The “therapeutic range” of the one-stage prothrombin time in the control of anticoagulant therapy: The effect of different thromboplastin preparations

Commercially available thromboplastin reagents and two human brain preparations have been compared using the one-stage prothrombin time and plasma samples from patients receiving long-term oral anticoagulant therapy. Considerable variation is noted between various thromboplastins using the same plasma sample. The commercially available thromboplastins give shorter prothrombin times than do human brain preparations. With the latter, the “therapeutic range” is represented by a prothrombin time of about 1.8 to 3.0 times the normal control value, whereas with commercial preparations the “therapeutic range” is about 1.25 to 1.75 times normal. The implications of these observations are discussed; the desirability of standardization of the one-stage prothrombin time is emphasized.     

Simultaneous determination of functional coagulation factors and competitive (PIVKA-) inhibitors based on enzyme kinetics

For a plasma containing the competitive (PIVKA-) inhibitors induced by anticoagulant treatment the coagulation time t is related to the concentrations of functional coagulation factors S (substrates) and competitive inhibitors I by t = tmin + el/S + gamma I/S with tmin being the minimum possible coagulation time and e and gamma the sensitivities of the test procedure towards a change in the concentration of functional coagulation factors and competitive inhibitors, respectively. The calibration of the test procedure can be achieved by performing a series of dilutions on an inhibitor-free plasma (determination of tmin and e) and, after that, on a plasma of known inhibitor content (determination of gamma) in both cases recording the parametrizing straight line which results from multiplying the respective equation by S. The content of functional coagulation factors and competitive inhibitors in the plasmas of anticoagulated patients then can be determined simultaneously by treating the patient's plasma like in the calibration for gamma. The proposed method should allow the complete metrological characterization of thromboplastin time reagents without any need for reference thromboplastins.     

Oral anticoagulant therapy and international normalized ratios in swine

While monitoring coagulation testing in Yucatan miniature swine being given oral anticoagulants, we noticed instances of high international normalized ratios (INR) without clinical complications in our animal model. All pigs (n = 17) weighed approximately 35.2 kg and were dosed daily with 2 to 3 mg of coumadin. Plasma samples were obtained and assayed for prothrombin time (PT), calculated INR, and activated partial thromboplastin time (APTT) at baseline, and after 7 and 14 days of coumadin therapy. Results of initial testing indicated high INR values after anticoagulation and short APTT values at baseline, which led us to consider the activity of vitamin K-dependent coagulation factors in the pig. This information was not available in literature concerning this strain of swine, and was surprising given that pigs are frequently used cardiac research models. Using the same plasma samples, we repeated the PT, INR, and APTT determinations using different reagents and a different analyzer. We also determined activities of coagulation factors II, VII, IX, and X. Large PT and INR differences were seen between the two instrument/reagent combinations, possibly due to the differences in the thromboplastins used and differences in the photo-optic versus manual clot-detection method of the instruments. Vitamin K-dependent factors in all pigs responded to coumadin by decreasing to < 30.0% activity, except for factor IX. The high INR values were not as pronounced when the second instrument/reagent combination was used, and the results seemed more in line with the animals' clinical condition. With this instrument/reagent combination, the pig can be considered a good model for research requiring oral anticoagulant medication.     

Implications of Using Different Methods to Characterise Anticoagulant Control in Patients with Second Generation Mechanical Heart Valve Prostheses

Objective

Characterisation of anticoagulant control is fundamental to investigations of its association with clinical outcome. Anticoagulant control depends on several factors. This paper aims to illustrate the implications of different methods for measuring and analysing anticoagulant control in patients with second generation mechanical heart valve prostheses.

Methods

International normalised ratio (INR) data collected during the 10-year follow-up of a randomised controlled trial were analysed. We considered the influence of: 3 different target INR ranges; anticoagulant control expressed as the proportion of INR readings (PoR) vs. anticoagulant control follow-up time (PoT); 3 ways of describing the profile of anticoagulant control over time.

Results

Different target INR ranges dramatically influenced derived measures of anticoagulant control; the PoT within the target range varied from 88% for the widest to 28% for narrowest range. Overall distributions of PoR and PoT observations were similar but differed by up to ±20% for individuals; PoT exceeded PoR when control was good but was less than PoR when control was poor. Classifying PoT outside the target range showed that widely varying combinations of PoT too high and too low are possible across individuals.

Conclusions

Researchers'' choices about methods for measuring and quantifying anticoagulant control markedly influence the values derived from INR readings. The use of different methods across studies makes it difficult or impossible to compare findings and to establish an evidence base for clinical practice. Methods for quantifying anticoagulant control should be standardised.     

Thromboplastin standards

The Prothrombin Time (PT) test is used for monitoring of treatment with Vitamin K-antagonists (VKA). The result of the PT test should be expressed as the International Normalized Ratio (INR). Calculation of INR is based on the availability of International Standards (IS) for thromboplastin and a calibration model. Calibration of a new PT test system is performed with the appropriate IS and fresh plasma samples of healthy (normal) volunteers and patients treated with VKA. The calibration model is based on the assumption of a linear relationship between the log(PT)'s obtained with the new PT system and the reference IS for both normal and patients' samples. Patients' samples for calibration should be selected by rejecting samples beyond the 1.5–4.5 INR range. Outliers should be rejected defined as points with a perpendicular distance greater than three residual standard deviations from the line of relationship. Selection of patients' samples and rejection of outliers result in a reduction of the between-laboratory variation of calibration. In addition to monitoring of VKA, the PT is used for management of patients with chronic liver disease. Likewise, INR_liver should be based on calibration with an IS using samples from patients with chronic liver disease.     

Homogeneous, structurally defined heparin-oligosaccharides with low anticoagulant activity inhibit the generation of the amplification pathway C3 convertase in vitro

This paper demonstrates that heparin-oligosaccharides with low anticoagulant activity have a high capacity to inhibit activation of the amplification pathway of complement in vitro. We prepared heparin-oligosaccharides by partial depolymerization of heparin using purified flavobacterial heparinase. The resulting oligosaccharide mixture was then fractionated using strong anion exchange-high pressure liquid chromatography to produce individual oligosaccharide components of this mixture, with degree of polymerization ranging from 2 to 16. These heparin-oligosaccharides were examined for both their anticoagulant activity and capacity to inhibit activation of the amplification pathway of complement. Although there was little difference among commercial heparins, a correlation between molecular weight and activity to inhibit convertase generation was clearly established for heparin-oligosaccharides between degree of polymerization 2 through 16. Heparin-oligosaccharides of degree of polymerization 10-16 (Mr 3888-5320) demonstrated up to 54% of heparin's activity on a molar basis (and up to 163% of heparin's activity on a weight basis) in inhibiting the amplification pathway of complement in vitro while showing almost no anticoagulant activity. These studies, for the first time, completely separate heparin's ability to inhibit complement activation from its anticoagulant activity.     

Anticoagulants and the propagation phase of thrombin generation

The view that clot time-based assays do not provide a sufficient assessment of an individual''s hemostatic competence, especially in the context of anticoagulant therapy, has provoked a search for new metrics, with significant focus directed at techniques that define the propagation phase of thrombin generation. Here we use our deterministic mathematical model of tissue-factor initiated thrombin generation in combination with reconstructions using purified protein components to characterize how the interplay between anticoagulant mechanisms and variable composition of the coagulation proteome result in differential regulation of the propagation phase of thrombin generation. Thrombin parameters were extracted from computationally derived thrombin generation profiles generated using coagulation proteome factor data from warfarin-treated individuals (N = 54) and matching groups of control individuals (N = 37). A computational clot time prolongation value (cINR) was devised that correlated with their actual International Normalized Ratio (INR) values, with differences between individual INR and cINR values shown to derive from the insensitivity of the INR to tissue factor pathway inhibitor (TFPI). The analysis suggests that normal range variation in TFPI levels could be an important contributor to the failure of the INR to adequately reflect the anticoagulated state in some individuals. Warfarin-induced changes in thrombin propagation phase parameters were then compared to those induced by unfractionated heparin, fondaparinux, rivaroxaban, and a reversible thrombin inhibitor. Anticoagulants were assessed at concentrations yielding equivalent cINR values, with each anticoagulant evaluated using 32 unique coagulation proteome compositions. The analyses showed that no anticoagulant recapitulated all features of warfarin propagation phase dynamics; differences in propagation phase effects suggest that anticoagulants that selectively target fXa or thrombin may provoke fewer bleeding episodes. More generally, the study shows that computational modeling of the response of core elements of the coagulation proteome to a physiologically relevant tissue factor stimulus may improve the monitoring of a broad range of anticoagulants.     

Developing and validating a model to predict the dry matter intake of grazing lactating beef cows

Current techniques for measuring the dry matter intake (DMI) of grazing lactating beef cows are invasive, time consuming and expensive making them impractical for use on commercial farms. This study was undertaken to explore the potential to develop and validate a model to predict DMI of grazing lactating beef cows, which could be applied in a commercial farm setting, using non-invasive animal measurements. The calibration dataset used to develop the model was comprised of 94 measurements recorded on 106 beef or beef–dairy crossbred cows (maternal origin). The potential of body measurements, linear type scoring, grazing behaviour and thermal imaging to predict DMI in combination with known biologically plausible adjustment variables and energy sinks was investigated. Multivariable regression models were constructed for each independent variable using SAS PROC REG and contained milk yield, BW, parity, calving day and maternal origin (dairy or beef). Of the 94 variables tested, 32 showed an association with DMI (P < 0.25) upon multivariable analysis. These variables were incorporated into a backwards linear regression model using SAS PROC REG. Variables were retained in this model if P < 0.05. Five variables; width at pins, full body depth, ruminating mastications, central ligament and rump width score, were retained in the model in addition to milk yield, BW, parity, calving day and maternal origin. The inclusion of these variables in the model increased the predictability of DMI by 0.23 (R² = 0.68) when compared to a model containing milk yield, BW, parity, calving day and maternal origin only. This model was applied to data recorded on an independent dataset; a herd of 60 lactating beef cows two years after the calibration study. The R² for the validation was 0.59. Estimates of DMI are required for measuring feed efficiency. While acknowledging challenges in applicability, the findings suggest a model such as that developed in this study may be used as a tool to more easily and less invasively estimate DMI on large populations of commercial beef cows, and therefore measure feed efficiency.     

Modified calibration protocol evaluated in a model-based testing of SBR flexibility

The purpose of this paper is to refine the BIOMATH calibration protocol for SBR systems, in particular to develop a pragmatic calibration protocol that takes advantage of SBR information-rich data, defines a simulation strategy to obtain proper initial conditions for model calibration and provides statistical evaluation of the calibration outcome. The updated calibration protocol is then evaluated on a case study to obtain a thoroughly validated model for testing the flexibility of an N-removing SBR to adapt the operating conditions to the changing influent wastewater load. The performance of reference operation using fixed phase length and dissolved oxygen set points and two real-time control strategies is compared to find optimal operation under dynamic conditions. The results show that a validated model of high quality is obtained using the updated protocol and that the optimization of the system’s performance can be achieved in different manners by implementing the proposed control strategies.     

Increased tissue-plasminogen activator (t-PA) levels in patients under oral anticoagulant therapy

The fibrinolytic system was investigated in 30 patients under oral anticoagulant therapy, and in 23 control patients not receiving oral anticoagulants. Patients under oral anticoagulant therapy had significantly higher tissue-plasminogen activator (t-PA) antigen levels than patients in the control group. Mean t-PA levels before venous occlusion were 18.4 ng/ml in the anticoagulated patients vs. 7.9 ng/ml in the control patients (p less than 0.001). After venous occlusion for 10 minutes, t-PA levels were 45.0 ng/ml in the anticoagulated patients and 24.2 ng/ml in the control patients (p less than 0.01). Plasminogen activator inhibitor (PAI) capacity was not significantly different in the two groups before venous occlusion (VO) but differed slightly (p less than 0.05) after VO. The net decrease in euglobulin lysis time (ELT) after venous occlusion (= ELT before VO - ELT after VO), indicating the relative potency of the fibrinolytic activity in blood, was also significantly higher in the anticoagulated patients (median 240 min vs. 125 min, p less than 0.001). These data indicate that oral anticoagulant therapy increases the fibrinolytic activity in blood, and thus may have an additional therapeutic effect in addition to anticoagulation.     

Standardization: a Progress Report

The introduction of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products requires the availability of well-characterized reference reagents. Working reagents for hepatitis C virus RNA, hepatitis B virus DNA, HIV-1 RNA and human parvovirus B19 DNA have been established at NIBSC and at many other laboratories (both official medicinal control laboratories and commercial laboratories). However, as these reagents have been characterised independently, it is difficult to compare results from assays using different working reagents. Recently, a WHO International Standard was established for HCV RNA NAT assays. This standard has been calibrated in International Units (IU) and provides a common standard against which all working reagents can be calibrated. Collaborative studies to characterise two further candidate International Standards for HBV DNA and HIV-1 RNA NAT assays have been completed.     

The congenital factor VII abnormalities (dysproconvertinemias). The genetic plot thickens

Congenital factor VII deficiency is a well known clotting disorder first identified in 1951. Several congenital abnormalities of factor VII have been described during the past decade. These abnormalities were all characterized by the presence of a discrepancy between factor VII clotting activity and factor VII cross reacting material or antigen. Recently other factor VII abnormalities have been described which showed peculiar sensitivities to ox-brain thromboplastins as compared to thromboplastins of other origin. These discoveries have complicated the differential diagnosis of prothrombin complex factors deficiences and abnormalities. A correct diagnosis may be reached only by means of a battery of tests which employ tissue thromboplastins of different origin.     

NIST/ISAC standardization study: Variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads

Results from a standardization study cosponsored by the International Society for Advancement of Cytometry (ISAC) and the US National Institute of Standards and Technology (NIST) are reported. The study evaluated the variability of assigning intensity values to fluorophore standard beads by bead manufacturers and the variability of cross calibrating the standard beads to stained polymer beads (hard-dyed beads) using different flow cytometers. Hard dyed beads are generally not spectrally matched to the fluorophores used to stain cells, and spectral response varies among flow cytometers. Thus if hard dyed beads are used as fluorescence calibrators, one expects calibration for specific fluorophores (e.g., FITC or PE) to vary among different instruments. Using standard beads surface-stained with specific fluorophores (FITC, PE, APC, and Pacific Blue?), the study compared the measured intensity of fluorophore standard beads to that of hard dyed beads through cross calibration on 133 different flow cytometers. Using robust CV as a measure of variability, the variation of cross calibrated values was typically 20% or more for a particular hard dyed bead in a specific detection channel. The variation across different instrument models was often greater than the variation within a particular instrument model. As a separate part of the study, NIST and four bead manufacturers used a NIST supplied protocol and calibrated fluorophore solution standards to assign intensity values to the fluorophore beads. Values assigned to the reference beads by different groups varied by orders of magnitude in most cases, reflecting differences in instrumentation used to perform the calibration. The study concluded that the use of any spectrally unmatched hard dyed bead as a general fluorescence calibrator must be verified and characterized for every particular instrument model. Close interaction between bead manufacturers and NIST is recommended to have reliable and uniformly assigned fluorescence standard beads. ? 2012 International Society for Advancement of Cytometry.     

Determination of viable yeast cells by gravitational field-flow fractionation with fluorescence detection

Vinification processing is largely related to yeast performance and depends on the initial cell viability. To optimize the quality of wine fermentation, control of the yeast quality is mandatory. The present paper describes a new method using gravitational field flow fractionation (GrFFF) with fluorescence detection for the determination of yeast cell viability before the fermentation process. A GrFFF calibration procedure was developed using commercial yeast to prepare standards of viable cells and propidium iodide (PI) as fluorescent probe for nonviable cells. The suitability of the new method was tested with several commercial yeast strains with a g/L content ranging from 1 to 3. The validation of the method was performed by comparing GrFFF viability values with those obtained using Coulter counter and flow cytometry techniques.     

Multiplex genome editing of mammalian cells for producing recombinant heparin

Heparin is an essential anticoagulant used for treating and preventing thrombosis. However, the complexity of heparin has hindered the development of a recombinant source, making its supply dependent on a vulnerable animal population. In nature, heparin is produced exclusively in mast cells, which are not suitable for commercial production, but mastocytoma cells are readily grown in culture and make heparan sulfate, a closely related glycosaminoglycan that lacks anticoagulant activity. Using gene expression profiling of mast cells as a guide, a multiplex genome engineering strategy was devised to produce heparan sulfate with high anticoagulant potency and to eliminate contaminating chondroitin sulfate from mastocytoma cells. The heparan sulfate purified from engineered cells grown in chemically defined medium has anticoagulant potency that exceeds porcine-derived heparin and confers anticoagulant activity to the blood of healthy mice. This work demonstrates the feasibility of producing recombinant heparin from mammalian cell culture as an alternative to animal sources.     

Structural basis for the anticoagulant activity of heparin. 1. Relationship to the number of charged groups.

This study was undertaken to provide further information concerning the chemical heterogeneity of heparins and the relationships between the anticoagulant activity (USP assay) and the anionic density of the heparin. A sample of commercial heparin was fractionated into 13 fractions by sequential extraction in a two-phase system of 1-butanol-aqueous NaCl containing excess hexadecylpyridinium chloride. The anionic density distribution was characterized by the fractional distribution of uronate among the fractions. The fractions were characterized by several molar ratios of constituents, molecular weight, charge density, and anticoagulant activity in recalcified sheep plasma. The heparin was broadly distributed among the last 10 fractions; the first three contained impurities which were completely separated from the heparin fractions. The heparin fractions differ systematically in anionic density but are of substantially the same molecular weight. Anticoagulant activity increased markedly with anionic density, ranging from 81 units/mg for the heparin fraction with the lowest anionic density up to a high of 243 units/mg. The relationship between anticoagulant activity and either anionic density or its square is nonlinear. However, in the latter case an initial linear relationship was observed for anticoagulant activities of less than 200 units/mg.     

A new application of isoindole fluorophore derivative in sitagliptin antidiabetic medication assay: Application to dosage forms and biological fluid evaluation

Dipeptidyl peptidase-4 enzyme suppressant is a unique category of oral antidiabetic medication. Sitagliptin (STG) is a perfect member of this category and is pharmaceutically marketed alone or in combination with metformin. Here, the ideal application of an isoindole derivative for STG assay was developed using a feasible, easy-to-use, economic, and affordable method. STG as an amino group donor can form a luminescent derivative: isoindole on interaction with o-phthalaldehyde and the existence of (2-mercaptoethanol) 0.02% (v/v) as a thiol group donor. Excitation (339.7 nm) and emission (434.6 nm) wavelengths were used to monitor the isoindole fluorophore yield; moreover, each experimental variable was carefully investigated and adjusted. The calibration graph was constructed by plotting fluorescence intensities against STG concentrations, and controlled linearity was observed at concentrations ranging from 50 to 1000 ng/ml. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines were analyzed in depth to prove the technique validation. The implementation of the present technique was extended successfully to the evaluation of various types of STG dose forms and spiking samples of human plasma and urine. The developed technique was shown to be an effective, simple, and quick replacement for quality control and clinical study evaluation of STG.     

Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays

The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1^st International Standard for Ebola virus RNA.     

Comparison of different camera calibration approaches for underwater applications

The purpose of this study was to compare three camera calibration approaches applied to underwater applications: (1) static control points with nonlinear DLT; (2) moving wand with nonlinear camera model and bundle adjustment; (3) moving plate with nonlinear camera model. The DVideo kinematic analysis system was used for underwater data acquisition. The system consisted of two gen-locked Basler cameras working at 100 Hz, with wide angle lenses that were enclosed in housings. The accuracy of the methods was compared in a dynamic rigid bar test (acquisition volume-4.5×1×1.5 m(3)). The mean absolute errors were 6.19 mm for the nonlinear DLT, 1.16 mm for the wand calibration, 1.20 mm for the 2D plate calibration using 8 control points and 0.73 mm for the 2D plane calibration using 16 control points. The results of the wand and 2D plate camera calibration methods were less associated to the rigid body position in the working volume and provided better accuracy than the nonlinear DLT. Wand and 2D plate camera calibration methods presented similar and highly accurate results, being alternatives for underwater 3D motion analysis.