<abstract xmlns="http://www.w3.org/1999/xhtml"><bold>Objective</bold>: Peripheral blood mononuclear cells (PBMC) are extremely important in the body’s immune response. Their isolation represents a major step in many immunological experiments. In this two phase study, we aimed to establish an optimum protocol for PBMC isolation by density-gradient centrifugation.<bold>Methods</bold>: During Phase-1, we compared two commercially available PBMC isolation protocols, Stemcell Technologies (ST) and Miltenyi Biotec (MB), in terms of PBMC recovery and purity. Twelve blood samples were assigned to each protocol. Each sample was divided in three subsamples of 1ml, 2ml and 3ml in order to assess the influence of blood sample volume on isolation performance. During Phase-2, a hybrid protocol was similarly tested, processing six blood samples. Additionally, we performed a flow cytometric analysis using an Annexin-V/Propidium-Iodide viability staining protocol.<bold>Results</bold>: Phase-1 results showed that, for all subsample volumes, ST had superior PBMC recovery (mean values: 56%, 80% and 87%, respectively) compared to MB (mean values: 39%, 54% and 43%, respectively). However, platelet removal was significantly higher for MB (mean value of 96.8%) than for ST (mean value of 75.2%). Regarding granulocyte/erythrocyte contamination, both protocols performed similarly, yielding high purity PBMC (mean values: 97.3% for ST and 95.8% for MB). During Phase-2, our hybrid protocol yielded comparable results to MB, with an average viability of 89.4% for lymphocytes and 16.9% for monocytes.<bold>Conclusions</bold>: ST yields higher cell recovery rates and MB excels at platelet removal, while the hybrid protocol is highly similar to MB. Both cell recovery and viability increase with blood sample volume.</abstract>

Objective: Peripheral blood mononuclear cells (PBMC) are extremely important in the body’s immune response. Their isolation represents a major step in many immunological experiments. In this two phase study, we aimed to establish an optimum protocol for PBMC isolation by density-gradient centrifugation.Methods: During Phase-1, we compared two commercially available PBMC isolation protocols, Stemcell Technologies (ST) and Miltenyi Biotec (MB), in terms of PBMC recovery and purity. Twelve blood samples were assigned to each protocol. Each sample was divided in three subsamples of 1ml, 2ml and 3ml in order to assess the influence of blood sample volume on isolation performance. During Phase-2, a hybrid protocol was similarly tested, processing six blood samples. Additionally, we performed a flow cytometric analysis using an Annexin-V/Propidium-Iodide viability staining protocol.Results: Phase-1 results showed that, for all subsample volumes, ST had superior PBMC recovery (mean values: 56%, 80% and 87%, respectively) compared to MB (mean values: 39%, 54% and 43%, respectively). However, platelet removal was significantly higher for MB (mean value of 96.8%) than for ST (mean value of 75.2%). Regarding granulocyte/erythrocyte contamination, both protocols performed similarly, yielding high purity PBMC (mean values: 97.3% for ST and 95.8% for MB). During Phase-2, our hybrid protocol yielded comparable results to MB, with an average viability of 89.4% for lymphocytes and 16.9% for monocytes.Conclusions: ST yields higher cell recovery rates and MB excels at platelet removal, while the hybrid protocol is highly similar to MB. Both cell recovery and viability increase with blood sample volume.

Optimization of a Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells

University of Medicine and Pharmacy of Tîrgu Mureş

Acta Marisiensis - Seria Medica

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Objective: Peripheral blood mononuclear cells (PBMC) are extremely important in the body’s immune response. Their isolation represents a major step in many...

Optimization of a Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells

Published Online: Jul 07, 2018

Page range: 83 - 90

Received: May 29, 2018

Accepted: Jun 15, 2018

DOI: https://doi.org/10.2478/amma-2018-0011

Keywords
density gradient centrifugation, PBMC isolation, PBMC viability

© 2018 Georgiana Mihaela Şerban et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Optimization of a Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells

Published Online: Jul 07, 2018

Page range: 83 - 90

Received: May 29, 2018

Accepted: Jun 15, 2018

DOI: https://doi.org/10.2478/amma-2018-0011

Keywordsdensity gradient centrifugation, PBMC isolation, PBMC viability

© 2018 Georgiana Mihaela Şerban et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Keywords
density gradient centrifugation, PBMC isolation, PBMC viability