International collaborative study for the calibration of proposed International Standards for thromboplastin, rabbit, plain, and for thromboplastin, recombinant, human, plain.

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.

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

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.

Calidad de las tromboplastinas utilizadas en el laboratorio clínico y en los equipos POCT y su impacto en la dosificación de acenocumarol en pacientes con terapia anticoagulante oral / Types of thromplastin used for INR determination in patients under chronic oral anticoagulant treatment: discrepancies that may affect the dose of coumarin derivatives needed for treatment safety

Antecedentes: El monitoreo del tratamiento con anticoagulantes cumarínicos se realiza a través del INR (International Normalized Ratio) que es el parámetro estandarizado del Tiempo de Protrombina. Las recomendaciones de la OMS indican que la precisión en el cálculo del INR puede ser mejorada usando reactivo de tromboplastina con Indice de Sensibilidad Internacional (ISI) bajo, considerándose como ISI de referencia internacional el valor 1,0. Debido a incongruencias observadas en los INR de pacientes controlados en el Servicio de Salud Metropolitano Occidente, comparando valores de muestra venosa con resultados de INR capilar obtenidos en el mismo paciente el mismo día y hora (con reactivos Tromboplastina de distinto ISI), se efectuó un ensayo clínico cruzado entre los distintos métodos. Materiales y métodos: En 100 pacientes se comparó INR venoso con dos tromboplastinas de diferente ISI (1,3 y 1,0) vs aquel efectuado con muestra capilar (ISI 1,0). Resultados: Los resultados del estudio muestran que a partir de valores de INR 3,0 las determinaciones obtenidas usando Tromboplastina de cerebro de conejo ISI=1,3 subestiman el valor de INR para un mismo paciente y una misma muestra. Conclusiones: El uso de Tromboplastina recombinante humana ISI 1,0 permite evitar la subestimación del INR en pacientes con mayor riesgo tromboembóli-co (indicación de INR objetivo más alto). Por ello, este método se adoptó en el control del TACO en pacientes controlados en el Servicio de Salud Occidente.

Background: INR (International Normalized Ratio) is the standard Prothrombin Time parameter for monitoring anticoagulant treatment with coumarin derivatives Recommendations of WHO indicate that precision in the calculation of the INR can be improved using thromboplastins with a low Index of International Sensibility (ISI=1,0). Discrepancies in INR obtained using either this technique or conventional rabbit brain derived reagents in the same sample in patients attending the Servicio de Salud Metropolitano Occidente (West Metropolitan Health Service) were observed. Our objective was to evaluate these discrepancies in a systematic way. Materials and methods: A comparative study was conducted using two thromboplastins of different ISI (1.0 and 1.3) for the calculation of venous INR in comparison with capillary INR in 100 patients. Results: The study showed that INR values may differ significantly according to the method used. In particular, rabbit brain thromboplastin ISI = 1.3 underestimates the value of INR in the range of INR ≥3.0. Conclusions: The use of human recombinant thromboplastin ISI= 1.0. for determination of INR may significantly decrease the risk of hemorrhagic complications in patients requiring higher levels of anticoagulation.

Factor VII assay performance: an analysis of the North American Specialized Coagulation Laboratory Association proficiency testing results.

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.

Point-of-care monitoring of vitamin K-antagonists: validation of CoaguChek XS test strips with International Standard thromboplastin.

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.

Thromboplastin standards.

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

A modified international normalized ratio as an effective way of prothrombin time standardization in hepatology.

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.

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

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.

European Concerted Action on Anticoagulation. A multicentre calibration study of WHO international reference preparations for thromboplastin, rabbit (RBT/90) and human (rTF/95).

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.

Usefulness of lyophilized calibration plasmas for International Normalized Ratio determination with the bovine combined thromboplastin (Thrombotest): results of a collaborative study.

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.

Poor agreement among prothrombin time international normalized ratio methods: comparison of seven commercial reagents.

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.

Effect of magnesium contamination in evacuated blood collection tubes on the prothrombin time test and ISI calibration using recombinant human thromboplastin and different types of coagulometer.

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.

Guidelines on preparation, certification, and use of certified plasmas for ISI calibration and INR determination.

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.