scholarly journals The High PMNs Phagocytosis Resistance of Enterococcal Isolates from RTx Patients

2015 ◽  
Vol 2015 ◽  
pp. 1-5 ◽  
Author(s):  
Tomasz Jarzembowski ◽  
Agnieszka Daca ◽  
Jacek M. Witkowski ◽  
Ewa Bryl ◽  
Bolesław Rutkowski
Keyword(s):  
Flow Cytometry ◽  
High Risk ◽  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
Culture Media ◽  
Immunocompromised Patients ◽  
Bacterial Cells ◽  
Opportunistic Pathogens ◽  
Fluorescent Reporter ◽  
Significant Difference

Infections caused by opportunistic pathogens such as enterococci remain difficult to manage, especially in immunocompromised patients. Because of infections’ limited symptoms in such patients the additional problems are to find proper diagnostic criteria and the management of infection. Here we aimed to compare the resistance of commensal enterococcal strains and RTx patients’ isolates, to PMNs phagocytosis. Thirty-six enterococcal urine and faecal isolates from RTx patients and 17 faecal isolates from healthy volunteers were cultured in planktonic and biofilm forms in 37°C or 42°C. Another tested variable was the addition of immunosuppressant to the culture media. Bacterial cells were stained with fluorescent reporter (CFDA, PI) and incubated with PMNs. Results of phagocytosis were estimated as a mean fluorescence intensity (MFI) of PMNs using flow cytometry. Commensal enterococci cultured in all abovementioned (37°C and 42°C/the addition of immunosuppressant) conditions were less resistant to phagocytosis compared to RTx isolates. Observed significant difference in phagocytosis resistance suggests that patients in immunosuppression are colonized with high risk strains which may lead to the development of infection.

Standardization of Functional Immune Cell-Based Assays: An Integral Aspect to Vaccine and Biologic Development and Validation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3933-3933
Author(s):  
Julie Wilkinson ◽  
Cecilia Smith ◽  
Sybil D’Costa ◽  
Enrique Rabellino
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Sample Preparation ◽  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
Immune Cell ◽  
Cell Populations ◽  
Surrogate Markers ◽  
Functional Assays ◽  
Variable Nature

Abstract The utility of the ex-vivo evaluation of immune cell functionality in the context of a) Determining an efficacious vaccine strategy for infectious diseases/cancer, b) Determining a tolerance profile in autoimmunity and transplantation, and c) understanding the basic mechanisms of immune cell responses in disease pathogenesis is well recognized. However, the benefit of these assays as surrogate markers of immune cell activity in vivo has not been fully realized due to the variable nature of these in vitro assays which is particularly pronounced in T cell functional assays. This variability arises from a variety of factors ranging from choice of assay, source of the cells, the sample processing methodology (isolation, freezing, thawing, and culturing), sample staining protocol for the chosen assay and ultimately data analysis, and data reduction. With a view to reducing variability and standardizing targeted steps of T cell functional assays, an automated methodology for simultaneous staining and analysis of multiple intracellular cytokines and cytotoxicity markers via flow cytometry was developed and validated. A 5-color flow cytometry assay (2–3 surface markers; 2 intracellular markers) was developed to characterize the restricted polyclonal (SEB/CD28) and antigen specific (CEF peptide pool) cytokine and cytotoxic profile response in human PBMCs. A modification to available sample preparation instruments was performed that enabled the automated pipetting, incubation, and staining of intracellular and surface molecules of stimulated human peripheral blood mononuclear cell populations (PBMC) for flow cytometric analysis. Statistically significant reductions in both inter and intra assay variability was observed in the automated methodology as compared to the manual assay with improvements in CVs for positive cell numbers and mean fluorescence intensity. For example, the inter assay CVs for IFNg cytokine producing CD4+ T cell populations improved from approximately 15 to 5, while the mean fluorescence intensity improved nearly 5 fold with automation. Importantly, the automated methodology furnished comparable responses in percent positive cytokine/cytotoxicity profiles as compared to the manual method while reducing the “handson” sample preparation and analysis time from 2 hours to 20 minutes. With the standardization of functional assays, other sources of variability in assays result can now be addressed specifically e.g. specimen handling, freezing, thawing, culturing, or biological. Standardized multiparametric functional profiling of the cells thus reveals the complex nature of the immune response and lends credence to their use as surrogate markers of efficacy and functionality.

scholarly journals Evaluation of Influenza-Specific Humoral Response by Microbead Array Analysis

2011 ◽  
Vol 22 (1) ◽  
pp. 25-29 ◽  
Author(s):  
Yoav Keynan ◽  
Tavis Bodnarchuk ◽  
Stephen Wayne ◽  
Yan Li ◽  
Keith R. Fowke
Keyword(s):  
Influenza Vaccination ◽  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
New Caledonia ◽  
Small Sample ◽  
Antigen Specificity ◽  
Serum Samples ◽  
P Values ◽  
Significant Difference ◽  
Microbead Array

RATIONALE: Quantitation and determination of antigen specificity of systemic and mucosal immune responses to influenza vaccination is beneficial for future vaccine development. Previous methods to acquire this information were costly, time consuming and sample exhaustive. The benefits of suspension microbead array (MBA) analysis are numerous. The multiplex capabilities of the system conserve time, money and sample, while generating statistically powerful data.OBJECTIVE: To demonstrate the use of the assay by comparing the humoral influenza-specific responses of two cohorts from two countries that differed in circulating influenza strains and rates of influenza vaccination.METHODS: Influenza hemagglutinin (HA) from different strains were coated on microbeads and incubated with serum samples to capture immunoglobulin (Ig) A1and IgG1host antibodies.RESULTS: Statistically significant differences in IgA1and IgG1exist between the serum samples from Winnipeg (Manitoba) donors and those from Kenyan (Africa) donors. Data were compared using Mann-Whitney nonparametric tests. The Winnipeg donors had higher mean fluorescence intensity values, with significant P values for anti-HA IgA1to A/Wyoming/3/2003 (P=0.044), A/Vietnam/1203/2004 (P=0.0179), A/New Caledonia/20/99 (P<0.0001) and B/Tokyo/53/99 (P=0.0002). No differences were seen between the groups in their response to B/Jilin/20/2003. The Winnipeg donors had higher mean fluorescence intensity values, with significant P values for anti-HA IgG1to A/Wyoming/3/2003 (P=0.0135), B/Tokyo/53/99 (P=0.006) and B/Jilin20/2003 (P=0.026).CONCLUSION: Influenza-specific IgA1and IgG1antibodies were successfully detected using MBA technology. A significant difference in antibody response was observed between Winnipeg and Kenyan donor serums. MBA analysis is a relatively quick and cost-effective method for serum antibody analysis. The potential to simultaneously assay small sample volumes for a multitude of antigens makes this method invaluable for future vaccine response monitoring.

95-P: Post transplant monitoring of mean fluorescence intensity (MFI) for high risk renal transplant patients

2009 ◽  
Vol 70 ◽  
pp. S59
Author(s):  
Carrera Kostur ◽  
Luz Stamm ◽  
Lee Ann Tibbles ◽  
Kevin McLaughlin ◽  
Wenjie Wang ◽  
...  
Keyword(s):  
High Risk ◽  
Renal Transplant ◽  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
Post Transplant ◽  
Transplant Patients ◽  
Renal Transplant Patients

scholarly journals Increased Lipid Peroxidation May Be Linked to Ferritin Levels Elevation in Adult-Onset Still’s Disease

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1508
Author(s):  
Po-Ku Chen ◽  
Kai-Jieh Yeo ◽  
Po-Hao Huang ◽  
Shih-Hsin Chang ◽  
Ching-Kun Chang ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Flow Cytometry ◽  
Fluorescence Intensity ◽  
Plasma Levels ◽  
Mean Fluorescence Intensity ◽  
Inflammatory Responses ◽  
Flow Cytometry Analysis ◽  
Inflammatory Parameters ◽  
Healthy Control ◽  
The Relationship

Lipid peroxidation (LPO) and hyper-ferritinemia are involved in inflammatory responses. Although hyper-ferritinemia is a characteristic of AOSD, its link to LPO remains unclear. We investigated the association between LPO and ferritin expression, and evaluated the relationship between LPO-related metabolites and inflammatory parameters. Mean fluorescence intensity (MFI) of LPO (C11-Biodipy581/591)-expressing PBMCs/monocytes in AOSD patients and healthy control (HC) subjects was determined by flow-cytometry analysis. Expression of ferritin and cytokines on PBMCs/macrophages was examined by immunoblotting. Plasma levels of LPO-related metabolites and cytokines were determined by ELISA and the MULTIPLEX platform, respectively. LPO MFI on PBMCs/monocytes were significantly higher in patients (median 4456 and 9091, respectively) compared with HC (1900, p < 0.05, and 4551, p < 0.01, respectively). Patients had higher ferritin expression on PBMCs (mean fold, 1.02) than HC (0.55, p < 0.05). Their ferritin expression levels on PBMCs stimulated with LPO inducers erastin or RSL3 (2.47 or 1.61, respectively) were higher than HC (0.84, p < 0.05, or 0.74, p < 0.01). Ferritin expression on erastin-treated/IL-1β-treated macrophages from patients were higher than those from HC (p < 0.001). The elevated levels of LPO-related metabolites, including malondialdehyde and 4-hydroxyalkenals, were positively correlated with disease activity scores, suggesting LPO involvement in AOSD pathogenesis. Increased ferritin expression on PBMCs/macrophages stimulated with LPO inducers indicates a link between LPO and elevated ferritin.

scholarly journals VS38 Identifies Myeloma Cells With Dim CD38 Expression and Plasma Cells Following Daratumumab Therapy, Which Interferes With CD38 Detection for 4 to 6 Months

2019 ◽  
Author(s):  
Elizabeth L Courville ◽  
Sophia Yohe ◽  
Paula Shivers ◽  
Michael A Linden
Keyword(s):  
Flow Cytometry ◽  
Plasma Cell ◽  
Fluorescence Intensity ◽  
Plasma Cells ◽  
Myeloma Cells ◽  
Flow Cytometry Data ◽  
Cd38 Expression ◽  
Cell Percentage ◽  
Significant Difference ◽  
Institutional Experience

Abstract Objectives We report our institutional experience using VS38 to evaluate plasma cells by flow cytometry. Methods Flow cytometry data were reanalyzed to compare plasma cell percentages between the standard panel and VS38 panel. Natural killer (NK) and plasma cell CD38 median fluorescence intensity (MFI) values were calculated. Results Our cohort included 63 specimens from 38 patients. Twenty-six had received daratumumab (monoclonal anti-CD38 therapy) between less than 1 month and 17 months prior. For NK and plasma cells, CD38 MFI values were suppressed for 0 to 4 months and started to increase 4 to 6 months after last exposure. There was no significant difference in clonal plasma cell percentage calculated by the VS38 and standard panels; however, identification and quantification using the VS38 panel were easier. Conclusions VS38 is a viable alternative to bright CD38 to identify plasma cells and particularly helpful in myeloma cases with dim CD38 and after daratumumab. Daratumumab interference with CD38 identification persists 4 to 6 months after the last exposure.

scholarly journals Expression of CD14 and toll-like receptors 2 and 4 by milk neutrophils in bovine mammary glands infected with Corynebacterium bovis

2015 ◽  
Vol 35 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Maiara G. Blagitz ◽  
Fernando N. Souza ◽  
Camila F. Batista ◽  
Bruna P. Santos ◽  
Andrea C. Parra ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Fluorescence Intensity ◽  
Somatic Cell Count ◽  
Mammary Glands ◽  
Negative Control ◽  
Median Fluorescence Intensity ◽  
Toll Like Receptor ◽  
Significant Difference ◽  
Culture Negative

This study evaluated the expression of CD14, toll-like receptor (TLR) 2 and TLR4 on the surface of milk neutrophils in bovine mammary glands infected with Corynebacterium bovis. Here, we used 23 culture-negative control quarters with no abnormal secretion on the strip cup test and milk somatic cell count lower than 1x105 cells/mL, and 14 C. bovis infected quarters. The identification of neutrophils, as well as, the percentage of neutrophils that expressed CD14, TLR2 and TLR4 were analyzed by flow cytometry using monoclonal antibodies. The present study encountered no significant difference in the percentages of milk neutrophils that expressed TLR2 and TLR4 or in the expression of TLR4 by milk neutrophils. Conversely, a lower median fluorescence intensity of TLR2 in milk neutrophils was observed in C. bovis-infected quarters. The percentage of neutrophils that expressed CD14 and the median fluorescence intensity of CD14 in milk neutrophils was also lower in C. bovis-infected quarters.

scholarly journals Protistan Grazing Analysis by Flow Cytometry Using Prey Labeled by In Vivo Expression of Fluorescent Proteins

2003 ◽  
Vol 69 (11) ◽  
pp. 6848-6855 ◽  
Author(s):  
Yutao Fu ◽  
Charles O'Kelly ◽  
Michael Sieracki ◽  
Daniel L. Distel
Keyword(s):  
Flow Cytometry ◽  
Prey Species ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Heterotrophic Flagellate ◽  
Broad Host Range ◽  
Bacterial Cells ◽  
Protein Markers ◽  
Significant Difference ◽  
Paraphysomonas Imperforata

ABSTRACT Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells.

Function of Large and Small Platelets Differs, Depending on Extracellular Calcium Availability and Type of Inductor

2020 ◽  
Vol 120 (07) ◽  
pp. 1075-1086
Author(s):  
Stefan Handtke ◽  
Jan Wesche ◽  
Raghavendra Palankar ◽  
Andreas Greinacher ◽  
Thomas Thiele
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
Thrombin Generation ◽  
Adenosine Diphosphate ◽  
Thrombin Receptor ◽  
Functional Studies ◽  
Functional Differences ◽  
Calcium Availability ◽  
The Impact

AbstractIt is widely anticipated that large platelets are more reactive than small platelets. This was mainly shown in Ca2+-poor media albeit extracellular Ca2+ is utilized by platelets for activation. We determined the impact of extracellular Ca2+ on functional differences between large and small platelets in response to thrombin receptor activating peptide 6 (TRAP-6), adenosine diphosphate (ADP), and epinephrine. In Ca2+-poor buffer, large platelets responded stronger to TRAP-6 which equalized in Ca2+ containing buffer. Large platelets contained and mobilized more Ca2+ from their intracellular stores upon TRAP-6 stimulation explaining their better reactivity in Ca2+-poor media. Stronger aggregation of large platelets in response to ADP also equalized in presence of Ca2+, whereas large platelets responded weaker to ADP in flow cytometry (CD62P-expression: 9.7 mean fluorescence intensity [MFI] [4.4–17.9] vs. 17.5 MFI [6.1–45.6], p = 0.0234) and PAC-1 binding (11.1 MFI [5.7–19.6] vs. 20.5 MFI [14.4–35.0], p = 0.0078). Epinephrine response was stronger in large platelets (CD62P-expression: 11.8 MFI [6.8–33.0] vs. 6.8 MFI [2.5–15.2], p = 0.0078; PAC-1 binding 18.9 MFI [13.6–38.4] vs. 13.0 MFI [6.8–22.4], p = 0.0234; max. aggregation 82.9% [58.7–94.8] vs. 77.2% [19.8–88.8], p = 0.0313), which expressed more α2A receptors. Epinephrine further increased phosphatidylserine (PS) exposure especially in large platelets. PS-positive platelets progressively divided into two subpopulations with high or basic intracellular Ca2+ dependent on extracellular Ca2+. Thrombin generation was faster with small, but accelerated by PS exposure and epinephrine-coactivated large platelets. We show that responses of large and small platelets differ depending on extracellular Ca2+ availability and the inductor. Careful control of extracellular Ca2+ is necessary in functional studies with large and small platelets.

scholarly journals Features of immunophenotypic finding B-cell lymphoproliferative diseases by flow cytometry

2020 ◽  
Vol 101 (1) ◽  
pp. 145-152
Author(s):  
J J Chuksina ◽  
E V Kataeva ◽  
T A Mitina
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Information Content ◽  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
Differential Diagnostics ◽  
Lymphoproliferative Diseases ◽  
Color Flow ◽  
Positive Expression ◽  
Hodgkin's Lymphomas

Aim. To assess the information content of conventional and additional immunophenotypic markers (CD200, CD305) in the differential diagnosis B-cell lymphoproliferative diseases by flow cytometry. Methods. An immunophenotypic study using 4-color flow cytometry was performed in 204 patients with different variants of B-cell non-Hodgkin's lymphomas. The study material included peripheral blood and bone marrow. The expression of CD45, CD19, CD20, CD22, CD79b, CD79a, CD5, CD10, CD23, FMC7, CD43, CD38, CD11c, CD103, CD25, CD 200, CD 305, light chains of immunoglobulins (kappa/lambda) using monoclonal antibodies (Becton Dickinson, USA) was evaluated. The intensity of antigen expression was assessed using mean fluorescence intensity (y. e.). Results. Conventional FMC7-positive expression revealed only half patients with different variants of leukemization of non-Hodgkin's lymphomas, whereas atypical positive expression of CD23 was observed in patients with marginal spleen lymphoma and follicular lymphoma in 27.3 and 28.6% of cases, respectively. In mantle cell lymphoma, expression of CD200 in B-cell was detected in a significantly smaller number of observations, accompanied by a significant decrease in the average intensity of CD200 fluorescence compared to B-cell chronic lymphocytic leukemia (B-CLL) cells. The mean fluorescence intensity (MFI) of CD305 in hairy cell leukemia is significantly higher than in splenic marginal zone lymphoma (SMZL) with villous lymphocytes. Conclusion. Different levels of the information content of some conventional markers were revealed in differential immunophenotypic diagnosis of B-cell lymphoproliferative diseases by flow cytometry; the use of additional markers CD200 and CD305 was highly informative in differential diagnostics between different variants of B-cell lymphoproliferative diseases with similar immunophenotypic and morphological characteristics of lymphoid elements.

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