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

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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.

1. Mesko B., Poliska S., Nagy L.- Gene expression profiles in peripheral blood for the diagnosis of autoimmune diseases. Trends Mol Med 2011;17(4):223-233.

2. Olsen N.J., Moore J.H., Aune T.M. - Gene expression signatures for autoimmune disease in peripheral blood mononuclear cells. Arthritis Res Ther 2004;6(3):120-128.

3. Shi G., Zhang Z., Li Q. - New Biomarkers in Autoimmune Disease. Hindawi, J Immunol Res 2017;https://doi.org/10.1155/2017/8702425.

4. Harouaka R., Kang Z., Zheng S. et al. - Circulating tumor cells: advances in isolation and analysis, and challenges for clinical applications. Pharmacol Ther. 2014;141(2):209–221.

5. Esmaeilsabzali H., Beischlag T.V., Cox M.E. et al. - Detection and isolation of circulating tumor cells: Principles and methods. Biotechnol Adv, 2013;31(7):1063-1084.

6. Masucci G.V., Cesano A., Hawtin R. et al. - Validation of biomarkers to predict response to immunotherapy in cancer: Volume I - pre-analytical and analytical validation. J Immunother Cancer 2016;4:76.

7. Farkona S., Diamandis E.P., Blasutig I.M.- Cancer immunotherapy: the beginning of the end of cancer?. BMC Med. 2016;14:73.

8. Vile R.G.- Socializing Individualized T-Cell Cancer Immunotherapy.

9. Mol. Ther 2016;24(7):1170-1173.

10. Yuan J., Hegde P.S., Clynes R. et al. - Novel technologies and emerging biomarkers for personalized cancer immunotherapy. J Immunother Cancer 2016;4:3.

11. Kohrt H.E., Tumeh P.C., Benson D. et al. - Immunodynamics: a cancer immunotherapy trials network review of immune monitoring in immuno-oncology clinical trials. J Immunother Cancer 2016;4:15.

12. Kumar S., Sunagar R., Pham G. et al. - Ex vivo antigen-pulsed PBMCs generate potent and long lasting immunity to infection when administered as a vaccine. Vaccine 2017;35(7):1080-1086.

13. Sambor A., Garcia A., Berrong M.et al. - Establishment and maintenance of a PBMC repository for functional cellular studies in support of clinical vaccine trials. J Immunol Methods 2014;0:107–116.

14. Bürdek M., Spranger S., Wilde S.et al. - Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation. J Transl Med 2010;8:90.

15. Frijters R., van Vugt M., Smeets R.et al. - Literature Mining for the Discovery of Hidden Connections between Drugs, Genes and Diseases. PLoS Comput Biol 2010;6(9):e1000943.

16. Heath J.R., Ribas A., Mischel P.S.- Single cell analytic tools for drug discovery and development. Nat Rev Drug Discov. 2016;15(3):204–216.

17. Pourahmad J., Salimi A. - Isolated Human Peripheral Blood Mononuclear Cell (PBMC), a Cost Effective Tool for Predicting Immunosuppressive Effects of Drugs and Xenobiotics. Iran J Pharm Res 2015;14(4):679-980.

18. Dagur P.K., McCoy Jr. J.P. - Collection, Storage, and Preparation of Human Blood Cells. Curr Protoc Cytom. 2016;73:5.1.1–5.1.16.

19. Rahmanian N., Bozorgmehr M., Torabi M. et al. - Cell separation: Potentials and pitfalls. Prep Biochem Biotechnol 2017;47(1):38-51.

20. Grievink H.W., Luisman T., Kluft C. et al. - Comparison of Three Isolation Techniques for Human Peripheral Blood Mononuclear Cells: Cell Recovery and Viability, Population Composition, and Cell Functionality. Biopreserv Biobank 2016;14(5):410-415.

21. Stone M., Murcia K., Dimapasoc M. et al. - Maximizing PMBC Recovery and Viability. A Method to Optimize and Streamline Peripheral Blood Mononuclear Cell Isolation, Cryopreservation, and Thawing. Bioprocess Int 2015;13(4)s.

22. Nilsson C., Aboud S., Karle K. et al. - Optimal Blood Mononuclear Cell Isolation Procedures for Gamma Interferon Enzyme-Linked Immunospot Testing of Healthy Swedish and Tanzanian Subjects. Clin. Vaccine Immunol. 2008;15(4):585–589.

23. Ruitenberg J.J., Mulder C.B., Maino V.C. et al. - VACUTAINER® CPT and Ficoll density gradient separationperform equivalently in maintaining the quality and function of PBMC from HIV seropositive blood samples. BMC Immunol. 2006;7(11).

24. Apithy M.-J., Desoutter J., Guillaume N.- Comparison of two peripheral mononuclear cell isolation protocols for flow cytometry crossmatching. doi: 10.1111/tan.13192.

25. Debey S., Schoenbeck U., Hellmich M. et al. - Comparison of different isolation techniques prior gene expression profiling of blood derived cells: impact on physiological responses, on overall expression and the role of different cell types. Pharmacogenomics J. 2004;4(3):193–207.

26. Palmirotta R., De Marchis M. L., Ludovici G. et al. - Impact of preanalytical handling and timing for peripheral blood mononuclear cells isolation and RNA studies: the experience of the Interinstitutional Multidisciplinary BioBank (BioBIM). Int. J. Biol. Markers 2012;27(2):90-98.

27. Berhanu D., Mortari F., De Rosa S.C. et al. - Optimized lymphocyte isolation methods for analysis of chemokine receptor expression. J Immunol Methods 2003;279(1-2):199 – 207.

28. Ramachandran H., Laux J., Moldovan I. et al. - Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies. Cells 2012;1(3):313-324.

29. Hønge B. L., Petersen M. S., Olesen R. et al. - Optimizing recovery of frozen human peripheral blood mononuclear cells for flow cytometry. PLOS ONE 2017;12(11):e0187440.

30. Yang J., Diaz N., Adelsberger J. et al. - The effects of storage temperature on PBMC gene expression. BMC Immunol. 2016;17:6.

31. Boyum A. - Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. Suppl. 1968;97:77–89.

32. Boyum A. - Separation of lymphocytes, lymphocyte subgroups and monocytes: A review. Lymphology 1977;10(2):71–76.

Acta Medica Marisiensis

The Journal of The University of Medicine and Pharmacy of Targu-Mures

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