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Introduction: The definition of the International Normalized Ratio (INR) depends on a reference measurement procedure for the prothrombin time (PT) determined with international standards for thromboplastins. The agreed water bath temperature for PT determination in the reference measurement procedure is 37°C. The aim of the study was to assess the influence of small deviations of the agreed reaction temperature on PT and INR determined with World Health Organization international standards for thromboplastins rTF/16 (recombinant human) and RBT/16 (rabbit brain)., Methods: Prothrombin time was determined, with a manual hook technique, in glass test tubes in a water bath at a controlled temperature. The PT reaction temperatures were varied between 28 and 40°C. Pooled normal plasma and pooled coumarin plasma (INR ≈ 2.8) were used as test plasmas. The data were fitted to a quadratic relationship between PT and temperature., Results: Prothrombin times with rTF/16 were shortened by increasing the reaction temperature up to approximately 39-40°C. PTs with RBT/16 were shortened by increasing the reaction temperature up to approximately 34-37°C, but were prolonged at higher temperatures. The apparent INR change of the coumarin plasma at 37.0°C was 0.06/°C and 0.11/°C for rTF/16 and RBT/16, respectively., Conclusions: Reaction temperature had a significant effect on PT and the apparent INR with the International Standards. At 37.0°C, the apparent INR of coumarin plasma determined with RBT/16 was more responsive to temperature change than the apparent INR determined with rTF/16. The required accuracy of the water bath temperature should be 37.0 ± 0.1°C., (© 2021 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)

Background: The target ranges (TR) for anticoagulation with vitamin K antagonists (VKA) in the Netherlands were changed in 2016 from INR 2.0-3.5 ('low intensity') and INR 2.5-4.0 ('high intensity') to INR 2.0-3.0 and INR 2.5-3.5, respectively., Aim: To assess the effect of the TR change on therapeutic quality control (TQC) in a Dutch regional thrombosis center taking care of approximately 3600-5500 patients annually., Methods: TQC of chronically treated patients was assessed as the average time in therapeutic range (TTR). Evaluations were performed for non-self-management (NSM), as well as self-management patients. INR percentiles were assessed from all INR determinations in all patients, i.e. including those of induction episodes and patients treated for a short-term., Results: The number of NSM patients treated chronically decreased gradually, while their average age increased, with a marginal but significant gradual increase in bleeding complications. In the period 2011-2015, i.e. before the TR change, there was a gradual increase of the TTR in NSM patients from 77.5% to 88.9% (low intensity) and from 75.3% to 84.1% (high intensity). In the same period, the median INR of all patients in the low and high intensity ranges decreased from 2.9 to 2.7, and from 3.3 to 3.2, respectively. The TTR in self-management patients remained virtually constant. After TR changes from 2016 on, the TTR of all NSM patients in the low and high intensity groups decreased to 77% and 70%, respectively, and median INRs decreased to 2.6 and 3.0, respectively., Conclusions: Introduction of internationally harmonized target ranges in 2016 resulted in further lowering of median INR values in both target ranges. As expected, TTR was reduced slightly. These findings, together with a slight increase in average age and concomitant bleeding complications, suggest that the patients on long-term VKA treatment will require intensified monitoring and treatment., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Whole blood point-of-care PT/INR test devices, e.g. CoaguChek XS, are calibrated by their manufacturers. In the Netherlands, each new lot of test strips for CoaguChek XS is validated by a group of anticoagulant clinics collaborating with a Coagulation Reference Laboratory. In 2017, a new International Standard for recombinant human thromboplastin (coded rTF/16) has been established by the World Health Organization., Aim: To assess uncertainty of the validation procedure and the magnitude of the INR bias of a series of consecutive lots of test strips imported in the Netherlands., Methods: CoaguChek XS test strip INR results were compared to INRs determined with the new International Standard rTF/16. Comparisons were made with variable numbers of blood samples obtained from patients treated with vitamin K-antagonists. Relationships between CoaguChek XS and rTF/16 results were determined with orthogonal regression analysis. The relationships were used to assess bias and uncertainty of bias., Results: Average bias between CoaguChek XS test results and rTF/16 depends on the INR level. Overall, there was a trend of increasing bias and increasing uncertainty with increasing INR values. Along the sequence of 47 consecutive lots, a temporary fluctuation of bias was observed. At an INR level of 3.0 the average bias was less than 10% in all cases, but at an INR of 4.0 there were 5 lots with average bias between 10 and 15%., Conclusion: Validation of test strips is useful to assess bias but depends on availability of fresh patients' samples and traceability to an accepted Reference Measurement System., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

Background: International normalized ratio (INR) is traceable to World Health Organization (WHO) International Standards for thromboplastins. International Standards must be used with a manual tilt tube technique (MTT) for prothrombin time (PT) determination. An important part of the total variability of INR is due to poor harmonization of MTT across WHO reference laboratories., Objectives: To determine the origins of PT differences between operators performing MTT and to develop a harmonized MTT., Methods: Two workshops were held where WHO reference laboratory operators could compare their PTs using MTT and the same equipment. A harmonized MTT was used by seven operators in the second workshop., Results: Differences have been observed in tilting frequency and in the height of pipetting plasma in the test tube. At the beginning of the first workshop, the tilting cycle time varied between 1.1 and 2.7 seconds. The mean PT of normal plasma obtained by pipetting plasma at the top of the tube was 14.3 seconds but was 12.9 seconds when plasma was pipetted at the bottom of the tube. When using the harmonized MTT for WHO International Standard rTF/16, the differences between operators were not greater than 1.1 seconds in normal plasma, and not greater than 1.3 seconds in patient plasma with average INR of 3.0. INR between-operator coefficient of variation was 2.3%., Conclusion: Application of a harmonized MTT in three reference laboratories resulted in substantial reduction of between-operator variation of PT and INR. The harmonized MTT is proposed as Candidate Reference Measurement Procedure., (2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background In the Netherlands, each new lot of test strips for the CoaguChek XS is validated by a group of collaborating centers. The purpose of this study was to assess the accuracy of the international normalized ratio (INR) measured with consecutive test strip lots and the suitability of frozen plasma pools for accuracy evaluation. Methods Each year, a particular lot of CoaguChek XS test strips is used as reference lot. The reference lots have been validated with the International Standard for thromboplastin rTF/09, yielding a mathematical relationship (R1) between reference lot INR and International Standard INR. New lots are compared to the reference lot using patients' capillary blood samples, yielding a relationship (R2) between the new lot INR and the reference lot INR. INRs of the blood samples were within the 1.5-4.5 interval. In parallel, three frozen plasmas pools are analyzed with the test strips. The distance of each plasma point to the line of relationship R2 was assessed. Results Fifty-four test strip lots have been evaluated during 3 years (2014-2016). Mean INR differences between test strip lot and International Standard rTF/09 varied between -0.14 and +0.20 (-4% and +8%, respectively). A positive trend with strip lot sequence number was observed (p<0.001). In several cases, the distance of the frozen plasmas to the whole blood relationship (R2) was greater than the critical value for commutability. Conclusions Using whole blood, all evaluated test strip lots met the analytical bias criterion of ±10%. Frozen plasma pools behave differently compared to whole blood and are not suitable for assessing absolute accuracy of new CoaguChek XS test strips.

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: Many clinical laboratories use a clotting rate assay according to Clauss for the determination of fibrinogen in citrated plasma. The aim of the present study was to assess the commutability of the current International Standard for fibrinogen (coded 09/264), three commercial fibrinogen standards, and 10 freeze-dried plasma quality control samples from various sources., Methods: Clotting rate assays according to Clauss were performed on three automated instruments (Sysmex CA1500, STA-Rack Evolution and ACL-Top 700), using three commercial thrombin reagents (Siemens, Stago, and Instrumentation Laboratory). Relationships between the results obtained with the three instruments were determined with 25 fresh-frozen plasma samples obtained from patients. The deviations of the assay results obtained with the freeze-dried samples were compared with the deviations obtained with the fresh-frozen samples, according to approved CLSI guideline C53A., Results: Freezing and thawing had no influence on the assay results. There were significant differences in the mean assay results (fibrinogen, g/L) for the fresh-frozen plasma samples between the three automated instruments: 2.51 (STA-Rack Evolution), 2.25 (ACL-Top 700) and 2.20 (Sysmex CA1500). Similar differences were observed for several freeze-dried plasma samples. Some freeze-dried plasma samples, including the International Standard, were out of the 95% confidence interval for the relationship between STA-Rack Evolution and Sysmex CA1500., Conclusions: Some freeze-dried plasmas including the international standard for fibrinogen are not commutable among automated instruments for fibrinogen clotting rate assays according to Clauss. Our results have consequences for all interested parties in the traceability chain (WHO, industry, external quality assessment schemes, clinical laboratories).