Defining a metrologically traceable and sustainable calibration hierarchy of international normalized ratio for monitoring of vitamin K antagonist treatment in accordance with International Organization for Standardization (ISO) 17511:2020 standard: communication from the International Federation of Clinical Chemistry and Laboratory Medicine-SSC/ISTH working group on prothrombin time/international normalized ratio standardization.

Calibration of prothrombin time (PT) in terms of international normalized ratio (INR) has been outlined in "Guidelines for thromboplastins and plasmas used to control oral anticoagulant therapy" (World Health Organization, 2013). The international standard ISO 17511:2020 presents requirements for manufacturers of in vitro diagnostic (IVD) medical devices (MDs) for documenting the calibration hierarchy for a measured quantity in human samples using a specified IVD MD. The objective of this article is to define an unequivocal, metrologically traceable calibration hierarchy for the INR measured in plasma as well as in whole blood samples. Calibration of PT and INR for IVD MDs according to World Health Organization guidelines is similar to that in cases where there is a reference measurement procedure that defines the measurand for value assignment as described in ISO 17511:2020. We conclude that, for PT/INR standardization, the optimal calibration hierarchy includes a primary process to prepare an international reference reagent and measurement procedure that defines the measurand by a value assignment protocol conforming to clause 5.3 of ISO 17511:2020. A panel of freshly prepared human plasma samples from healthy adult individuals and patients on vitamin K antagonists is used as a commutable secondary calibrator as described in ISO 17511:2020. A sustainable metrologically traceable calibration hierarchy for INR should be based on an international protocol for value assignment with a single primary reference thromboplastin and the harmonized manual tilt tube technique for clotting time determination. The primary international reference thromboplastin reagent should be used only for calibration of successive batches of the secondary reference thromboplastin reagent.

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' samples and traceability to an accepted Reference Measurement System.

Paving the way for establishing a reference measurement system for standardization of plasma prothrombin time: Harmonizing the manual tilt tube method.

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.

Accuracy assessment of consecutive test strip lots for whole blood INR point-of-care instruments: clarifying the role of frozen plasma pools.

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.

Fibrinogen determination according to Clauss: commutability assessment of International and commercial standards and quality control samples.

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).