Abstract 4474: A quantitative flow cytometry method for the measurement of HLA-DR expression level on monocyte subsets

2020 ◽  
Author(s):  
Wei Huang
Keyword(s):  
Flow Cytometry ◽  
Expression Level ◽  
Monocyte Subsets ◽  
Hla Dr ◽  
Quantitative Flow

scholarly journals Alveolar compartmentalization of inflammatory and immune cell biomarkers in pneumonia-related ARDS

Critical Care ◽  
2021 ◽  
Vol 25 (1) ◽  
Author(s):  
Inès Bendib ◽  
Asma Beldi-Ferchiou ◽  
Frédéric Schlemmer ◽  
Mathieu Surenaud ◽  
Bernard Maitre ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Hospital Mortality ◽  
Distress Syndrome ◽  
Immune Cell ◽  
Routine Care ◽  
Clinical Endpoint ◽  
Blood Samples ◽  
Hla Dr ◽  
Serum Ratio ◽  
Immunocompetent Patients

Abstract Background Biomarkers of disease severity might help individualizing the management of patients with the acute respiratory distress syndrome (ARDS). Whether the alveolar compartmentalization of biomarkers has a clinical significance in patients with pneumonia-related ARDS is unknown. This study aimed at assessing the interrelation of ARDS/sepsis biomarkers in the alveolar and blood compartments and explored their association with clinical outcomes. Methods Immunocompetent patients with pneumonia-related ARDS admitted between 2014 and 2018 were included in a prospective monocentric study. Bronchoalveolar lavage (BAL) fluid and blood samples were obtained within 48 h of admission. Twenty-two biomarkers were quantified in BAL fluid and serum. HLA-DR+ monocytes and CD8+ PD-1+ lymphocytes were quantified using flow cytometry. The primary clinical endpoint of the study was hospital mortality. Patients undergoing a bronchoscopy as part of routine care were included as controls. Results Seventy ARDS patients were included. Hospital mortality was 21.4%. The BAL fluid-to-serum ratio of IL-8 was 20 times higher in ARDS patients than in controls (p < 0.0001). ARDS patients with shock had lower BAL fluid-to-serum ratio of IL-1Ra (p = 0.026), IL-6 (p = 0.002), IP-10/CXCL10 (p = 0.024) and IL-10 (p = 0.023) than others. The BAL fluid-to-serum ratio of IL-1Ra was more elevated in hospital survivors than decedents (p = 0.006), even after adjusting for SOFA and driving pressure (p = 0.036). There was no significant association between alveolar or alveolar/blood monocytic HLA-DR or CD8+ lymphocytes PD-1 expression and hospital mortality. Conclusions IL-8 was the most compartmentalized cytokine and lower BAL fluid-to-serum concentration ratios of IL-1Ra were associated with hospital mortality in patients with pneumonia-associated ARDS.

Typing for all known HLA-DR alleles by group-specific PCR and flow cytometry-based multiplexed single nucleotide extension

2003 ◽  
Vol 64 (10) ◽  
pp. S38
Author(s):  
Yanzheng Zhang ◽  
Charlie Costin ◽  
Christopher Giang ◽  
Robert Vorhaben ◽  
Peter Stastny
Keyword(s):  
Flow Cytometry ◽  
Single Nucleotide ◽  
Specific Pcr ◽  
Hla Dr

Vitamin D modulates the expression of HLA-DR and CD38 after in vitro activation of T-cells

2017 ◽  
Vol 29 (3) ◽  
Author(s):  
Simon Villegas-Ospina ◽  
Wbeimar Aguilar-Jimenez ◽  
Sandra M. Gonzalez ◽  
María T. Rugeles
Keyword(s):  
Vitamin D ◽  
Flow Cytometry ◽  
Healthy Subjects ◽  
Mononuclear Cells ◽  
Activation Markers ◽  
Hla Dr ◽  
In Vitro Activation ◽  
Cells Cultured

AbstractObjective:Vitamin D (VitD) is an anti-inflammatory hormone; however, some evidence shows that VitD may induce the expression of activation markers, such as CD38 and HLA-DR. We explored its effect on the expression of these markers on CD4Materials and methods:CD38 and HLA-DR expression was measured by flow cytometry in PHA/IL-2-activated mononuclear cells cultured under VitD precursors: three cholecalciferol (10Results:Cholecalciferol at 10Conclusion:Although no significant correlations were observed in vivo in healthy subjects, VitD treatment in vitro modulated immune activation by increasing the expression of CD38 and decreasing the proliferation of HLA-DR

scholarly journals Predictive value of tyrosine phosphatase receptor gamma for the response to treatment tyrosine kinase inhibitors in chronic myeloid leukemia patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohamed A. Ismail ◽  
Marzia Vezzalini ◽  
Hisham Morsi ◽  
Ahmad Abujaber ◽  
Ali Al Sayab ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Tyrosine Phosphatase ◽  
Expression Level ◽  
Response To Treatment ◽  
Healthy Individuals

AbstractProtein tyrosine phosphatase receptor gamma (PTPRG) is a member of the receptor-like family protein tyrosine phosphatases and acts as a tumor suppressor gene in different neoplasms. Recent studies reported the down-regulation of PTPRG expression levels in Chronic Myeloid Leukemia disease (CML). In addition, the BCR-ABL1 transcript level is currently a key predictive biomarker of CML response to treatment with Tyrosine Kinase Inhibitors (TKIs). The aim of this study was to employ flow cytometry to monitor the changes in the expression level of PTPRG in the white blood cells (WBCs) of CML patients at the time of diagnosis and following treatment with TKIs. WBCs from peripheral blood of 21 CML patients were extracted at diagnosis and during follow up along with seven healthy individuals. The PTPRG expression level was determined at protein and mRNA levels by both flow cytometry with monoclonal antibody (TPγ B9-2) and RT-qPCR, and BCR-ABL1 transcript by RT-qPCR, respectively. PTPRG expression was found to be lower in the neutrophils and monocytes of CML patients at time of diagnosis compared to healthy individuals. Treatment with TKIs nilotinib and Imatinib Mesylate restored the expression of PTPRG in the WBCs of CML patients to levels observed in healthy controls. Moreover, restoration levels were greatest in optimal responders and occurred earlier with nilotinib compared to imatinib. Our results support the measurement of PTPRG expression level in the WBCs of CML patients by flow cytometry as a monitoring tool for the response to treatment with TKIs in CML patients.

Immunophenotypic Study of Basophils by Multiparameter Flow Cytometry

2008 ◽  
Vol 132 (5) ◽  
pp. 813-819
Author(s):  
Xiaohong Han ◽  
Jeffrey L. Jorgensen ◽  
Archana Brahmandam ◽  
Ellen Schlette ◽  
Yang O. Huh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Myelogenous Leukemia ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Color Flow ◽  
Aberrant Expression ◽  
Flow Cytometric ◽  
Hla Dr

Abstract Context.—The immunophenotypic profile of basophils is not yet fully established, and the immunophenotypic changes in chronic myelogenous leukemia are not fully characterized. Objective.—To establish a comprehensive immunophenotypic spectrum of normal basophils and to assess the range of immunophenotypic aberrations of basophils in chronic myelogenous leukemia. Design.—Using 4-color flow cytometry, we compared the immunophenotypic profile of basophils in peripheral blood or bone marrow samples from 20 patients with no evidence of neoplasia to basophils from 15 patients with chronic myelogenous leukemia. Results.—Basophils in control cases were all positive for CD9, CD13, CD22, CD25 (dim), CD33, CD36, CD38 (bright), CD45 (dimmer than lymphocytes and brighter than myeloblasts), and CD123 (bright), and were negative for CD19, CD34, CD64, CD117, and HLA-DR. Basophils in all chronic myelogenous leukemia patients possessed 1 to 5 immunophenotypic aberrancies. The most common aberrancies were underexpression of CD38, followed by aberrant expression of CD64 and underexpression of CD123. CD34 and CD117 were present in cases with basophilic precursors. Myeloblasts showed a distinct immunophenotypic profile, as they typically expressed CD34 and CD117, showed dimmer expression (compared with basophils) of CD38, CD45, and CD123, and lacked expression of CD22. Conclusions.—Flow cytometric immunophenotyping can identify immunophenotypic aberrations of basophils in chronic myelogenous leukemia, and discriminate basophils from myeloblasts.

scholarly journals Phagocytic activity of blood monocytes in response to methicillin-resistant strains of Staphylococcus aureus

2020 ◽  
Vol 10 (3) ◽  
pp. 551-557
Author(s):  
O. A. Kolenchukova ◽  
N. I. Sarmatova ◽  
A. V. Moshev
Keyword(s):  
Respiratory Burst ◽  
Peripheral Blood ◽  
Phagocytic Activity ◽  
Fluorescein Isothiocyanate ◽  
Phagocytic Index ◽  
Blood Monocytes ◽  
Monocyte Subsets ◽  
Superoxide Anion Production ◽  
Hla Dr ◽  
Burst Intensity

Current study performed to estimate the phagocytic activity of blood monocytes of varying phenotypes exposed to MRSA and MSSA strains.  Objects: Blood monocytes were collected from 25 healthy adults (age: 25–45 years). Live suspensions of MRSA/MSSA strains were used at concentration of 106 colony-forming units (CFU)/mL.  Metods. Phagocytic functions were estimated by using fluorescein isothiocyanate (FITC)-labelled MRSA and MSSA strains followed by running flow cytometry on FC 500 series flow cytometer (Beckman Coulter, USA). Whole peripheral blood cells were directly labelled with immunofluorescently tagged monoclonal CD14-PE/CD45-ECD/HLA-DR-PC5/CD16-PC7 antibodies (Beckman Coulter, USA). Respiratory burst intensity was evaluated in monocytes by measuring activity of lucigeninand luminol-dependent spontaneous and induced chemiluminescence. Monocytes were induced by using live suspension of MRSA/MSSA strains at a concentration of 106 CFU/mL. Results and discussion. While studying luminol-dependent monocyte activities after exposure to MRSA vs. MSSA, it was observed a 3.5-fold decreased curve square, whereas lucigenin-dependent chemiluminescence was increased by 6-fold. Compared to MSSA exposure, index of activation (IA) was decreased by 1.1-fold in response to MRSA exposure that was confirmed by lowered release of reactive oxygen species (ROS) from monocytes in response to MRSA exposure. Moreover, IRSS increased by 1.3-fold upon MRSA exposure. Examining monocyte oxygen-independent phagocytosis against MRSA vs. MSSA revealed significantly increased phagocytic number and concomitantly decreased phagocytic index. An evaluation of the activities of various monocyte subsets in response to MRSA vs. MSSA revealed increased phagocytic index by 1.5-fold for CD14lowCD16+ and CD14+CD16+ monocyte subsets as well as 3-fold for CD14+CD16– monocytes. Counts for all phagocytic subsets were decreased (1.4-, 1.5- and 4-fold for CD14lowCD16+, CD14+CD16+ and CD14+CD16– monocytes, respectively). To summarize, intensity of the respiratory burst was lowered upon MRSA exposure and percentage of monocyte subsets. Overall deficiency of superoxide anion production was observed in response to MRSA. In contrast, oxygen-independent event revealed phenotypic changes in frequency of peripheral blood monocytes upon MRSA exposure. We observed that CD14+CD16– classical monocytes were more rapidly activated. Conclusion. Thus, we concluded that CD14+CD16– monocytes became more rapidly activated but exhibited less effective phagocytosis, whereas CD14+CD16+ and CD14lowCD16+ monocytes were more slowly activated and demonstrated stronger phagocytic activity.

scholarly journals Developing a Simple Algorithm Based on Multiparameter Flow Cytometry for Fast Screening of Acute Promyelocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-26
Author(s):  
Vitória Ceni Ceni ◽  
Katia B Pagnano ◽  
Gislaine B O Duarte ◽  
Marina DB Pellegrini ◽  
Bruno Kosa Duarte ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Discriminant Analysis ◽  
Acute Promyelocytic Leukemia ◽  
Simple Algorithm ◽  
Diagnostic Algorithm ◽  
Promyelocytic Leukemia ◽  
Advisory Board ◽  
Multiparameter Flow Cytometry ◽  
Fast Screening ◽  
Hla Dr

Introduction: Acute promyelocytic leukemia (APL) is a genetically and molecularly well-defined type of acute leukemia that is curable but has a frequent early mortality due to bleeding. So, there is a need for a fast diagnostic screening in order to start appropriate therapy. Multiparameter flow cytometry (MFC) is usually performed in all types of acute myeloid leukemias (AMLs) but only few features have been described as characteristic of APL. Aim: to develop a diagnostic algorithm based on the intensity of expression of several antigens examined by MFC in AML that could reliably discriminate between APL and the other types of AML. Material and Methods: Consecutive newly diagnosed AMLs treated in our Institution during the last 2 years entered the study. Immunophenotyping was included in the diagnostic workup. An 8-color platform based on the Euroflow recommendations was used. The mean fluorescence intensity (MFI) of each antigen tested was assessed and those best discriminating between APL and all other types of AML were obtained by a discriminant analysis. Phenotypic characteristics of normal myeloblasts taken from examinations of bone marrow (BM) MFC performed for the diagnosis of cytopenias were used as controls. Results: 24 cases of APL and 56 cases of other primary AML entered the study. Median age: 39 (23-56) and 62(26-81) years respectively. Concerning ELN risk groups of non-APL cases, 13 were favorable risk, 26 were intermediate and 09 were adverse risk. In 8 cases risk assessment was not possible due to the absence of cytogenetics. Moreover, among APL patients, 7 cases had a FLT3-ITD mutation. Among non-APL AMLs, 4 had FLT3-ITD mutation, 4 had NPM1 and 10 had FLT3-ITD and NPM1mutation. Concerning antigen expression, CD34 was expressed in only 1/24 APL samples, and in 18/56 samples from non-APL AMLs. The following flow features were differentially expressed in both groups: SSC (p &lt;0.0001), CD45 (p=0.02), CD13 (p=0.001), CD64 (p=0.004), HLA-DR (p&lt;0.0001) and CD33 (p&lt;0.0001) (Table 1). In the discriminant analysis, MFI CD34 and MFI HLA-DR were able to accurately classify APL and non-APL AML in only 62.5%. However, after the addition of the ratio of SSC between blasts and lymphocytes, these 3 parameters were able to differentiate APL from non-APL AML in 91.2% of the cases. Conclusion: MFC was adequate for a fast screening of APL in most cases. Expression of CD34 was not very useful, as many AMLs do not express this antigen, similar to APL, but SSC, together with HLA-DR could discriminate both types of leukemia in most cases. Disclosures Pagnano: Astellas: Other: Advisory Board and lecture; Novartis: Other: Advisory Board; Pintpharma: Other: Lecture; EMS: Other: Lecture. Duarte:Janssen: Other: Lecture; Astellas: Other: Lecture.

scholarly journals METTL3-mediated M6a Modification Regulates Cell Cycle Progression of Dental Pulp Stem Cells

2021 ◽  
Author(s):  
Haiyun Luo ◽  
Wenjing Liu ◽  
Yanli Zhang ◽  
Xiao Jiang ◽  
Shiqing Wu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tissue Engineering ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Differentially Expressed Genes ◽  
Dental Pulp ◽  
Cell Cycle Control ◽  
Dental Pulp Stem Cells ◽  
Differentially Expressed ◽  
Expression Level

Abstract Background: Dental pulp stem cells (DPSCs) exhibited self-renewal, pluripotency capacity and served as promising cells source in endodontic regeneration and tissue engineering. Meanwhile, the regenerative capacity of DPSCs is limited and reduced in long lifespan. N6-methyladenosine (m6A) is the most prevalent, reversible internal modification in RNAs. The methyltransferases complex and demethylases mediated m6A methylation and cooperated to impact various biological processes associated with stem cell fate determination. However, the biological effect of m6A methylation in DPSCs remained unclear. Methods: Cell surface markers and differentiation potential of primary DPSCs were identified and m6A immunoprecipitation with deep sequencing (m6A RIP-seq) was used to uncover characteristics of m6A modifications in DPSCs transcriptome. Expression level of m6A-related genes were evaluated in immature/mature pulp tissues and cells. Lentiviral vectors were constructed to knockdown or overexpress methyltransferase like 3 (METTL3). Cell morphology, viability, senescence and apoptosis were further analyzed by β-galactosidase, TUNEL staining and flow cytometry. Bioinformatic analysis combing m6A RIP and shMETTL3 RNA-seq was used to functionally enrich overlapped genes and screen target of METTL3. Cell cycle distributions were assayed by flow cytometry and m6A RIP-qPCR was used to confirm METTL3 mediated m6A methylation in DPSCs. Results: Here, m6A peaks distribution, binding area and motif in DPSCs were first revealed by m6A RIP-seq. We also found a relative high expression level of METTL3 in immature DPSCs with superior regenerative potential and METTL3 knockdown induced cell apoptosis and senescence. Furthermore, Conjoint analysis of m6A RIP and RNA-sequencing showed differentially expressed genes affected by METTL3 depletion was mainly enriched in cell cycle, mitosis and alteration of METTL3 expression resulted in cell cycle arrest which indicated METTL3 make essential effect in cell cycle control. To further investigate underlying mechanisms, we explored proteins interaction network of differentially expressed genes and Polo-like Kinase 1 (PLK1), a critical cycle modulator was identified as target of METTL3-mediated m6A methylation in DPSCs. Conclusions: These results revealed m6A methylated hallmarks in DPSCs and a regulatory role of METTL3 in cell cycle control. Our study shed light on therapeutic approaches in vital pulp therapy and serve new insight in stem cells based tissue engineering.

Quantitative Flow Cytometry-Based Assays for Measuring

2020 ◽  
pp. 115-129
Author(s):  
David E. Gordon ◽  
Amber S. Shun-Shion ◽  
Asral W. Asnawi ◽  
Andrew A. Peden
Keyword(s):  
Flow Cytometry ◽  
Quantitative Flow
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