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.

Immune Subsets From Ficoll Density Gradient Separation in Kidney Transplant Recipients

“…In contrast to previous studies that showed a significant increase in the proportion of granulocytes in patients suffering from inflammation compared with controls, 7 , 10 there was no statistically significant difference in the cellular make up of the PBMC fraction of kidney transplant recipients with acute TCMR compared with nonrejecting kidney transplant recipients. The results in our study show that acute rejection might therefore be considered as a relatively “milder” state of inflammation in the peripheral blood compartment compared with septicemia or other systemic inflammatory diseases.…”

“…9 The biological validity of these studies could therefore be affected if based on faulty methodological assumptions. 7,10 Surprisingly, no studies report on whether the proportion of PBMC subpopulations from transplant recipients differ from healthy controls, or if rejection affects cellular PBMC composition. We compared the proportions of PBMC subsets in kidney transplant recipients during a biopsy-proven acute T cell-mediated rejection (TCMR) episode (n = 11) with those from kidney transplant recipients without acute rejection (n = 12) and healthy controls (kidney transplant donors before transplantation) (n = 10).…”

“… 6 However, the PBMC fraction consists of various leukocyte subpopulations, including T, B, and NK lymphocytes and monocytes, with potential contamination by granulocytes. By using Ficoll density gradient separation to isolate the PBMCs, the proportion of low-density granulocytes is increased in inflammatory conditions such as sepsis, 7 burn wounds, 8 or in autoimmune disease. 9 The biological validity of these studies could therefore be affected if based on faulty methodological assumptions.…”

“… 9 The biological validity of these studies could therefore be affected if based on faulty methodological assumptions. 7 , 10 …”

Phenotypic characteristics of peripheral immune cells of Myalgic encephalomyelitis/chronic fatigue syndrome via transmission electron microscopy: A pilot study

“…PBMCs encompass a heterogeneous cell population comprising monocytes (which can be differentiated to macrophages and dendritic cells) and lymphocytes (T cells, natural killer cells, and B-cells). However, most PBMC purification methods contain a considerable amount of platelet contamination [ 57 ] as well as traces of erythrocytes, and low-density granulocytes (neutrophils, basophils, and eosinophils) [ 58 , 59 ]. The existence of a round nucleus in PBMCs can help easily distinguish most immune cells under TEM from erythrocytes and platelets, which have no nuclei and from granulocytes, which show a lobulated segmented nucleus [ 60 ] as well as various types of cytoplasmic granules.…”