scholarly journals A QUICK AND EASY METHOD FOR THE ISOLATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS FROM WHOLE BLOOD BY DENSITY GRADIENT CENTRIFUGATION USING HISEP LSM MEDIUM.

2016 ◽  
Vol 4 (9) ◽  
pp. 1774-1778 ◽  
Author(s):  
Brahmaji Sontyana ◽  
◽  
Suresh Govatati ◽  
RaghavaRao Tamanam ◽  
◽  
...  
Keyword(s):  
Density Gradient ◽  
Peripheral Blood Mononuclear Cells ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Easy Method ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

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 Monitoring Tacrolimus Concentrations in Whole Blood and Peripheral Blood Mononuclear Cells: Inter- and Intra-Patient Variability in a Cohort of Pediatric Patients

2021 ◽  
Vol 12 ◽  
Author(s):  
Amedeo De Nicolò ◽  
Michele Pinon ◽  
Alice Palermiti ◽  
Antonello Nonnato ◽  
Alessandra Manca ◽  
...  
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Pediatric Patients ◽  
Mononuclear Cells ◽  
Individual Variability ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Intracellular Concentrations ◽  
The One

Tacrolimus (TAC) is a first-choice immunosuppressant for solid organ transplantation, characterized by high potential for drug-drug interactions, significant inter- and intra-patient variability, and narrow therapeutic index. Therapeutic drug monitoring (TDM) of TAC concentrations in whole blood (WB) is capable of reducing the incidence of adverse events. Since TAC acts within lymphocytes, its monitoring in peripheral blood mononuclear cells (PBMC) may represent a valid future alternative for TDM. Nevertheless, TAC intracellular concentrations and their variability are poorly described, particularly in the pediatric context. Therefore, our aim was describing TAC concentrations in WB and PBMC and their variability in a cohort of pediatric patients undergoing constant immunosuppressive maintenance therapy, after liver transplantation. TAC intra-PBMCs quantification was performed through a validated UHPLC–MS/MS assay over a period of 2–3 months. There were 27 patients included in this study. No significant TAC changes in intracellular concentrations were observed (p = 0.710), with a median percent change of −0.1% (IQR −22.4%–+46.9%) between timings: this intra-individual variability was similar to the one in WB, −2.9% (IQR −29.4–+42.1; p = 0.902). Among different patients, TAC weight-adjusted dose and age appeared to be significant predictors of TAC concentrations in WB and PBMC. Intra-individual seasonal variation of TAC concentrations in WB, but not in PBMC, have been observed. These data show that the intra-individual variability in TAC intracellular exposure is comparable to the one observed in WB. This opens the way for further studies aiming at the identification of therapeutic ranges for TAC intra-PBMC concentrations.

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