Abstract
BackgroundThe purification of peripheral blood mononuclear cells (PBMCs) by means of density gradient (1.07 g/mL) centrifugation is one of the most commonly used methods in diagnostics and research laboratories as well as in biobanks. Here, we evaluated whether it was possible to set up an automated protocol for PBMC purification using a programmable liquid handling robotized workstation (Tecan, Freedom EVO 150). We selected a population composed of 30 subjects for whom it was possible to dispose of two ethylenediaminetetraacetic acid (EDTA) vacutainer tubes containing unfractionated peripheral blood. The purification of PBMCs was performed in parallel using automated and manual workflows.ResultsAn automated workflow using the Freedom EVO 150 liquid handling workstation was generated for the isolation of PBMCs. This protocol allowed blood dilution in Dulbecco’s phosphate-buffered saline (DPBS), stratification onto the density gradient, and the collection of PBMC rings after centrifugation. The comparison between the automated and manual methods revealed no significant differences after separation in terms of total mononuclear cell enrichment, red blood cell contamination, or leucocyte formula, including the percentage of lymphoid subpopulations as B, T and natural killer (NK) lymphocytes.ConclusionsOur results show that it is possible to set up an automated protocol for the isolation of PBMCs using a robotized liquid handling workstation. This automated protocol provided comparable results to the routinely used manual method. This automatic method could be of interest for those working in biobanking or industries involved in diagnostics and therapeutics field, to avoid operator-dependent errors as well as procedures standardization.
Highlights
The purification of peripheral blood mononuclear cells (PBMCs) by means of density gradient (1.07 g/ mL) centrifugation is one of the most commonly used methods in diagnostics and research laboratories as well as in biobanks
Different companies, such as Hamilton, Qiagen, Brooks Life Sciences and Tecan, provide solutions for the customization of automatic procedures for biological sample processing. Due to their versatility, automated liquid handling systems are commonly used for different laboratory applications [8,9,10], when the pipetting steps must be as accurate and precise as possible, as in the cases of biopharmaceutical products preparation [11, 12], serial dilutions, and reagent transfers [13, 14]. We propose these complex technological systems for the automatic purification of peripheral blood mononuclear cells (PBMCs) from whole blood collected in ethylenediaminetetraacetic acid (EDTA) tubes
Because the density gradient purification of PBMCs is a procedure with high operator-dependent variability, we believe that the methodology presented in this paper could be useful for the standardization of PBMC isolation in biobanking
Summary
The purification of peripheral blood mononuclear cells (PBMCs) by means of density gradient (1.07 g/ mL) centrifugation is one of the most commonly used methods in diagnostics and research laboratories as well as in biobanks. Biobanks provide high-quality samples for the setting up of research protocols dedicated to the translation of important discoveries coming from basic research laboratories to clinical practice [1] One example of their usefulness comes from the generation of The Cancer Genome Atlas (TCGA) [2]. Different companies, such as Hamilton (https://www.hamiltoncompany.com), Qiagen (https:// www.qiagen.com/us/products/instruments-and-automation/?akamai-feo=off ), Brooks Life Sciences (https:// www.brookslifesciences.com/about-brooks-life-sciences) and Tecan (https://www.tecan.com/), provide solutions for the customization of automatic procedures for biological sample processing Due to their versatility, automated liquid handling systems are commonly used for different laboratory applications [8,9,10], when the pipetting steps must be as accurate and precise as possible, as in the cases of biopharmaceutical products preparation [11, 12], serial dilutions, and reagent transfers [13, 14]. Because the density gradient purification of PBMCs is a procedure with high operator-dependent variability, we believe that the methodology presented in this paper could be useful for the standardization of PBMC isolation in biobanking
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