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Essentials Two candidate International Standards for thromboplastin (coded RBT/16 and rTF/16) are proposed. International Sensitivity Index (ISI) of proposed standards was assessed in a 20-centre study. The mean ISI for RBT/16 was 1.21 with a between-centre coefficient of variation of 4.6%. The mean ISI for rTF/16 was 1.11 with a between-centre coefficient of variation of 5.7%., Summary: Background The availability of International Standards for thromboplastin is essential for the calibration of routine reagents and hence the calculation of the International Normalized Ratio (INR). Stocks of the current Fourth International Standards are running low. Candidate replacement materials have been prepared. This article describes the calibration of the proposed Fifth International Standards for thromboplastin, rabbit, plain (coded RBT/16) and for thromboplastin, recombinant, human, plain (coded rTF/16). Methods An international collaborative study was carried out for the assignment of International Sensitivity Indexes (ISIs) to the candidate materials, according to the World Health Organization (WHO) guidelines for thromboplastins and plasma used to control oral anticoagulant therapy with vitamin K antagonists. Results Results were obtained from 20 laboratories. In several cases, deviations from the ISI calibration model were observed, but the average INR deviation attributabled to the model was not greater than 10%. Only valid ISI assessments were used to calculate the mean ISI for each candidate. The mean ISI for RBT/16 was 1.21 (between-laboratory coefficient of variation [CV]: 4.6%), and the mean ISI for rTF/16 was 1.11 (between-laboratory CV: 5.7%). Conclusions The between-laboratory variation of the ISI for candidate material RBT/16 was similar to that of the Fourth International Standard (RBT/05), and the between-laboratory variation of the ISI for candidate material rTF/16 was slightly higher than that of the Fourth International Standard (rTF/09). The candidate materials have been accepted by WHO as the Fifth International Standards for thromboplastin, rabbit plain, and thromboplastin, recombinant, human, plain., (© 2017 International Society on Thrombosis and Haemostasis.)

Background: Long-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., Methods: Part 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., Results: There 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., Conclusion: The 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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The performance of factor VII (FVII) assays currently used by clinical laboratories was examined in North American Specialized Coagulation Laboratory Association (NASCOLA) proficiency tests. Data from 12 surveys conducted between 2008 and 2010, involving 20 unique specimens plus four repeat-tested specimens, were analyzed. The number of laboratories per survey was 49-54 with a total of 1224 responses. Numerous reagent/instrument combinations were used. For FVII > 80 or <40 U/dL, 99.5% of results (859/863) were correctly classified by laboratories as normal/abnormal. Classification of specimens with 40-73 U/dL FVII was heterogeneous. Interlaboratory precision was better for normal specimens (coefficient of variation (CV) 10.7%) than for FVII<20 U/dL (CV 33.1%), with a mean CV of 17.2% per specimen. Intralaboratory precision for repeated specimens demonstrated no significant difference between the paired survey results (mean absolute difference 2.5-5.0 U/dL). For specimens with FVII >50 U/dL, among commonly used methods, one thromboplastin and one calibrator produced results 5-6 U/dL higher and another thromboplastin and calibrator produced results 5-6 U/dL lower than all other methods, and human thromboplastin differed from rabbit by +7.6 U/dL. Preliminary evidence suggests these differences could be due to the calibrator. For FVII <50 U/dL, differences among the commonly used reagents and calibrators were generally not significant., (© 2013 Blackwell Publishing Ltd.)

Aims: Many patients treated with vitamin K-antagonists (VKA) use point-of-care (POC) whole blood coagulometers for self-testing. The majority of patients in the Netherlands use one type of POC coagulometer, that is, the CoaguChek XS. Each new lot of test strips for the CoaguChek XS is validated by a group of collaborating thrombosis centres. We assessed the International Normalised Ratio (INR) differences between each of 51 new lots of test strips and the International Standard for thromboplastin rTF/95 or its successor rTF/09., Methods: Each year, a particular lot of CoaguChek XS test strips was used as reference lot. The reference lot was validated by comparison to the International Standard, yielding a relationship between the reference lot INR and International Standard INR. Successive lots of test strips were compared to the reference lot by three centres using 19-29 capillary blood samples obtained from VKA-treated patients. Each patient provided two blood drops from the same finger prick, one for the reference lot strip and one for the new lot., Results: The mean INR differences between each lot and the International Standard varied between -8% and +4%. The mean absolute values of the relative differences varied between 2.4% and 8.1%. There were small but clinically unimportant differences in INR between the first and second drop of blood., Conclusions: Accuracy of CoaguChek XS INR determinations can be assessed by a group of collaborating centres using a limited number of capillary blood samples. As the mean INR differences with the International Standard were smaller than 10%, the lots were approved for use by the Netherlands Thrombosis Services.

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., (2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: International Normalized Ratio (INR), which standardizes prothrombin time (PT) during oral anticoagulation, has been extended to standardize PT in liver diseases and is included in prognostic models such as the Model for End stage Liver Disease (MELD). However, mechanisms of PT prolongation in liver diseases differ from those involved in oral anticoagulation, and the thromboplastin reagents differ in their sensitivities to these 2 mechanisms. Our aim was to determine whether, in the calibration model for thromboplastins proposed by the World Health Organization, the use of plasmas from patients with liver diseases instead of plasmas from patients on oral anticoagulation could lead to a new INR specific for liver diseases (INR "LD"), achieving a real standardization of PT. First, 5 thromboplastins were calibrated against an international reference using 60 plasmas of patients with liver failure and, in a second step, the variation of PT reported as seconds, the ratio of patient PT to normal PT, INR, and INR"LD" was assessed in 34 other patients. MELD scores were calculated with the INR values obtained with the 5 thromboplastins. Only INR"LD" eliminated variability in PT results observed with the different thromboplastins. The discrepancy between MELD scores were up to 4 and 7 points in 52% and 17% of the patients, respectively., Conclusion: INR "LD" may provide a common international scale of PT reporting in hepatology. Its adoption would be an important step because of the significant impact on MELD score induced by interlaboratory variability in INR determination.

Background: 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., Results: 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., Conclusions: 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.

A 10 centre calibration was performed after six years to determine the international sensitivity index (ISI) of rTF/95 relative to RBT/90, and to assess any international normalised ratio (INR) bias compared with the original multicentre calibration. After exclusion of one outlying centre, the follow up calibration gave a mean ISI for rTF/95 of 0.99, which although a small difference, is significantly greater than the mean ISI of 0.94 obtained previously. The change in ISI for international reference preparation (IRP) rTF/95 relative to RBT/90 would lead to a slight bias in INR for human compared with rabbit thromboplastins. At a theoretical INR of 3.0, the INR bias is 6.0%, and this is below the accepted 10% level of clinical relevance. Ongoing stability monitoring of World Health Organisation thromboplastin IRP is advised.

The logical solution to account for the influence of coagulometers on the International Sensitivity Index (ISI) is local calibration with freeze-dried plasmas. However, because of their unpredictable behavior these plasmas must be validated before large-scale implementation. We report on a collaborative exercise designed to evaluate the suitability of a set of such plasmas used with Thrombotest in combination with a coagulometer provided by the manufacturer to be used with that reagent. This was a two-step study. First, one lot of reagent was calibrated against the international standard OBT/79 in two expert laboratories. The calibrated lot was then used as an intermediate standard to calibrate two additional lots of the same reagent in four field laboratories where the ISI was determined for both plasma and native blood. The International Normalized Ratio (INR) for the patient plasmas tested in each laboratory were calculated using two algorithms: the World Health Organization-recommended ISI mode (gold standard), and the simplified calibration plasma mode. In the latter, the INR was derived from the local calibration curve constructed by plotting the certified INR versus local coagulation times obtained with calibration plasmas. The between-algorithm INR differences indicate that this set of calibration plasmas may be employed for local INR calibration of the investigated reagent/instrument combination, especially when plasma is used for INR determination where the average INR (range) difference is 5% (3-13%) or 2% (3-8%) according to whether the INRs to calibration plasmas were assigned by the manufacturer or by the two expert laboratories. A slight but measurable difference of the INR may be predicted [9% (6-20%) or 6% (8-15%)] if this set of calibration plasmas is used for local calibration when native blood is employed for INR determination. Whether this bias is of practical significance is to be determined.

Background: Prothrombin time (PT) has long been the most popular test for monitoring oral anticoagulation therapy. The International Normalized Ratio (INR) was introduced to overcome the problem of marked variation in PT results among laboratories and the various recommendations for patient care. According to this principle, all reagents should be calibrated to give identical results and the same patient care globally. This is necessary for monitoring of single patients and for application of the results of anticoagulation trials and guidelines to clinical practice., Methods: We took blood samples from 150 patients for whom oral anticoagulation had been prescribed. Plasmas were separated and PTs determined by use of seven commercial reagents and four calibrator sets. The differences in results were assessed by plotting, for each possible pair of methods, the differences in INR values for each sample against the mean INR value (Bland-Altman difference plots)., Results: Mean results differed significantly (P <0.001) for 17 of 21 possible paired comparisons of methods. Only two pairs of methods produced very similar results when assessed for problems of substantial differences in INR values; a significant, systematic increase in the difference with INR; and a significant systematic increase in the variation in difference with increasing INR values., Conclusions: The agreement among several (and perhaps most) commercial INR methods is poor. The failure of current calibration strategies may severely compromise both the monitoring of individual patients and the application of oral anticoagulation guidelines and trial results to clinical practice.

Unlabelled: The purpose of the present study was to assess the effect of two types of evacuated blood collection tube on the prothrombin time and international sensitivity index (ISI) of Recombiplastin, a recombinant human thromboplastin. Vacutainer tubes were compared with Venoject II tubes. Magnesium contamination was detected in the sodium citrate solutions contained in the Vacutainer tubes with concentrations ranging from 1.1 to 1.5 mmol/l. In contrast, magnesium ions could not be detected in the Venoject II tubes. The prothrombin ratio was decreased by contamination with magnesium ions and, hence, the ISI was increased. The magnitude of the effect of magnesium contamination on the ISI was influenced by the type of coagulometer and increased in the order: ACL Advance (3%), ACL-300 (4%), Electra-1000 (6%). The ISI bias is transmitted to the international normalized ratio (INR). In the case of the Electra-1000, the INR bias would be approximately 6% at INR 3.0 if the two types of blood collection tubes would be used without distinction. In a secondary study, the effect of magnesium contamination on the prothrombin time was assessed with the current World Health Organization international reference preparation for recombinant human thromboplastin (rTF/95). Magnesium chloride added to patients' blood (0.2 mmol/l) induced 2.3% reduction of the INR determined with rTF/95 and the manual technique., Conclusion: The magnitude of the influence of blood collection tubes contaminated with magnesium on ISI and INR determined with recombinant human thromboplastin depends on the coagulometer.

Reliable international normalized ratio (INR) determination depends on accurate values for international sensitivity index (ISI) and mean normal prothrombin time (MNPT). Local ISI calibration can be performed to obtain reliable INR. Alternatively, the laboratory may determine INR directly from a line relating local log(prothrombin time [PT]) to log(INR). This can be done by means of lyophilized or frozen plasmas to which certified values of PT or INR have been assigned. Currently there is one procedure for local calibration with certified plasmas which is a modification of the WHO method of ISI determination. In the other procedure, named 'direct' INR determination, certified plasmas are used to calculate a line relating log(PT) to log(INR). The number of certified plasmas for each procedure depends on the method of preparation and type of plasma. Lyophilization of plasma may induce variable effects on the INR, the magnitude of which depends on the type of thromboplastin used. Consequently, the manufacturer or supplier of certified plasmas must assign the values for different (reference) thromboplastins and validate the procedure for reliable ISI calibration or 'direct' INR determination. Certification of plasmas should be performed by at least three laboratories. Multiple values should be assigned if the differences between thromboplastin systems are greater than 10%. Testing of certified plasmas for ISI calibration may be performed in quadruplicate in the same working session. It is recommended to repeat the measurements on three sessions or days to control day-to-day variation. Testing of certified plasmas for 'direct' INR determination should be performed in at least three sessions or days. Correlation lines for ISI calibration and for 'direct' INR determination should be calculated by means of orthogonal regression. Quality assessment of the INR with certified plasmas should be performed regularly and should be repeated whenever there is a change in reagent batch or in instrument. Discrepant results obtained by users of certified plasmas should be reported to manufacturers or suppliers.

RBT 1010, a rabbit brain thromboplastin, plain, was prepared at the Thrombosis Reference Centre, Withington Hospital, Manchester, in 1989. The batch has been stored at -20 degrees C, in rubber-stoppered ampoules, for 13 years. The material was unchanged after this time. This was confirmed in a stability study, in which the reagent was used to test the prothrombin times of a panel of plasmas stored in liquid nitrogen, one from a normal volunteer and two from stable anticoagulated patients. RBT 1010 was also tested in calibrations, according to World Health Organization recommendations, against the reference thromboplastin preparations CRM 149S and RBT 90.

The international sensitivity index (ISI) of the first working standard of Simplastin HTF, a new human tissue factor thromboplastin derived from cultured human cells, has been assessed in a calibration exercise in two Canadian and five European laboratories. Calibrations against international reference preparations (IRP) were performed for the manual method and six types of automated coagulometers that cover the majority of clotting endpoint principles in routine use. The ISI was method-dependent and varied between 1.03 and 1.29 when calibrated against rTF/95 (human IRP). The ISI was also dependent on the route of calibration. Compared with calibration against rTF/95, the ISIs obtained by calibration against RBT/90 (rabbit IRP) were on average 4.4% higher (P < 0.005). Considering the principle of 'like vs. like', the ISIs obtained by calibration against rTF/95 should be preferred.

Background: For monitoring of treatment with oral anticoagulants, the clotting time obtained in the prothrombin time (PT) test is transformed to the International Normalized Ratio (INR) with use of a system-specific International Sensitivity Index (ISI). The calibrant plasma procedure (CPP) is an alternative approach to INR calculation based on the use of a set of lyophilized plasmas with assigned INRs., Methods: With the CPP, a linear relationship is established between log(PT) and log(INR), using orthogonal regression. CPP was validated for Simplastin HTF, a new human tissue factor reagent derived from cultured human cells. CPP precision was assessed as the CV of the slope of the regression line. The accuracy of the CPP was determined by comparing the INR obtained with the CPP with that obtained with the established ISI-based reference method. INRs of the calibrants were assigned by different routes: by manufacturer (consensus labeling) or by use of Simplastin HTF or International Reference Preparations (IRPs; rTF/95 or RBT/90)., Results: The mean CV of the CPP regression slope ranged from 1.0% (Simplastin HTF reagent-specific INR) to 2.4% (INR assigned with rTF/95). INRs calculated with the CPP were similar to those obtained with the reference method, but when the routes for assigning INRs to the calibrant plasmas were compared, the mean difference in INR between CPP and the reference method was smaller with Simplastin HTF reagent-specific values. In several (but not all) cases, this difference was significant (P <0.05, t-test)., Conclusion: CPP can be used for local INR determination, but better precision and accuracy are obtained with reagent-specific INRs compared with INR assignment by consensus labeling or IRP.

To improve the standardization of the factor VII clotting activity (FVII:C) assay in patients treated with recombinant activated factor VII (rFVIIa), we conducted a multicentre study on plasma samples from four patients with haemophilia A, before and at various times after injection of a single dose of rFVIIa. FVII:C and prothrombin time were measured with the methods and reagents routinely used in each laboratory. Strong inter-laboratory variability of FVII:C values was found. The main source of variability was the type of thromboplastin. FVII:C values measured using rabbit thromboplastin were very close to activated factor VII clotting activity values (FVIIa:C) measured with a commercial assay (Staclot VIIa-TF). FVII:C values obtained with human placental thromboplastin were about three times lower than those obtained with rabbit and recombinant thromboplastins, and with the FVIIa:C assay. There was a good relationship between FVIIa:C and activated factor VII antigen values measured using a commercial immunoassay (Imubind FVIIa ELISA). In conclusion, rFVIIa at pharmacological concentrations can be easily monitored on the basis of FVII:C, using rabbit and probably also recombinant thromboplastin; equivalent results are obtained with a specific activated factor VII bioassay.

International sensitivity index (ISI) calibration of whole blood prothrombin time (PT) monitors is too complex. We previously simplified the method by using European Concerted Action on Anticoagulation (ECAA) lyophilized plasma samples with the TAS PT-NC (Bayer AG, Leverkusen, Germany) and the CoaguChek Mini (Roche Diagnostics, Mannheim, Germany) whole blood PT monitoring systems. The TAS PT-NC required a correction derived from the line of equivalence. Monte Carlo bootstrap analysis of reducing numbers of test samples was performed with both systems. Plasma samples from patients receiving coumarin (coumarin samples), healthy subjects (normal samples), and plasma samples artificially depleted of coagulation factors were used. With the TAS PT-NC, 20 coumarin samples or 20 artificially depleted samples with 7 normal samples gave reliable ISI and international normalized ratio and satisfactory precision. With the CoaguChek Mini, 30 coumarin and 10 normal samples were required. Simplification of ISI calibration of the 2 monitoring systems is possible using fewer ECAA lyophilized plasma samples than the 80 required according to the World Health Organization guidelines for conventional PT systems and previously recommended for fresh plasma samples tested on the same 2 monitoring systems.

Magnesium ions can shorten the tissue factor-induced coagulation time. Some blood collection systems with sodium citrate are contaminated with variable amounts of magnesium and influence the results of the prothrombin time (PT) test. The aim of the study was to determine the dose-response relationship between exogenous magnesium chloride added to blood and the PT and the international normalized ratio (INR). Blood specimens from twenty patients on oral anticoagulant therapy were investigated. Four different types of thromboplastin reagents were used: recombinant human, human placenta, rabbit brain, and bovine brain combined with adsorbed bovine plasma. With all four reagents, exogenous magnesium induced a reduction of the apparent INR. Bovine thromboplastin was not as responsive to magnesium as the human and rabbit reagents. The magnitude of the INR deviation induced by 0.1 mmol/l magnesium in the blood was smaller than 10% in all patient samples. At 0.5 mmol/l magnesium in the blood, 10-35% of the patient samples had INR deviations greater than 10%, depending on the thromboplastin reagent used.

Background and Objectives: We tested the principle of local International Normalized Ratio (INR) calibration using INR calibrator plasmas (PT Calibration Plasma Kit, Behring), two thomboplastin reagents (Neoplastin plus, rabbit brain, Stago, and Recombiplastin, recombinant human tissue factor, Ortho Diagnostics) and the same coagulometer (STA, Stago) on 92 patients on stable oral anticoagulant treatment., Design and Methods: A four-point calibration was obtained with each reagent by linear regression (sec/INR) on a log-log scale (r > or = 0.999). The bias between the two reagents (Recombiplastin - Neoplastin Plus) was reduced from 31.7% to 17.5% and 7.5% (p=0.001) when results were expressed, respectively, as PT ratio (using the mean normal prothrombin time as denominator term), INR (using instrument-specific ISI supplied by the manufacturers) and calibrated INR, but there was a consistently significant regression of the differences over the average values even after log transformation (r > or = 0.586). The bias between the reagents was reduced to 1% (p=ns) when assuming Recombiplastin as the reference thromboplastin and applying Tomenson's correction, but limits of agreements were as large as 20%. Factor VII, X, V and II activity was measured with the two thromboplastin reagents in all plasma samples using immunodepleted plasmas (Stago)., Results: Statistically significant biases were observed for all clotting factors with the two reagents (Recombiplastin Neoplastin Plus) and ranged from 3.5 % (FII) to 37.2% (FVII). In addition, for FVII and FV there was a significant regression of the difference over the average value (after log-transformation, r > or = 0.282). The patients were divided into 3 groups according to their degree of anticoagulation (INR <2.0; INR between 2.0 and 3.5; INR >3.5). Factor levels differed significantly with the two reagents throughout the 3 groups of patients. In addition, the relative distributions of the 3 vitamin K-dependent factors also differed in the 3 groups with the two thromboplastin reagents., Interpretation and Conclusions: The discrepant sensitivity to factor VII, X and V levels of the two thromboplastin reagents explored in this study prevents INR calibration with commercially available calibrator plasmas and is responsible for a significant variability in INR values even under optimal conditions of INR calibration.

Background: The International Sensitivity Index (ISI) calibration of whole-blood prothrombin time (PT) monitors for point-of-care testing (POCT) described by Tripodi et al. (Thromb Haemost 1993;70:921-4) has been shown to be dependable but is too complex and demanding. The use of plasma would simplify calibration of whole-blood POCT PT monitors, but important differences may exist between the ISI for whole blood and plasma calibrations., Methods: In a 10-center calibration study of two POCT whole-blood monitoring systems (CoaguChek Mini and TAS PT-NC), we characterized the relationship between the log PT for whole blood and fresh plasma with use of single lots of test strips/cards. This relationship (linear) was used to correct the difference between the whole-blood and plasma ISI. The reliability of the correction with different lots of test strips/cards was assessed at three centers. The linear relationship was used to correct the difference in the whole-blood and plasma ISI with four other lots of TAS PT-NC cards and with two additional lots of CoaguChek Mini test strips., Results: The correction decreased the ISI difference from 13.3% to 0.9% for the TAS PT-NC and from 5.7% to 0.6% for the CoaguChek Mini. In assessments at three centers, which included different lots of test strips/cards, the mean ISI difference was markedly decreased with the TAS PT-NC but not with the CoaguChek Mini, for which the mean ISI difference increased slightly., Conclusions: The proposed correction resolves the discrepancy between whole-blood and fresh plasma ISI calibrations with TAS PT-NC test cards. The CoaguChek Mini systems could be calibrated without this correction.

Two established international reference preparations (IRP) for thromboplastins, i.e. RBT/90 (rabbit, plain) and rTF/95 (recombinant human, plain) have been calibrated against each other in a 7-centre exercise performed in 2000. The purpose of the study was to compare the calibration results with those of the original calibration study performed in 1995. The international sensitivity index (ISI) for rTF/95 was calculated relative to the ISI for RBT/90. The mean ISI for rTF/95 was 0.961 (between-lab SD 0.028) which was 2% greater than the historical value determined in 1995. There is no indication that the two IRP have deteriorated, but further monitoring of the calibration relationship is recommended.

Clinical evaluation of thromboplastins manufactured by RENAM (Moscow) with international index of sensitivity (IIS) 1.1-1.5 and comparison of the results with the data obtained in the same patients with foreign thromboplastins (Dade, USA; Behring, Germany; Diamed, Switzerland) and Mediolab thromboplastins (Russia) manufactured from different raw materials (rabbit brain and human placenta) showed that by sensitivity to indirect anticoagulants, Russian thromboplastins made by RENAM and Mediolab firms are not inferior to the best foreign analogs.

The international sensitivity index (ISI) calibration of point-of-care-test (POCT) prothrombin time (PT) whole blood monitors is complex, requiring manual PT testing of 60 patients' and 20 healthy subjects' plasma samples. The possibility of reducing these numbers was studied by a Monte Carlo Bootstrap study for 2 POCT PT systems. For reduced sample numbers, this consisted of 50,000 calibrations using whole blood and plasma samples tested on the monitors with manual PT testing of plasma samples from the same blood donations. There was little effect on mean ISI by reduction of sample numbers to a total of 7, but there was progressively less certainty regarding the reliability of the calibration. Precision of the calibrations and international normalized ratio deviation were not affected markedly by reducing numbers to half As ISI calibration with the 2 POCT systems was less precise than conventional manual testing, for maximum confidence, reduction of numbers is not advised.

A procedure for using citrated fresh plasmas for International Sensitivity Index (ISI) calibration of two types of whole blood point-of-care test (POCT) prothrombin time (PT) monitor systems has been assessed in a multicentre study. The CoaguChek Mini and TAS PT-NC systems gave higher ISI with whole blood samples than with fresh plasma calibrations. However. there was good agreement between whole blood and fresh plasma monitor system International Normalised Ratio (INR) and the reference INR of target samples. Reliable INR can therefore be obtained with both whole blood and plasma samples on these two POCT systems based on their respective ISI. With the CoaguChek Mini system, the plasma calibration ISI can also be used to derive reliable INR with whole blood PT results. This was not possible with the TAS PT-NC system.

Prothrombin time (PT) is the control test for oral anticoagulant therapy as well as the screening test for defects of the extrinsic pathway of coagulation. Its responsiveness to decreased extrinsic clotting factors depends on the source and type of tissue factor thromboplastin extract. In 1994, a rabbit brain thromboplastin - Thromboplastin Bilbao (TBi) - was introduced as a replacement for a human brain preparation used since 1983, with the aim of establishing a national standard. The purpose of this study was to check the reproducibility, the inter-assay/intra-assay accuracy and the stability of this reagent under temperature changes and over time. A method modified from Frei et al. [World Health Organisation Regional Publications, Eastern Mediterranean Series, Alexandria, 1995] was used for the preparation of thromboplastin extract. Thirty-five batches of human TBi were prepared from 1983 to 1988, while from 1993 to 1999 13 batches of rabbit TBi were produced. The inter-assay reproducibility of rabbit TBi exhibited a coefficient of variation (CV) of 1.07-1.57% for normal plasma and of 1.25-2.56% for anticoagulated plasma. The intra-assay CV was 0.06-1.30% for normal plasma and 1.23-2.66% for anticoagulated plasma. The stability of the reagent to temperature changes and time was also estimated, with similar results for the two thromboplastins. As a result of the Oral Anticoagulant Treatment Quality Assessment Scheme in the Basque Country, an in-house rabbit thromboplastin with good sensitivity and reproducibility was developed., (Copyright 2002 S. Karger AG, Basel)

Background and Objectives: A new test for screening the procoagulant capacity of plasma is described and evaluated. This test is based on the coagulation of plasma initiated by thromboplastin (Tp) in the presence of thrombomodulin (TM). In a previous paper we reported that this test had a significant phenotypic and genetic correlation with thrombosis susceptibility. The present report describes the characteristics of the test and its sensitivity to the concentration of some hemostasis factors., Design and Methods: Plasma from normal subjects, from individuals with various disorders of hemostasis and plasma with different concentrations of factors II, V, VII, VIII, X, fibrinogen, protein C and protein S were studied. The thromboplastin-thrombomodulin-mediated time (Tp-TMT) is measured after mixing 100 mL of plasma diluted 1/10 at 37 C with 100 mL of a solution composed of 2 parts of thromboplastin, 1 part of thrombomodulin at 30 U/mL and 1 part of Owren's buffer. The results are expressed as the ratio of the patient's clotting time to that of the control. Values were compared with Student's t test and the Mann-Whitney test. Differences were considered statistically significant when p<0.05., Results: In the control group women showed significantly lower values than men. Raised levels of factors II, V, VII and X reduced the coagulation time obtained with Tp-TM. Elevated concentrations of fibrinogen and factor VIII did not influence the test. The Tp-TMT was sensitive to protein S deficiencies, but not to protein C deficiencies., Interpretation and Conclusions: These results indicate that the effect of protein S on the test is through its anti-prothrombinase activity., In Conclusion: Tp-TMT, which is correlated with thrombosis susceptibility, is sensitive to raised levels of factors II, V, VII and X, as well as to low levels of protein S, and may be an indicator of thrombosis risk.

The Basque Country Quality Assessment Scheme on Oral Anticoagulant Treatment (PEEC-CAV) was first launched in 1984. This project combined prothrombin time (PT) quality controls and a new in-house standard reagent called Thromboplastin Bilbao (TBi). Originally, this reagent was of human origin, but in 1994, it was replaced by one derived from rabbit brain following WHO recommendations. Nine hospitals in the area collaborated in carrying out quality controls (a) to assess different systems of PT test performance in the Basque Country and (b) to evaluate TBi response in these controls. Evaluation over the period 1994-1999 yielded an INR coefficient of variation (CV) for the plasmas used in these controls of 8.29+/-4.18% using the laboratories' routine system (reagent and coagulometer), and 9.7+/-2.70% using TBi and the manual technique. These results were similar to those obtained in the CV of PT ratio with human brain thromboplastin from 1984 to 1988 (9.26+/-2.84%).

An alternative approach to INR estimation is for laboratories to calibrate their own local system using calibrant plasmas supplied by manufacturers or reference laboratories. The purpose of the present study was to investigate the within-laboratory variability of a calibrant plasma procedure using various sets of lyophilized plasmas. INR had been assigned to 13 calibrant plasmas in a previous multi-center study. Each of 10 other ("field") laboratories measured PTs in the 13 calibrant plasmas and in 15 local fresh coumarin plasmas, using three different thromboplastin reagents. Each fresh coumarin PT was converted to INR using a calibration procedure with a set of 4 calibrants (1 normal + 3 abnormals). The abnormals of each set were either coumarin or artificial and were used with different assigned INR. When the INR had been assigned with a thromboplastin brand identical to the thromboplastin in the field laboratory, the procedure was named "reagent-specific" calibration. Otherwise the procedure was named "dissimilar" calibration. Using "reagent-specific" calibration procedures, relatively homogeneous INR were obtained for the fresh coumarin plasmas, whatever type of calibrant was used. In contrast, discrepant INR were obtained when "dissimilar" cross-species calibration procedures were used. The study was limited to 9 laboratories using the same type of coagulometer and one using a different type.

Background: To simplify International Sensitivity Index (ISI) calibration, the possibility of substituting fresh plasma for fresh whole-blood samples with point-of-care testing (POCT) whole-blood monitors was investigated in a three-center study of three different POCT systems., Methods: A modified full WHO calibration procedure based on 20 healthy controls and 60 coumarin-treated patients was performed on three monitoring systems with whole-blood and plasma samples against plasma tested using the European Concerted Action on Anticoagulation (ECAA) rabbit reference plain thromboplastin and the manual prothrombin time (PT) method., Results: With one of the three systems, the mean ISI was 1.51 for whole blood and 1.49 for plasma; with the second system, the mean ISI was 1.08 for both whole blood and plasma. With the third system, however, the difference between the mean ISI for whole blood and that for plasma was greater (1.15 and 1.01, respectively). Overall, the precision of the calibrations was less than with traditional manual plasma PT testing., Conclusions: Provided that an appropriate calcium chloride concentration is used, the plasma PT results can be used for accurate ISI calibration of two of these three whole-blood POCT systems. Precision criteria need to be modified for POCT monitors.

Recombinant factor VIII and factor IX concentrates, human-plasma-derived albumin, and samples from previously untreated patients with hemophilia were examined for the presence of TT virus (TTV) by using polymerase chain reaction testing. Blood samples from the patients were obtained prospectively before and every 3 to 6 months after therapy was begun. TTV was detected in 23.5% of the recombinant-product lots and 55.5% of the albumin lots tested. Only first-generation factor VIII recombinant concentrates stabilized with human albumin were positive for TTV, whereas all second-generation (human protein-free) concentrates were negative for the virus. In 59% of patients treated with either first- or second-generation recombinant factor concentrates, TTV infection developed at some point after the initial infusion. Infection with TTV in these patients before and after treatment did not appear to be clinically important. Thus, first-generation recombinant factor VIII concentrates may contain TTV and the source of the viral contamination may be human albumin.

Prothrombin time (PT) assays are clotting methods that measure the activity of vitamin K-dependent coagulation factors (F) II, VII, and X. There are three main types of PT assays in general usage, namely the Quick assay, Owren's assay and PT dry chemistry test cards. PT assays were initially developed to monitor dose-adjustments of vitamin K antagonists such as warfarin. The aim of the present study was to investigate whether commercially available PT assays are suitable for evaluating the anticoagulant activity of direct thrombin inhibitors. Melagatran, a reversible direct thrombin inhibitor, was added to human plasma at concentrations ranging from 0.1 to 2.0 micromol/l. Seventeen different commercially available PT kits were used, including thirteen Quick reagents, two Owren reagents and two PT test cards. The sensitivity of the different reagents, expressed as the concentration of melagatran that doubled the prothrombin time (IC50) varied widely, with Thromboplastin S and Thromboplastin HS being the most sensitive (IC50 = 0.9 micromol/l). The reagents with apparently the lowest sensitivity were the two Owren reagents Nycotest PT and SPA 50 with an IC50 of 2.2 and 2.9 micromol/L, respectively. This is most likely due to a higher dilution of melagatran in these assays compared to the dilution in the Quick assays. The results were also dependent on the International Sensitivity Index (ISI) of each reagent. The concentration of melagatran that produced an International Normalized Ratio (INR) of 2 was calculated from dose-response curves for each assay, and these results revealed that reagents with a high ISI value gave an INR of 2 at much lower concentrations of melagatran (0.5-0.7 micromol/L) than those with an ISI-values around one (0.9-1.2 micromol/L). It was found that INR depends not only on the plasma concentration of melagatran, but also on the sensitivity of the PT reagent and on the final dilution of the plasma sample in the prothrombin time assay. Thus, since the same melagatran concentration can be associated with widely varying PT/INR results depending on the specific assay used it is concluded that PT assays and INR can not be used to monitor melagatran activity.

Multiple studies have shown that the two different citrate concentrations in common use as the anticoagulant in blood collection for haemostasis assays can affect the results obtained with the prothrombin time assay. It is clear from the literature that there is considerable variability in the results obtained using different instrument-reagents combinations, but the clinical relevance of these differences is unclear. Most of the studies have used an optical system for end-point detection. This study reports on the citrate sensitivity using mechanical end-point detection. Using two different reagents, one previously shown to be citrate sensitive on optical systems (Neoplastin CI plus) and a citrate-insensitive reagent (Neoplastin CI), we demonstrate that the effect of using different citrate concentrations (0.105 or 0.129 mol/l) has statistically significant but clinically irrelevant effects on the International Normalized Ratio using a mechanical instrument (STA)-reagent combination (mean percentage difference in results, 1.9 and 3.8% respectively). This demonstrates that the citrate effect is both instrument type and reagent dependent. Every reagent and instrument combination needs to be tested to see whether any citrate effect exists. In a secondary study, it was shown that the international reference rabbit thromboplastin (CRM 149(s)) was not citrate-concentration sensitive.

Comparative analysis of foreign and Russian thromboplastins demonstrated differences in the notions "activity" and "sensitivity" and the significance of low value of International Index of Sensitivity (IIS). Sensitivity of thromboplastin manufactured by RENAM Firm, Russia (IIS = 1.5) to deficiency of prothrombin factors II, VII, X has been demonstrated, which means that this thromboplastin can be used for diagnosis of deficiency of each of these factors. The authors consider that testing of thromboplastin sensitivity to factor VII is the main criterion of its fitness for monitoring anticoagulant therapy.

We report two cases of high-titre IgM and moderate-titre IgA anticardiolipin antibodies (ACA) in whom prothrombin times (PT) derived using recombinant thromboplastins (rTP) were prolonged but were normal when measured with conventional thromboplastins. The anticoagulant nature of these antibodies cannot be categorized as the classical lupus type. We suggest that routine screening for the presence of antiphospholipid antibodies (APA) should be performed in patients who fall into this category.

Oral anticoagulant therapy is usually monitored with the prothrombin time (PT) on citrate plasma samples. In recent years instruments have been developed for measurement of the PT in non-citrated whole blood. In the present study, the manufacturer's calibration of one type of device (CoaguChek) in terms of the international normalized ratio (INR) was evaluated by one laboratory. Three subsequent lots of test strips for the CoaguChek were investigated using blood samples from 56 coumarin-treated patients. Citrated plasma samples from the same patients were analysed with two international reference preparations for thromboplastin (IRP), i.e., rTF/95 (recombinant human) and RBT/90 (rabbit brain). There were statistically significant INR differences between CoaguChek and the international reference preparations (p <0.001), but the mean relative deviation of the INR was not greater than 0.104. Clinically relevant criteria were used to assess the agreement between the CoaguChek and the IRP results. Standard agreement ranged from 82% to 95%. It is concluded that these test strips achieved a clinically acceptable level of accuracy. Further studies of patient management with these strips are justified.

Our study compared point-of-care (POC) device monitoring with traditional clinical laboratory methods device of patients on oral anticoagulant therapy. The POC devices used in the study were Coumatrak, CoaguChek, CoaguChek Plus, Thrombolytic Assessment System (TAS) PT-One, TAS PTNC, TAS PT, Hemachron Jr. Signature, ProTime Microcoagulation System, and Medtronics ACT II. The clinical laboratory method used thromboplastins with different ISI values: Innovin and Thromboplastin C Plus (TPC). All POC INRs showed strong correlation with both laboratory methods, with correlation coefficients of >0.900. All POC methods demonstrated a significant (p <0.05) difference in INR values, except the TAS PTNC and ACT II INRs (p: 0.12 and 0.71 respectively) when compared with Innovin INRs. All POC INRs were significantly different from TPC generated INRs (p <0.05). Comparisons of the POC INRs to the group mean of the POC methods, show higher correlation (R>0.93), but there were still significant (p<0.05) differences noted between the POC group INR mean and CoaguChek Plus, ACT II, TAS PT-One, TAS PTNC, and Hemachron Jr Signature INRs. These data indicate that POC INR biases exist between laboratory methods and POC devices. Until a suitable whole blood INR standardization method is available, we conclude that clinicians using point-of-care anticoagulation monitoring should be aware of differences between POC and parent laboratory values.

The prothrombin time is a common method of monitoring patients undergoing oral anticoagulant therapy. The proliferation of commercial thromboplastin brands with different international sensitivity indices (ISI) in conjunction with wider availability of automated coagulation analyzers has elevated the need for standardization in monitoring therapy.

The possibility of reduction of numbers of fresh coumarin and normal plasmas has been studied in a multicentre manual prothrombin (PT) calibration of high international sensitivity index (ISI) rabbit and low ISI human reference thromboplastins at 14 laboratories. The number of calibrant plasmas was reduced progressively by a computer program which generated random numbers to provide 1000 different selections for each reduced sample at each participant laboratory. Results were compared with those of the full set of 20 normal and 60 coumarin plasma calibrations. With the human reagent, 20 coumarins and seven normals still achieved the W.H.O. precision limit (3% CV of the slope), but with the rabbit reagent reduction < 50 coumarins with 17 normal plasmas led to unacceptable CV. Little reduction of numbers from the full set of 80 fresh plasmas appears advisable. For maximum confidence, when calibrating the ISI of a new reagent, it would always seem worthwhile to use the full number of patients' and normal plasmas at each centre for the initial calibration, i.e. 60 fresh coumarin and 20 fresh normal plasmas. Smaller numbers should achieve the required precision with the lower ISI reagent.

This report presents the recommendations of a WHO Expert Committee commissioned to coordinate activities leading to the adoption of international requirements for the production and control of vaccines and other biologicals and the establishment of international biological reference materials. The report starts with a discussion of general issues brought to the Committee's attention and provides information on the status and development of reference materials for various antibodies, antibiotics, antigens, blood products and related substances, cytokines and growth factors and other substances for which the Committee has discerned a need for international reference materials. The second part of the report, of particular relevance of manufacturers and national control authorities, contains guidelines for the production and control of synthetic peptide vaccines, requirements for tick-borne encephalitis vaccine (inactivated), guidelines for thromboplastins and plasma used to control oral anticoagulant therapy, an amendment to the requirements for hepatitis B vaccine made by recombinant DNA techniques and a report on the standardization and calibration of cytokine immunoassays.

The introduction of international reference preparations (IRP) of thromboplastins was an important step forward in the standardisation of laboratory monitoring of coumarin therapy for the treatment and prevention of thrombosis. In more recent years, the standard manual technique for prothrombin time (PT), which was used to monitor coumarin therapy, has been replaced gradually by a variety of different automated techniques in the laboratory. The international sensitivity index (ISI), assigned to a working thromboplastin by comparison to the IRP, is invalidated by the use of automated techniques, and is not necessarily consistent among analysers of the same type. Consequently, current recommendations are that individual laboratories should standardise their own thromboplastin with their routine method, using calibrated lyophilised plasmas which have international normalised ratios (INR) assigned to them.