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

2021 ◽  
Vol 12 ◽  
Author(s):  
Judith Schenz ◽  
Manuel Obermaier ◽  
Sandra Uhle ◽  
Markus Alexander Weigand ◽  
Florian Uhle
Keyword(s):  
Healthy Volunteers ◽  
Density Gradient ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Low Density ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

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.

scholarly journals Separation and Optimisation of a Sucrose Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells (PBMC)

2018 ◽  
pp. 1-4
Author(s):  
Sudeep Nagaraj ◽  
Shubha Nivargi ◽  
Leelavathy Nanjappa ◽  
Jagadish Tavarekere Venkataravanappa
Keyword(s):  
Density Gradient ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Mononuclear Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
One Step

One step centrifugation procedure used commonly for separation of blood cells is the ficoll gradient centrifugation. In this method, after centrifugation, the peripheral blood mononuclear cells (PBMCs) are located on the top of the separation fluid, whereas other blood cells erythrocytes and granulocytes sediment to the bottom. In the present study 75% of lymphocyte suspension could be separated by using a one-step density gradient centrifugation of sodium heparin blood with Sucrose. Sucrose was diluted into different concentrations using miliQ water (10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%,). 4 mL of diluted blood was layered on 4 mL of each sucrose solution and centrifuged for 45 minutes at 1000 rpm. Clear separation of PBMCs could be observed in solution with 40% sucrose. The separated PBMCs were analysed in haeme analyser which showed 75% lymphocytes, 23% monocytes and 2% of other cells.

scholarly journals Impact of SARS-CoV-2 infection on the recovery of peripheral blood mononuclear cells by density gradient

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maria D. I. Manunta ◽  
Giuseppe Lamorte ◽  
Francesca Ferrari ◽  
Elena Trombetta ◽  
Mario Tirone ◽  
...  
Keyword(s):  
Density Gradient ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Early Phase ◽  
Mononuclear Cells ◽  
Early Stage ◽  
Gradient Centrifugation ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Circulating Lymphocytes

AbstractSARS-CoV-2 virus infection is responsible for coronavirus disease (COVID-19), which is characterised by a hyperinflammatory response that plays a major role in determining the respiratory and immune-mediated complications of this condition. While isolating peripheral blood mononuclear cells (PBMCs) from whole blood of COVID-19 patients by density gradient centrifugation, we noticed some changes in the floating properties and in the sedimentation of the cells on density medium. Investigating this further, we found that in early phase COVID-19 patients, characterised by reduced circulating lymphocytes and monocytes, the PBMC fraction contained surprisingly high levels of neutrophils. Furthermore, the neutrophil population exhibited alterations in the cell size and in the internal complexity, consistent with the presence of low density neutrophils (LDNs) and immature forms, which may explain the shift seen in the floating abilities and that may be predictive of the severity of the disease. The percentage of this subset of neutrophils found in the PBMC band was rather spread (35.4 ± 27.2%, with a median 28.8% and IQR 11.6–56.1, Welch’s t-test early phase COVID-19 versus blood donor healthy controls P < 0.0001). Results confirm the presence of an increased number of LDNs in patients with early stage COVID-19, which correlates with disease severity and may be recovered by centrifugation on a density gradient together with PBMCs.

scholarly journals Impact of SARS-CoV-2 infection on the recovery of peripheral blood mononuclear cells by density gradient

2020 ◽  
Author(s):  
Maria D. I. Manunta ◽  
Giuseppe Lamorte ◽  
Francesca Ferrari ◽  
Elena Trombetta ◽  
Mario Tirone ◽  
...  
Keyword(s):  
Density Gradient ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Early Phase ◽  
Mononuclear Cells ◽  
Early Stage ◽  
Gradient Centrifugation ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Circulating Lymphocytes

Abstract SARS-CoV-2 virus infection is responsible for coronavirus disease (COVID-19), which is characterised by a hyperinflammatory response that plays a major role in determining the respiratory and immune-mediated complications of this condition. While isolating peripheral blood mononuclear cells (PBMCs) from whole blood of COVID-19 patients by density gradient centrifugation, we noticed some changes in the floating properties and in the sedimentation of the cells on density medium. Investigating this further, we found that in early phase COVID-19 patients, characterised by reduced circulating lymphocytes and monocytes, the PBMC fraction contained surprisingly high levels of neutrophils. Furthermore, the neutrophil population exhibited alterations in the cell size and in the internal complexity, consistent with the presence of low density neutrophils (LDNs) and immature forms which may explain the shift seen in the floating abilities and that may be predictive of the severity of the disease. The percentage of this subset of neutrophils found in the PBMC band was rather spread (35.4±27.2%, with a median 28.8% and IQR 11.6-56.1, Welch’s t-test early phase COVID-19 versus blood donor healthy controls P<0.0001). Results confirm the presence of an increased number of LDNs in patients with early stage COVID-19, which correlates with disease severity and may be recovered by centrifugation on a density gradient together with PBMCs.

scholarly journals Effect of Lamivudine on the Plasma and Intracellular Pharmacokinetics of Apricitabine, a Novel Nucleoside Reverse Transcriptase Inhibitor, in Healthy Volunteers

2007 ◽  
Vol 51 (8) ◽  
pp. 2943-2947 ◽  
Author(s):  
T. Holdich ◽  
L. A. Shiveley ◽  
J. Sawyer
Keyword(s):  
Reverse Transcriptase ◽  
Healthy Volunteers ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Transcriptase Inhibitor ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Reverse Transcriptase Inhibitor

ABSTRACT Apricitabine is a novel deoxycytidine analog reverse transcriptase inhibitor. In vitro apricitabine competes with other deoxycytidine analogues for intracellular phosphorylation mediated by deoxycytidine kinase. The topic of this study, the effect of concomitant administration of apricitabine and lamivudine on the plasma and intracellular pharmacokinetics of the two compounds, was investigated in healthy volunteers. Participants (n = 21; age, 18 to 30 years) received apricitabine at 600 mg twice daily, lamivudine at 300 mg once daily, and the two treatments in combination for 4 days each in random order. Plasma, urine, and intracellular pharmacokinetics were assessed on day 4 of each treatment period. Apricitabine was rapidly absorbed after oral administration, with peak concentrations being attained after a mean of 1.76 h. Coadministration with lamivudine had no significant effect on the plasma and urine pharmacokinetics of apricitabine. However, the formation of apricitabine triphosphate in peripheral blood mononuclear cells was markedly reduced after the coadministration of apricitabine and lamivudine than after the administration of apricitabine alone: the area under the concentration-time curve from 0 to 12 h for apricitabine triphosphate during combination treatment was ca. 15% of that seen after the administration of apricitabine alone. In contrast, apricitabine had no effect on the plasma pharmacokinetics of lamivudine or on the formation of lamivudine triphosphate in peripheral blood mononuclear cells. These results are consistent with in vitro findings that lamivudine inhibits the intracellular phosphorylation of apricitabine. In conjunction with similar in vitro observations for emtricitabine and apricitabine, these results suggest that apricitabine should not be coadministered with other deoxycytidine analogues for the treatment of human immunodeficiency virus infection.

scholarly journals Red Wine Intake Prevents Nuclear Factor-κB Activation in Peripheral Blood Mononuclear Cells of Healthy Volunteers During Postprandial Lipemia

Circulation ◽  
2000 ◽  
Vol 102 (9) ◽  
pp. 1020-1026 ◽  
Author(s):  
Luis Miguel Blanco-Colio ◽  
Mónica Valderrama ◽  
Luis Antonio Alvarez-Sala ◽  
Carmen Bustos ◽  
Mónica Ortego ◽  
...  
Keyword(s):  
Healthy Volunteers ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Red Wine ◽  
Mononuclear Cells ◽  
Postprandial Lipemia ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells
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