The technique of measuring thrombin generation with fluorogenic substrates: 1. Necessity of adequate calibration

2008 ◽  
Vol 100 (08) ◽  
pp. 343-349 ◽  
Author(s):  
Erik De Smedt ◽  
Raed Al Dieri ◽  
Henri M. H. Spronk ◽  
Karli Hamulyak ◽  
Hugo ten Cate ◽  
...  
Keyword(s):  
Fluorescence Quenching ◽  
Thrombin Generation ◽  
Systematic Errors ◽  
Peak Height ◽  
Fluorogenic Substrates ◽  
Parallel Sample ◽  
Coefficients Of Variation ◽  
Individual Calibration ◽  
Source Of Error ◽  
Preanalytical Conditions

SummaryIn fluorogenic thrombin generation (TG) measurement the concentrations of thrombin are obtained from the course of fluorescence intensity. Because of fluorescence quenching, in one series of normal plasmas (n=60), the rate of fluorescence increase at fixed thrombin activity was 70 ± 13% of that in buffer, in another (n= 139) 75 ± 8%. Using a calibration factor (CF) measured in buffer therefore underestimates thrombin concentrations in plasma and introduces a source of error. A fixed CF also neglects the 25 – 35% increase of CF during the experiment and thus distorts the form of the TG curve so that the ETP cannot be determined. Continuous individual calibration (CIC), in which CF is determined continuously in a parallel sample, avoids such systematic errors but adds random error because the thrombin course is calculated from two different measurements. We determined the intra-individual coefficients of variation (CV) of the peak-height and ETP as obtained with CIC to those obtained with a fixed CF measured in buffer. With the fixed CF, the CVs varied between 18% and 49%; with CIC they lowered to 4–7% (n=5x12), i.e. in a range allowing clinical application. It is shown that CIC can be discarded for the measurement of peak thrombin values and replaced by comparison to a reference plasma only if quenching is not a systematic confounder. This was shown to be the case in the set of 139 normal plasmas but not in the set used for determining the intraindividual CVs, a difference that may depend upon preanalytical conditions.

Thrombin generation in paediatric patients with congenital heart disease

2008 ◽  
Vol 28 (S 01) ◽  
pp. S61-S66 ◽  
Author(s):  
G. Cvirn ◽  
A. Rosenkranz ◽  
B. Leschnik ◽  
W. Raith ◽  
W. Muntean ◽  
...  
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
Negative Correlation ◽  
Congenital Heart ◽  
Thrombin Generation ◽  
Significant Negative Correlation ◽  
Peak Height ◽  
Global Test ◽  
Paediatric Patients ◽  
Age Range

SummaryThrombin generation was studied in paediatric patients with congenital heart disease (CHD) undergoing cardiac surgery using the calibrated automated thrombography (CAT) in terms of the lag time until the onset of thrombin formation, time to thrombin peak maximum (TTP), endogenous thrombin potential (ETP), and thrombin peak height. The suitability to determine the coagulation status of these patients was investigated. Patients, material, methods: CAT data of 40 patients with CHD (age range from newborn to 18 years) were compared to data using standard coagulation parameters such as prothrombin (FII), antithrombin (AT), tissue factor pathway inhibitor (TFPI), prothrombin fragment 1.2 (F 1.2), thrombin-antithrombin (TAT), activated partial thromboplastin time (aPTT), and prothrombin time (PT). Results: A significant positive correlation was seen between ETP and FII (p < 0.01; r = 0.369), as well as between peak height and F II (p < 0.01; r = 0.483). A significant negative correlation was seen between ETP and TFPI values (p < 0.05; r = –0.225) while no significant correlation was seen between peak height and TFPI. A significant negative correlation was seen between F 1.2 generation and ETP (p < 0.05; r = –0.254) and between F 1.2 generation and peak height (p < 0.05; r = –0.236). No correlation was seen between AT and ETP or peak. Conclusions: CAT is a good global test reflecting procoagulatory and inhibitory factors of the haemostatic system in paediatric patients with CHD.

Gas-Liquid Chromatographic Method for Analysis of Pentachloronitrobenzene Formulations: Collaborative Study

1982 ◽  
Vol 65 (1) ◽  
pp. 110-114
Author(s):  
Alan R Hanks
Keyword(s):  
Chromatographic Method ◽  
Collaborative Study ◽  
Internal Standard ◽  
Peak Height ◽  
Matched Pairs ◽  
Commercial Products ◽  
Coefficients Of Variation ◽  
Liquid Chromatographic Method ◽  
Wettable Powder ◽  
Liquid Chromatographic

Abstract A collaborative study has been conducted on a gasliquid chromatographic (GLC) method for determining pentachloronitrobenzene (PCNB) in formulations. Wettable powder, liquid, and granular matched pairs of commercial products were analyzed by 17 laboratories using peak height measurements and by 12 laboratories using integrator area measurements. Samples were dissolved in chloroform and aliquots were mixed with internal standard before GLC analysis on a 5% SE-30 column. Mean coefficients of variation for the completed study were 1.54% for integrator area measurements and 1.35% for peak height measurements. The method has been adopted official first action.

Interaction between pyridine nucleotide coenzymes and heme proteins as a possible source of error in assay of activities of coenzyme-linked enzyme.

1989 ◽  
Vol 35 (10) ◽  
pp. 2129-2133 ◽  
Author(s):  
M D Jeyasingham ◽  
O E Pratt ◽  
H K Roopral
Keyword(s):  
Pyridine Nucleotide ◽  
Peak Height ◽  
Heme Group ◽  
Ultraviolet Absorbance ◽  
The Third ◽  
Spectral Shifts ◽  
Muscle Breakdown ◽  
Activity Of Enzymes ◽  
Source Of Error ◽  
Absorbance Spectra

Abstract The ultraviolet absorbance spectra of pyridine nucleotide coenzymes change in the presence of heme-containing proteins. The positions of each of the two main absorbance peaks of NADH are shifted progressively towards shorter wavelengths in the presence of increasing concentrations of hemoglobin, and the third peak, at 220 nm, disappears altogether. Similar changes are seen in the spectra of NAD+ and NADPH, and similar effects on these spectra are produced by myoglobin and cytochrome c, but not by comparable concentrations of albumin. The spectral shifts are generally accompanied by a decreased peak height. This finding may help explain problems reported by previous workers in the measurement of the activity of enzymes such as transketolase or lactate dehydrogenase in erythrocyte hemolysates. Errors may be considerable if allowance is not made for this effect, especially if the concentration of heme protein in the spectrophotometer cuvette much exceeds 1 g/L. The interaction appears to indicate some form of bonding, occurring generally between pyridine nucleotide coenzymes and the heme group in proteins. We relate the findings to measurement of activities of pyridine nucleotide-linked enzymes in erythrocyte lysates and in plasma containing myoglobin after muscle breakdown.

Improved liquid-chromatographic method for determination of serum cortisol.

1979 ◽  
Vol 25 (7) ◽  
pp. 1293-1296 ◽  
Author(s):  
P M Kabra ◽  
L L Tsai ◽  
L J Marton
Keyword(s):  
Limit Of Detection ◽  
Internal Standard ◽  
Serum Cortisol ◽  
Peak Height ◽  
Reversed Phase ◽  
Column Temperature ◽  
Coefficients Of Variation ◽  
Liquid Chromatographic Method ◽  
Liquid Chromatographic

Abstract We describe a specific and precise method for measuring concentrations of cortisol in serum or plasma by liquid chromatography. Cortisol, together with an internal standard, equilenin, is extracted from 1 mL of serum or plasma and analyzed isocratically on a reversed-phase column with a mobile phase of acetonitrile/phosphate buffer (30/70, by vol.), at a flow rate of 2.0 mL/min. The eluted cortisol is detected by its absorption at 254 nm and quantitated by peak height measurements. Each analysis requires no longer than 15 min at the optimum column temperature of 50 degrees C. The lower limit of detection for cortisol is about 2 ng/sample for a standard solution; sensitivity is routinely 5 micrograms/L of serum. Analytical recoveries exceeded 95%, with good day-to-day precision (coefficients of variation between 4 and 7%). Of more than 50 drugs and steroids tested for possible interference, only the steroids cortisone, prednisone, and prednisolone may interfere with the analysis of cortisol.

Increased thrombin generation and fibrinogen level after therapeutic plasma transfusion: Relation to bleeding

2008 ◽  
Vol 99 (01) ◽  
pp. 64-70 ◽  
Author(s):  
Paola E. J. van der Meijden ◽  
René van Oerle ◽  
Joyce Curvers ◽  
Elisabeth C. M. van Pampus ◽  
Saskia E. M Schols ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Fibrinogen Level ◽  
Coagulation Factor ◽  
Peak Height ◽  
Fresh Frozen Plasma ◽  
Plasma Transfusion ◽  
Perioperative Bleeding ◽  
Coagulation Tests ◽  
Fresh Frozen ◽  
Dilutional Coagulopathy

SummaryIn a clinical setting, fresh frozen plasma (FFP) is transfused to diluted patients with complicated surgery or trauma, as guided by prolonged conventional coagulation times or low fibrinogen levels. However, the limited sensitivity of these coagulation tests may restrict their use in measuring the effect of transfusion and hence predicting the risk of perioperative bleeding. We used the more sensitive, calibrated automated thrombogram (CAT) method to evaluate the result of therapeutic FFP transfusion to 51 patients with dilutional coagulopathy. Thrombin generation was measured in pre- and post-transfusion plasma samples in the presence of either platelets or phospholipids. For all patients, the transfusion led to higher plasma coagulation factor levels, a shortened activated partial thromboplastin time, and a significant increase in thrombin generation (peak height and endogenous thrombin potential). Interestingly, thrombin generation parameters and fibrinogen levels were higher in posttransfusion plasmas from patients who stopped bleeding (n=32) than for patients with ongoing bleeding (n=19). Plasmas from 15 of the 19 patients with ongoing bleeding were markedly low in either thrombin generation or fibrinogen level. We conclude that the thrombin generation method detects improved haemostatic activity after plasma transfusion. Furthermore, the data suggest that thrombin generation and fibrinogen are independent determinants of the risk of perioperative bleeding in this patient group.

Collaborative Study of High Pressure Liquid Chromatographic Method for Determining Methyl Parathion

1977 ◽  
Vol 60 (3) ◽  
pp. 724-727
Author(s):  
Edwin R Jackson
Keyword(s):  
Methyl Parathion ◽  
Collaborative Study ◽  
Internal Standard ◽  
Peak Height ◽  
Emulsifiable Concentrate ◽  
Coefficients Of Variation ◽  
Liquid Chromatographic Method ◽  
Technical Material ◽  
Water Saturated ◽  
Liquid Chromatographic

Abstract Methyl parathion in technical material, 4 lb/gal. emulsifiable concentrate, and 6 lb toxaphene-3 lb methyl parathion/gal. emulsifiable concentrate was chromatographed on microparticulate silica with 40% water-saturated chloroform mobile phase and 254 nm ultraviolet detection. Ten collaborators reported results on 3 matched pairs of samples by peak height measurements with acetophenone as internal standard. The mean of the coefficients of variation for the 6 samples was 1.71%. Results for 4 of the samples were compared with gas-liquid chromatographic results on the same samples. No difference in the means by the 2 methods was noted at the 95% confidence level. The method has been adopted as official first action.

Evaluations of Global Wave Prediction at the Fleet Numerical Meteorology and Oceanography Center*

2005 ◽  
Vol 20 (5) ◽  
pp. 745-760 ◽  
Author(s):  
W. Erick Rogers ◽  
Paul A. Wittmann ◽  
David W. C. Wang ◽  
R. Michael Clancy ◽  
Y. Larry Hsu
Keyword(s):  
Primary Source ◽  
Wave Model ◽  
Systematic Errors ◽  
Wind Forcing ◽  
Evaluation Methodology ◽  
Northeast Pacific ◽  
Internal Error ◽  
Source Of Error ◽  
Insight Into

Abstract It is a major challenge to determine whether bias in operational global wave predictions is predominately due to the wave model itself (internal error) or due to errors in wind forcing (an external error). Another challenge is to characterize bias attributable to errors in wave model physics (e.g., input, dissipation, and nonlinear transfer). In this study, hindcasts and an evaluation methodology are constructed to address these challenges. The bias of the wave predictions is evaluated with consideration of the bias of four different wind forcing fields [two of which are supplemented with the NASA Quick Scatterometer (QuikSCAT) measurements]. It is found that the accuracy of the Fleet Numerical Meteorology and Oceanography Center’s operational global wind forcing has improved to the point where it is unlikely to be the primary source of error in the center’s global wave model (WAVEWATCH-III). The hindcast comparisons are specifically designed to minimize systematic errors from numerics and resolution. From these hindcasts, insight into the physics-related bias in the global wave model is possible: comparison to in situ wave data suggests an overall positive bias at northeast Pacific locations and an overall negative bias at northwest Atlantic locations. Comparison of frequency bands indicates a tendency by the model physics to overpredict energy at higher frequencies and underpredict energy at lower frequencies.

Elevated Procoagulant Microparticles Expressing Endothelial and Platelet Markers in Essential Thrombocythemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2793-2793
Author(s):  
Marijke Trappenburg ◽  
Muriel van Schilfgaarde ◽  
Marina Marchetti ◽  
Henri Spronk ◽  
Hugo ten Cate ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Thrombin Generation ◽  
Cell Types ◽  
Endothelial Activation ◽  
Peak Height ◽  
Cellular Origin ◽  
Thrombin Generation Assay ◽  
Von Willebrand ◽  
Design And Methods ◽  
Willebrand Factor

Abstract Background: Most cell types, including blood - and vascular cells, produce microparticles (MPs) upon activation. Since cellular MPs are known to be elevated in thromboembolic diseases, we hypothesized a role for MPs in the pathogenesis of thrombosis in Essential Thrombocythemia (ET). Design and methods: In plasma samples from 21 ET patients and 10 healthy subjects, the levels and the cellular origin of MPs were determined by flowcytometric analysis, while the MP-associated procoagulant activity was measured by the thrombin generation assay. Results: ET patients had significantly higher numbers of circulating AnnexinV-positive MPs than controls (median 4500 vs 2500×106 events/L; p=0.039), including significantly higher number of MPs positive for the platelet marker CD61 (median 4000 vs 2400×106/L; p=0.043) the endothelial marker CD62E (median 875 vs 14×106/L; p=0.009), and for Tissue Factor (median 1.8 vs 0.9×106/L; p=0.036). CD62E was co-expressed with the platelet marker CD41 on MPs, suggesting a bilineal origin of such MPs, that were observed only in patients with risk factors for thrombosis. ET patients had higher plasma mature von Willebrand factor (vWF) levels (median 50 vs 35 nM, p=0.045) but similar propeptide levels (median 7 vs 5 nM, p=0,07) compared to controls, indicating chronic endothelial activation. In thrombin generation analyses, MP rich plasma from ET patients had a shorter lag time (9.7 min, 95%CI: 8.7–10.7 versus 15.9 min, 95%CI: 10.9–20.9, p=0.001) and higher peak height (215 nM, 95%CI: 189–241 versus 142 nM, 95%CI: 87–189, p=0.038) than from controls. Peak height correlated significantly with the total number of MPs (R=0.634, p&lt;0.001). Conclusions: ET patients showed higher number of circulating MPs with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This is confirmed by an increased mature vWF level and an abnormal mature vWF/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for MPs in thrombosis in ET.

scholarly journals Effects of tissue factor, thrombomodulin and elevated clotting factor levels on thrombin generation in the calibrated automated thrombogram

2009 ◽  
Vol 102 (11) ◽  
pp. 936-944 ◽  
Author(s):  
Kellie Machlus ◽  
Emily Colby ◽  
Jogin Wu ◽  
Gary Koch ◽  
Nigel Key ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Relative Sensitivity ◽  
Epidemiological Studies ◽  
Peak Height ◽  
Clotting Factor ◽  
Time To Peak ◽  
High Throughput Method ◽  
Assay Conditions

SummaryElevated procoagulant levels have been correlated with increased thrombin generation in vitro and with increased venous thromboembolism (VTE) risk in epidemiological studies. hrombin generation tests are increasingly being employed as a high throughput method to provide a global measure of procoagulant activity in plasma samples. The objective of this study was to distinguish the effects of assay conditions [tissue factor (TF), thrombomodulin, platelets/lipids] and factor levels on thrombin generation parameters, and determine the conditions and parameters with the highest sensitivity and specificity for detecting elevated factor levels. Thrombin generation was measured using calibrated automated thrombography (CAT) in corn trypsin inhibitor (CTI)-treated platelet-free plasma (PFP) and plateletrich plasma (PRP). Statistical analysis was performed using logarithms of observed values with analysis of variance that accounted for experiment and treatment. he relative sensitivity of lag time (LT), time to peak (TTP), peak height and endogenous thrombin potential (ETP) to elevated factors XI, IX,VIII, X, and prothrombin was as follows: PFP initiated with 1 pM TF > PFP initiated with 5 pM TF > PRP initiated with 1 pM TF. For all conditions, inclusion of thrombomodulin prolonged the LT and decreased the peak and ETP; however, addition of thrombomodulin did not increase the ability of CAT to detect elevated levels of individual procoagulant factors. In conclusion, CAT conditions differentially affected the sensitivity of thrombin generation to elevated factor levels. Monitoring the peak height and/ or ETP following initiation of clotting in PFP with 1 pM TF was most likely to detect hypercoagulability due to increased procoagulant factor levels.

Quantitative phase analysis of α- and β-silicon nitrides. I. Estimation of errors

1999 ◽  
Vol 32 (4) ◽  
pp. 704-715 ◽  
Author(s):  
H. Toraya
Keyword(s):  
Phase Analysis ◽  
Rietveld Method ◽  
Systematic Errors ◽  
Peak Height ◽  
Quantitative Phase Analysis ◽  
Silicon Nitrides ◽  
Profile Function ◽  
Quantitative Phase ◽  
Counting Statistics ◽  
Integrated Intensities

Errors in the quantitative phase analysis (QPA) of α- and β-silicon nitrides (Si3N4) using the mean normalized intensity (MNI) method and the Rietveld method have been estimated by theory and experiments. A total error for a weight fraction (w) in a binary system can be expressed in the formE(w) =w(1 −w)S, whereSis the quadratic sum of statistical and systematic errors. Random errors associated with counting statistics for integrated intensities in the MNI method are below 0.1∼0.2 wt% if the studied reflections have average peak heights of more than ∼1000 counts. Such errors will become approximately twice as large if peak-height intensities are used. The error associated with particle statistics in the studied samples was smaller than the counting-statistics error. Among various sources of systematic errors examined, incorrect choice of constrained/unconstrained full width at half-maximum (FWHM) parameters gave the largest error. The choice of the background function had little influence on the QPA, whereas the choice of the profile function had a large influence. Truncation errors in profile function calculations and the 2θ range of the observed data are below ±0.1 wt% when appropriate criteria are applied. Systematic errors in the measurement of peak-height intensity arise primarily from the overestimation of intensities of weak peaks that overlap the tails of strong peaks, as well as from line broadening of β-phase reflections in the studied samples. Errors caused by ignoring the difference in density between the two phases were negligibly small. Estimated errors of the methods followed the order: the MNI method using peak-height intensities < the MNI method using integrated intensities ≃ the Rietveld method.

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