Granulocyte Contamination of Ficoll-Hypaque Preparations of Mononuclear Cells following Thermal Injury May Lead to Substantial Overestimation of Lymphocyte Recovery

Calvano, Steve E.; Greenlee, Paul G.; Reid, Alyce M. M.; Shires, G T; Antonacci, Anthony C.

doi:10.1097/00005373-198803000-00011

Cited by 18 publications

(8 citation statements)

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“…However, even the best designed studies lose their biological validity if based on wrong methodological assumptions. In this article we would therefore like to draw attention to a phenomenon that has been known for a long time but has received little attention: In certain pathological conditions apart from sepsis (8) including burn patients (9,10), chronical infections (11,12), and autoimmune diseases (13), but also in healthy pregnancies (14), increased amounts of granulocytes with a lower density compared to normal neutrophils can be found. The detailed elucidation of their origin, function, and role in these conditions is part of current research efforts (15).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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“…Two granulocyte markers were reported in this study, CD10 and CD66c [41,42]. Granulocytes in patients with infections, burns, allergies, and other inflammatory disorders are thought to change their density and are therefore more difficult to separate from mononuclear cells [43]. This may explain why granulocyte markers appear in the mononuclear cell preparations.…”

Section: Variations Between Healthy Donors and Patients With Cadmentioning

confidence: 59%

Stable and unstable angina: Identifying novel markers on circulating leukocytes

Brown

Lattimore

McGrady

et al. 2008

Proteomics Clinical Apps

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There is currently no blood-based test that can rapidly and objectively distinguish between chest pain which is initiated by increased myocardial oxygen demand (stable angina pectoris (SAP)) and chest pain initiated due to decreased coronary blood flow (unstable angina pectoris (UAP)). Since leukocytes play an active role in the progression of coronary artery disease (CAD), we hypothesize these can provide novel markers of SAP and UAP. Here we use a microarray of 82 cluster of differentiation (CD) antibodies (plus controls) to selectively immobilize peripheral blood mononuclear cells. We find that the pattern of leukocyte immobilization from patients with CAD significantly differs from healthy donors. Within the CAD group, 15 SAP patients exhibited significant (p<0.05) changes in 8 of 82 CD antibody spots compared to 19 age-matched healthy blood donors. An additional ten CD antigens differed between healthy donors and patients with UAP (p<0.05). Furthermore, seven CD antibody spots are significantly different between SAP and UAP patients. These preliminary data suggest it is now appropriate to undertake a larger clinical trial to test the hypothesis that these antibody microarrays can monitor the progression from SAP to UAP.

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“…Ficoll density centrifugation may not be optimal in separating pure mononuclear cells [40], and methods other than bioassays are available for cytokine assay. However, the methods employed in this study approximate those used in studies quoted, discussing links between impaired lymphocyte function, metabolic disturbance, TNF, and impaired survival in various conditions [7][8][9]13,14,20,21,[33][34][35]39].…”

Section: Discussionmentioning

confidence: 99%

Metabolic disturbance and sensitivity to endotoxin in patients with advanced cancer: relationship to lymphocyte reactivity, tumour necrosis factor (TNF) production and survival

Gough

Fearon

Carter

1996

Clinical and Experimental Immunology

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SUMMARYIncreased TNF production and impaired lymphocyte function have been individually linked with metabolic disturbance, endotoxaemia and mortality in humans. The inter-relationship between these observations was investigated in humans with cancer. In 13 patients with metastatic colorectal cancer and seven healthy volunteers, observations (n 23) included peripheral blood mononuclear cell (PBMC) TNF production, IL-2 production and phytohaemagglutinin (PHA) response; the acutephase protein response (APPR) (serum C-reactive protein (CRP), albumin, CRP/albumin ratio), and survival. APPR correlated with survival (CRP, r

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Granulocyte Contamination of Ficoll-Hypaque Preparations of Mononuclear Cells following Thermal Injury May Lead to Substantial Overestimation of Lymphocyte Recovery

Cited by 18 publications

References 0 publications

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

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

Stable and unstable angina: Identifying novel markers on circulating leukocytes

Metabolic disturbance and sensitivity to endotoxin in patients with advanced cancer: relationship to lymphocyte reactivity, tumour necrosis factor (TNF) production and survival

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