Luminol-enhanced, whole blood chemiluminescence of human neutrophils evaluated by means of an automated, computer-assisted, and high-sensitivity luminescence analyzer

1995 ◽  
Vol 25 (4) ◽  
pp. 216-221 ◽  
Author(s):  
G. Carulli ◽  
S. Minnucci ◽  
C. Angiolini ◽  
A. Azzarà ◽  
F. Ambrogi
Keyword(s):  
Whole Blood ◽  
High Sensitivity ◽  
Human Neutrophils ◽  
Computer Assisted ◽  
Luminescence Analyzer

3-D organization of fibrinogen receptors using computer reconstruction and whole-mount microscopy

1988 ◽  
Vol 46 ◽  
pp. 30-31
Author(s):  
E. T. O'Toole ◽  
R. R. Hantgan ◽  
J. C. Lewis
Keyword(s):  
Whole Blood ◽  
Colloidal Gold ◽  
Blood Platelets ◽  
Cell Contact ◽  
Computer Assisted ◽  
Adherent Cells ◽  
Differential Centrifugation ◽  
Computer Reconstruction ◽  
Sds Page ◽  
Whole Mount

Thrombocytes (TC), the avian equivalent of blood platelets, support hemostasis by aggregating at sites of injury. Studies in our lab suggested that fibrinogen (fib) is a requisite cofactor for TC aggregation but operates by an undefined mechanism. To study the interaction of fib with TC and to identify fib receptors on cells, fib was purified from pigeon plasma, conjugated to colloidal gold and used both to facilitate aggregation and as a receptor probe. Described is the application of computer assisted reconstruction and stereo whole mount microscopy to visualize the 3-D organization of fib receptors at sites of cell contact in TC aggregates and on adherent cells.Pigeon TC were obtained from citrated whole blood by differential centrifugation, washed with Ca++ free Hank's balanced salts containing 0.3% EDTA (pH 6.5) and resuspended in Ca++ free Hank's. Pigeon fib was isolated by precipitation with PEG-1000 and the purity assessed by SDS-PAGE. Fib was conjugated to 25nm colloidal gold by vortexing and the conjugates used as the ligand to identify fib receptors.

Development of a point-of-care assay system for high-sensitivity C-reactive protein in whole blood

2003 ◽  
Vol 332 (1-2) ◽  
pp. 51-59 ◽  
Author(s):  
Jae Soon Ahn ◽  
Sunga Choi ◽  
Sang Ho Jang ◽  
Hyuk Jae Chang ◽  
Jae Hoon Kim ◽  
...  
Keyword(s):  
Whole Blood ◽  
Point Of Care ◽  
High Sensitivity ◽  
Assay System ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Point Of Care Assay

scholarly journals Clinical Validation of a Volumetric Absorptive Micro-Sampling Device for Pharmacokinetic Studies With Tranexamic Acid

2021 ◽  
Vol 12 ◽  
Author(s):  
Stanislas Grassin-Delyle ◽  
Elodie Lamy ◽  
Michaela Semeraro ◽  
Iléana Runge ◽  
Jean-Marc Treluyer ◽  
...  
Keyword(s):  
Tranexamic Acid ◽  
Whole Blood ◽  
Plasma Concentrations ◽  
High Sensitivity ◽  
Pharmacokinetic Studies ◽  
Limits Of Agreement ◽  
Sampling Device ◽  
Average Bias ◽  
Alternative Routes ◽  
Matched Samples

We assessed the accuracy of tranexamic acid (TXA) concentrations measured in capillary whole blood using volumetric absorptive micro-sampling (VAMS) devices. Paired venous and VAMS capillary blood samples were collected from 15 healthy volunteers participating in a pharmacokinetic study of alternative routes (oral, IM and IV) of administering TXA. To assess accuracy across a range of concentrations, blood was drawn at different times after TXA administration. We measured TXA concentrations in plasma, whole blood from samples collected by venepuncture and whole blood from venous and capillary samples collected using VAMS devices. TXA was measured using a validated high sensitivity liquid chromatography - mass spectrometry method. We used Bland-Altman plots to describe the agreement between the TXA concentrations obtained with the different methods. In the 42 matched samples, the mean plasma TXA concentration was 14.0 mg/L (range 2.6–36.5 mg/L) whereas the corresponding whole blood TXA concentration was 7.7 mg/L (range 1.6–17.5 mg/L). When comparing TXA concentrations in VAMS samples of venous and capillary whole blood, the average bias was 0.07 mg/L (lower and upper 95% limits of agreement: −2.1 and 2.2 mg/L respectively). When comparing TXA concentrations in venous whole blood and VAMS capillary whole blood, the average bias was 0.7 mg/L (limits of agreement: −2.7 and 4.0 mg/L). Volumetric absorptive micro-sampling devices are sufficiently accurate for use in pharmacokinetic studies of tranexamic acid treatment in the range of plasma concentrations relevant for the assessment of fibrinolysis inhibition.

scholarly journals Monitoring DOACs with a Novel Dielectric Microsensor: A Clinical Study

2020 ◽  
Author(s):  
Debnath Maji ◽  
Aman Opneja ◽  
Michael A. Suster ◽  
Kara L. Bane ◽  
Brigid M. Wilson ◽  
...  
Keyword(s):  
Whole Blood ◽  
Point Of Care ◽  
Oral Anticoagulants ◽  
Characteristic Curve ◽  
Direct Oral Anticoagulants ◽  
Unmet Need ◽  
Area Under The Curve ◽  
High Sensitivity ◽  
Diagnostic Study ◽  
Coagulation Tests

Abstract Background There are acute settings where assessing the anticoagulant effect of direct oral anticoagulants (DOACs) can be useful. Due to variability among routine coagulation tests, there is an unmet need for an assay that detects DOAC effects within minutes in the laboratory or at the point of care. Methods We developed a novel dielectric microsensor, termed ClotChip, and previously showed that the time to reach peak permittivity (T peak) is a sensitive parameter of coagulation function. We conducted a prospective, single-center, pilot study to determine its clinical utility at detecting DOAC anticoagulant effects in whole blood. Results We accrued 154 individuals: 50 healthy volunteers, 49 rivaroxaban patients, 47 apixaban, and 8 dabigatran patients. Blood samples underwent ClotChip measurements and plasma coagulation tests. Control mean T peak was 428 seconds (95% confidence interval [CI]: 401–455 seconds). For rivaroxaban, mean T peak was 592 seconds (95% CI: 550–634 seconds). A receiver operating characteristic curve showed that the area under the curve (AUC) predicting rivaroxaban using T peak was 0.83 (95% CI: 0.75–0.91, p < 0.01). For apixaban, mean T peak was 594 seconds (95% CI: 548–639 seconds); AUC was 0.82 (95% CI: 0.73–0.91, p < 0.01). For dabigatran, mean T peak was 894 seconds (95% CI: 701–1,086 seconds); AUC was 1 (p < 0.01). Specificity for all DOACs was 88%; sensitivity ranged from 72 to 100%. Conclusion This diagnostic study using samples from “real-world” DOAC patients supports that ClotChip exhibits high sensitivity at detecting DOAC anticoagulant effects in a disposable portable platform, using a miniscule amount of whole blood (<10 µL).

scholarly journals The chemokinetic response of human neutrophils

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1036-1042 ◽  
Author(s):  
TH Howard
Keyword(s):  
Dose Dependence ◽  
Human Neutrophils ◽  
Maximal Response ◽  
Computer Assisted ◽  
Cellular Inflammatory Response ◽  
Blood Neutrophils ◽  
Response Increase ◽  
Flow Through ◽  
The Mean ◽  
Serial Addition

Abstract A computer-assisted single cell assay that allows quantification of the locomotive behavior of individual cells and a flow-through system that allows study of response of individual cells to stimulation were utilized to study the chemokinetic response of neutrophils. The range of basal mean rate of locomotion (mROL) and chemokinetic response to 10(-9) mol/L formylmethionyl leucyl phenylalanine (FMLP) was determined for neutrophils of eight normal adults. The basal mROL was 8.2 +/- 1.5 um/min and 6.2 +/- 1.0 um/min; the rate after 10(-9) mol/L fMLP was 12.1 +/- 2.1 and 9.5 +/- 1.8 um/min in 2.0 g% and 0.05 g% HSA, respectively. The mean increase in ROL for neutrophils was 50%. Assay with the flow-through system shows that the chemokinetic response-- increase in mROL of a population of neutrophils in response to 10(-9) mol/L--is due to an increase in ROL when cells are actively moving and not due to a decrease in the amount of time the cell spends inactive. Studies of individual cells within the populations show that chemokinetic response to 10(-9) mol/L fMLP is highly variable. The majority of cells (77%) respond with an increase in ROL; the minority (23%) are nonresponders that characteristically move at ROL greater than or equal to 14 um/min prior to stimulation and do not change ROL or exhibit a net decline in ROL in response to 10(-9) mol/L fMLP. The dose response of a population of neutrophils and of individual neutrophils to serial addition of 10(-10) to 10(-6) mol/L fMLP shows that the fMLP dose dependence for maximal chemokinetic response is highly variable among individual cells. Seventeen percent of cells do not respond to any fMLP concentration; 25% of neutrophils exhibit maximal response to 10(-10) mol/L fMLP, while 50% and 25% of cells showed peak chemokinetic response to 10(-9) mol/L and greater than or equal to 10(-8) mol/L fMLP, respectively. These studies document the variability in the locomotive responses of peripheral blood neutrophils. Understanding the causes of variability in the chemokinetic responsiveness of individual neutrophils may improve our understanding of how the cellular inflammatory response in man can be modulated.

Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

2003 ◽  
Vol 285 (2) ◽  
pp. H589-H596 ◽  
Author(s):  
Kazuyoshi Kirima ◽  
Koichiro Tsuchiya ◽  
Hiroyoshi Sei ◽  
Toyoshi Hasegawa ◽  
Michiyo Shikishima ◽  
...  
Keyword(s):  
Endothelial Dysfunction ◽  
Whole Blood ◽  
Computer Assisted ◽  
Subtraction Method ◽  
Epr Spectrum ◽  
Systemic Blood ◽  
Paramagnetic Resonance ◽  
Species Overlap ◽  
Epr Signal ◽  
Electron Paramagnetic

The measurement of hemoglobin-nitric oxide (NO) adduct (HbNO) in whole blood by the electron paramagnetic resonance (EPR) method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and unknown radical species overlap the same magnetic field as that of HbNO. To reveal the EPR spectrum of HbNO, we then introduced the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of sample blood using the software. Rats were treated with Nω-nitro-l-arginine methyl ester (l-NAME; 120 mg · kg–1 · day–1) for 1 wk to obtain HbNO-depleted blood. When this method was applied to the analysis of untreated fresh whole blood, the five-coordinate state of HbNO was observed. HbNO concentration in pentobarbital-anesthetized rats was augmented (change in [HbNO] = 1.6–5.5 μM) by infusion of l-arginine (0.2–0.6 g/kg) but not d-arginine. Using this method, we attempted to evaluate the effects of temocapril on HbNO dynamics in an l-NAME-induced rat endothelial dysfunction model. The oral administration of l-NAME for 2 wk induced a serious hypertension, and the HbNO concentration was reduced (change in [HbNO] = 5.7 μM). Coadministration of temocapril dose dependently improved both changes in blood pressure and the systemic HbNO concentration. In this study, we succeeded in measuring the blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril improves abnormalities of NO dynamics in l-NAME-induced endothelial dysfunction rats using the EPR HbNO signal subtraction method.

scholarly journals Elucidation of a PTS–Carbohydrate Chemotactic Signal Pathway inEscherichia coliUsing a Time-resolved Behavioral Assay

1999 ◽  
Vol 10 (4) ◽  
pp. 1133-1146 ◽  
Author(s):  
Renate Lux ◽  
V. Ranjit N. Munasinghe ◽  
Fred Castellano ◽  
Joseph W. Lengeler ◽  
John E. T. Corrie ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
High Sensitivity ◽  
Signal Pathway ◽  
Response Threshold ◽  
Computer Assisted ◽  
Time Resolved ◽  
Response Range ◽  
Transport Mutants ◽  
Signaling Activity

Chemotaxis of Escherichia coli toward phosphotransferase systems (PTSs)–carbohydrates requires phosphoenolpyruvate-dependent PTSs as well as the chemotaxis response regulator CheY and its kinase, CheA. Responses initiated by flash photorelease of a PTS substrates d-glucose and its nonmetabolizable analog methyl α-d-glucopyranoside were measured with 33-ms time resolution using computer-assisted motion analysis. This, together with chemotactic mutants, has allowed us to map out and characterize the PTS chemotactic signal pathway. The responses were absent in mutants lacking the general PTS enzymes EI or HPr, elevated in PTS transport mutants, retarded in mutants lacking CheZ, a catalyst of CheY autodephosphorylation, and severely reduced in mutants with impaired methyl-accepting chemotaxis protein (MCP) signaling activity. Response kinetics were comparable to those triggered by MCP attractant ligands over most of the response range, the most rapid being 11.7 ± 3.1 s−1. The response threshold was <10 nM for glucose. Responses to methyl α-d-glucopyranoside had a higher threshold, commensurate with a lower PTS affinity, but were otherwise kinetically indistinguishable. These facts provide evidence for a single pathway in which the PTS chemotactic signal is relayed rapidly to MCP–CheW–CheA signaling complexes that effect subsequent amplification and slower CheY dephosphorylation. The high sensitivity indicates that this signal is generated by transport-induced dephosphorylation of the PTS rather than phosphoenolpyruvate consumption.

scholarly journals Whole blood immunophenotyping uncovers immature neutrophil-to-VD2 T-cell ratio as an early marker for severe COVID-19

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Guillaume Carissimo ◽  
Weili Xu ◽  
Immanuel Kwok ◽  
Mohammad Yazid Abdad ◽  
Yi-Hao Chan ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Whole Blood ◽  
Roc Analysis ◽  
Γδ T Cells ◽  
High Sensitivity ◽  
The Novel ◽  
Cellular Mechanisms ◽  
Cell Counts ◽  
Novel Coronavirus

Abstract SARS-CoV-2 is the novel coronavirus responsible for the current COVID-19 pandemic. Severe complications are observed only in a small proportion of infected patients but the cellular mechanisms underlying this progression are still unknown. Comprehensive flow cytometry of whole blood samples from 54 COVID-19 patients reveals a dramatic increase in the number of immature neutrophils. This increase strongly correlates with disease severity and is associated with elevated IL-6 and IP-10 levels, two key players in the cytokine storm. The most pronounced decrease in cell counts is observed for CD8 T-cells and VD2 γδ T-cells, which both exhibit increased differentiation and activation. ROC analysis reveals that the count ratio of immature neutrophils to VD2 (or CD8) T-cells predicts pneumonia onset (0.9071) as well as hypoxia onset (0.8908) with high sensitivity and specificity. It would thus be a useful prognostic marker for preventive patient management and improved healthcare resource management.

scholarly journals Ultrarapid, Ultrasensitive One-Step Kinetic Immunoassay for C-Reactive Protein (CRP) in Whole Blood Samples: Measurement of the Entire CRP Concentration Range with a Single Sample Dilution

2002 ◽  
Vol 48 (2) ◽  
pp. 269-277 ◽  
Author(s):  
Piia Tarkkinen ◽  
Tom Palenius ◽  
Timo Lövgren
Keyword(s):  
Whole Blood ◽  
Solid Phase ◽  
Point Of Care ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Cardiac Complications ◽  
Fluorescence Signal ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
One Step

Abstract Background: Recently, measurement of very low concentrations of C-reactive protein (CRP) has gained popularity as a potential new means for predicting the risk of future cardiac complications. In this study, we demonstrate the feasibility of a kinetic, one-step microparticle assay for quantitative determination of extremely low and high CRP concentrations in the limited timeframe typical for point-of-care testing. Methods: A noncompetitive, kinetic CRP immunoassay was developed that uses individual, porous microparticles as the solid phase. The microparticles were covalently coated with a monoclonal capture antibody, and the monoclonal detection antibody was labeled with europium. The one-step binding reaction was stopped by washing after 2 min of incubation, and the fluorescence signal of individual particles was measured. Results: The analytical detection limit (mean of zero calibrator + 3 SD) was 0.00016 mg/L CRP. Clinical samples were diluted 400-fold before assay to cover the CRP concentration range of 0.064–1200 mg/L. The assay correlated well with the Dade Behring N High Sensitivity CRP assay (for 0–10 mg/L, r = 0.969, Sy|x = 0.68, n = 54; for 0–350 mg/L, r = 0.969, Sy|x = 11.7, n = 100). The within- and between-run CVs based on calculated concentrations were, respectively, 9–16% and 14% at 0.11 mg/L, 4.5–12% and 8.2% at 4.2 mg/L, and 3.5–6.3% and 4.4% at 105 mg/L, with a CV &lt;15% at 0.2 mg/L and above. Conclusions: Use of the kinetic microparticle approach combined with time-resolved fluorometry allows ultrasensitive quantification of CRP in whole blood in 2 min with a linear assay range spanning more than four orders of magnitude.

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