scholarly journals Optimized multiparametric flow cytometric analysis of circulating endothelial cells and their subpopulations in peripheral blood of patients with solid tumors: a technical analysis

2018 ◽  
Vol Volume 10 ◽  
pp. 447-464
Author(s):  
Fangbin Zhou ◽  
Yaying Zhou ◽  
Ming Yang ◽  
Jinli Wen ◽  
Jun Dong ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Solid Tumors ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Technical Analysis ◽  
Circulating Endothelial Cells ◽  
Flow Cytometric

Flow Cytometric Phenotyping and Analysis of CD8 Regulatory and Suppressor Cells In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2947-2947
Author(s):  
Karthick Raja Muthu Raja ◽  
Lucie Kovarova ◽  
Ivana Buresova ◽  
Roman Hajek ◽  
Jaroslav Michalek
Keyword(s):  
Multiple Myeloma ◽  
Solid Tumors ◽  
Peripheral Blood ◽  
Monoclonal Gammopathy ◽  
Hematological Malignancies ◽  
Flow Cytometric Analysis ◽  
Suppressor Cells ◽  
Color Flow ◽  
Flow Cytometric ◽  
Significant Difference

Abstract Abstract 2947 In solid tumors and hematological malignancies, elevated proportions of CD4 regulatory (CD4 Tregs) cells were reported and the function was also documented well. In the context of CD8 regulatory cells (CD8 Tregs) existence has been revealed in various solid tumors including colorectal, prostate and ovarian cancers. In hematological malignancies or multiple myeloma (MM) the presence of CD8 Tregs are yet to be revealed. To unveil and understand the existence of CD8 Tregs, we characterized and quantified CD8 Tregs along with CD8 suppressor cells in MM. A total of 91 monoclonal gammopathy patients were screened for CD8 Tregs and suppressor cells in peripheral blood (PB) and bone marrow (BM). For comparison, 21 healthy volunteers (HVs) PB were also analyzed. Clinical diseases of the patients were represented as monoclonal gammopathy of undetermined significance (MGUS)-14% (13/91), untreated MM- 60% (55/91) and relapsed MM (Rel MM)-26% (23/91). A simple three-color flow cytometric analysis was used to phenotype the CD8 Tregs and suppressor cells. CD8 Tregs were determined as CD8+FoxP3+ (Frisullo et al, 2010, Hum Immunol) and CD8 suppressor cells were identified by negative expression of co-stimulatory molecule CD28 (CD8+CD28-). All the results were summarized as percentage median and range. Our results showed strong significant increase in CD8 Tregs and suppressor cells in PB of patient cohort compared to HVs [CD8 Tregs- 0.39% (0.03%–2.41%) vs. 0.18% (0.04%–0.61%); P=0.002 and CD8 suppressor cells- 69.42% (10.99%–95.91%) vs. 27.58% (9.08%–70.52%); P<0.0001]. Additionally, strong significant difference was also noticed between HVs vs. MGUS, MM, and Rel MM for PB CD8 Tregs and suppressor cells; the data with statistical results has been depicted in table 1. More or less similar frequencies of PB CD8 Tregs were found between MGUS vs. MM vs. Rel MM. In term of PB CD8 suppressor cells Rel MM had insignificant increase compared to MGUS and MM. Surprisingly, MM cohort had insignificantly decreased frequency of CD8 suppressor cells compared to MGUS. As similarly to PB, BM CD8 Tregs and suppressor cells did not show any statistical significance in between patient groups. The BM CD8 Tregs frequency was observed respectively as: MGUS-0.32% (0.08%–1.04%), MM-0.44% (0.06%–1.82%) and Rel MM-0.27% (0.11%–1.16%). Respective BM CD8 suppressor cells frequency was noticed as: MGUS-63.32% (24.83%–94.43%), MM-62.15% (6.19%–93.45%) and Rel MM-67.29% (18.55%–87.61%). Subgroup of patients with < 95% of abnormal plasma cells [ANPCs- 59.21% (13.35%–91.83%)] had significantly decreased level of PB CD8 suppressor cells compared to counter group [≥95% of ANPCs- 74.23% (10.99%–95.91%)]; P=0.046. In line with PB, BM CD8 suppressor cells also showed significantly reduced frequency in <95% of ANPCs cohort [56.99% (16.48%–94.43%)] compared to ≥ 95% ANPCs cohort [69.61% (6.19%–93.45%)]; P=0.013. No relevant association was noticed between CD8 Tregs and suppressor cells with other clinical features including β2M, albumin, creatinine, lactate dehydrogenase, hemoglobulin and monoclonal protein level. In conclusion, our results disclose that as similar to CD4 Tregs, CD8 Tregs are also deregulated in MM compared to HVs; this is also in line with CD8 suppressor cells. This data suggest that in MM immune dysfunction is also enhanced by the shift in favor of CD8 immune regulatory and suppressor cells. This study was supported by the respective grants-MSM0021622434, LC06027, IGA NS10406, IGA NS10408, and GACR P304/10/1395. Table 1: Comparison of Peripheral Blood CD8 Tregs and Suppressor Cells between Hvs versus MGUS, MM and Relapsed MM Disclosures: No relevant conflicts of interest to declare.

scholarly journals Circulating endothelial cells transiently increase in peripheral blood after kidney transplantation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
H. Tejeda-Mora ◽  
J. G. H. P. Verhoeven ◽  
W. Verschoor ◽  
K. Boer ◽  
D. A. Hesselink ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Kidney Transplantation ◽  
Kidney Transplant ◽  
Allograft Rejection ◽  
Flow Cytometric Analysis ◽  
Principal Component ◽  
Circulating Endothelial Cells ◽  
Clinical Markers ◽  
Kidney Transplant Recipients ◽  
Post Transplant

AbstractThe diagnosis of kidney allograft rejection is based on late histological and clinical markers. Early, specific and minimally-invasive biomarkers may improve rejection diagnosis. Endothelial cells (EC) are one of the earliest targets in kidney transplant rejection. We investigated whether circulating EC (cEC) could serve as an earlier and less invasive biomarker for allograft rejection. Blood was collected from a cohort of 51 kidney transplant recipients before and at multiple timepoints after transplantation, including during a for cause biopsy. The number and phenotype of EC was assessed by flow-cytometric analysis. Unbiased selection of EC was done using principal component (PCA) analysis. Paired analysis revealed a transient cEC increase of 2.1-fold on the third day post-transplant, recovering to preoperative levels at seventh day post-transplant and onwards. Analysis of HLA subtype demonstrated that cEC mainly originate from the recipient. cEC levels were not associated with allograft rejection, allograft function or other allograft pathologies. However, cEC in patients with allograft rejection and increased levels of cEC showed elevated levels of KIM-1 (kidney injury marker-1). These findings indicate that cEC numbers and phenotype are affected after kidney transplantation but may not improve rejection diagnosis.

Detection of Circulating Endothelial Cells: CD146-Based Magnetic Separation Enrichment or Flow Cytometric Assay?

2007 ◽  
Vol 25 (5) ◽  
pp. e1-e2 ◽  
Author(s):  
Françoise Dignat-George ◽  
Florence Sabatier ◽  
Andrew Blann ◽  
Alexander Woywodt
Keyword(s):  
Endothelial Cells ◽  
Magnetic Separation ◽  
Circulating Endothelial Cells ◽  
Flow Cytometric Assay ◽  
Flow Cytometric

scholarly journals POS0424 DETERMINING CIRCULATING ENDOTHELIAL CELLS USING CELLSEARCH SYSTEM IN SYSTEMIC SCLEROSIS PATIENTS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 441.1-441
Author(s):  
F. Pignataro ◽  
L. Zorzino ◽  
W. Maglione ◽  
A. Minniti ◽  
G. Clericuzio ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Systemic Sclerosis ◽  
Peripheral Blood ◽  
Direct Evidence ◽  
Endothelial Damage ◽  
Circulating Endothelial Cells ◽  
Cellsearch System ◽  
The Mean ◽  
Standardized Method ◽  
Active Disease

Background:Endothelial damage and fibroproliferative vasculopathy of small vessels are pathological hallmarks of Systemic Sclerosis (SSc). Detection and analysis of circulating endothelial cells (CECs) detached from affected blood vessels may be an informative tool to study vascular dysfunction and could be considered a novel biomarker of scleroderma vasculopathy. Our group first showed the presence of CECs in SSc by fluorescence-activated cell sorting (FACS), demonstrating that a raised counts of active CECs may represent direct evidence of active vascular disease in SSc. Despite these interesting data, issues related to difficulties in CEC counting through FACS analysis, due their very low concentration in peripheral blood, prevented further investigations in this field. Recently, a specific kit for the detection of CECs has been developed through the CellSearch System (CS), a semi-automated device for the standardized analysis of rare cells, such as CECs, in peripheral blood.Objectives:To assess the counts of CECs determined by the CS in SSc patients and to evaluate their clinical implication and potential as vascular biomarker in SSc.Methods:10mL of blood samples were collected from 29 subjects (19 SSc patients and 10 healthy donors - HDs) and stored in tubes containing a specific preservative, to allow the analysis of 4mL of blood within 72 hours, according to manufacturer instructions. Out of 19 SSc patients, 18 were female, 10 had the limited form and 9 the diffuse cutaneous variant of SSc. CS uses a proprietary kit containing a ferrofluid-based reagent, that target CD146 to magnetically capture CECs, and the immunofluorescent reagents to stain the CECs, defined as CD146+, CD105-PE+, DAPI+ and CD45-APC-. Clinical, laboratoristic and demographic data were also collected.Results:The mean number of CECs in patients with SSc was significantly higher in comparison to HDs (554/4mL vs. 53.5/4 mL, p=0.0042). When analyzed according to disease subset, both lcSSc and dcSSc showed significantly increased levels of CECs in comparison with HDs (p=0.003 and p=0.005, respectively). No statistical difference was observed in the mean number of CECs in patients with lcSSc compared to those with dcSSc. Regarding vascular involvement, the CECs counts strictly correlated with the presence of digital ulcers (DUs) (p=0.0001) showing a median of 863cells/4mL for the SSc patients with DUs versus a median of 276.2/4mL for the SSc patients without DUs. No statistical correlation was found between CECs and serological autoantibody pattern, skin parameters, or joint and muscle involvement. Patients with active disease, according to the EUSTAR Activity Index, showed a higher CECs value than those with inactive disease (p=0.0012).Conclusion:The amount of CECs detectable in peripheral blood has been recently proposed as a marker of endothelial damage in different vascular diseases, including SSc. However, currently no standardized method is available to determine CEC counts, which makes reported data on CECs reliable and suitable. The CS system is a commercially available semi-automated system that enables standardized determination of CECs. Thus, we examined clinical utility of CECs count by this system in SSc patients. Our results confirm that baseline CEC counts, evaluated by a new standardized method, may represent direct evidence of endothelial damage in SSc and could be a promising tool for monitoring active disease and evaluating therapeutic responses to vascular and immunosuppressive treatments.References:[1]Del Papa N, Pignataro F. Front Immunol. 2018 Jun 18;9:1383[2]De Simone C et al. J Eur Acad Dermatol Venereol. 2014 May;28(5):590-6[3]Del Papa N et al. Arthritis Rheum. 2004 Apr;50(4):1296-304Disclosure of Interests:Francesca Pignataro: None declared, Laura Zorzino: None declared, Wanda Maglione: None declared, Antonina Minniti: None declared, Giulia Clericuzio: None declared, Marco Picozzi: None declared, Cecilia Simonelli Employee of: Menarini Silicon Biosystems, Francesco Picardo Employee of: Menarini Silicon Biosystems, Roberto Caporali: None declared, Nicoletta Del Papa: None declared

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