Low-Density Granulocyte Contamination From Peripheral Blood Mononuclear Cells of Patients With Sepsis and How to Remove It - A Technical Report

Abstract

Elucidating the mechanisms contributing to the dysregulated host response to infection as part of the syndrome is a current challenge in sepsis research. Peripheral blood mononuclear cells are widely used in immunological studies. Density gradient centrifugation, a common method, is of limited use for blood drawn from patients with sepsis. A significant number of low-density granulocytes co-purify contributing to low purity of isolated peripheral blood mononuclear cells. Whole blood anticoagulated with lithium heparin was drawn from patients with sepsis (n=14) and healthy volunteers (n=11). Immediately after drawing, the plasma fraction was removed and PBMC were isolated from the cellular fraction by density gradient centrifugation. Samples derived from patients with sepsis were subsequently incubated with cluster of differentiation 15 MicroBeads and granulocytes were depleted using magnetic-activated cell sorting. Core cellular functions as antigen presentation and cytokine secretion were analyzed in cells isolated from healthy volunteers (n=3) before and after depletion to confirm consistent functionality. We report here that depleting CD15⁺ cells after density gradient centrifugation is a feasible way to get rid of the low-density granulocyte contamination. Afterwards, the purity of isolated, functionally intact peripheral blood mononuclear cells is comparable to healthy volunteers. Information on the isolation purity and identification of the containing cell types are necessary for good comparability between different studies. Depletion of CD15⁺ cells after density gradient centrifugation is an easy but highly efficient way to gain a higher quality and more reliability in studies using peripheral blood mononuclear cells from septic patients without affecting the functionality of the cells.

Keywords: Ficoll; MACS; PBMC (peripheral blood mononuclear cells); Sepsis; cell isolation; centrifugation; density gradient; low-density granulocytes.

Figure 1

Depletion of CD15⁺ cells leads to pure PBMC from patients with sepsis. (A) Representative gating strategy for the identification of low-density granulocytes. Single cells were gated from all cellular events. B cells were identified as CD19⁺. T cells were identified as CD3⁺. Monocytes were identified as CD14⁺. Low-density granulocytes were identified as CD3^-, CD14^-, CD19^-, SSC^hi. (B) Granulocyte percentage in patients with sepsis after density gradient centrifugation. Density plots from three samples selected for analysis are shown. Numbers indicate the proportion of low-density granulocytes in all single cells. (C) Flow cytometric quality control of low-density granulocyte depletion. Representative density plots are shown for patients with sepsis after depletion of CD15⁺ cells from PBMC isolated by density gradient centrifugation respectively for healthy volunteers after density gradient centrifugation. Numbers indicate the proportion of low-density granulocytes in all single cells. For total cohort, relative percentages of low-density granulocytes after depletion of CD15⁺ cells are shown (in case of healthy volunteers after density gradient centrifugation). Each data point represents an individual patient (sepsis d1: n = 14; d8: n = 12/healthy: n = 11) and horizontal line marks the median. Group comparisons (sepsis vs. healthy) were performed using Mann-Whitney test (****P < 0.0001, **P ≤ 0.01).

Figure 2

Low-density granulocytic contamination is also reflected in the number of isolated cells. Cell yield per mL blood after in case of patients with sepsis (A) density gradient centrifugation and (B) additional depletion of CD15⁺ cells, in case of healthy volunteers’ density gradient centrifugation. (C) Calculated total number of cells removed by the depletion process per mL blood in patients with sepsis. Each data point represents an individual patient (sepsis d1: n = 14; d8: n = 12/healthy: n = 11). Horizontal lines mark the median. Group comparisons (sepsis vs. healthy) were performed using Mann-Whitney test (****P < 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05).

Figure 3

Depletion of CD15⁺ cells leads to a small loss of PBMC. Flow cytometric results after staining of the fraction containing the depleted CD15⁺ cells shown as density plots (sepsis: n = 2). Numbers indicate the proportion of B cells, T cells, monocytes, and low-density granulocytes respectively in all single cells.

Figure 4

Handling during depletion has no impact on cellular function. Proportion of (A) low-density granulocytes, (B) monocytes, B cells, and T cells in all single cells before and after depletion of CD15⁺ cells. (C) Mitochondrial ROS expression level and (D) number of HLA-DR molecules per CD14⁺ monocyte before and after depletion of CD15⁺ cells. (E) Induced IL-6 production in response to stimulation with LPS, flagellin, or zymosan and induced IFNγ production in response to stimulation with human T-Activator CD3/CD28. Each data point represents a separate healthy volunteer (n=3). Paired results from the same donor before and after depletion of CD15⁺ cells are connected by a line. Group comparisons were performed using Mann-Whitney test (n.s., not significant).