wash medium temperature are critical. Summary To avoid biased results in PBMC assays, these critical preanalytical factors must be standardized and/or documented. Additionally, participation in external quality assurance programs is strongly recommended.

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Section: Pbmc Isolationmentioning

confidence: 99%

Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications

et al. 2019

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“…Nucleic acids with little or no PCR or RT-qPCR product indicate tissue degradation and poor quality 6 . Our results showed acceptable Ct values, which is a good indicator of the suitability of the material for subsequent use in biomedical research 35 .…”

Section: Discussionmentioning

confidence: 57%

“…Moreover, it has been shown that time from venipuncture to PBMC isolation and cryopreservation may affect the cell quantity, recovery, viability, white blood cells subpopulation distribution, gene expression, and lymphoblastoid cell lines transformation 35 and also have impact in the viability and performance of T cells in downstream immunological assays [36][37] and in the cytotoxic activity of natural killer cells 38 . In addition, it has also been shown that a 24-h delay between blood collection and processing, especially at 4ºC, significantly increases granulocyte contamination, which may inhibit T cell function 22 .…”

Section: Discussionmentioning

confidence: 99%

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Quality Assurance of Samples and Processes in the Spanish Renal Research Network (REDinREN) Biobank

Calleros-Basilio

Cortés

García-Jérez

et al. 2016

Biopreservation and Biobanking

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“…Another potential solution is to integrate automation to the sample processing workflow. Instruments such as liquid handlers offer protocols for automated processing which have been shown to perform as efficiently as the goldstandard Ficoll density gradient method, as measured by overall yield, viability, recovery, white blood cell subpopulation distribution, and gene expression (16)(17)(18)(19).…”

Section: Sample Preparation and Storage: The Crucial First Stepmentioning

confidence: 99%

Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

et al. 2019

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The rapid advancement of immunotherapy strategies has created a need for technologies that can reliably and reproducibly identify rare populations, detect subtle changes in modulatory signals, and assess antigenic expression patterns that are time-sensitive. Accomplishing these tasks requires careful planning and the employment of tools that provide greater sensitivity and specificity without demanding extensive time. Flow Cytometry has earned its place as a preferred analysis platform. This technology offers a flexible path to the interrogation of protein expression patterns and detection of functional properties in cell populations of interest. Mass Cytometry is a newcomer technology that has generated significant interest in the field. By incorporating mass spectrometry analysis to the traditional principles of flow cytometry, this innovative tool promises to significantly expand the ability to detect multiple proteins on a single cell. The use of these technologies in a manner that is consistent and reproducible through multiple sample sets demands careful attention to experiment design, reagent selection, and instrumentation. Whether applying flow or mass cytometry, reaching successful, reliable results involves many factors. Sample preparation, antibody titrations, and appropriate controls are major biological considerations that impact cytometric analysis. Additionally, instrument voltages, lasers, and run quality assessments are essential for ensuring comparability and reproducibility between analyses. In this article, we aim to discuss the critical aspects that impact flow cytometry, and will touch on important considerations for mass cytometry as well. Focusing on their relevance to immunotherapy studies, we will address the importance of appropriate sample processing and will discuss how selection of suitable panels, controls, and antibodies must follow a carefully designed plan. We will also comment on how educated use of instrumentation plays a significant role in the reliability and reproducibility of results.Through this work, we hope to contribute to the effort toward establishing higher standards for rigor and reproducibility of cytometry practices by researchers, operators, and general cytometry users employing cytometry-based assays in their work.

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Method Validation for Automated Isolation of Viable Peripheral Blood Mononuclear Cells

Abstract: We validated the first fully automated method for isolating viable PBMCs, including RNA analysis and generation of LCLs. We recommend processing within 8 h of blood collection.

Cited by 22 publications

References 39 publications

Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications

Biospecimen Science of Blood for Peripheral Blood Mononuclear Cell (PBMC) Functional Applications

Quality Assurance of Samples and Processes in the Spanish Renal Research Network (REDinREN) Biobank

Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

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