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Time and Temperature Dependent Ficoll Separation of Aged Whole Blood Neutrophils
Author(s) -
Thompson William C,
Gu Dongsheng,
Johnstone Brian,
Sherry Aubrey,
LaFontaine Michael,
Woods Erik
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.496.63
Subject(s) - ficoll , buffy coat , bone marrow , degranulation , haematopoiesis , immunology , centrifugation , progenitor cell , andrology , differential centrifugation , peripheral blood mononuclear cell , biology , chemistry , stem cell , microbiology and biotechnology , medicine , chromatography , biochemistry , receptor , in vitro
Emerging clinical applications of bone marrow often require separation and purification of hematopoietic stem/progenitor cells (HSPC). Ficoll density gradient centrifugation is a well‐known and effective method for separating mononuclear cells in fresh blood and bone marrow to facilitate cell selection. Often these cell selection processes are performed in centralized locations, requiring transport and some amount of subsequent ischemia. As blood and bone marrow ages, neutrophil characteristics change causing interference in standard cell separations. Over time granulocytes degranulate and become less dense. Thus, degranulated granulocytes are no longer able to sediment through the Ficoll layer and they become trapped in the buffy coat with lymphocytes and HSPCs. Using blood as a surrogate, this study aims to define the critical time points of the degranulation process and its temperature dependence to optimize parameters of Ficoll separation for aged samples. Blood was collected from three healthy donors and stored at 4 and 20 °C. This experiment was repeated for an n of 2. At zero, three, and twenty‐four hours after collection, blood was centrifuged at 400g for 40 minutes with no acceleration or brake over a 1.077 g/ml Ficoll density gradient. The buffy coat and pellet were collected from each sample and characterized using flow cytometry. Cell marker CD45 was used to identify white blood cells of hematopoietic lineage. Cell marker CD66b was used to identify granulocytes. Results indicated that blood stored at 4 °C exhibited more degranulation of neutrophils compared to blood stored at 20 °C [Table 1]. After three hours, blood stored at 4 °C had more granulocytes trapped in the buffy coat following Ficoll density gradient centrifugation as compared to 20 °C. After twenty‐four hours, blood stored at 20 °C also failed to separate adequately. In conclusion, the results of this study indicate that neutrophil stability decreases rapidly following low temperature exposure, which impacts subsequent cell separation techniques and must be taken into account with hypothermic transport of samples intended for cell selection. Support or Funding Information Marian University and Ossium Health This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .