Functional Flow Cytometric Assay for Reliable and Convenient Heparin-Induced Thrombocytopenia Diagnosis in Daily Practice.
Ontology highlight
ABSTRACT:
SUBMITTER: Tardy-Poncet B
PROVIDER: S-EPMC8064483 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Glucose-6-phosphate dehydrogenase (G6PD) activity is essential for redox equilibrium of red blood cells (RBCs) and, when compromised, the RBCs are more susceptible to haemolysis. 8-aminoquinolines (primaquine and tafenoquine) are used for the radical curative treatment of Plasmodium vivax malaria and can cause haemolysis in G6PD deficient subjects. Haemolytic risk is dependent on treatment dose and patient G6PD status but ultimately it correlates with the number of G6PD deficient RBCs. The G6PD spectrophotometric assay reliably identifies deficient subjects but is less reliable in heterozygous females, especially when other blood conditions are present. In this work we analysed samples with a range of G6PD phenotypes and haematologic conditions from 243 healthy volunteers of Asian or African-American heritage using both the spectrophotomeric assay and the G6PD flow-cytometric assay. Overall 18.5% of subjects (29.3% of Asian females) presented with anaemia, associated with decreased RBCs volume (MCV) and reticulocytosis; the flow-cytometric assay showed good correlation with the spectrophotometric assay (Pearson's r 0.918-0.957) and was less influenced by haemoglobin concentration, number of RBCs and number of reticulocytes. This resulted in more precise quantification of the number of G6PD deficient RBCs and presumably higher predictive power of drug induced haemolytic risk.
| S-EPMC5575121 | biostudies-literature
Project description:Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria) vaccine. The putative mechanism involves formation of pathological anti-platelet factor 4 (PF4) antibodies that activate platelets via the low-affinity immunoglobulin G receptor FcγRIIa to drive thrombosis and thrombocytopenia. Functional assays are important for VITT diagnosis, as not all detectable anti-PF4 antibodies are pathogenic, and immunoassays have varying sensitivity. Combination of ligand binding of G protein-coupled receptors (protease-activated receptor-1) and immunoreceptor tyrosine-based activation motif-linked receptors (FcγRIIa) synergistically induce procoagulant platelet formation, which supports thrombin generation. Here, we describe a flow cytometry-based procoagulant platelet assay using cell death marker GSAO and P-selectin to diagnose VITT by exposing donor whole blood to patient plasma in the presence of a protease-activated receptor-1 agonist. Consecutive patients triaged for confirmatory functional VITT testing after screening using PF4/heparin ELISA were evaluated. In a development cohort of 47 patients with suspected VITT, plasma from ELISA-positive patients (n = 23), but not healthy donors (n = 32) or individuals exposed to the ChAdOx1 nCov-19 vaccine without VITT (n = 24), significantly increased the procoagulant platelet response. In a validation cohort of 99 VITT patients identified according to clinicopathologic adjudication, procoagulant flow cytometry identified 93% of VITT cases, including ELISA-negative and serotonin release assay-negative patients. The in vitro effect of intravenous immunoglobulin (IVIg) and fondaparinux trended with the clinical response seen in patients. Induction of FcγRIIa-dependent procoagulant response by patient plasma, suppressible by heparin and IVIg, is highly indicative of VITT, resulting in a sensitive and specific assay that has been adopted as part of a national diagnostic algorithm to identify vaccinated patients with platelet-activating antibodies.
| S-EPMC9198924 | biostudies-literature
Project description:BackgroundImmunoassays are crucial in the work-up of patients with suspected heparin-induced thrombocytopenia (HIT) and rapid tests have been recently developed. However, comparative data on diagnostic accuracy, reproducibility, and analytical costs of different immunoassays in clinical practice are limited.MethodsSamples of 179 consecutive patients evaluated for suspected HIT in clinical practice using a polyspecific enzyme-linked immunoabsorbent assay (GTI diagnostics; ELISA) and a rapid particle gel immunoassay (PaGIA), were additionally analysed with a IgG-specific chemiluminescent immunoassay (AcuStar HIT-IgG). Presence of HIT was defined as a positive functional heparin-induced platelet aggregation test. Diagnostic accuracy was determined for low, intermediate and high thresholds as previously established (ELISA: optical density 0.4, 1.3, and 2.0 respectively; PaGIA: positive/negative, titre of 4, titre of 32; AcuStar HIT-IgG: 1.0 U/ml, 2.8, 9.4) and reproducibility was assessed by repeated measurements. Costs of test determination were calculated taking reagents, controls, and working time of technicians according to Swiss health care system into account.ResultsData on PaGIA results were available for 171 patients (95.5%), ELISA for 144 patients (80.4%), and AcuStar HIT-IgG for 179 patients (100%). Sensitivity was above 95% for all assays at low and intermediate thresholds. Specificity increased with higher thresholds and was above 90% for all assays with intermediate and high thresholds. Specificity of AcuStar HIT-IgG (92.8%; 95% CI 87.7, 96.2) was significantly higher than PaGIA (83.0%; 95% CI 76.3, 88.5) and higher than ELISA (81.8%, 95% CI 74.2, 88.0) at low threshold (p<0.05). Reproducibility was adequate for all assays. Total costs per test were CHF 51.02 for ELISA, 117.70 for AcuStar HIT-IgG, and 83.13 for PaGIA.ConclusionsWe observed favourable diagnostic accuracy measures and a high reproducibility for PaGIA and AcuStar HIT-IgG. Implementation into 24-hours-service might improve patient care but the results must be confirmed in other settings and larger populations as well.
| S-EPMC5464550 | biostudies-literature
Project description:Interactions between DNA and transcription factors (TFs) guide cellular function and development, yet the complexities of gene regulation are still far from being understood. Such understanding is limited by a paucity of techniques with which to probe DNA-protein interactions. We have devised magnetic protein immobilization on enhancer DNA (MagPIE), a simple, rapid, multi-parametric assay using flow cytometric immunofluorescence to reveal interactions among TFs, chromatin structure and DNA. In MagPIE, synthesized DNA is bound to magnetic beads, which are then incubated with nuclear lysate, permitting sequence-specific binding by TFs, histones and methylation by native lysate factors that can be optionally inhibited with small molecules. Lysate protein-DNA binding is monitored by flow cytometric immunofluorescence, which allows for accurate comparative measurement of TF-DNA affinity. Combinatorial fluorescent staining allows simultaneous analysis of sequence-specific TF-DNA interaction and chromatin modification. MagPIE provides a simple and robust method to analyze complex epigenetic interactions in vitro.
| S-EPMC3554230 | biostudies-literature
Project description:The use of surface-enhanced Raman spectroscopy (SERS) to determine spectral markers for the diagnosis of heparin-induced thrombocytopenia (HIT), a difficult-to-diagnose immune-related complication that often leads to limb ischemia and thromboembolism, is proposed. The ability to produce distinct molecular signatures without the addition of labels enables unbiased inquiry and makes SERS an attractive complementary diagnostic tool. A capillary-tube-derived SERS platform offers ultrasensitive, label-free measurement as well as efficient handling of blood serum samples. This shows excellent reproducibility, long-term stability and provides an alternative diagnostic rubric for the determination of HIT by leveraging machine-learning-based classification of the spectroscopic data. We envision that a portable Raman instrument could be combined with the capillary-tube-based SERS analytical tool for diagnosis of HIT in the clinical laboratory, without perturbing the existing diagnostic workflow.
| S-EPMC7547846 | biostudies-literature
Project description:Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell's ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 10(5) -10(10) doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell's ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4-12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug's single-cell potency and can be used for any fluorescent or fluorescently labeled drug, including nanoparticles or antibody-drug conjugates.
| S-EPMC4706798 | biostudies-literature
Project description:Notch signaling is often involved in cancer cell initiation and proliferation. Aberrant Notch activation underlies more than 50% of T-cell acute lymphoblastic leukemia (T-ALL); accordingly, chemicals disrupting Notch signaling are of potential to treat Notch-dependent cancer. Here, we developed a flow cytometry-based high-throughput assay to identify compounds that disrupt the interactions of DNA and RBPJ, the major downstream effector of Notch signaling. From 1492 compounds, we identified 18 compounds that disrupt RBPJ-DNA interactions in a dose-dependent manner. Cell-based assays further revealed that auranofin downregulates Notch-dependent transcription and decreases RBPJ-chromatin interactions in cells. Most strikingly, T-ALL cells that depend on Notch signaling for proliferation are more sensitive to auranofin treatment, supporting the notion that auranofin downregulates Notch signaling by disrupting RBPJ-DNA interaction. These results validate the feasibility of our assay scheme to screen for additional Notch inhibitors and provide a rationale to further test the use of auranofin in treating Notch-dependent cancer.
| S-EPMC7442708 | biostudies-literature
Project description:Current methods for monitoring E3 ubiquitin ligase activity in cell culture or in vivo are limited. As a result, the degradation of cellular targets by many E3 ubiquitin ligases in live cells has not yet been examined. For this study, a target of an E3 ubiquitin ligase was expressed as a fluorescently labeled protein in cell culture. If the E3 ubiquitin ligase mediates the degradation of a target protein in cell culture, it is expected that the target will show a reduced fluorescence signal by FCM analysis. We initially used the E3 ubiquitin ligase, herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) and one of its targets, promyelocytic leukemia (PML) protein, to determine the feasibility of our approach. Cells expressing a PML-GFP fusion protein were selected by cell sorting and infected with an adenoviral vector expressing ICP0. In contrast to mock-infected cells, only PML-GFP-expressing cells infected with the ICP0 adenoviral vector led to a significant decrease in the fluorescence signal of PML-GFP when examined by fluorescence microscopy and FCM analysis. Our results suggest that it is possible to examine the live activity of an E3 ubiquitin ligase (via one of its targets) in cell culture by FCM analysis.
| S-EPMC2750075 | biostudies-literature
Project description:Yersinia pestis is the causative agent of plague and is considered a category A priority pathogen due to its potential for high transmissibility and the significant morbidity and mortality it causes in humans. Y. pestis is endemic to the western United States and much of the world, necessitating programs to monitor for this pathogen on the landscape. Elevated human risk of plague infection has been spatially correlated with spikes in seropositive wildlife numbers, particularly rodent-eating carnivores, which are frequently in contact with the enzootic hosts and the associated arthropod vectors of Y. pestis In this study, we describe a semiautomated bead-based flow cytometric assay developed for plague monitoring in wildlife called the F1 Luminex plague assay (F1-LPA). Based upon Luminex/Bio-Plex technology, the F1-LPA targets serological responses to the F1 capsular antigen of Y. pestis and was optimized to analyze antibodies eluted from wildlife blood samples preserved on Nobuto filter paper strips. In comparative evaluations with passive hemagglutination, the gold standard tool for wildlife plague serodiagnosis, the F1-LPA demonstrated as much as 64× improvement in analytical sensitivity for F1-specific IgG detection and allowed for unambiguous classification of IgG status. The functionality of the F1-LPA was demonstrated for coyotes and other canids, which are the primary sentinels in wildlife plague monitoring, as well as felids and raccoons. Additionally, assay formats that do not require species-specific immunological reagents, which are not routinely available for several wildlife species used in plague monitoring, were determined to be functional in the F1-LPA.
| S-EPMC6018325 | biostudies-literature
Project description:Neutrophil extracellular traps (NETs) contribute to innate immunity as well as numerous diseases processes such as deep vein thrombosis, myocardial ischemia, and autoimmune disease. To date, most knowledge on NETs formation has been gathered via the qualitative microscopic examination of individual neutrophils in vitro, or aggregate structures in vivo. Here we describe a novel flow cytometry (FLOW)-based assay to identify and quantify NETs using antibodies against key NETs constituents, specifically DNA, modified histones, and granular enzymes. This method is applicable to both murine and human samples for the assessment of induced NETs in vitro, or detection of NETosis in vivo in blood samples. This FLOW-based method was validated by comparison with the well-established microscopy assay using two genetic mouse models previously demonstrated to show defective NETosis. It was then used on healthy human neutrophils for detection of ex vivo induced NETs and on blood samples from patients with sepsis for direct assessment of in vivo NET-forming neutrophils. This new methodology allows rapid and robust assessment of several thousand cells per sample and is independent of potential observer-bias, the two main limitations of the microscopic quantification. Using this new technology facilitates the direct detection of in vivo circulating NETs in blood samples and purification of NETting neutrophils by fluorescence-activated cell sorting (FACS) for further analysis.
| S-EPMC4715743 | biostudies-literature