scholarly journals Development of an Improved Whole Blood Assay for Diagnosis of Latent and Active Tuberculosis Cases

2021 ◽  
pp. 197-209
Author(s):  
Rupam R. Nashine ◽  
Amit R. Nayak ◽  
Aliabbas Husain ◽  
Gargi D. Mudey ◽  
Hatim F. Daginawala ◽  
...  
Keyword(s):  
Whole Blood ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Interferon Gamma Release Assay ◽  
Antigenic Peptides ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Variable Sensitivity ◽  
Latent Tb Infection

Background: Latent TB infection (LTBI) is an infection where the presence of disease causing organism M. tuberculosis is there without any sign and symptoms of the disease hence mostly remains undiagnosed, though Tuberculin skin test (TST) and Interferon Gamma Release Assay (IGRA) were used to diagnose the LTBI. They have their limitations, TST gives major cross-reactivity with BCG vaccine and gives inaccurate results in individuals who have taken BCG and IGRA are very costly and variable sensitivity is repeated in various populations hence the modifications are needed in the IGRA for proper diagnosis of LTBI. Objectives: In the proposed study we aimed to develop an improved whole blood assay                    towards a diagnosis of latent and active TB infection as an alternative to the Quantiferon QFT assay Methodology: Synthetic antigenic peptides against latency specific antigens will be designed and synthesized. Designed peptides will be screened for LTBI specific cytokine by in-vitro experiments. Development & production of Whole assay using selected peptides evaluation of developed assay through ELISA in clinical samples. Expected Results: Latent specific peptides will be identified and peptide-based whole blood assay for detection and diagnosis of tuberculosis will be developed as an indigenous alternative for the existing QFT assay. Conclusion: An improved whole blood assay will be developed for screening of LTBI in the Indian population.

scholarly journals Whole blood assay as a model for in vitro evaluation of inflammasome activation and subsequent caspase-mediated interleukin-1 beta release

PLoS ONE ◽  
2019 ◽  
Vol 14 (4) ◽  
pp. e0214999 ◽  
Author(s):  
Thi Anh Thu Tran ◽  
Hendrika W. Grievink ◽  
Katarzyna Lipinska ◽  
Cornelis Kluft ◽  
Jacobus Burggraaf ◽  
...  
Keyword(s):  
Whole Blood ◽  
Interleukin 1 ◽  
Inflammasome Activation ◽  
Interleukin 1 Beta ◽  
Whole Blood Assay ◽  
In Vitro Evaluation ◽  
Blood Assay

Abstract 571: A Novel High-Throughput Whole Blood Immuno-Counting Assay is Useful in the Assessment of Platelet Responses to Antiplatelet Therapy

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Marie Lordkipanidze ◽  
Natalia Dovlatova ◽  
Mohammad Algahtani ◽  
Martina H Lundberg ◽  
Timothy D Warner ◽  
...  
Keyword(s):  
Platelet Count ◽  
Antiplatelet Therapy ◽  
Platelet Function ◽  
Whole Blood ◽  
Dual Antiplatelet Therapy ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Treated Sample ◽  
Function Testing

Background: The current gold standard in platelet function testing, light transmission aggregometry, is time- and labor-intensive, and uses platelet-rich plasma which makes it sub-optimal for high throughput testing. In order to reduce blood manipulation prior to platelet function testing and to study multiple platelet activation pathways simultaneously, we have developed a 96-well plate-based assay carried out in whole blood, where aggregation is measured as a decrease in the number of fluorescently-labeled single platelets by flow cytometry. Aim: To investigate whether a 96-well plate-based whole blood assay can be used to assess platelet function. Methods: Platelet function in response to 5 concentrations of lyophilized arachidonic acid (AA), ADP, collagen, epinephrine, TRAP, U46619, and ristocetin, was assessed in healthy volunteers (n=20) to establish normal ranges. The effect of antiplatelet drugs was assessed in vitro by incubation with aspirin (100 μM), cangrelor (1 μM) or both (n=20), and in patients on dual antiplatelet therapy (n=20). After addition of 40 μl of whole blood per well, the plate was shaken for 5 min at 1000 rpm at 37°C; a fixative solution (Platelet Solutions, Nottingham) was applied to stop platelet aggregation and allow analysis in a central laboratory. Fixed whole blood samples (stable for up to 9 days) were labeled with FITC-conjugated CD42a and assessed by flow cytometry. Aggregation was calculated as (Platelet count in vehicle-treated sample - Platelet count in agonist-stimulated sample) / Platelet count in vehicle-treated sample x 100. Results: Dose-response curves were readily assessable for all agonists and intra-individual variability was minimal in healthy volunteers (CV<10%). In vitro addition of aspirin alone resulted in inhibition of AA- and collagen-induced aggregation, whereas cangrelor induced a shift in dose-response to most agonists in addition to profound inhibition of ADP responses. In patients on dual antiplatelet therapy, the pattern of response was consistent with the results obtained with in vitro agents. Conclusions: A 96-well plate-based whole blood assay with a minimal blood volume requirement (<2 ml) could be used to provide a global portrait of platelet responses to antiplatelet agents.

scholarly journals Ultrafast and Predictive Mass Spectrometry–Based Autotaxin Assays for Label-Free Potency Screening

2017 ◽  
Vol 22 (4) ◽  
pp. 425-432 ◽  
Author(s):  
Tom Bretschneider ◽  
Andreas Harald Luippold ◽  
Helmut Romig ◽  
Daniel Bischoff ◽  
Klaus Klinder ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Whole Blood ◽  
Label Free ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Data Points ◽  
Set Up

Autotaxin (ATX) is a promising drug target for the treatment of several diseases, such as cancer and fibrosis. ATX hydrolyzes lysophosphatidyl choline (LPC) into bioactive lysophosphatidic acid (LPA). The potency of ATX inhibitors can be readily determined by using fluorescence-based LPC derivatives. While such assays are ultra-high throughput, they are prone to false positives compared to assays based on natural LPC. Here we report the development of ultrafast mass spectrometry–based ATX assays enabling the measurement of data points within 13 s, which is 10 times faster than classic liquid chromatography–mass spectrometry. To this end, we set up a novel in vitro and whole-blood assay. We demonstrate that the potencies determined with these assays are in good agreement with the in vivo efficacy and that the whole-blood assay has the best predictive power. This high-throughput label-free approach paired with the translatable data quality is highly attractive for appropriate guidance of medicinal chemists for constructing strong structure-activity relationships.

Inhibition of STAT5 Phosphorylation by Anti-GM-CSF Monoclonal Antibodies: Validation of a New Whole Blood Assay

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4728-4728
Author(s):  
Karine Grivel ◽  
Laurent Magnenat ◽  
Maxime Moulard
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Signaling ◽  
Cell Lines ◽  
Whole Blood ◽  
Receptor Activation ◽  
Whole Blood Assay ◽  
Stable Cell Lines ◽  
Gm Csf ◽  
Blood Assay

Abstract Abstract 4728 Introduction. Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates survival, proliferation, and differentiation of monocytes/macrophages. The GM-CSF receptor (CD116) is expressed on primary monocytes and mediates cell signaling through STAT5 phosphorylation. Therefore STAT5 is central to GM-CSF action. BioCytex has developed a new semi-quantitative flow cytometry (FC) assay to measure the phosphorylation status of STAT5 in human primary monocytes and lymphocytes in whole blood samples. The assay was used to investigate the potency of recombinant monoclonal antibodies (MAbs) for the neutralization of human GM-CSF. Methods. Engineered anti-GM-CSF MAbs were generated and characterized by Merck Serono. Several clones and preparation were tested, including MAbs 343 (low and high LPS), 467; 340; 339; 366, and MAb 285 as isotype control. Their different in vitro potencies for blocking recombinant GM-CSF activity in stable cell lines was determined using the TF-1 proliferation and IL-8 secretion in U937 cell assays. Whole blood FC assay was designed and validated at BioCytex using either a rabbit MAb or a rabbit polyclonal antibody specific for phospho-STAT5 (Tyr694). Monocytes were gated using a PE-labeled anti-CD14 probe. Inhibition of STAT5 phosphorylation by anti-GM-CSF MAbs was performed using recombinant GM-CSF at EC90. Results. EC90 of GM-CSF was determined at 2.3 pM from n=5 healthy volunteers. All GM-CSF specific MAbs inhibited STAT5 phosphorylation in monocytes, whereas the control (MAb 285) did not. More interestingly, there was a marked effect of LPS content since inhibition of STAT5 phosphorylation was not completed in presence of high LPS load (MAb 343). IC50 and IC90 for MAbs 339 and 366 were 10-fold increased as compared to MAbs 467 and 340. IC50 were ranging from 0.12 to 0.19 nM for clones 343 (low LPS), 467 and 340. IC50 and IC90 for clone 339 were 0.016 and 0.034 nM, respectively. Conclusion. Inhibition of GM-CSF mediated monocyte activation by neutralizing MAbs can be monitored in a whole blood assay. STAT5 phophorylation could be viewed as a potential biomarker for GM-CSF receptor activation. The cell-signaling inhibition potency in primary cells correlated with in vitro potency using stable cell lines. In summary, the new validated FC could be used for research and clinical development purposes. Disclosures: Grivel: LFB Biotechnologies: Consultancy. Magnenat:Merck Serono S.A.: Employment. Moulard:BioCytex: Consultancy, Employment.

Inter- and intraindividual variation of endotoxin- and β(1 → 3)-glucan-induced cytokine responses in a whole blood assay

2002 ◽  
Vol 18 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Inge M Wouters ◽  
Jeroen Douwes ◽  
Peter S Thorne ◽  
Dick Heederik ◽  
Gert Doekes
Keyword(s):  
Whole Blood ◽  
Large Scale ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Intraindividual Variation ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Within Subjects ◽  
Cytokine Responsiveness

Inflammatory airway responses to bioaerosols and to their active compounds, such as endotoxin and β(1 → 3)- glucan, vary between individuals. These differences may be explained by variation in cytokine responsiveness, which can be assessed by in vitro stimulation tests with isolated blood leukocytes or lung macrophages. In large- scale population studies, ex vivo induced cytokine production may also be tested with a more simple `whole blood assay’ (WBA). However, applicability of a WBA to characterize a subject’s responsiveness depends largely on its reproducibility. This study was conducted to: 1) assess the within- and between-subject variability in cytokine production in a WBA after stimulation with endotoxin or β(1 → 3)-glucan; and 2) to determine under which conditions this test is most discriminating between subjects and most reproducible within subjects. Blood was collected from 14 healthy volunteers, of whom 10 also participated on a second occasion. Each blood sample was used in two WBA tests; the first WBA was initiated two hours and the second 26 hours after venapuncture. The WBA test itself comprised overnight incubation with serial dilutions of endotoxin [lipopolysaccharide (LPS)] and curdlan (a β(1 → 3)-glucan), after which blood cell supernatant was collected. Interleukin(IL)-1, IL6, IL8 and tumor necrosis factor (TNF) were determined in the supernatant. In all individuals, a dose-dependent production of cytokines was observed for both LPS and curdlan. For all cytokines, variation between subjects was higher than within subjects, and this was most pronounced for IL1 and IL6. There was moderate-to-high correlation in the induced release of all four cytokines, and between cytokine release induced by LPS or curdlan. Optimal stimulation concentrations were 6.25 and 12.5 ng/mL for endotoxin and 12 500 and 25 000 ng/mL for curdlan. Cytokine production in WBA initiated 26 hours after venapuncture showed lower between-subject and larger within-subject variance, thus favoring an early initiation of the assay. In conclusion, measuring endotoxin-or glucan-induced cytokine production in a WBA initiated within two hours after venapuncture appears to be an effective method to determine a person’s cytokine responsiveness, at least in healthy naive subjects. Toxicology and Industrial Health 2002; 18: 15-27.

scholarly journals A Human In Vitro Whole Blood Assay to Predict the Systemic Cytokine Response to Therapeutic Oligonucleotides Including siRNA

PLoS ONE ◽  
2013 ◽  
Vol 8 (8) ◽  
pp. e71057 ◽  
Author(s):  
Christoph Coch ◽  
Christian Lück ◽  
Anna Schwickart ◽  
Bastian Putschli ◽  
Marcel Renn ◽  
...  
Keyword(s):  
Whole Blood ◽  
Cytokine Response ◽  
Whole Blood Assay ◽  
Blood Assay

A Whole Blood Assay of Inhibition of Platelet Aggregation by Glycoprotein IIb/IIIa Antagonists: Comparison with other Aggregation Methodologies

1999 ◽  
Vol 82 (10) ◽  
pp. 1307-1311 ◽  
Author(s):  
Robert Storey ◽  
Jane May ◽  
Stan Heptinstall ◽  
Robert Wilcox
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Impedance Aggregometry ◽  
In Vitro Effects ◽  
To Receive ◽  
Oral Prodrug ◽  
Antagonist Effect

SummaryWe have used a whole blood single-platelet counting assay (WBSPC) that is sensitive to microaggregation for monitoring GPIIb/IIIa antagonists and have compared this with other methodologies. In vitro effects of the GPIIb/IIIa antagonist fradafiban on ADP-induced platelet aggregation were determined using WBSPC and PRP turbidimetry, comparing citrate and hirudin anticoagulation. Fradafiban was a more potent inhibitor of aggregation assessed by PRP turbidimetry compared to WBSPC. Citrate showed only a trend towards enhancing fradafiban potency (p = 0.087). Citrated blood from 8 patients with unstable angina, randomised to receive oral lefradafiban (the oral prodrug of fradafiban) or placebo, was studied before and during treatment using WBSPC, PRP turbidimetry, impedance aggregometry and Rapid Platelet Function Assay (RPFA, Accumetrics). RPFA, PRP turbidimetry and WBSPC measurements correlated well. Impedance aggregometry responses were oversensitive to GPIIb/IIIa blockade. WBSPC was most discriminating at high levels of inhibition and offered a rapid means of monitoring GPIIb/IIIa antagonist effect within the therapeutic range of inhibition.

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