International Sensitivity Index Values Research Articles

Estimating the measurement uncertainties of the international sensitivity index of 12 thromboplastins through Monte Carlo simulation

BackgroundMeasurement uncertainty (MU) estimation has become an important process in clinical laboratories; however, calculating the MUs of the international sensitivity index (ISI) of thromboplastins is difficult because of the complex mathematical calculations required in calibration. Therefore, this study quantifies the MUs of ISIs through the Monte Carlo simulation (MCS), which involves random sampling of numerical values to solve a complex mathematical calculation. MethodsEighty blood plasmas and commercially available certified plasmas (ISI Calibrate) were used to assign the ISIs of each thromboplastin. Prothrombin times were measured using reference thromboplastin and 12 commercially available thromboplastins (Coagpia PT-N, PT Rec, ReadiPlasTin, RecombiPlasTin 2G, PT-Fibrinogen, PT-Fibrinogen HS PLUS, Prothrombin Time Assay, Thromboplastin D, Thromborel S, STA-Neoplastine CI Plus, STA-Neoplastine R 15, and STA-NeoPTimal) with two automated coagulation instruments: ACL TOP 750 CTS (ACL TOP; Instrumentation Laboratory, Bedford, MA, USA) and STA Compact (Diagnostica Stago, Asnières-sur-Seine, France). Then, the MUs of each ISI were simulated through MCS. ResultsThe MUs of ISIs ranged from 9.7 % to 12.1 % and 11.6 % to 12.0 % when blood plasma and ISI Calibrate were used, respectively. For some thromboplastins, the ISI claimed by manufacturers significantly differed from the estimated results. ConclusionsMCS is adequate to estimate the MUs of ISI. These results would be clinically useful for estimating the MUs of the international normalized ratio in clinical laboratories. However, the claimed ISI significantly differed from the estimated ISI of some thromboplastins. Therefore, manufacturers should provide more accurate information about the ISI value of thromboplastins.

Standardization of Prothrombin Time/International Normalized Ratio (PT/INR).

The prothrombin time (PT) represents the most commonly used coagulation test in clinical laboratories. The PT is mathematically converted to the international normalized ratio (INR) for use in monitoring anticoagulant therapy with vitamin K antagonists such as warfarin in order to provide test results that are adjusted for thromboplastin and instrument used. The INR is created using two major PT 'correction factors', namely the mean normal PT (MNPT) and the international sensitivity index (ISI). Manufacturers of reagents and coagulometers have made some efforts to harmonizing INRs, for example, by tailoring reagents to specific coagulometers and provide associated ISI values. Thus, two types of ISIs may be generated, with one being a 'general' or 'generic' ISI and others being reagent/coagulometer-specific ISI values. Although these play a crucial role in improving INR results between laboratories, these laboratories reported INR values are known to still differ, even when laboratories use the same thromboplastin reagent and coagulometer. Moreover, ISI values for a specific thromboplastin can vary among different models of coagulometers from a manufacturer using the same method for clot identification. All these factors can be sources of error for INR reporting, which in turn can significantly affect patient management. In this narrative review, we provide some guidance to appropriate ISI verification/validation, which may help decrease the variability in cross laboratory reporting of INRs.

Assignment of international normalized ratio to frozen and freeze-dried pooled plasmas.

Objectives Frozen and freeze-dried plasmas may be used for local prothrombin time system calibration, for direct international normalized ratio (INR) determination, and for quality assessment. The purpose of the present study was to evaluate the usefulness of INRs assigned with various types of thromboplastins to frozen and freeze-dried pooled plasmas obtained from patients treated with vitamin K antagonists. Methods INRs were calculated according to the international sensitivity index (ISI) model using various thromboplastins and instruments, i.e. International Standards for thromboplastin as well as six commercial reagents prepared from rabbit and bovine brain, and recombinant human tissue factor. The uncertainty of the INRs was assessed using the standard deviations of clotting times and ISI values. Commutability of the plasmas was assessed according to the approved Clinical and Laboratory Standards Institute (CLSI) Guideline EP30-A. Validation of a set of six frozen plasma pools for direct INR determination was performed according to the Subcommittee on Control of Anticoagulation of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (SSC/ISTH) guidelines. Results For all frozen and freeze-dried plasmas, the INRs calculated with bovine thromboplastin Thrombotest were lower than the INRs assigned with other thromboplastins. With a few exceptions, the frozen and freeze-dried pooled plasmas were commutable. When the set of six frozen plasma pools was used for local calibration, the analytical bias of the INR was less than ±10% for all commercial reagents except Thrombotest. Conclusions Processing of fresh plasmas to prepare pooled frozen plasmas and freeze-dried plasmas may lead to different INR assignments depending on the thromboplastin used. Despite minor INR differences, a set of six frozen plasma pools could be used for local calibration by direct INR determination.

How to Generate a More Accurate Laboratory-Based International Normalized Ratio: Solutions to Obtaining or Verifying the Mean Normal Prothrombin Time and International Sensitivity Index.

Although the landscape of anticoagulation therapy is evolving, vitamin K antagonists (VKAs) such as warfarin remain an anticoagulant of choice for many clinicians and their patients. Nevertheless, management of VKA therapy remains challenging, largely because of patient variability and drug and food interactions; thus, VKA dosing has to be personalized. This is achieved by regular monitoring using a test called the prothrombin time (PT), mathematically converted to an international normalized ratio (INR). The INR system is meant to harmonize laboratory test results by taking into account reagent and instrumentation variability that is otherwise expected to give rise to variable PT values, but which should accordingly lead to less variable INR values. Of clinical importance, too low an INR is suggestive of increased thrombotic risk and typically means the VKA dose should be increased, whereas too high an INR is suggestive of increased bleeding risk and typically means the VKA dose should be temporarily withheld and/or decreased. However, evidence continues to show that variability in INR values between laboratories remains unacceptably high. Given that modern instrumentation provides for robust analytical values-meaning highly reproducible intralaboratory clotting times or PTs in this case-the most likely cause of high INR variability is inconsistency in the INR test components-meaning the MNPT (mean normal PT) and ISI (international sensitivity index) values used by laboratories to generate a given INR. In other words, there are doubts as to the accuracy of some INR values because there are corresponding doubts about the accuracy of MNPT and/or ISI values that have been assigned by some laboratories for their reagent/instrument combination. The current report is intended to provide some solutions around the problems of inaccurate INRs, ISIs, and MNPTs, thus aiming to drive laboratory INRs closer to "truth," and thus promote better patient management. The novel strategies include a primary process of transference to obtain/verify MNPT and/or ISI values for a new reagent using an existing reagent as reference, and a secondary process whereby external quality assessment data can be used to correct bias or existing errors in assigned MNPT and/or ISI values.

Misguided by INR in Liver Disease Patients? Implications for Clinicians Including Pain Proceduralists.

THE ROSY PAST The prothrombin time (PT) assay, developed in 1935, was used in the identification of the anticoagulants dicumarol and warfarin. Currently, this assay is most commonly used to measure the therapeutic activity of warfarin. It was well known that PT results experienced a variation among centers using different reagents, making comparisons challenging. Around the same time came the invention of the international sensitivity index (ISI), allowing for calibration of different batches of thromboplastins to an international standard. To address interlaboratory PT variation, the international normalized ratio (INR), defined as the ratio of a patient’s PT to a control sample raised to the power of the ISI value, was invented in the 1980s. The INR became widely accepted after endorsement by the World Health Organization. This endorsement was only for control of oral anticoagulant therapy, as ISI calibration was specifically developed for comparing samples of patients with normal coagulation profiles to those who were anticoagulated with Coumadin (warfarin; Bristol-Myers-Squibb, New York, NY) for at least 6 weeks.1 Historically, plasma transfusions have been recommended for patients whose PT was found to be 1.5× longer than normal. Early thromboplastins in the United States had an ISI >2.0; therefore, a 1.5× increase in PT would equal an INR of 2.25. Currently, many thromboplastin reagents have an ISI close to 1.0 (World Health Organization standard); therefore, the same sample with a PT 1.5× longer than normal will equal an INR of 1.5. In the past, a PT 1.5× longer than normal indicated a moderate to severe factor deficiency (factor levels <30%), whereas today, the same value indicates only a mild factor deficiency (factor levels >40%).2 Figure 1.: The cascade model of fibrin formation. This model divides the coagulation system into separate redundant pathways (extrinsic and intrinsic) either of which can result in generation of FXa. The common pathway results in generation of thrombin and subsequent cleavage of fibrinogen to fibrin. Many of the enzymes and enzymatic complexes require Ca2+ and binding to active membrane surfaces (PL) for full activity. For simplicity, feedback activation of procofactors to cofactors and the many inhibitors of the various enzymes have been omitted. Reprinted with permission from Smith.4 Ca2+ indicates calcium; Fb, fibrin; Fg, fibrinogen; FXa, activated form of factor X; HMWK, high molecular weight kininogen; K, kallikrein; PK, prekallikrein; PL, active membrane surfaces; TF, tissue factor.

Simplified Method for Local Correction of System International Normalized Ratio.

International normalized ratio (INR) derivation is dependent on the international sensitivity index (ISI) of thromboplastin. It varies with instrument and reagents used. To evaluate the role of a correction factor in the derivation of INR. We studied prothrombin time (PT) and INR from patients using 3 thromboplastins of varying ISI values. The correction factor was applied to the observed INR to obtain a corrected INR. The difference between corrected INR and observed INR values varies from -0.8 through 0.96. Corrected INR is dependent on PT only. It can be applied to all patients irrespective of cause for elevated PT.

Long-term stability of international standards for thromboplastin stored at -20 °C, -70 °C, and -150 °C

BackgroundLong-term stability is an essential requirement for all international biological standards. The main stocks of the current international standards for thromboplastin, i.e. RBT/05 (rabbit brain thromboplastin) and rTF/09 (recombinant human tissue factor), are stored at -20°C. The aim of the present study is to assess the long-term stability of the international sensitivity index (ISI) for RBT/05 and rTF/09. MethodsPart of the main stocks of RBT/05 and rTF/09 were stored at -70°C and -150°C, up to 38months. At various time points samples were taken from the materials stored at -20°C, -70°C, and -150°C. The samples were reconstituted and analysed in the prothrombin time (PT) test using plasma samples derived from healthy subjects and patients treated with vitamin K-antagonists (VKA). The PT’s obtained with the standards stored at -20°C were compared to the PT’s obtained with the standards stored at -70°C and at -150°C. The PT’s were used to calculate relative ISI values by means of orthogonal regression. ResultsThere were no important differences between the ISI values for the materials stored at -20°C, -70°C, and -150°C. There was no significant trend with storage time. ConclusionThe ISI values for the international standards RBT/05 and rTF/09 appear to be stable at storage temperatures of -20°C, -70°C, and -150°C.

Significance of Local International Sensitivity Index Systems for Monitoring Warfarin and Liver function

Use of a local calibrator has been recommended for standardization of the international normalized ratio (INR) and international sensitivity index (ISI). We investigated the performance of two commercial local calibrators for warfarin monitoring and determined the significance of liver-specific INR. ISI values were determined using the World Health Organization (WHO) method and two commercial local calibrators. Liver-specific ISI was determined using plasma samples from patients with liver cirrhosis and normal controls. In warfarin monitoring, the two local ISIs determined by the two local calibrators showed better consistency than uncorrected ISI, although they were inferior to the ISIs calibrated using the WHO method. Alternative calibration using calibration plasma from patients with liver cirrhosis instead of warfarinized plasma reduced the INR variability. Local ISI determined by a commercial local calibrator improved INR standardization among thromboplastins. The alternative ISI calibration using liver-specific calibration plasma is expected to reduce INR variability for the evaluation of liver function.

Local Verification and Assignment of Mean Normal Prothrombin Time and International Sensitivity Index Values across Various Instruments: Recent Experience and Outcome from North America

Warfarin dosing relies on accurate measurements of international normalized ratio (INR), which is calculated from the prothrombin time (PT), International Sensitivity Index international sensitivity index (ISI) of the thromboplastin, and the geometric mean of normal PT (MNPT). However, ISI assignments of certain reagent/instrument combinations are frequently unavailable, especially when the reagent and instrument are not from the same manufacturer. The effort to be in compliance with widely endorsed Clinical and Laboratory Standards Institute (CLSI) guidelines by locally verifying or assigning an ISI to an unsupported reagent/instrument combination is further hindered by the lack of US Food and Drug Administration (FDA)-approved certified plasmas designated for a particular reagent/instrument combination. The objectives of the study include development of a process to verify/assign ISI and MNPT of a single thromboplastin reagent from one manufacturer across multiple instruments including several from another manufacturer and across several campuses of a single institution, the Mayo Clinic. In this study, RecombiPlasTin 2G (R2G), was evaluated on the ACL TOP 700 (IL), STA-R Evolution, STA Compact, and STA Satellite. Random normal donor samples (n = 25) were used to verify/assign MNPT. A subset of the normal donors (n = 8) and 13 warfarin pools (INR range: 1.3-3.9), created from stable warfarin patient plasma, were used for ISI verification/assignment. The manufacturer's assigned ISI was first verified on the ACL TOP 700 (reference method), then assigned on three unsupported instruments using orthogonal regression analysis. The MNPT and manufacturer assigned ISI (11.0, 0.95) were verified on the ACL TOP 700 and subsequently assigned on the STA-R Evolution (11.6, 1.04); STA Compact (11.5, 1.02); and STA Satellite (10.9, 0.99). Linear correlations of the INR results from all the four instruments demonstrated an r2 > 0.99. This process provides a reproducible approach to assigning ISIs on unsupported reagent/instrument combinations. Our data also confirm that ISIs of the same PT reagent differ significantly on different instruments, thus confirming the requirement for evaluations and validation of ISIs for different reagent/instrument combinations.

Improving the Inter-Laboratory Harmonization of the International Normalized Ratio (INR): Utilizing the Concept of Transference to Estimate and/or Validate International Sensitivity Index (ISI) and Mean Normal Prothrombin Time (MNPT) Values and/or to Eliminate Measurement Bias

The Prothrombin Time (PT) assay is clinically the most often requested coagulation test, as used primarily for monitoring of Vitamin K antagonist therapy where results are typically expressed as an International Normalized Ratio (INR). The INR reflects the patient's PT adjusted for the specific test reagent and instrument combination used by applying two correction factors, namely the International Sensitivity Index (ISI) and the Mean Normal Prothrombin Time (MNPT), according to the formula: INR = (patient PT/MNPT)ISI. When the manufacturer provides an ISI, laboratories are encumbered to check or locally validate the assigned value. Where a manufacturer does not provide an ISI, the laboratory needs to define its own (local ISI) value. The MNPT typically has to be locally defined, based on the population being tested. The main current CLSI recommendation for defining ISI values comprises use of commercial reference ('certified') plasma calibration sets, but FDA cleared material is limited, and different results may arise using different products. The MNPT can be defined using a WHO/CLSI recommended procedure requiring 20 normal individuals or with some calibration sets. Overall, there is limited data to validate the performance of these processes in laboratory practice, and ongoing evidence from external quality assurance (proficiency testing) programs indicates continued failure in INR harmonization, suggesting that ISI and MNPT values used by laboratories (and presumably assessed using current recommended processes) continue to be inaccurate. To assess some novel approaches to the laboratory estimation and/or validation of ISI and MNPT values for use in the INR calculation, and including the process of 'transference', normally used to assess the comparability of analytical systems or to transfer reference intervals between comparable systems. We have successfully adapted these comparative procedures, including 'transference', to permit ongoing estimation and/or validation of ISI and MNPT values for use in INR calculations for a range of instrumentation, which has led to improved harmonization of INR values obtained in our pathology network. These processes do not require the use of any normal individual plasmas or calibrator sets and greatly simplifies the INR process. Evidence for validation of the processes used is provided by ongoing satisfactory performance in external quality assurance (proficiency testing).

Do You Report an Accurate International Normalized Ratio? Find Out Using Local Verification and Calibration

The current method for monitoring vitamin K antagonist (AVK) anticoagulant therapy is the international normalized ratio (INR) that provides consistency and standardization for the prothrombin time (PT) assay value. Even after the standardization of the INR, inaccuracies of this value have still been reported. To make the INR even more accurate, better local assessments of INR parameters are becoming available. These new methods use plasmas with certified INR values to locally verify and, if necessary, recalculate the international sensitivity index (ISI) for the local laboratory’s reagent and instrument system. This CE Update will discuss the concepts of local verification and calibration to better define the manufacturer’s assigned ISI value, thus reporting more accurate INR results. * INR : international normalized ratio; ISI : international sensitivity index; PT : prothrombin time; aPTT : activated partial thromboplastin time; IRP : international reference preparation; DTI : direct thrombin inhibitor; MNPT : mean normal prothrombin time; AVK : vitamin K antagonists; WHO : World Health Organization; CLSI : Clinical and Laboratory Standards Institute; CAP : College of American Pathologists * Activated partial thromboplastin time (aPTT): : The time in seconds required for a fibrin clot to form in a plasma sample after a negative surface activator and optimal amount of phospholipid and calcium ions have been added to the sample. The aPTT measures the intrinsic pathway (factors VIII, IX, XI, and XII) and the common pathway (factors X and V, and prothrombin and fibrinogen). Calibration: : Set of operations that establishes, under specified conditions, the relationship between values of quantities indicated by a measuring instrument or measuring system, or values represented by a material measure or a reference material, and the corresponding values realized by standards. Certified plasma: : Normal or abnormal plasma samples assigned a PT/INR value by a manufacturer, using a manual method based on a WHO-accepted protocol determined against an appropriate thromboplastin international reference preparation (IRP) (or manufacturer or standard reference reagent) that has been calibrated against the appropriate WHO standard in a multicenter study. Direct thrombin inhibitor (DTI): : A class of drugs (either oral or intravenous) directly inhibiting the enzyme thrombin (without the need for a cofactor). Generic ISI: : An ISI determined for a thromboplastin that is not instrument specific (ie, determined for a group of instruments using the same general method for endpoint detection, such as manual, photo-optical, or mechanical methods). International normalized ratio (INR): : The patient’s prothrombin time (PT) test result expressed as a ratio to a mean normal prothrombin time (MNPT), which has been standardized (or normalized) for the potency of the thromboplastin used in the assay. International reference preparation (IRP): : A thromboplastin with defined biological activity used to calibrate other reference preparations and secondary or manufacturer’s standards. The thromboplastin reagent is a commercially prepared, lyophilized reagent packaged in individual 1 mL vials available from the WHO. It is usually purchased for a nominal fee by commercial manufacturers of PT reagents. Its shelf life is between 3 to 8 years. International sensitivity index (ISI): : A quantitative measure, in terms of the IRP of Human Thromboplastin, of the responsiveness of a prothrombin-time system to the defect induced by oral anticoagulants. Instrument-specific ISI: : A thromboplastin ISI generated by the reagent manufacturer specific for the particular reagent/instrument combination. Mean normal prothrombin time (MNPT): : The geometric mean of the PTs of the healthy adult population. Prothrombin time (PT): : The time in seconds required for a fibrin clot to form in a plasma sample after tissue thromboplastin and an optimal amount of calcium chloride have been added to the sample. Verification: : Confirmation through the provision of objective evidence that specified requirements have been fulfilled. Vitamin K antagonists (AVK): : A class of compounds that, when taken orally, act as AVK and, depending on the dose administered, result in diminished levels of functional factors II, VII, IX, and X, creating an in vivo anticoagulant effect. World Health Organization (WHO): : A specialized agency of the United Nations acting as a coordinating authority on international health. In regard to INR, it accepts and maintains the IRP for the standardized thromboplastin reagent for ISI assignment.

Prothrombin time standardisation in canine samples with regard to inter-batch and inter-reagent variability

The objectives of this study were to evaluate the batch-to-batch and inter-reagent variability of prothrombin time (PT) measurements in canine plasma and to investigate the effect of different methods of standardisation. PT standard test (PT(ST)) and PT modified test (PT(MT); 1:20 sample dilution, fibrinogen supplementation) were measured using five different batches of each of two commercial reagents (the first a human placenta thromboplastin and the second a rabbit brain thromboplastin). In addition, inter-reagent variability was studied for PT(MT) in 86 canine samples (four standardised reagents) based on clotting times and after calculation of percentage activity, prothrombin ratio (PTR) or International Normalised Ratio (INR). PT(ST) and PT(MT) measurements showed only a low variation between different batches of the two reagents (CV≤5%). Calculation of percentage activity or PTR reduced differences between different reagents when compared to results reported as coagulation times. Calculation of INR using the International Sensitivity Index values provided by manufacturers for use in humans showed less favourable results. In conclusion, standardisation to reduce inter-batch variability seems unnecessary for the studied reagents. Possibilities for PT standardisation in order to achieve a reagent-independent result are limited.

Standardization of the INR: How Good Is Your Laboratory's INR and Can It Be Improved?

The prothrombin time (PT) assay is the most clinically ordered coagulation test and is most often used for monitoring of vitamin K antagonist (VKA) therapy (e.g., warfarin), where results are expressed as an international normalized ratio (INR). The INR is in essence the patient's PT "mathematically adjusted" to a standardized value by taking into account the peculiarities of the test system through applying two "correction factors" defined by an international sensitivity index (ISI) and mean normal prothrombin time (MNPT). Although some manufacturers provide assigned ISI values for specific PT reagents and instrumentation, it is still recommended practice that laboratories check or locally validate these ISIs, as well as estimate the MNPT based on the population being tested. Where a manufacturer does not provide an ISI, the laboratory needs to define its own (local ISI) value. Current recommendations suggest the use of commercial reference-plasma calibration sets, but there is limited information to validate their performance in laboratory practice. We report our personal experience with use of some of these materials, as well as review alternate or supplementary procedures for calibration and/or validation of ISI and for determination and validation of MNPT. In brief, our data and experience suggests that further verification checks should be performed prior to acceptance of ISI and MNPT estimates generated from commercial reference-plasma calibration sets. We detail various strategies to ensure that laboratory practices are optimized to provide INRs that accurately reflect a patient's true anticoagulant status and to thus assist their clinical therapeutic management.

Time to think outside the box? Prothrombin time, international normalised ratio, international sensitivity index, mean normal prothrombin time and measurement of uncertainty a novel approach to standardisation

The prothrombin time (PT) assay is the most clinically ordered coagulation test, and most often used for monitoring of vitamin K antagonist therapy (e.g., warfarin), where results are expressed as an international normalised ratio (INR). The INR is in essence the patient's PT 'mathematically adjusted' to a standardised value taking into account the peculiarities of the test system as defined by an ISI (international sensitivity index) and MNPT (mean normal prothrombin time). Although some manufacturers provide assigned ISI values for specific PT reagents and instrumentation, it is still recommended practice that laboratories check or validate these ISIs, as well as estimate the MNPT. Where an ISI is not provided by a manufacturer, the laboratory needs to estimate its own value. Current recommendations suggest the use of commercial reference-plasma calibration sets, but there is limited information on the performance of these in the field. We report a comparative study that assessed the utility of three such commercial calibration plasma sets, used as recommended, as well as alternate or supplementary procedures for estimation of ISI and MNPT. The latter included one novel approach using comparative data of 'existing' versus 'replacement' reagent, as well as assessment of external quality assurance data. Although MNPT value estimates were not grossly disparate, a wide variety of ISI values (e.g., 1.12-1.30 for our primary instrument) was obtained with the different plasma sets. Because of the above, further verification checks are required prior to acceptance of ISI and MNPT estimates generated from commercial plasma calibration sets. We also provide some recommendations regarding the process of standardisation of INR testing.

How to implement the modified international normalized ratio for cirrhosis (INRliver) for model for end-stage liver disease calculation

How to implement the modified international normalized ratio for cirrhosis (INRliver) for model for end-stage liver disease calculation

Influence of Blood Collection Systems on the Prothrombin Time and International Sensitivity Index Determined With Human and Rabbit Thromboplastin Reagents

Three brands of blood collection tubes were studied for their influence on the prothrombin time (PT) and international sensitivity index (ISI) for 5 commercial thromboplastin reagents. With all reagents, PTs were shorter in Vacutainer (Becton Dickinson Vacutainer Systems, Plymouth, England) samples than in S-Monovette (Sarstedt, Nümbrecht, Germany) or Venosafe (Terumo Europe, Leuven, Belgium) samples. ISI values were higher with Vacutainer samples than with S-Monovette or Venosafe samples. The ISI differences between the tubes were small for Thromborel-S (2.1%; Dade Behring, Marburg, Germany) and Hepato Quick (1.1%; Diagnostica Stago, Asnières, France; Roche Diagnostics Nederland, Almere, the Netherlands) but greater for Neoplastin Plus (5.5%; Diagnostica Stago; Roche Diagnostics Nederland), Simplastin HTF (8.3%; bioMérieux, Durham, NC), and Innovin (8.8%; Dade Behring). The PT and ISI differences between the tubes could be explained mostly by the effect of magnesium ion contamination in the sodium citrate solutions. When PT ratios were transformed into international normalized ratios (INRs) using crossover ISI (ie, samples collected with one type of tube and ISI determined with another collection system for the PT reagent), the differences in mean INRs could be approximately 10%. For ISI calibration of reference thromboplastins, blood collection tubes should be used with minimal divalent metal ion contamination of the citrate solution.

Multicentre ISI assignment and calibration of the INR measuring range of a new point-of-care system designed for home monitoring of oral anticoagulation therapy

The new CoaguChek XS system is designed for use in patient selftesting. It is the successor of the current CoaguChek S system. The detection principle is based on the amperometric measurement of the thrombin activity initiated by starting the coagulation cascade using a human recombinant thromboplastin. This study was performed to assign the International SEnsitivity Index (ISI) to the new test according to the WHO guidelines for thromboplastins and plasmas used to control anticoagulant therapy, and to establish the measuring range of the new system. At four study sites a total of 90 samples of normal donors and 291 samples of warfarin-, phenprocoumon- or acenocoumarol-treated patients were included in the study. The ISI value of the new test was assigned against the human recombinant reference thromboplastin rTF/95 at each site using the samples from stabilized patients in the International Normalized Ratio (INR) range between 1.5 and 4.5 only. The new point-of-care system's measuring range between 0.8 and 8 INR was calibrated against the mean INR of rTF/95 and AD149 using polynomial regression. ISIs were (CV of the slope): Site 1: ISI 0.99 (1.1%); Site 2: ISI 1.02 (2.0%); Site 3: ISI 1.03 (1.1%); Site 4: ISI 1.00 (1.4%). All regression lines calculated from patient-only data pass through the normal donor data points. All CVs of the slopes of the orthogonal regression lines are well below 3%, thus fulfilling the requirements of the WHO guidelines. The mean ISI for the new CoaguChek XS PT Test is 1.01.

International collaborative study for the calibration of a proposed international standard for thromboplastin, rabbit, plain

A preparation of rabbit brain thromboplastin, provisionally coded 04/162, is proposed as a candidate for the World Health Organization (WHO) International Standard (IS) for thromboplastin (rabbit, plain), meant to replace the IS coded RBT/90 (rabbit, plain), stocks of which are now exhausted. The preparation was calibrated in an international collaborative study involving 21 laboratories from 13 countries and the calibration was performed against the existing WHO-IS (i.e. rTF/95 and OBT/79) and other Certified Reference Materials from the Institute for Reference Materials and Measurements of the European Commission (i.e. CRM149 S) and from the European Action on Anticoagulation (i.e. EUTHR-01). An additional candidate rabbit brain thromboplastin coded as 04/106 was also included in the study. On the basis of predefined criteria (the within- and between-laboratory precision of the calibration and the conformity to the calibration model), 04/162 was the preferred candidate. The assigned International Sensitivity Index value was 1.15 and the inter-laboratory SD and coefficient of variation were 0.057% and 4.9%, respectively.

Phospholipid composition controls thromboplastin sensitivity to individual clotting factors.

Tissue factor is the active ingredient in thromboplastin reagents used to perform prothrombin time (PT) clotting tests to monitor oral anticoagulant therapy and to screen for clotting factor deficiencies. Thromboplastins are complex mixtures prepared from extracts of brain or placenta, although newer thromboplastins contain recombinant tissue factor incorporated into phospholipid vesicles. Thromboplastins can vary widely in their sensitivity to reductions in the levels of vitamin K-dependent clotting factors. A system to compensate for this, the International Sensitivity Index (ISI) and International Normalized Ratio (INR), has revolutionized the monitoring of oral anticoagulant therapy. The INR system is also sometimes used to monitor coagulopathies in patients with sepsis or liver failure, applications for which it was not originally designed and for which it has not been rigorously validated. To better understand thromboplastin performance, we systematically investigated which properties of recombinant thromboplastins influence their sensitivities to changes in the levels of specific clotting factors. We now report that relative sensitivities to changes in the plasma levels of factors V, VII, X (FV, FVII, FX) and prothrombin are differentially influenced by a recombinant thromboplastin's content of phospholipid and sodium chloride. Furthermore, thromboplastins of similar ISI values may exhibit quite different sensitivities to each of these clotting factors. Differing sensitivities of thromboplastin reagents to individual clotting factor levels have implications for monitoring of oral anticoagulant therapy and interpreting results of the PT assay.

Fluorogenic Assays for Functional Factor VIIa and Tissue Factor.

Blood coagulation is initiated when cryptic tissue factor (TF) becomes exposed on the surface of vascular cells where it can bind circulating factor VIIa (fVIIa). The resulting fVIIa/TF enzyme complex catalyzes the activation of certain blood zymogens that propagate the coagulation event. As a key enzyme for the initiation of coagulation, circulating fVIIa levels have been viewed as an indicator of hemostatic potential and may also be an indicator for the risk of developing cardiovascular disease. The formation of the fVIIa/TF complex is also the basis of specific coagulation assays. Natural or synthetic TF is employed in the prothrombin time (PT assay) where it is used to initiate coagulation in vitro. Therefore, fVIIa and TF play important roles both in vivo and in vitro. Despite such important roles, there have been no simple methods described for measuring the active forms of either protein. This report describes the initial development of an assay for fVIIa and TF that employs the use of an Aminonaphthalenesulfonamide-based (ANSN) fluorogenic substrate. The ANSN substrate is hydrolyzed by fVIIa in a TF-dependent manner, and by titrating fVIIa into a system containing excess TF, the assay becomes sensitive to the fVIIa concentration. Inversely, by titrating TF into a system containing excess fVIIa, the assay becomes sensitive to the TF concentration. This approach has led to the development of both a rapid kinetic and an end-point assay method for each analyte providing detection ranges down to 156 and 1.56 pM respectively. The utility of these assays is displayed by the following applications. Thromboplastin reagents are generally qualified against a W.H.O. standard and are assigned a numerical value known as the International Sensitivity Index (ISI). Theoretically the ISI value should be affected by the quantity and quality of TF in each preparation. An inverse relationship between ISI value and the amount of functional TF would support this theory. To prove this relationship the TF assay was employed to examine two thromboplastin reagents with ISI values of 1.84 and 1.01. The concentration of functional TF measured by this assay was 10.8 nM and 14.6 nM respectively. Because most commercial thromboplastin reagents are prepared by adding TF on a mass basis (as opposed to a functional basis), target ISI values are often missed, leading to an excessive number of failed lots. The TF functional assay provides a method for assigning a specific activity to each TF preparation. By adding TF on a functional basis, lot-to-lot variability would be minimized and target ISI values would be easily achieved. To substantiate this claim, we have determined the functional activity of seven different lots of purified recombinant human TF. With each lot analyzed at a fixed concentration, we observed a broad range of specific activity (range: 8,250 U/sec/mg -14,500 U/sec/mg). These data support the need for a functional TF assay to be incorporated in the manufacturing process of thromboplastin reagents. Lastly, the utility of the fVIIa assay was demonstrated in an experiment using factor VII-deficient plasma samples that were spiked with known quantities of fVIIa. Plasma samples spiked with fVIIa in the range of 0.5-2nM returned mean assay values within 71–100% of the expected values. These data demonstrate the feasibility of using the fVIIa assay to monitor fVIIa levels in hemophilia patients receiving recombinant fVIIa therapy. Further studies will address the circulating concentrations of fVIIa and TF in “normal” and disease state plasma samples.