Abstract
BackgroundWithin the last years, the interest in physical exercise as non-invasive stimulus influencing circulating hematopoietic stem and progenitor cell (CPC) concentrations has constantly grown. Cell estimates are often derived by determining the subgroup of CPC as percent lymphocytes (LYM) or mononuclear cells (MNC) via flow cytometry and back calculation over whole blood (WB) cell counts. However, results might depend on the used cell isolation technique and/or gating strategy. We aimed to investigate MNC loss and apoptosis during the flow cytometry sample preparation process preceded by either density gradient centrifugation (DGC) or red blood cell lysis (RBCL) and the potential difference between results derived from back calculation at different stages of cell isolation and from WB.MethodsHuman blood was subjected to DGC and RBCL. Samples were stained for flow cytometry analysis of CPC (CD34+/CD45dim) and apoptosis analysis (Annexin V) of MNC and CPC subsets. MNC and LYM gating strategies were compared.ResultsBoth DGC as well as RBCL yielded comparable CPC concentrations independent of the gating strategy when back calculated over WB values. However, cell loss and apoptosis differed between techniques, where after DGC LYM, and monocyte (MONO) concentrations significantly decreased (p < 0.01 and p < 0.05, respectively), while after RBCL LYM concentrations significantly decreased (p < 0.05) and MONO concentrations increased (p < 0.001). LYM apoptosis was comparable between techniques, but MONO apoptosis was higher after DGC than RBCL (p < 0.001).ConclusionsInvestigated MNC counts (LYM/MONO ratio) after cell isolation and staining did not always mimic WB conditions. Thus, final CPC results should be corrected accordingly, especially when reporting live CPC concentrations after DGC; otherwise, the CPC regenerative potential in circulation could be biased. This is of high importance in the context of non-invasively induced CPC mobilization such as by acute physical exercise, since these cell changes are small and conclusions drawn from published results might affect further applications of physical exercise as non-invasive therapy.
Highlights
Within the last years, the interest in physical exercise as non-invasive stimulus influencing circulating hematopoietic stem and progenitor cell (CPC) concentrations has constantly grown
Whole blood lymphocyte and monocyte concentrations compared to values after density gradient centrifugation and red blood cell lysis Directly after DGC and buffy coat isolation (Fig. 1, DGC unwashed (DGCun)), LYM and MONO concentrations measured by a hemocytometer were decreased by 50% (p < 0.001) and 42% (p > 0.05) in comparison to whole blood (WB) values, respectively (Fig. 4a)
After red blood cell lysis (RBCL), LYM concentration declined by 61% and was significantly lower compared to the control (WB) condition (p = 0.029; Fig. 4a), but was comparable to DGCuw and DGC washed (DGCw) samples
Summary
The interest in physical exercise as non-invasive stimulus influencing circulating hematopoietic stem and progenitor cell (CPC) concentrations has constantly grown. We aimed to investigate MNC loss and apoptosis during the flow cytometry sample preparation process preceded by either density gradient centrifugation (DGC) or red blood cell lysis (RBCL) and the potential difference between results derived from back calculation at different stages of cell isolation and from WB. The influence of acute physical exercise on circulating hematopoietic stem and progenitor cell (CPC) concentrations has become an intensely researched topic within the last years [1]. Two of the few flow cytometers that do include volumetric data are the AttuneTM Nxt [15, 16] or the MACSQuant [17] Machines such as the BD FACSCaliburTM [3] or BD FACSCantoTM II [18], only report cell proportions
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