Variability of INR in patients on stable long-term treatment with phenprocoumon and acenocoumarol and implications for analytical quality requirements.

Within each patient treated with vitamin K antagonist (VKA), variation of the international normalised ratio (INR) occurs over the treatment period. The purpose of the present study was to assess INR variation in selected patients on long-term treatment in whom the dose of VKA was not changed. This type of variation is considered as "biological variation" which is caused by many factors but not VKA dose changes or other medication. Four groups of long-term patients were examined: each group with a different VKA (acenocoumarol or phenprocoumon) or a different target intensity (INR 2.0-3.5 or 2.5-4.0). All patients were monitored with the same PT system (Hepato Quick, STA-R Evolution coagulation instrument) by one laboratory. The variation of the INR within each patient was expressed as coefficient of variation (CV, in %). The CV was corrected for the average imprecision of the INR measurement (CV, 2.4%). The mean corrected CV values for the four groups were: 10.9% (acenocoumarol, target INR 2.0-3.5); 10.5% (acenocoumarol, target INR 2.5-4.0); 10.4% (phenprocoumon, target INR 2.0-3.5); 9.1% (phenprocoumon, target INR 2.5-4.0). The analytical performance goal for the INR measurement (imprecision) can be derived from the within-subject biological variation. Desirable INR imprecision goals are <4.9% and <5.3% CV for monitoring of phenprocoumon and acenocoumarol, respectively. These goals were achieved using the aforesaid PT system.

A multicentre randomised assessment of the DAWN AC computer-assisted oral anticoagulant dosage program.

Computer-assisted oral anticoagulant dosage is being increasingly used to meet growing demands for oral anticoagulation. The DAWN AC is one of the most widely used computer-dosage programs. Evidence of its value and that of other computer programs has been based previously only on laboratory evidence of "time in target INR range" (TIR) not on clinical safety in practice. A five-year international randomised clinical study of computer assistance with the DAWN AC program compared with manual dosage in 2,631 patients has been performed at 13 centres with established expertise in oral anticoagulation mainly in the EU. Safety assessment have been based on the comparison of bleeding or thrombotic events with DAWN AC compared with manual dosage in a randomised study. Safety of the DAWN AC program has been demonstrated. Clinical events of bleeding and thrombosis were almost identical with the experienced manual dosage group. Therapeutic control improved with DAWN AC to 66.8% from 63.4% TIR. The program failed to provide a dosage recommendation on only 5.7% of occasions. At a group of experienced centres with a special interest in oral anticoagulation, the DAWN AC computer-dosage program proved as safe clinically as manual dosage by experienced medical staff. With DAWN AC, laboratory control was improved, the difference being highly significant. The results should reassure hospitals and community clinics that the DAWN AC program is safe and facilitate greater and longer provision of warfarin treatment where required.

Influence of haematocrit on international normalised ratio (INR) differences between a whole blood point-of-care coagulation monitor and reference prothrombin time in plasma.

The CoaguChek S system is designed for use in patient self-testing. In this system a whole blood sample without sodium citrate is applied to a test strip containing thromboplastin and iron oxide particles. The detection principle is based on fibrin formation which inhibits and finally stops the movement of the iron oxide particles. In the classic prothrombin time (PT) test, citrate plasma is mixed with thromboplastin and an excess of calcium ions. In the monitoring of vitamin K-antagonist (VKA) treatment, all results are expressed on a common scale, i.e. international normalised ratio (INR). In patients on long-term VKA treatment, INRs were determined by the CoaguChek S system and reference methods for the classic PT. Four different CoaguChek S strip lots were evaluated. The difference in INR between the reference PT and the CoaguChek S system was negatively correlated to the haematocrit of the patients. We conclude that INR differences between CoaguChek S and plasma PT may be explained in part by the haematocrit. The magnitude of the effect of haematocrit, within the reference interval of 0.37 - 0.51, on the INR difference was not greater than approximately 10% for the combined data of the four strip lots. A bias of less than 10% seems to be acceptable clinically.

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

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

Influence of blood collection systems on the prothrombin time and international sensitivity index determined with human and rabbit thromboplastin reagents.

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

Long-term stability of relationships between reference materials for thromboplastins.

Reference materials for thromboplastins are available from the World Health Organization (WHO) and the European Commission (EC). The long-term stability of the reference materials is an essential requirement and must be monitored. The relationship between two reference materials for rabbit thromboplastin, i.e. ERM-AD149 (EC) and RBT/90 (WHO), has been monitored in the period 1996-2002. No significant trend with time was detected. In addition, the relationship between ERM-AD149 and the reference material for bovine thromboplastin (i.e. OBT/79) has been determined in 1994 and in 2005 in multicentre studies (n = 11 and n = 9, respectively). No significant changes were observed in the relationships between these reference materials when all results were included (5% significance level).

A national field study of quality assessment of CoaguChek point-of-care testing prothrombin time monitors.

A system for quality assessment (QA) of the CoaguChek (Roche Diagnostics, Mannheim, Germany) point-of-care testing prothrombin time monitor has been developed by the European Concerted Action on Anticoagulation. Hitherto there has not been an adequate rapid method for CoaguChek QA. Sets of 5 certified international normalized ratio (INR) plasma samples were tested on 539 CoaguChek monitors by experienced staff at 9 Netherlands Thrombosis Centers and results compared with certified INR. A 15% or more deviation has been classified as significant deviation. Overall mean and certified INR values were similar, but 20.3% of participants showed a 15% or more deviation from the certified INR on at least 1 of the 5 QA plasma samples. Statistically significant differences in results with different lots of CoaguChek test strips were found. There is need for large scale QA of CoaguChek monitors. The importance of the 5 CoaguChek certified INR QA plasma samples being tested on a single occasion is demonstrated.

Long-term stability and reproducibility of CoaguChek test strips.

Whole-blood point-of-care testing prothrombin time monitors are being used on an increasing scale for the monitoring of oral anticoagulant therapy. These monitors are used with disposable test strips containing tissue factor reagent. The purpose of the present study was to assess the stability of different CoaguChek-S strip lots for a period of one year. Ten strip lots introduced successively in the Netherlands were obtained from the manufacturer's representative and stored at 4-8 degrees C. Six deep-frozen pooled plasmas were analyzed for the prothrombin time and INR with the strips at six or seven occasions spread over one year. The test plasmas were recalcified immediately before application to the CoaguChek-S system. Regression analysis was performed on the clotting times obtained with each plasma. In the majority of cases (i.e.95%), no significant change was observed at the 5% significance level. A significant change was observed in only 3 cases. In addition, a ranking statistic was used as test of a monotonic relationship in the two-way analysis of variance. The results of the ranking statistic were not significant for any strip lot, indicating that the test strips were stable under these storage conditions. The reproducibility of INR measured with the CoaguChek-S was assessed. The mean within-run coefficient of variation (CV) of INR ranged from 2.58% to 3.36% (CV). The between-lot variation of the mean INR ranged from 3.2 to 4.5 % (CV). The over-all variation of single INR measurements, i.e. including between-lot and within-lot, ranged from 5.0 to 6.0 % (CV).

The influence of exogenous magnesium chloride on the apparent INR determined with human, rabbit, and bovine thromboplastin reagents.

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.

Minimum numbers of fresh whole blood and plasma samples from patients and healthy subjects for ISI calibration of CoaguChek and RapidPointCoag monitors.

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.

European concerted action on anticoagulation. Minimum numbers of lyophilized plasma samples for ISI calibration of CoaguChek and TAS point-of-care whole blood prothrombin time monitors.

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.

A comparative study of conventional versus new, magnesium-poor Vacutainer® Sodium Citrate blood collection tubes for determination of prothrombin time and INR.

INTRODUCTION: Conventional Vacutainer® Sodium Citrate blood collection tubes contain a relatively high concentration of contaminating magnesium ions, which may result in shortening of the prothrombin time (PT) and the International Normalized Ratio (INR). Recently the manufacturer of Vacutainer® Sodium Citrate tubes introduced new tubes with a magnesium-poor stopper. The magnesium concentration in the new low-Mg tubes is significantly lower than that in the conventional plastic tubes. The purpose of the present study was to compare PT and INR determined in specimens drawn with the new tubes to those drawn with the conventional tubes. MATERIALS AND METHODS: Venous blood specimens were collected from 22 healthy persons and 65 patients treated with vitamin K-antagonists using conventional Vacutainer® Sodium Citrate tubes and new, low-Mg Vacutainer® tubes. PT and INR were determined with four thromboplastin reagents, i.e., three brands of recombinant human tissue factor and one brand of combined rabbit brain reagent. Magnesium concentrations were determined in the citrate plasmas with a colorimetric method. RESULTS: The differences in PT, INR and International Sensitivity Index (ISI) between the two tubes were significant when using three recombinant human thromboplastin reagents, but were not significant when using the rabbit thromboplastin. The PT and INR differences between the tubes correlated with the magnesium concentration differences (P<0.001). The INR bias between the four reagents was greater for specimens drawn with conventional tubes than the INR bias for specimens drawn with the new tubes. CONCLUSION: Agreement of INR between reagents is improved by using magnesium-poor tubes.

Preparation of control blood for external quality assessment of point-of-care international normalized ratio testing in the Netherlands.

OBJECTIVES: The aim of this study was to prepare control blood for an external quality assessment scheme (EQAS) for international normalized ratio (INR) point-of-care testing (POCT) in the Netherlands and to assess the performance of the participants. METHODS: Control blood was prepared from dialyzed pooled patient plasma and washed human erythrocytes. Samples of control blood were mailed to participants of the Netherlands EQAS from October 2006 through December 2012. RESULTS: Most participants used CoaguChek XS (Roche Diagnostics, Mannheim, Germany) devices for POCT. The median between-center coefficient of variation (CV) of the reported INR decreased from 4.5% in 2006 to 2.6% in 2012. A few participants used the ProTime Microcoagulation System (ITC, Edison, NJ) for POCT. The median CV (per year) of the INR with the latter system was 7.0% to 10.6%. CONCLUSIONS: The control blood samples were useful for external quality assessment in the Netherlands. The participants' performance with the CoaguChek XS system improved with time, demonstrating the value of external quality assessment.

Long-term stability of frozen pooled plasmas stored at -70°C, -40°C, and -20°C for prothrombin time and International Normalized Ratio (INR) assessment.

BACKGROUND: Frozen pooled plasmas may be used for quality assessment and local calibration of prothrombin time (PT) and International Normalized Ratio (INR) measurement systems. The purpose of the present study is to estimate the long-term stability of frozen pooled plasmas stored for at least three years. METHODS: Six pooled plamas with different INR levels were stored at -70°C, -40°C, and -20°C for various time periods up to 1453days. PT of stored samples were measured with two thromboplastin reagents on a single automated coagulation instrument. INR was calculated from PT and plotted against plasma storage time. Linear regression lines of INR on storage time were used to estimate the percentage increase of INR. RESULTS: INR of plasma stored at -40°C or -20°C increased significantly with time. INR of plasma stored at -70°C did not change significantly in 11 out of 12 cases. The INR change of pooled plasmas stored at -70°C for 3years varied between 0.07% and 2.03%. CONCLUSION: Long-term storage of plasmas at -40°C or -20°C should be avoided. Pooled plasmas stored at -70°C can be used for quality assessment and local calibration for at least 3years.

Biological variation of INR in stable patients on long-term anticoagulation with warfarin.

Within-individual biological variation of INR (CV(B)) was assessed in 245 selected stable warfarin-treated patients monitored by three thrombosis centers. Selection criteria were: treatment period of six months or longer before the observation period; at least six consecutive INRs within the therapeutic range of 2.0 - 3.0; interval between consecutive INR measurements of two weeks or longer; no change in warfarin dose; no changes in the patient's circumstances which may influence the INR, such as intercurrent diseases, invasive procedures, starting or stopping drugs interacting with warfarin. The minimum, maximum and mean within-individual coefficient of variation CV(B) of the INR measurements in the 245 selected patients were 0.4%, 14.5%, and 9.0%, respectively Analytical performance goals for the INR measurement (imprecision) could be derived from the mean CV(B). For a therapeutic range of 2.0 - 3.0 with warfarin, the desirable and optimum imprecision of INR determination is <4.5% CV and <2.25% CV, respectively. The biological variation and analytical performance goals have been derived using classic laboratory methods but should be applicable to point-of-care testing as well.

Harmonization and external quality assessment of antithrombin activity assays.

UNLABELLED: In the framework of a Dutch project named "Calibration 2000" harmonization of antithrombin activity assays was studied. The commutability of potential calibrators for antithrombin was assessed by means of a twin-study design, which is a multicentre, split-patient sample, between-field-methods protocol. The twin-study consisted of simultaneous analysis of fresh-frozen patient plasmas and three potential calibrators for antithrombin by 30 Dutch laboratories forming 15 couples. The state-of-the-art intralaboratory standard deviation (SD(SA)) was used to assess the commutability of the potential calibrators. The regression line residuals for the potential calibrators were normalized by expressing them as multiples of SD(SA). All residuals of the potential calibrators were within the 3×SD(SA) limit. One potential calibrator was used in an attempt to harmonize antithrombin assay results in a Dutch field study. The interlaboratory coefficient of variation (CV) of the antithrombin results for three test samples could be reduced from 6.9 - 13.2% (before harmonization) to 5.6 - 9.8% using the common calibrator. CONCLUSION: The potential calibrators were commutable. Limited harmonization was achieved by using a common calibrator for all participants.

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.

Comparative study of blood collection tubes and thromboplastin reagents for correction of INR discrepancies: a proposal for maximum allowable magnesium contamination in sodium citrate anticoagulant solutions.

International normalized ratio (INR) discrepancies were noted between clinical laboratories using various prothrombin time (PT) systems. We studied the influence of different commercial blood collection tubes and different PT systems on INR measurements. INRs of fresh patient samples were determined by 3 laboratories, each using different PT systems. In the first part of the study, samples were drawn with Vacutainer tubes and in the second part with Monovette tubes. In the first part of the study, the maximum bias for all patients amounted to 0.46 INR (14%), and in the second part, to 0.14 INR (4.9%). The maximum bias for all patients could be reduced further by local system calibration using frozen pooled plasma specimens. The sodium citrate solutions in the blood collection tubes were contaminated with magnesium ions (approximately 2.7 mmol/L and 0.3 mmol/L in the Vacutainer and Monovette, respectively). INR discrepancies could be explained largely by this influence of blood collection tubes. The maximum allowable magnesium contamination in sodium citrate anticoagulant solutions should be less than 1 mmol/L.

Harmonisation of factor VIII:C assay results: study within the framework of the Dutch project 'Calibration 2000'.

In a Dutch project for harmonisation of factor VIII coagulant activity (FVIII:C) assays, the commutability of potential calibrators for FVIII:C was assessed by means of a 'twin-study design', which is in essence a multi-centre, split-patient sample, between-field-methods protocol. Commutability was defined as the degree to which a material yielded the same numerical relationships between results of measurements by a given set of measurement procedures as those between the expectations of the relationships for the same procedures applied to those types of material for which the procedures were intended. The study consisted of the simultaneous analysis of fresh frozen patient plasmas and three potential calibrators for FVIII:C by 16 Dutch laboratories forming eight couples. The state-of-the-art intra-laboratory standard deviation was used to assess the commutability of the potential calibrators. One potential calibrator was used to harmonise FVIII:C assay results in a Dutch field study. The inter-laboratory coefficient of variation of two test samples could be reduced significantly, but no significant effect was observed with three other test samples. We recommend that at least three different sample dilutions be used in each FVIII:C assay, in agreement with previous recommendations.