P233 On the performance characteristics of luminex single antigen bead (LSAB) assay mean fluorescence intensity cutpoint in predicting flow cytometry crossmatch (FCXM) results

2017 ◽  
Vol 78 ◽  
pp. 226
Author(s):  
Prabhakar Putheti ◽  
Rex Friedlander ◽  
Cindy H. Park ◽  
Darshana Dadhania ◽  
Vijay K. Sharma ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Intensity ◽  
Mean Fluorescence Intensity ◽  
Performance Characteristics ◽  
Single Antigen

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 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.

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.

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.

73-P: Evaluation of CDC-AHG, flow cytometry and Luminex crossmatch techniques to detect donor specific antibody by Luminex single antigen beads

2009 ◽  
Vol 70 ◽  
pp. S48
Author(s):  
Lauren Wetter ◽  
D. Phelan ◽  
G. Morris ◽  
M. Jendrisak ◽  
T. Mohanakumar
Keyword(s):  
Flow Cytometry ◽  
Specific Antibody ◽  
Single Antigen

Pretransplant Donor-Specific Antibodies Detected by Single-Antigen Bead Flow Cytometry Are Associated With Inferior Kidney Transplant Outcomes

2010 ◽  
Vol 90 (10) ◽  
pp. 1079-1084 ◽  
Author(s):  
Neeraj Singh ◽  
Arjang Djamali ◽  
David Lorentzen ◽  
John D. Pirsch ◽  
Glen Leverson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Kidney Transplant ◽  
Specific Antibodies ◽  
Transplant Outcomes ◽  
Single Antigen ◽  
Donor Specific Antibodies

scholarly journals A flow cytometric assay of neutrophil degranulation.

1983 ◽  
Vol 31 (6) ◽  
pp. 737-744 ◽  
Author(s):  
W R Abrams ◽  
L W Diamond ◽  
A B Kane
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Intensity ◽  
Ionophore A23187 ◽  
Flow Cytometric Assay ◽  
Elastase Activity ◽  
Flow Cytometric ◽  
Specific Granules ◽  
Neutrophil Degranulation ◽  
High Fluorescence Intensity ◽  
High Fluorescence

A quantitative assay of neutrophil degranulation was developed using flow cytometry. Dog neutrophils were purified to greater than 95% purity and viability by isopyknic density centrifugation in an isosmotic medium. These cells concentrated the fluorochrome acridine orange (AO) in their azurophilic granules, but not in specific granules. Also contained in the azurophilic granules are elastase, myeloperoxidase, and approximately 50% of the lysozyme activity. The fluorochrome was released concomitantly with elastase activity, as shown by flow cytometry, fluorescence microscopy, and biochemical assay in response to the ionophore A23187. By flow cytometry, unstimulated cells are distributed in a single broad peak of high fluorescence intensity. With increasing concentrations of A23187 (0.48-4.80 microM), a greater proportion of the cells shifted to a single peak of low fluorescence intensity. Few cells with intermediate fluorescence were observed. These analyses revealed that the neutrophils degranulated in a quantal, all-or-none response.

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