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 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 Identification of BHLHE40 expression in peripheral blood mononuclear cells as a novel biomarker for diagnosis and prognosis of hepatocellular carcinoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pattapon Kunadirek ◽  
Chaiyaboot Ariyachet ◽  
Supachaya Sriphoosanaphan ◽  
Nutcha Pinjaroen ◽  
Pongserath Sirichindakul ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Early Stage ◽  
High Sensitivity ◽  
Peripheral Blood Mononuclear ◽  
Diagnosis And Prognosis ◽  
Transcription Profiles ◽  
Blood Mononuclear Cells

AbstractNovel and sensitive biomarkers is highly required for early detection and predicting prognosis of hepatocellular carcinoma (HCC). Here, we investigated transcription profiles from peripheral blood mononuclear cells (PBMCs) of 8 patients with HCC and PBMCs from co-culture model with HCC using RNA-Sequencing. These transcription profiles were cross compared with published microarray datasets of PBMCs in HCC to identify differentially expressed genes (DEGs). A total of commonly identified of 24 DEGs among these data were proposed as cancer-induced genes in PBMCs, including 18 upregulated and 6 downregulated DEGs. The KEGG pathway showed that these enriched genes were mainly associated with immune responses. Five up-regulated candidate genes including BHLHE40, AREG, SOCS1, CCL5, and DDIT4 were selected and further validated in PBMCs of 100 patients with HBV-related HCC, 100 patients with chronic HBV infection and 100 healthy controls. Based on ROC analysis, BHLHE40 and DDIT4 displayed better diagnostic performance than alpha-fetoprotein (AFP) in discriminating HCC from controls. Additionally, BHLHE40 and DDIT4 had high sensitivity for detecting AFP-negative and early-stage HCC. BHLHE40 was also emerged as an independent prognostic factor of overall survival of HCC. Together, our study indicated that BHLHE40 in PBMCs could be a promising diagnostic and prognostic biomarker for HBV-related HCC.

Isolation of Peripheral Blood Mononuclear Cells Using Glycerol Density Gradient

2013 ◽  
Vol 68 (3) ◽  
pp. 583-585 ◽  
Author(s):  
I. A. Aimola ◽  
H. M. Inuwa ◽  
A. J. Nok ◽  
A. I. Mamman ◽  
N. Habila ◽  
...  
Keyword(s):  
Density Gradient ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

scholarly journals Similar dysregulation of lupus-associated miRNAs in peripheral blood mononuclear cells and splenic lymphocytes in MRL/lpr mice

2018 ◽  
Vol 5 (1) ◽  
pp. e000290 ◽  
Author(s):  
Zhuang Wang ◽  
Bettina Heid ◽  
Rujuan Dai ◽  
Sattar Ansar Ahmed
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Lupus Erythematosus ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Gradient Centrifugation ◽  
Splenic Lymphocytes ◽  
Murine Lupus ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Mirnas Expression

ObjectiveMicroRNAs (miRNAs) play an important role in the pathogenesis of various autoimmune diseases including systemic lupus erythematosus (SLE; lupus). We have previously reported a common pattern of miRNA dysregulation in splenic lymphocytes from several mouse models of lupus. In this study, we investigated whether there is a similar miRNAs expression dysregulation in peripheral blood mononuclear cells (PBMCs) and splenocytes in a classical murine lupus model, MRL/lpr.MethodPBMCs were isolated from blood samples of control MRL and lupus MRL/lpr mice aged 14–15 weeks by gradient centrifugation with Histopaque 1083 density media. miRNA TaqMan assays were performed to analyse the expression of 10 lupus-associated miRNAs including miR-182-96-183 cluster, miR-146a, miR-148a, miR-21, miR-31, miR-127, miR-155, and miR-411 in MRL and MRL/lpr PBMCs.ResultIn this study, we found that 8 out of 10 examined miRNAs (miR-21, miR-31, miR-127, miR-155, miR-96, miR-182, miR-183 and miR-411) were similarly dysregulated in both PBMCs and splenocytes of MRL/lpr mice when compared with MRL control mice. Only two miRNAs (miR-146a and miR-148a) showed different dysregulation pattern in the PBMCs and splenocytes of MRL/lpr mice. By comparing with the published miRNA data in human lupus, we demonstrated similarity in miRNA dysregulation in murine and human lupus PBMCs.ConclusionThe findings in this study suggest that the miRNA changes observed in PBMCs largely reflect the miRNA dysregulation in cells from the lymphoid organ spleen. Analysis of miRNAs in PBMCs has an advantage over the splenocytes since it allows for monitoring the kinetics of lupus-associated miRNAs expression with peripheral blood cell samples during the development of the disease or after instituting treatment. The similar dysregulation of miRNAs in murine and human lupus PBMCs supports the importance and the feasibility of using murine lupus models to study the pathogenic and therapeutic function of miRNAs in human lupus.

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