Clinical applications of monitoring immune status with 90 immune cell subsets in human whole blood by 10‐color flow cytometry

2021 ◽  
Author(s):  
Weiwei Wang ◽  
Haibo Li ◽  
Lihua Zhang ◽  
Wenli Jiang ◽  
Lisong Shen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Immune Status ◽  
Immune Cell ◽  
Clinical Applications ◽  
Color Flow ◽  
Human Whole Blood ◽  
Immune Cell Subsets

scholarly journals Single-Cell RNA Sequencing Reveals the Expansion of Cytotoxic CD4+ T Lymphocytes and a Landscape of Immune Cells in Primary Sjögren’s Syndrome

2021 ◽  
Vol 11 ◽  
Author(s):  
Xiaoping Hong ◽  
Shuhui Meng ◽  
Donge Tang ◽  
Tingting Wang ◽  
Liping Ding ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Immune Cell ◽  
Susceptibility Genes ◽  
Primary Sjögren’S Syndrome ◽  
Healthy Controls ◽  
Immune Cell Subsets ◽  
Single Cell Rna Sequencing

ObjectivePrimary Sjögren’s syndrome (pSS) is a systemic autoimmune disease, and its pathogenetic mechanism is far from being understood. In this study, we aimed to explore the cellular and molecular mechanisms that lead to pathogenesis of this disease.MethodsWe applied single-cell RNA sequencing (scRNA-seq) to 57,288 peripheral blood mononuclear cells (PBMCs) from five patients with pSS and five healthy controls. The immune cell subsets and susceptibility genes involved in the pathogenesis of pSS were analyzed. Flow cytometry was preformed to verify the result of scRNA-seq.ResultsWe identified two subpopulations significantly expand in pSS patients. The one highly expressing cytotoxicity genes is named as CD4+ CTLs cytotoxic T lymphocyte, and another highly expressing T cell receptor (TCR) variable gene is named as CD4+ TRAV13-2+ T cell. Flow cytometry results showed the percentages of CD4+ CTLs, which were profiled with CD4+ and GZMB+ staining; the total T cells of 10 patients with pSS were significantly higher than those of 10 healthy controls (P= 0.008). The expression level of IL-1β in macrophages, TCL1A in B cells, as well as interferon (IFN) response genes in most cell subsets was upregulated in the patients with pSS. Susceptibility genes including HLA-DRB5, CTLA4, and AQP3 were highly expressed in patients with pSS.ConclusionsOur data revealed disease-specific immune cell subsets and provided some potential new targets of pSS. Specific expansion of CD4+ CTLs may be involved in the pathogenesis of pSS, which might give valuable insights for therapeutic interventions of pSS.

DETECTION OF ACTIVATED PLATELETS IN WHOLE BLOOD BY FLOW CYTOMETRY

1987 ◽  
Author(s):  
S J Shattil ◽  
J A Hoxie ◽  
M Cunningham ◽  
C S Abrahms ◽  
J O’Brien ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Platelet Activation ◽  
Whole Blood ◽  
Thrombus Formation ◽  
Membrane Surface ◽  
White Blood Cells ◽  
Color Flow ◽  
Platelet Surface ◽  
Activated Platelets

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We have developed a direct test for activated platelets in whole blood that utilizes dual-color flow cytometry and requires no washing steps. Platelets were distinguished from erythrocytes and white blood cells in the flow cytometer by labeling the platelets with biotin-AP1, an antibody specific for membrane glycoprotein lb, and analyzing the cells for phycoerythrin-streptavidin fluorescence. Membrane surface changes resulting from platelet activation were detected with three different FITC-labeled monoclonal antibodies: 1) PAC1, an antibody specific for the fibrinogen receptor on activated platelets; 2) 9F9, which binds to the D-domain of fibrinogen and detects platelet-bound fibrinogen; and 3) S12, which binds to an alpha-granule membrane protein that associates with the platelet surface during secretion. Unstimulated platelets demonstrated no PAC1, 9F9, or S12-specific fluorescence, indicating that they did not bind these antibodies. Upon stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in FITC-fluorescence. The binding of 9F9 to activated platelets required fibrinogen. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 or 9F9 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate that evokes full platelet aggregation and secretion induced maximal binding of all three antibodies. When blood samples containing activated and non-activated platelets were mixed, as few as 0.8% activated platelets could be detected by this technique. There was a direct correlation between ADP-induced FITC-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = 0.99; p < 0.001). These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This method may be useful to assess the degree of platelet activation and the efficacy platelet inhibitor therapy in thrombotic disorders.

scholarly journals P-MAPA, a Fungi-Derived Immunomodulatory Compound, Induces a Proinflammatory Response in a Human Whole Blood Model

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Mariana Torrente Gonçalves ◽  
Carla Cristina Squaiella-Baptistão ◽  
Giselle Pidde ◽  
Priscila Hess Lopes ◽  
Iseu da Silva Nunes ◽  
...  
Keyword(s):  
Animal Models ◽  
Complement Activation ◽  
Whole Blood ◽  
Cell Activation ◽  
Immune Cell ◽  
Ex Vivo ◽  
Proinflammatory Response ◽  
Human Whole Blood ◽  
Human Leukocytes ◽  
Immunomodulatory Properties

P-MAPA is a complex compound, derived from Aspergillus oryzae cultures, that has shown immunomodulatory properties in infection and cancer animal models. Despite promising results in these models, the mechanisms of cellular activation by P-MAPA, suggested to be Toll-like receptor- (TLR-) dependent, and its effect on human immune cells, remain unclear. Using an ex vivo model of human whole blood, the effects of P-MAPA on complement system activation, production of cytokines, and the expression of complement receptors (CD11b, C5aR, and C3aR), TLR2, TLR4, and the coreceptor CD14 were analyzed in neutrophils and monocytes. P-MAPA induced complement activation in human blood, detected by increased levels of C3a, C5a, and SC5b-9 in plasma. As a consequence, CD11b expression increased and C5aR decreased upon activation, while C3aR expression remained unchanged in leukocytes. TLR2 and TLR4 expressions were not modulated by P-MAPA treatment on neutrophils, but TLR4 expression was reduced in monocytes, while CD14 expression increased in both cell types. P-MAPA also induced the production of TNF-α, IL-8, and IL-12 and oxidative burst, measured by peroxynitrite levels, in human leukocytes. Complement inhibition with compstatin showed that P-MAPA-induced complement activation drives modulation of C5aR, but not of CD11b, suggesting that P-MAPA acts through both complement-dependent and complement-independent mechanisms. Compstatin also significantly reduced the peroxynitrite generation. Altogether, our results show that P-MAPA induced proinflammatory response in human leukocytes, which is partially mediated by complement activation. Our data contribute to elucidate the complement-dependent and complement-independent mechanisms of P-MAPA, which ultimately result in immune cell activation and in its immunomodulatory properties in infection and cancer animal models.

Acidosis in Human Whole Blood Does Not Necessarily Impair the Coagulation and the Effect of RFVIIa.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3930-3930
Author(s):  
Dorthe Viuff ◽  
Marianne Kjalke ◽  
Vivian Lind ◽  
Egon Persson ◽  
Mirella Ezban
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Whole Blood ◽  
Annexin A5 ◽  
In Vivo Models ◽  
Human Whole Blood ◽  
Major Interest ◽  
R Value

Abstract Introduction: Acidosis is associated with high mortality in trauma patients. Therefore there is a major interest in generating acidosis models in vitro and in vivo to determine the effect of acidosis on coagulation and to develop treatments. The aim of this study was to examine the effect of acidosis induction in human whole blood using HCl versus Hepes and to analyze the subsequent effect of rFVIIa (NovoSeven®). Materials and Methods: Native human whole blood was obtained from healthy volunteers (n=6) and pH was adjusted to 6.8 using 1 M HCl or 1 M Hepes (pH 6.8). Coagulation was triggered with kaolin or tissue factor (TF, Innovin, final dilution 1:42500) and measured by thrombelastography (TEG, Haemoscope®). Furthermore, the effect of rFVIIa (25nM ∼ 90 mcg/kg) was measured. The TEG parameters R (sec), angle (deg) and maximum amplitude (MA, mm) were recorded and presented as mean±SD. A shorter R and greater angle and MA values are indicative of a more robust clot formation. Statistical analysis was performed by a two-way ANOVA-model. Platelet function was analyzed by platelet aggregation using Multiplate (Dynabyte Medical). Exposure of P-selectin, negatively charged phospholipids (annexin A5 binding) and induction of the active conformation of the fibrinogen receptor GPIIb/IIIa (PAC-1 binding) on platelets after TRAP-stimulation of whole blood was analyzed using a FACS Canto flow cytometer (BD). Results: TEG, platelet aggregation and flow cytometry indicated that lowering the pH to 6.8 by HCl affected the blood significantly different than when pH was lowered by addition of Hepes. HCl-treated blood triggered with either kaolin or TF showed a significantly decreased R value (378±45 or 661±130 vs 539±98 or 888±353 in untreated controls), significantly decreased MA (52±6 or 51±9 vs 66±8 or 62±13) and decreased angle (50±7 or 36±10 vs control 57±10 or 44±19, not significant). Hepes-treated blood triggered with kaolin showed no difference in R (458±52), angle (64±4) and MA (58±9) compared to untreated controls, whereas blood triggered with TF showed significantly shortened R-value (461±91) and enhanced angle (63±5) compared to untreated controls. Hepes treatment had no effect on MA (64±12). rFVIIa significantly shortened R irrespective of the acidosis inducer or clot trigger(HCl/kaolin 283±34, HCl/TF 307±52; Hepes/kaolin 363±32, Hepes/TF 313±46). Although the other TEG parameters were also improved, the effect was only significant when blood was treated with HCl and clotting initiated with TF (angle 48±11, MA 56±10). HCl-induced acidosis abolished platelet aggregation, whereas Hepes-induced acidosis did not alter platelet aggregation compared to normal blood. Flow cytometry showed that platelets from HCl-treated blood were pre-activated as evidence by expression of P-selectin on 70% of the platelets, annexin A5 binding to 14% of the platelets and PAC-1 binding to 62% of the platelets before stimulation. TRAP-stimulation increased P-selectin expression, and PAC1 and Annexin A5 binding to platelets in HCl-treated blood. In contrast, Hepes-treatment did not pre-activate the platelets and the increase in P-selectin expression, and annexin A5 and PAC-1 binding after TRAP-stimulation was as seen for control blood. Conclusion: The method used to lower pH in human blood strongly influences the functionality of the platelets and coagulation factors independent of the final pH. It is therefore important in experimental in vitro and in vivo models to be aware of these dramatically different effects in order to draw correct conclusions.

Cellular Immunity in Hemodialysis Patients: A Quantitative Analysis of Immune Cell Subsets by Flow Cytometry

1995 ◽  
Vol 15 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Souzana Sabeva Deenitchina ◽  
Takash Ando ◽  
Seiya Okuda ◽  
Naoko Kinukawa ◽  
Hideki Hirakata ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Quantitative Analysis ◽  
Cellular Immunity ◽  
Immune Cell ◽  
Hemodialysis Patients ◽  
Immune Cell Subsets

Atlas of the immune cell repertoire in human atherosclerotic plaques characterized by single cell RNA-sequencing and multi-color flow cytometry

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
H Horstmann ◽  
A Lindau ◽  
S Hansen ◽  
P Stachon ◽  
I Hilgendorf ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cell Surface ◽  
Immune Cell ◽  
Surface Marker ◽  
Cell Surface Marker ◽  
Atherosclerotic Plaques ◽  
Color Flow ◽  
Single Cell Rna Sequencing ◽  
Human Carotid

Abstract Rationale Atherosclerosis is a chronic inflammatory disease that is driven by the accumulation of pro- and anti-inflammatory leukocytes in the intima of affected arteries. Yet, the cellular composition of human atherosclerotic plaques is only poorly understood. Here, we characterized immune cells to human carotid atherosclerotic plaques by multi-color flow cytometry and scRNAseq. Methods and results First, we compared a set of previously reported digestion protocols to liberate leukocytes from human carotid plaques after surgical thrombendarteriectomy. One digestion cocktail, containing Collagenase IV and DNase I, was superior regarding cell survival and cell surface marker preservation. Second, leukocytes from 56 surgical specimen were characterized by flow cytometry with a set of 16 parameters and cell surface markers capable of identifying principal hematopoietic leukocyte lineages. This protocol allowed to extract and analyze on average 4x103 viable CD45+ leukocytes from a mean of 988 mg plaque tissue. Surprisingly, we found that atherosclerotic plaques were dominated by T cells with 33.7±2.2% CD4+ T-helper cells and 25.6±2.5% CD8+ cytotoxic T cells. CD11b+ myeloid cells, including monocytes and macrophages, represented only 20.2±4.0% of all CD45+ leukocytes. CD19+B cells and CD56+ NK-cells accounted for 3.9±1.2 and 3.3±0.5%, respectively. TCR-g/d+ T cells and neutrophils were undetectable in atherosclerotic plaques. This cellular composition differed significantly from peripheral blood, but was not relevantly changed between different plaque locations, indicating that macrophage-rich necrotic cores mostly contain dead cells. We confirmed the principal composition of human plaques by single-cell RNA-sequencing from six patients. To allow an estimation of cellular heterogeneity independent of classical cell surface marker assignment, we performed an unsupervised cluster detection algorithm by t-distributed stochastic neighbor embedding (tSNE) and found more than 16 leukocyte clusters with unique cell surface marker expression, suggesting an unexpected high diversity of plaque leukocytes. Conclusion We developed an immune cell phenotyping protocol optimized for human carotid plaques. The definition of phenotypes and frequencies in atherosclerotic plaques will allow to build clinical associations between the immune cell composition and clinical outcomes in future. Funding Acknowledgement Type of funding source: None

Sign in / Sign up

Export Citation Format

Share Document