Effect of the reaction temperature on the prothrombin time and the apparent International Normalized Ratio determined with International Standards for thromboplastins

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 degrees 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 degrees C. Pooled normal plasma and pooled coumarin plasma (INR approximate to 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 degrees C. PTs with RBT/16 were shortened by increasing the reaction temperature up to approximately 34-37 degrees C, but were prolonged at higher temperatures. The apparent INR change of the coumarin plasma at 37.0 degrees C was 0.06/degrees C and 0.11/degrees 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 degrees 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 degrees C.Afdeling Klinische Chemie en Laboratoriumgeneeskunde (AKCL

Therapeutic quality control in a regional thrombosis center: the effect of changing the target intensity of anticoagulation with vitamin K antagonists

Background: The target ranges (TR) for anticoagulation with vitamin K antagonists (VKA) in the Netherlands were changed in 2016 from INR 2.0 & ndash;3.5 (& lsquo;low intensity & rsquo;) and INR 2.5 & ndash;4.0 (& lsquo;high intensity & rsquo;) to INR 2.0 & ndash;3.0 and INR 2.5 & ndash;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 & ndash;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 & ndash;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.Afdeling Klinische Chemie en Laboratoriumgeneeskunde (AKCL

Bias and uncertainty of the International Normalized Ratio determined with a whole blood point-of-care prothrombin time test device by comparison to a new International Standard for thromboplastin

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 & rsquo; samples and traceability to an accepted Reference Measurement System.Afdeling Klinische Chemie en Laboratoriumgeneeskunde (AKCL

Photochemical decomposition of dipyridamole in aqueous solution and the utilization of citrate-theophylline-adenosine-dipyridamole anticoagulant for monitoring of heparin

Thrombosis and Hemostasi

Artificially depleted plasmas are not necessarily commutable with native patient plasmas for International Sensitivity Index calibration and International Normalized Ratio derivation

Thrombosis and Hemostasi

Artificially depleted plasmas are not necessarily commutable with native patient plasmas for International Sensitivity Index calibration and International Normalized Ratio derivation: a reply to a rebuttal

Thrombosis and Hemostasi

A reassessment of the relationship between international reference preparations for human and rabbit thromboplastins

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

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 (CISI) 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 thrombo-plastin used. Despite minor INR differences, a set of six frozen plasma pools could be used for local calibration by direct INR determination.Afdeling Klinische Chemie en Laboratoriumgeneeskunde (AKCL