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1 AJCP /ORIGINAL ARTICLE Harmonizing the International Normalized Ratio (INR) Standardization of Methods and Use of Novel Strategies to Reduce Interlaboratory Variation and Bias Emmanuel J. Favaloro, PhD, FFSc (RCPA), 1 Wendy McVicker, 1 Michelle Lay, 1 Monica Ahuja, MSc, 1 Yifang Zhang, 1 Sayed Hamdam, 2 and Naomi Hocker 3 From the 1 Haematology Department, Institute of Clinical Pathology and Medical Research (ICPMR), Pathology West, NSW Health Pathology, Westmead Hospital, Westmead, NSW, Australia; 2 Pathology Department, Pathology West, NSW Health Pathology, Blacktown Hospital, Blacktown, NSW, Australia; and 3 Haematology Department, Pathology West, NSW Health Pathology, Wagga Wagga, NSW, Australia. Key Words: International normalized ratio; INR; Harmonization; Standardization; External quality assessment; EQA Am J Clin Pathol February 2016;145: DOI: /AJCP/AQV022 ABSTRACT Objectives: To reduce interlaboratory variation and bias in international normalized ratio (INR) results, as used to monitor patients receiving vitamin K antagonist therapy, including warfarin, in a large pathology network (n ¼ 27 laboratories) by procedural standardization and harmonization. Methods: Network consensus to standardize to common instrument and reagent platforms was established, following development of hemostasis test specifications. Subsequent installations and implementation occurred after conclusion of a government tender process. Network-wide application of simple novel process of verification harmonization of local international sensitive index and mean normal prothrombin time initiated for each new lot of INR reagent that does not require ongoing use of reference thromboplastin or calibration/certified plasma sets. : We achieved reduction of different instrument manufacturers (from four to one), instrument types (10 to three), reagent types (four to one), and instrument/reagent combinations (12 to three), plus substantial reduction in INR variability and bias. Conclusions: infer significant improvement in local patient management, with positive implications for other laboratories. For the United States in particular, lack of US Food and Drug Administration cleared certified plasmas may compromise INR accuracy, and our novel approach may provide a workable alternative for laboratories and networks. Upon completion of this activity you will be able to: define the separate components of the international normalized ratio. describe several options for local verification of mean normal prothrombin time and international sensitivity index. analyze laboratory-derived data to compute local mean normal prothrombin time and international sensitivity index values using one or more of these options. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit TM per article. Physicians should claim only the credit commensurate with the extent of their participation in the activùity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Exam is located at Patients at increased risk of thrombosis are prescribed one of several anticoagulants, depending on the condition being treated as well as other patient variables. 1 In particular, oral vitamin K antagonist (VKA) anticoagulants such as warfarin have been used clinically for over 50 years. 1 Although much attention has recently been placed on the so-called new, direct or non-vka oral anticoagulants (NOACs), VKAs remain in clinical use for many patients for a variety of reasons. 2,3 However, there remain many shortcomings to VKA therapy, including high interpatient variability and numerous drug and food interactions that mandate the need to monitor the efficacy of therapy. 2-5 Essentially, there is a requirement to maintain patients within narrow therapeutic ranges of a laboratory test called the international normalized ratio (INR), which is derived from a routine coagulation test, the prothrombin time (PT), CME/SAM American Society for Clinical Pathology, All rights reserved. For permissions, please journals.permissions@oup.com 191 Am J Clin Pathol 2016;145: DOI: /ajcp/aqv022

2 Favaloro et al /INRHARMONIZATION AND STANDARDIZATION after mathematically adjusting this result for varying instrument and test reagent sensitivity according to the following formula: INR ¼ (PT/MNPT) ISI, where the MNPT is the mean normal PT, and the ISI is an international sensitivity index (a correction factor applied to adjust for instrument and reagent sensitivity differences). Maintaining patients within these therapeutic ranges (typically 2-3 or 2.5-) is an ongoing challenge for clinicians. Indeed, in recent clinical trials for the NOACs, where it can be argued that patients are perhaps more closely monitored than normal practice, time within the therapeutic range (TTR) was reported as being 55% to 68%, 6 meaning that some patients reside outside these ranges (and, by inference, are at increased risk of bleeding or thrombosis) nearly as often as they are within the therapeutic range. Importantly, a recent study has shown high INR variability to represent an additional risk factor (in addition to TTR) for clinically adverse events. 7 The INR may be derived from a variety of methods, including manual methods, laboratory instrument-based testing (in turn using a wide variety of coagulation analyzers and reagents), and near-patient testing by means of point-of-care instruments. 8 However, laboratory instrument-based testing represents the current standard for INR monitoring. Although laboratory-based testing has been available for decades, with experienced laboratory personnel performing tests and quality issues perceived to be well controlled by the use of internal quality control and external quality assessment (EQA), there remains a large interlaboratory variation, as evidenced by various EQA studies Analytically, modern coagulation instrumentation is considered very precise, suggesting that the main reason for interlaboratory variation in INR testing can be explained by differences in thromboplastin/instrument combinations and ISI and MNPT values assigned to various reagents as used by different laboratories. Not only are different ISI and MNPT values assigned to different reagents; importantly, different ISI and MNPT values can be shown to be often applied to the same reagent/instruments by different laboratories. Thus, it can be inferred that many ISI and MNPT values are essentially incorrect, and there is much scope for improvement in assigned values to improve accuracy in INR testing. Overall, then, a proportion of interlaboratory variation in INR is due to use of different reagent/instrument combinations and noncommutability of INR values (despite the theoretical comparability) due to different behaviors and factor sensitivity of reagents, but some significant level of effect on INR variability can also be ascribed to inaccurate assignment of ISI and MNPT values by different laboratories. ISI values are often assigned by individual reagent/instrument manufacturers by using a World Health Organization (WHO) recommended procedure and/or WHO-assigned thromboplastin reference plasmas. 11,12 However, here it is still incumbent on laboratories to locally verify the manufacturerassigned ISI. Where an ISI value is not available for a particular reagent/instrument combination (eg, each are from different manufacturers), the laboratory has to assign and then verify its local ISI value. Given the complexity of the WHOrecommended procedure for defining or verifying ISI values, requiring at least 60 VKA-stabilized patient samples, 20 normal samples, a WHO reference thromboplastin, and a manual tilt method, it is doubtful if any modern laboratory still undertakes such a process. Accordingly, the main current Clinical and Laboratory Standards Institute (CLSI) recommendation comprises the use of commercial reference-plasma calibration sets (alternatively called certified plasmas ) for local ISI assignment 11,12 Figure 1 shows an example from our network. However, this is made very difficult in the United States since US Food and Drug Administration (FDA) cleared material is currently limited to only two manufacturer products for specific use on their reagent/instrument platforms Furthermore, a clear reduction of US-based interlaboratory INR variation was achieved in the recent past, due to increasing use of thromboplastin reagents with lower ISI values 9 ; however, further room for improvement will be difficult to achieve if laboratories encounter difficulties in applying correct ISI and MNPT values to local laboratory reagents. In any case, wider use of generic certified plasmas may still yield different ISI and MNPT values when different Log PT (Trial Reagent/Instrument) Figure 1 An example of the use of an international normalized ratio (INR) calibration/certified plasma set to obtain international sensitivity index (ISI) and mean normal prothrombin time (MNPT) values for a particular thromboplastin reagent and coagulation instrument. This particular set contained five INR plasmas ranging from approximately to 5.6. The slope is 0.99; the ISI value for the new reagent is 1/slope, which is 2. The intercept at x ¼ (ie, INR ¼ 1) is 902; MNPT ¼ antilog of y-intercept at x ¼, which is Am J Clin Pathol 2016;145: American Society for Clinical Pathology 192 DOI: /ajcp/aqv022

3 AJCP /ORIGINAL ARTICLE commercial products are used for the same reagent/instrument combination, 16 leading to uncertainty regarding which ISI value is most correct or should be adopted by the laboratory, as well as continued variability in INR values. The MNPT typically has to be locally defined, based on the population being tested. The MNPT can be defined using a WHO- and CLSI-recommended procedure requiring 20 normal individuals or with some certified plasma sets 11,12 (Figure 1 shows an example of the latter). Again, evidence indicates that different MNPT values will be generated using different methods, different commercial certified plasma products, and different sets of 20 normal individuals, 16 once more generating uncertainty regarding which MNPT value is most accurate or should be used by the laboratory, as well as contributing to interlaboratory variation in INR test results. Accordingly, our laboratory has previously reported on an additional novel approach to verification of local ISI and MNPT values that does not require ongoing use of certified plasmas or WHO reference thromboplastin reagent Using this approach, our main campus laboratory has evidenced ongoing comparative INRs that very closely match our national EQA peer group median values for nearly a decade (see and Discussion for details). In the current article, we have expanded this approach to our network of 27 laboratories. Combined with standardization of INR reagent and coagulation instrumentation, a substantive decrease in interlaboratory variationininrsisevidenced. Materials and Methods Setting Our pathology network (Pathology West) currently comprises 27 laboratories across a wide geographic region of our state of New South Wales (NSW), Australia. The main campus, the Institute of Clinical Pathology and Medical Research (ICPMR), was originally placed at Westmead to service the pathology needs of the then newest (and Australia s largest) tertiary-level academic teaching hospital (Westmead Hospital). Pathology West is currently the largest pathology network in NSW and a part of NSW Health Pathology, a publicly funded pathology health organization. The growth of our network from the position of individual laboratories, such as the ICPMR, to a network of 27 laboratories is shown in Figure 2. In 2010, Pathology West, faced with aging and varied coagulation equipment, made a decision to standardize reagents and equipment across its network. Although the expected outcome obviously anticipated cost savings based on economies of scale, reduced duplication, and ability to move staff between facilities, there was also a desire to improve the quality of testing across the network, and harmonization of practice and standardization of reagents/instrumentation was anticipated to reduce interlaboratory variability in test results and thus ultimately improve clinical management of patients across our geographic area. The prestandardization equipment and reagent sets, as used within our network, are shown in Table 1. Following a government tender process, requiring negotiated network agreement on test and instrument specifications, the network standardized to a single reagent (Neoplastine CI Plus; Diagnostica Stago, Asnières-sur-Seine, France) and a single instrument platform (STA line; Diagnostica Stago), comprising three instrument models, reflecting large, medium, and small test capacity for use in differently sized laboratories (Table 1). This achieved a reduction in instrument/reagent combinations pre- to poststandardization from 12 to three. The new instrument installation and combined reagent/instrument standardization process was coordinated and finalized within June/July Within our geographic region, EQA services are provided by the Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) Haematology. Participants for the General Haemostasis program, which includes the laboratory INR test module, mainly comprise Australasian laboratories, although about one-third of enrolled participants are from overseas countries such as Malaysia, Hong Kong, India, and South Africa. Participants (currently n ¼ 586 laboratories, but this represents more than 700 reported data sets given some multiple instruments per laboratory) comprise at least 16 different instrument groups and nine different reagents, with at least 62 different instrument/reagent combinations. 8 Lyophilized plasma samples (n ¼ 16/year, tested as two samples eight test periods/year) that cover the INR range of to are sent to participants for INR estimation, with results currently submitted online and then analyzed by the RCPAQAP using robust statistics. 8 The median of the laboratories specific reagent group is used for comparative statistical analysis, providing there are greater than 10 users in the group. Otherwise, results are compared with the overall median values. Figure 3 shows a summary of returned data for RCPAQAP INR test results from participant laboratories for years 2011 to 2014 inclusive. Data are shown as regression analysis of laboratory-returned INR values vs RCPAQAP median INR results. Considerable variation and bias from the line of equivalence for individual laboratories are evidenced. Some of this bias would reflect different instrument/reagent combinations, and some would reflect use of inaccurate ISI and/or MNPT values. American Society for Clinical Pathology Am J Clin Pathol 2016;145: DOI: /ajcp/aqv022

4 Favaloro et al /INRHARMONIZATION AND STANDARDIZATION New South Wales Western Australia Northern Territory South Australia Queensland Tasmania New South Wales Victoria New South Wales ICPMR 1975 Sydney Metropolitan Area Sydney Metropolitan Area Pathology West Figure 2 Setting for current report. The Institute of Clinical Pathology and Medical Research (ICPMR) was originally a standalone organization. Over four decades, it joined with other pathology laboratories to create a network that now comprises 27 laboratories spread across the state of New South Wales. Table 1 Summary of Methods in Place for INR Testing in Pathology West Laboratories a Prestandardization (2010) Poststandardization (2013) Instruments in Use Stago: STA-R x2; STA-Compact x2; STart4 x6 IL Werfen: ACL-7000 x1; ACL-1000 x1; ACL-9000 x2 Siemens: CA-540 x5; CA-1500 x1; BFT II x 2 BioMerieux/TCoag: Coagamate x7 Stago: STA-R Evolution x2; STA-Compact x10; STA-Satellite x23 PT Reagents in Use Stago: Neoplastine CI Plus x1 IL Werfen: Hemosil x2; Recombiplastin x5 Siemens: Thromborel S x26 Stago: Neoplastine CI Plus x27 Total Different Combinations (Reagent/Instrument) Different instrument manufacturers: n ¼ 4 Different instrument types: n ¼ 10 Total instruments: n ¼ 29 Different reagent types: n ¼ 4 Different instrument/reagent combinations: n ¼ 12 Different instrument manufacturers: n ¼ 1 Different instrument types: n ¼ 3 Total instruments: n ¼ 35 Different reagent types: n ¼ 1 Different instrument/reagent combinations: n ¼ 3 a Stago, Asnières-sur-Seine, France; IL Werfen, Bedford, MA; Siemens, Marburg, Germany; BioMerieux/TCoag, Wicklow, England. Definitions For the purpose of this report, we have essentially used CLSI definitions 11 for verification and harmonization, respectively, as follows: (1) verification is confirmation, through the provision of objective evidence, that specified requirements have been fulfilled and a one-time process completed to determine or confirm test performance characteristics before the test system is used for patient testing, and (2) harmonization is a process of recognizing, understanding, and explaining differences while taking steps to achieve uniformity. 194 Am J Clin Pathol 2016;145: American Society for Clinical Pathology 194 DOI: /ajcp/aqv022

5 AJCP /ORIGINAL ARTICLE A B 5.5 low 5.9 low C low high high A Novel Approach to Verification of Local ISI and MNPT Values The ICPMR laboratory has previously reported on a novel approach to assign and verify ISI and MNPT values that does not require the ongoing use of commercial (certified) plasma sets or WHO reference thromboplastin. 12,16,17 This process was later expanded to a small number of Pathology West laboratories to evaluate its effectiveness in a broader setting. 18 The current report in part describes the expansion of this process to the entire Pathology West network of 27 laboratories to harmonize processes, so as to hopefully reduce interlaboratory test variation. In brief, a process of comparative regression analysis is undertaken using the existing (but previously verified) thromboplastin/instrument combination as the reference method and the proposed thromboplastin/instrument combination as test method. 12,16-18 Indeed, using this D low high high Figure 3 Linear regression curves identifying data from all participant laboratories to the Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) Haematology reporting international normalized ratio (INR) data for years 2011 (A), 2012 (B), 2013 (C), and 2014 (D). Data from individual participant laboratories (y-axis) plotted vs overall RCPAQAP median values for different sets of 16 plasmas sent out in each year. The target INR ranges for these plasmas are approximately to. Considerable variation in results is evident, and variation generally increases as INR increases (predominantly an effect of international sensitivity index variation). approach, the ICPMR has managed to maintain evidence of very close comparability of local INR values to RCPAQAP median INR values as the gold standard for the past 7 years Figure 4, despite using a disparate reagent manufacturer (Thromborel S; Siemens, Marburg, Germany) and instrument (STA-R analyser; Diagnostica Stago) combination without any manufacturer-assigned ISI or MNPT values in the years 2008 to The current report, in part, expands the use of a similar approach to verification of local ISI and MNPT values for the current Pathology West network of 27 laboratories, as well as verifying current data for the ICPMR laboratory (see ). In summary, for the main ICPMR campus and largest analyzer (STA-R), the comparative analysis uses approximately 100 patient-derived samples comprising a wide range of INR values (recent example shown in Figure 5A ). American Society for Clinical Pathology Am J Clin Pathol 2016;145: DOI: /ajcp/aqv022

6 Favaloro et al /INRHARMONIZATION AND STANDARDIZATION INR at ICPMR RCPAQAP INR Line of equivalance Figure 4 Comparison of international normalized ratio (INR) data for Pathology West network main campus (Institute of Clinical Pathology and Medical Research [ICPMR]) primary coagulation analyzer for years 2008 to 2014 compared with Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) Haematology overall median INR values. The linear regression line (ICPMR vs RCPAQAP) closely follows the line of equivalence (RCPAQAP vs RCPAQAP). For the intermediate- and smaller-sized sites and mediumand smaller-sized analyzers (STA-C and STA-S, respectively), a smaller number of samples (between 20 and 50) but with a similar wide spread of INR values are used. The analysis may be repeated at several different network sites for comparability. As the current reagent manufacturer also provides the instruments, it assigns a reagent lot-specific ISI, so that currently, we use this process to verify the local ISI and to assign and verify the local MNPT Figure 5B. Any discrepancies in ISI and MNPT determined between evaluations at different sites are assessed for potential significance and a process of harmonization applied if differences are determined to be not significant. Statistical and Comparative Analysis The current report largely uses descriptive statistics such as medians, coefficient of variation (CV) to assess variability, and Bland-Altman difference plots to assess comparability of test systems. Differences between prestandardization and poststandardization are identified by relative reduction of variability between network-derived INRs and median RCPAQAP INRs (as gold standard) pre- vs postimplementation of this process. For the purpose of comparison, an identical period of 18 months of prestandardization data (January 2012 to June 2013) is compared with an 18-month period of poststandardization data (July 2013 to December 2014). In general, for ease of analysis and because of small peer groups for many previous reagent/instrument combinations, local data are compared with the median of overall RCPAQAP group data, rather than median peer group data for each reagent/instrument type. Notably, comparison with median peer group data for the current reagent/instrument combinations indicates similar medians to overall group data (see ). Examples of the variability of test results reported for INR by laboratories participating in the RCPAQAP INR program for the past 4 years are shown in Figure 3. These figures identify the laboratory-reported INR values for samples (n ¼ 16/year) distributed by the RCPAQAP Haematology for each year ( inclusive) vs the RCPAQAP group median values, which essentially are representative of the overall group performance (n ¼ ) and act here as surrogate reference or gold-standard INR values. Several main points are highlighted, in part better shown in Figure 6 using data for 2014 as the illustrative example. First, there is a large spread of laboratory-reported INR values around the line of equivalence, and the spread increases with increasing INR. This is characteristic of a variable ISI effect and due to the resultant INR calculation, which increases the relative INR with increasing ISI. The finding is similar in each survey year (Figure 3). In part, this spread of data reflects different instrument/reagent combinations (generating different sensitivities to plasma clotting factors and different ISI and MNPT values), as well as the use of inaccurate ISI and/or MNPT values. The ICPMR data for 2008 to 2014 are presented in Figure 4, as well as shown for 2014 in red in Figure 6A, both indicating good concordance of ICPMR main campus INR results and overall RCPAQAP median values. The ICPMR data for 2014 are also compared with that of the peer group median (Diagnostica Stago instrumentation and Neoplastine Plus reagent) in Figure 6B, again demonstrating good concordance and further highlighting that overall RCPAQAP median values are similar to RCPAQAP median values for this peer group. Occasionally, participant lines appear to show little resemblance to RCPAQAP medians (see arrowed lines in Figure 6C). These events are usually due to large analytical or typographical error(s). Data lines that generally follow but move away from the line of equivalence indicate too high or too low an ISI and/or MNPT value relative to the group (refer to Figure 6 legend for specific examples). Comparative variability for the Pathology West Network of 27 laboratories before standardization and harmonization Figure 7A shows a similar wide spread of data to overall RCPAQAP participants (Figure 5). A process of 196 Am J Clin Pathol 2016;145: American Society for Clinical Pathology 196 DOI: /ajcp/aqv022

7 AJCP /ORIGINAL ARTICLE A Log New [Trial] PT Reagent Log Reference INR standardization of reagent and equipment plus harmonization by adoption of our novel ISI/MNPT verification/harmonization method was subsequently applied (see Materials and Methods). An example of the process applied to verify manufacturer ISI values and to estimate and verify MNPT values is shown in Figure 5 (for the ICPMR site). Similar data were obtained for other network sites (data not shown). Subsequent to the application of this network process and harmonization of ISI and MNPT values for use in each instrument line, the comparative variability between network laboratories and RCPAQAP median values was substantially reduced Figure 7B.Thisim- provement is also highlighted by the Bland-Altman plots showing prestandardization/harmonization Figure 7C and poststandardization/harmonization Figure 7D. The greatest improvement was seen at high INR values, consistent with greatest original variation in this INR region. This improvement is also highlighted in Figure 8, which shows two examples of individual laboratory improvement with our network (Figures 8A and B), as well as CV differences (pre- vs poststandardization/harmonization) using Pathology West interlaboratory CVs compared with RCPAQAP overall INR New Neo (Calc ISI and MNPT) Current Neo (ISI and MNPT) New Neo (Stago ISI, WMD MNPT) INR Figure 5 Example of the process currently used to verify manufacturer-assigned international sensitivity index (ISI) values and to verify local mean normal prothrombin time (MNPT) values for a particular thromboplastin reagent and coagulation instrument (using Institute of Clinical Pathology and Medical Research [ICPMR]) primary coagulation analyzer for a recent evaluation of a new trial lot of Neoplastine CI Plus reagent (Diagnostica Stago, Asnières-sur-Seine, France) using the existing Neoplastine CI Plus reagent (with previously verified ISI and MNPT) as the reference. The approach undertaken is similar to that using an international normalized ratio (INR) certified plasma set (Figure 1), except the certified samples are replaced by a large sample set (100) of patient plasma samples with similarly wide range of prothrombin time (PT)/INR values. First, the log of the trial reagent PT (y-axis) is plotted against the log of the INR of the reference reagent (x-axis) (A)andtheISIandMNPTcalculatedasshown.ThecalculatedISIforthisexample (1.34), calculated by 1/slope of regression line in A, is marginally different from the manufacturer-assigned value (1.32), so we assessed the difference of using either ISI to calculate the INR for the same data set, using an MNPT of either 1 (existing MNPT for the reference reagent) or 13.8 (as the estimate from A using the antilog of the y-intercept when x ¼, or the antilog of 1.14), respectively, and the results are virtually identical over the INR range evaluated (B). Accordingly, for this example, an ISI of 13.2 (manufacturer assigned) was locally verified for use with an MNPT of 1 (which was identical to that of the previous reagent lot). These ISI and MNPT values were then assessed for further utility by ongoing performance in the Royal College of Pathologists of Australasia Quality Assurance Program external quality assessment process (eg, see Figures 4 and 6). WMD, Westmead ICPMR. B interlaboratory CVs at various INR values (refer to figure legend for full explanation). In total, RCPAQAP interlaboratory CVs ranged from 5.5% to 14.2% over the period of analysis; in comparison, the pre- vs poststandardization/harmonization Pathology West interlaboratory CVs ranged from 3.2% to 12.5% and 4.8% to 8.4%, respectively. Discussion In this report, we have been able to show substantial reduction in INR bias and variation from poststandardization and harmonization of instruments, reagents, and methods/ processes used to verify ISI and MNPT values across a network of 27 laboratories. Although each of these events has contributed in total to reducing variation of INR test results within our geographic region, covering nearly 85% of our state by accessible land mass, we wish to highlight the potential value of our novel approach to verification of ISI and MNPT values. Importantly, the process does not require the ongoing use of INR calibration/certified plasma sets, nor American Society for Clinical Pathology Am J Clin Pathol 2016;145: DOI: /ajcp/aqv022

8 Favaloro et al /INRHARMONIZATION AND STANDARDIZATION A 4.6 B 4.6 C low Slope = 0.98 Intercept = 2 high does it require use of a WHO thromboplastin reagent. The process does require, however, a previously verified thromboplastin/instrument combination with preverified ISI and MNPT values. In our study, the originating reference thromboplastin/instrument combination (with preverified ISI and MNPT values) has historically been derived as previously reported, 12,16-18 initially at the ICPMR campus using a combination of different manufacturer INR calibration/certified plasmas and a set of nearly 80 normal plasmas. 12,16,17 This process was subsequently expanded to a small set of laboratories within our network 18 and, in the current report, to the entire network of 27 laboratories. Your Low Your data High Method D 4.6 E 4.6 F low low high high low 4.6 low high high Figure 6 Linear regression curves identifying data from all participant laboratories to the Royal College of Pathologists of Australasia Quality Assurance Program Haematology reporting international normalized ratio (INR) data for the year 2014, to help highlight potential reasons for variable participant results. The results for the Institute of Clinical Pathology and Medical Research (ICPMR) primary coagulation analyzer are shown by the red line (A). The overall linear regression line closely follows the line of equivalence. Importantly, regression comparison between the ICPMR and peer group median values was also highly comparable (B). Sometimes individual participant-derived regression lines appear to bear no close relationship with the median INR values (potentially due to analytical or transcription error; examples arrowed in C). Some regression lines show higher movement away from the line of equivalence at high INR values; in these cases, the assigned international sensitivity index (ISI) values may be either too high (example, thick red line in D) or too low (green line). The thin red line shows the ICPMR primary coagulation analyzer result as comparison. Other regression lines run parallel to the line of equivalence; here, the assigned mean normal prothrombin time (MNPT) values may be either too high (example green line in E) or too low (thick red line), whereas other regression lines may show multiple effects of too high or low ISI and MNPT (examples in F). The thin red line shows the ICPMR primary coagulation analyzer result as comparison. In overview, there are several areas of potential error in INR results, including preanalytical and analytical issues. 8-12,15-19 In a real-world testing, sample integrity is key to accurate test results. In an EQA setting, this is better controlled and ensured by extensive homogeneity and stability testing of proficiency test material but still remains a potential source of error. Another potential source of variation in an EQA setting is sample reconstitution, which is under the control of the operator. Here, the main issues are use of appropriate quality water for reconstitution, standardized water temperature, use of well-calibrated pipettes and proper technique for dispensing the water for reconstituting samples, 198 Am J Clin Pathol 2016;145: American Society for Clinical Pathology 198 DOI: /ajcp/aqv022

9 AJCP /ORIGINAL ARTICLE A INR at Pathology West Site C Difference Line of equivalence RCPAQAP INR Average INR appropriate sample mixing to ensure a homogeneous mixture, and appropriate handling overall. Analytically, coagulation instruments, as used to generate INR values, can in general be identified as very robust. In the laboratory INR setting, there is additional preanalytical variation introduced by the laboratory assignment of the ISI and MNPT. Although the INR calculation was developed to reduce interlaboratory variability, differences between different reagent/instrument combinations still exist, as does the assignment of ISI and MNPT values for different as well as identical reagent/instrument combinations. The CVs for the interlaboratory INR data from the RCPAQAP over the past 4 years show an increase in INR variation from around 5% at INR to approximately 14% at INR. 8 This relationship is likely to reflect a combination of the various methods used by the manufacturer or laboratory for local establishment and application of their reagent ISI and MNPT, as well as noncommutability of INR values for different reagent/instrument combinations. However, we believe that a substantial element of the B INR at Pathology West Site D Difference Line of equivalence RCPAQAP INR Average INR Figure 7 Comparison of international normalized ratio (INR) values reported by Pathology West network sites (n ¼ 27 laboratories with instruments; y-axes) vs overall Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) median INR values (x-axes) before (A) vs after(b) standardization/harmonization and shown as linear regression data. C and D, Same data shown as Bland-Altman plots. Note the clear decrease in variation/bias poststandardization/harmonization. variation is due to incorrect assignment of ISI and MNPT values by many laboratories because of the issues previously noted. Importantly, many different ISI and MNPT values are reported to the RCPAQAP for identical reagent lots/identical instrument combinations (unpublished data from the RCPAQAP), and this will contribute to this trend as increasing INR values reflect increasing variation due to the INR formula being applied. As noted in the Materials and Methods section, the RCPAQAP laboratory INR participants currently comprise 586, with at least 62 different instrument/ reagent combinations. The situation in the United States is not entirely clear, as we could locate only a single recent report on College of American Pathologists (CAP) data. 9 Here, variation in INRreported values was seen to be reducing due to increasing use of thromboplastin reagents with ISI values closer to. 9 However, an evaluation of recent data from CAP by the authors indicates current CVs that are similar to those from other geographies (data not shown). Importantly, there are American Society for Clinical Pathology Am J Clin Pathol 2016;145: DOI: /ajcp/aqv022

10 Favaloro et al /INRHARMONIZATION AND STANDARDIZATION A INR at Pathology West Site RCPAQAP INR Line of equivalence Prestandardization Poststandardization C * PW prestandardization 2 PW poststandardization 1 RCPAQAP all participants RCPAQAP INR Interlaboratory CV (%) only two FDA-cleared certified plasma sets available in the United States, and these are only validated for use for specific manufacturer/instruments. 13 Accordingly, US laboratories are required to use different strategies to verify local ISI and MNPT values, especially when they use other, or disparate, manufactured products. As an example, the US Mayo Network recently reported a strategy that it employed. 15 Essentially, the manufacturer s assigned reagent (RecombiPlasTin 2G; Instrumentation Laboratory, Bedford, MA) ISI was first verified on an ACL TOP 700 (from the same manufacturer); this was defined as the reference method. The ISI was then assigned on three unsupported instruments using orthogonal regression analysis. The MNPT and ISI were subsequently assigned on the STA line of instruments from Stago. Maximum CV CV Minimum CV B INR at Pathology West Site RCPAQAP INR Line of equivalence Prestandardization Poststandardization Figure 8 A and B, Examples of improved international normalized ratio (INR) test performance at two laboratories in the Pathology West network pre- vs poststandardization/harmonization. Note that prestandardization, substantial bias away from Royal College of Pathologists of Australasia Quality Assurance Program (RCPQAP) median values was evident and that this bias was eliminated poststandardization/harmonization. C, Interlaboratory variation (shown as coefficient of variation; CV, %; y- axis) vs reported INR for all RCPAQAP participants, compared with Pathology West (PW) network laboratories pre- and poststandardization/harmonization (note reduction). CVs tend to increase with increasing INR (consistent with data in Figures 3, 6, and 7), but note an area of unusually high CVs (asterisk) where samples were later determined to also contain relatively low fibrinogen levels, causing difficulty for some optical test systems to accurately detect clot end points. The current study provides another option for laboratories. We do not consider our method to represent a definitive method, as validation of the outcome of such changes would require clinical studies assessing clinical outcomes, which is way beyond the scope of this report. The method should also only be used as part of the approach to assignment/verification of ISI and MNPT, as highlighted by Figure 9. We recognize several limitations to our study. First, our comparative assessments were in general made between the median of overall RCPAQAP group data, rather than the median peer group data for each reagent/instrument type, both for ease of analysis and because of small peer group numbers for many previous network reagent/instrument combinations. However, as evidenced for Westmead data (refer to Figures 4 and 6), median peer group data for 200 Am J Clin Pathol 2016;145: American Society for Clinical Pathology 200 DOI: /ajcp/aqv022

11 AJCP /ORIGINAL ARTICLE Manufacturer has assigned an ISI for your PT reagent/instrumentation combination Manufacturer has not assigned an ISI for your PT reagent/instrumentation combination Perform an ISI and MNPT estimation and a (Note: use different/independent procedures current reagent/instrument combinations are similar to overall median group data. Nonetheless, this may not hold true for all peer group medians (in particular, recombinant thromboplastins vary widely from each other and from other nonrecombinant thromboplastins). 19,20 Second, by inference, the ICPMR ISI and MNPT data set acted as the surrogate gold-standard or reference thromboplastin based on outcomes of previously published studies. 12,16-18 Other laboratories attempting these procedures also need to have a gold standard or reference starting point, and so adoption at some laboratories may be difficult. Third, the EQA data set used will have an obvious effect on the outcome of such evaluations and may be biased to particular reagent/instrument combinations. We believe that the larger the data set, the more likely the median value will represent the true value. Conclusions Current and published 12,16-18 reports Existing vs replacement reagent regression (ISI and MNPT) Audit laboratory performance testing (ISI and MNPT) validated with agreement in values using independent procedures plus evidence of Accept ISI and MNPT for lab use We report a substantial improvement in interlaboratory harmonization of INR-reported values in our CLSI recommended procedures Minimum 20 normal samples for (geometric) MNPT Commercial calibration procedure (ISI and sometimes MNPT) Recognized limitations for each individual approach use separate procedures validated (ie, limited or no agreement in values) and/or evidence of poor data to evaluate ongoing performance Recheck ISI and/or MNPT Figure 9 An algorithm that helps describe how the process outlined in this report (as well as our previous reports) 12,16-18 may be used to supplement alternate procedures to estimate/verify international sensitivity index (ISI) and mean normal prothrombin time (MNPT) and may be available at different localities. CLSI, Clinical and Laboratory Standards Institute; EQA, external quality assessment; PT, prothrombin time. network of 27 laboratories. This was achieved by standardization of reagents/equipment, reducing our previous combination of 12 to three currently (Table 1), as well as a novel approach of verification and harmonization of ISI and MNPT values. This reduction in interlaboratory variation in INR reporting should translate to a reduction in variability of reported INR values in patients receiving VKA therapy, who might be tested at different sites in our network, either because they may be traveling within our state or when samples are transferred between different facilities. We believe that this will provide better clinical utility for INR testing for our clients, although proving better clinical utility would require a major clinical study. Finally, these outcomes may also have implications with respect to improvements in maintaining patients within TTR, as well as reducing patient variabilityininr-reportedvalues,whichinturnmayimprove patient outcomes. 7 We also believe that our novel approach to verification of local ISI and MNPT, not requiring ongoing use of INR-certified plasma sets or WHO reference thromboplastin, may be easily adopted for use by other laboratories and networks, particularly in American Society for Clinical Pathology Am J Clin Pathol 2016;145: DOI: /ajcp/aqv022

12 Favaloro et al /INRHARMONIZATION AND STANDARDIZATION localities where provision of INR-certified plasmas may be limited. Corresponding author: Emmanuel J. Favaloro, PhD, FFSc (RCPA), Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, WESTMEAD, NSW, 2145, Australia; health.nsw.gov.au. Acknowledgments: We thank the Royal College of Pathologists of Australasia Quality Assurance Program Haematology for their general collaborative participation, as well as for making available some data reported in this article via their Internet access. We also thank various technical staff within our Pathology West Network who may have contributed technical assistance with testing of international normalized ratio samples as ultimately leading to the findings reported in this article. References 1. Gatt A, Chen D, Pruthi RK, et al. From vitamin K antagonists to liver international normalized ratio: a historical journey and critical perspective. Semin Thromb Hemost. 2014;40: Riva N, Ageno W. Pros and cons of vitamin K antagonists and non vitamin K antagonist oral anticoagulants. Semin Thromb Hemost. 2015;41: McMahon BJ, Kwaan HC. The new or non vitamin K antagonist oral anticoagulants: what have we learned since their debut. Semin Thromb Hemost. 2015;41: Lippi G, Franchini M, Favaloro EJ. Pharmacogenetics of vitamin K antagonists: useful or hype? Clin Chem Lab Med. 2009;47: Ageno W, Gallus AS, Wittkowsky A, et al. Oral Anticoagulant Therapy Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest. 2012;141:e44S-e88S. 6. Senoo K, Lip GYH. Comparative efficacy and safety of the non vitamin K antagonist oral anticoagulants for patients with nonvalvular atrial fibrillation. Semin Thromb Hemost. 2015;41: Razouki Z, Ozonoff A, Zhao S, et al. Improving quality measurement for anticoagulation: adding international normalized ratio variability to percent time in therapeutic range. Circ Cardiovasc Qual Outcomes. 2014;7: Bonar R,Mohammed S,Favaloro EJ. International normalized ratio (INR) monitoring of vitamin K antagonist (VKA) therapy: comparative performance of point of care (POC) and laboratory derived testing. Semin Thromb Hemost 2015;40: Olson JD, Brandt JT, Chandler WL, et al. Laboratory reporting of the international normalized ratio: progress and problems. Arch Pathol Lab Med. 2007;131: Kitchen DP, Jennings I, Kitchen S, et al. Bridging the gap between point of care testing and laboratory testing in haemostasis. Semin Thromb Hemost 2015;40: Clinical and Laboratory Standards Institute (CLSI). Procedures for Validation of INR and Local Calibration of PT/ INR Systems; Approved Guideline. H54-A, Vol. 25, No. 23. Wayne, PA: CLSI; Favaloro EJ, Adcock DM. Standardization of the INR: how good is your laboratory s INR and can it be improved? Semin Thromb Hemost. 2008;34: US Food and Drug Administration. Accessed March 7, Favaloro EJ, Plebani M, Lippi G. Regulation in hemostasis and thrombosis: part I in vitro diagnostics. Semin Thromb Hemost. 2013;39: Tange JI, Grill D, Koch CD, et al. Local verification and assignment of mean normal prothrombin time and international sensitivity index values across various instruments: recent experience and outcome from North America. Semin Thromb Hemost. 2014;40: Favaloro EJ, Hamdam S, McDonald J, et al. Time to think outside the box? Prothrombin time (PT), international normalised ratio (INR), international sensitivity index (ISI), mean normal prothrombin time (MNPT) and measurement of uncertainty (MU): a novel approach to standardisation. Pathology. 2008;40: Favaloro EJ, McVicker W, Hamdam S, Hocker N. Improving the harmonization of the international normalised ratio (INR): time to think outside the box? Clin Chem Lab Med. 2010;48: Favaloro EJ, McVicker W, Zhang Y, et al. Improving the inter-laboratory harmonization of the international normalized ratio (INR): utilizing the concept of transference to estimate and/or validate international sensitivity index (ISI) and mean normal prothrombin time (MNPT) values and/or to eliminate measurement bias. Clin Lab Sci. 2012;25: Smith SA, Morrissey JH. Properties of recombinant human thromboplastin that determine the international sensitivity index (ISI). J Thromb Haemost. 2004;2: Smith SA, Comp PC, Morrissey JH. Phospholipid composition controls thromboplastin sensitivity to individual clotting factors. J Thromb Haemost. 2006;4: Am J Clin Pathol 2016;145: American Society for Clinical Pathology 202 DOI: /ajcp/aqv022