The prothrombin time/international normalized ratio (PT/INR) Line: derivation of local INR with commercial thromboplastins and coagulometers – two independent studies

Summary. Background: The WHO scheme for prothrombin time (PT) standardization has been limited in application, because of its difficulties in implementation, particularly the need for mandatory manual PT testing and for local provision of thromboplastin international reference preparations (IRP). Methods: The value of a new simpler procedure to derive international normalized ratio (INR), the PT/INR Line, based on only five European Concerted Action on Anticoagulation (ECAA) calibrant plasmas certified by experienced centres has been assessed in two independent exercises using a range of commercial thromboplastins and coagulometers. INRs were compared with manual certified values with thromboplastin IRP from expert centres and in the second study also with INRs from local ISI calibrations. Results: In the first study with the PT/INR Line, 8.7% deviation from certified INRs was reduced to 1.1% with human reagents, and from 7.0% to 2.6% with rabbit reagents. In the second study, deviation was reduced from 11.2% to 0.4% with human reagents by both local ISI calibration and the PT/INR Line. With rabbit reagents, 10.4% deviation was reduced to 1.1% with both procedures; 4.9% deviation was reduced to 0.5% with bovine/combined reagents with local ISI calibrations and to 2.9% with the PT/INR Line. Mean INR dispersion was reduced with all thromboplastins and automated systems using the PT/INR Line. Conclusions: The procedure using the PT/INR Line provides reliable INR derivation without the need for WHO ISI calibration across the range of locally used commercial thromboplastins and automated PT systems included in two independent international studies.     

Coagulometer international sensitivity index (ISI) derivation,a rapid method using the prothrombin time/international normalized ratio (PT/INR) Line: a multicenter study

Summary. Background: The original WHO procedure for prothrombin time (PT) standardization has been almost entirely abandoned because of the universal use of PT coagulometers. These often give different international normalized ratio (INR) results from the manual method, between individual makes of instruments and with instruments from the same manufacture. Method A simple procedure is required to derive local INR with coagulometers. The PT/INR Line method has recently been developed using five European Concerted Action on Anticoagulation (ECAA) certified plasmas to derive local INR. This procedure has been modified to derive a coagulometer PT/INR Line providing International Sensitivity Index (ISI) and mean normal PT (MNPT) for coagulometers and give local INR. Results have been compared with conventional ISI calibrations at the same laboratories. Results: With human thromboplastins, mean ISI by local calibration was 0.93 (range: 0.77–1.16). With the PT/INR Line, mean coagulometer ISI was higher, for example 0.99 (0.84–1.23) but using the PT/INR Line derived MNPT there was no difference in local INR. Between‐centre INR variation of a certified validation plasma was reduced with human and bovine reagents after correction with local ISI calibrations and the PT/INR Line. Conclusion: The PT/INR Line–ISI with its derived MNPT is shown to provide reliable local INR with the 13 different reagent/coagulometer combinations at the 28 centres in this international study.     

International collaborative study for the calibration of proposed International Standards for thromboplastin,rabbit, plain,and for thromboplastin,recombinant, human,plain

Essentials

• Two candidate International Standards for thromboplastin (coded RBT/16 and rTF/16) are proposed.
• International Sensitivity Index (ISI) of proposed standards was assessed in a 20‐centre study.
• The mean ISI for RBT/16 was 1.21 with a between‐centre coefficient of variation of 4.6%.
• The mean ISI for rTF/16 was 1.11 with a between‐centre coefficient of variation of 5.7%.

Summary

Background

The availability of International Standards for thromboplastin is essential for the calibration of routine reagents and hence the calculation of the International Normalized Ratio (INR). Stocks of the current Fourth International Standards are running low. Candidate replacement materials have been prepared. This article describes the calibration of the proposed Fifth International Standards for thromboplastin, rabbit, plain (coded RBT/16) and for thromboplastin, recombinant, human, plain (coded rTF/16).

Methods

An international collaborative study was carried out for the assignment of International Sensitivity Indexes (ISIs) to the candidate materials, according to the World Health Organization (WHO) guidelines for thromboplastins and plasma used to control oral anticoagulant therapy with vitamin K antagonists.

Results

Results were obtained from 20 laboratories. In several cases, deviations from the ISI calibration model were observed, but the average INR deviation attributabled to the model was not greater than 10%. Only valid ISI assessments were used to calculate the mean ISI for each candidate. The mean ISI for RBT/16 was 1.21 (between‐laboratory coefficient of variation [CV]: 4.6%), and the mean ISI for rTF/16 was 1.11 (between‐laboratory CV: 5.7%).

Conclusions

The between‐laboratory variation of the ISI for candidate material RBT/16 was similar to that of the Fourth International Standard (RBT/05), and the between‐laboratory variation of the ISI for candidate material rTF/16 was slightly higher than that of the Fourth International Standard (rTF/09). The candidate materials have been accepted by WHO as the Fifth International Standards for thromboplastin, rabbit plain, and thromboplastin, recombinant, human, plain.     

Guidelines on preparation, certification, and use of certified plasmas for ISI calibration and INR determination.

Reliable international normalized ratio (INR) determination depends on accurate values for international sensitivity index (ISI) and mean normal prothrombin time (MNPT). Local ISI calibration can be performed to obtain reliable INR. Alternatively, the laboratory may determine INR directly from a line relating local log(prothrombin time [PT]) to log(INR). This can be done by means of lyophilized or frozen plasmas to which certified values of PT or INR have been assigned. Currently there is one procedure for local calibration with certified plasmas which is a modification of the WHO method of ISI determination. In the other procedure, named 'direct' INR determination, certified plasmas are used to calculate a line relating log(PT) to log(INR). The number of certified plasmas for each procedure depends on the method of preparation and type of plasma. Lyophilization of plasma may induce variable effects on the INR, the magnitude of which depends on the type of thromboplastin used. Consequently, the manufacturer or supplier of certified plasmas must assign the values for different (reference) thromboplastins and validate the procedure for reliable ISI calibration or 'direct' INR determination. Certification of plasmas should be performed by at least three laboratories. Multiple values should be assigned if the differences between thromboplastin systems are greater than 10%. Testing of certified plasmas for ISI calibration may be performed in quadruplicate in the same working session. It is recommended to repeat the measurements on three sessions or days to control day-to-day variation. Testing of certified plasmas for 'direct' INR determination should be performed in at least three sessions or days. Correlation lines for ISI calibration and for 'direct' INR determination should be calculated by means of orthogonal regression. Quality assessment of the INR with certified plasmas should be performed regularly and should be repeated whenever there is a change in reagent batch or in instrument. Discrepant results obtained by users of certified plasmas should be reported to manufacturers or suppliers.     

Properties of recombinant human thromboplastin that determine the International Sensitivity Index (ISI)

Prothrombin Time (PT) clotting tests are widely used to monitor oral anticoagulation therapy and to screen for clotting factor deficiencies. The active ingredient in PT reagents (thromboplastins) is tissue factor, the integral membrane protein that triggers the clotting cascade through the extrinsic pathway. Several years ago, a system for calibrating and using thromboplastin reagents, known as the International Sensitivity Index (ISI) and the International Normalized Ratio (INR), was developed to standardize monitoring of oral anticoagulant therapy. The ISI/INR method, while revolutionizing the monitoring of coumarin therapy, has been criticized for a number of perceived shortcomings. We have undertaken a series of studies aimed at achieving a detailed understanding of which parameters influence the ISI values of thromboplastin reagents, with an ultimate goal of creating 'designer thromboplastins' whose sensitivities to the various clotting factors can be individually tailored. In this study, we demonstrate that ISI values of thromboplastin reagents based on relipidated, recombinant human tissue factor can be controlled by a combination of changes in the phospholipid content (in particular, the levels of phosphatidylserine and phosphatidylethanolamine) and ionic strength. The sensitivity of a given thromboplastin reagent can be increased (i.e. its ISI value decreased) by decreasing the content of phosphatidylserine and/or increasing the ionic strength. The molar ratio of phospholipid to tissue factor, on the other hand, had essentially no impact on ISI value.     

Phospholipid composition controls thromboplastin sensitivity to individual clotting factors

BACKGROUND: Tissue factor is the active ingredient in thromboplastin reagents used to perform prothrombin time (PT) clotting tests to monitor oral anticoagulant therapy and to screen for clotting factor deficiencies. Thromboplastins are complex mixtures prepared from extracts of brain or placenta, although newer thromboplastins contain recombinant tissue factor incorporated into phospholipid vesicles. Thromboplastins can vary widely in their sensitivity to reductions in the levels of vitamin K-dependent clotting factors. A system to compensate for this, the International Sensitivity Index (ISI) and International Normalized Ratio (INR), has revolutionized the monitoring of oral anticoagulant therapy. The INR system is also sometimes used to monitor coagulopathies in patients with sepsis or liver failure, applications for which it was not originally designed and for which it has not been rigorously validated. OBJECTIVES: To better understand thromboplastin performance, we systematically investigated which properties of recombinant thromboplastins influence their sensitivities to changes in the levels of specific clotting factors. RESULTS: We now report that relative sensitivities to changes in the plasma levels of factors V, VII, X (FV, FVII, FX) and prothrombin are differentially influenced by a recombinant thromboplastin's content of phospholipid and sodium chloride. Furthermore, thromboplastins of similar ISI values may exhibit quite different sensitivities to each of these clotting factors. CONCLUSIONS: Differing sensitivities of thromboplastin reagents to individual clotting factor levels have implications for monitoring of oral anticoagulant therapy and interpreting results of the PT assay.     

凝血活酶参考品国际敏感度指数的标定

目的标定出凝血活酶参考品（ＷＲＰ），为国内凝血活酶产品国际敏感度指数（ＩＳＩ）的标定提供依据。方法以凝血活酶国际参考品为标准（ＩＲＰ），按照世界卫生组织（ＷＨＯ）推荐的凝血活酶国际参考品的使用方法进行测定，计算出被标定凝血活酶参考品的ＩＳＩ值和标定ＩＳＩ值的变异系数（ＣＶ）。结果标出的凝血活酶参考品的ＩＳＩ值为１．３５，标定ＩＳＩ值的ＣＶ为４．３％。符合ＷＨＯ的要求（ＣＶ≤５％）。结论用ＩＲＰ标定的凝血活酶参考品可作为国内凝血活酶试剂ＩＳＩ值标定的标准。     

European Concerted Action on Anticoagulation. Evaluation of a method for International Sensitivity Index calibration of two point-of-care prothrombin time (PT) monitoring systems (CoaguChek Mini and TAS PT-NC) with fresh plasmas based on whole-blood equivalent PT

BACKGROUND: The International Sensitivity Index (ISI) calibration of whole-blood prothrombin time (PT) monitors for point-of-care testing (POCT) described by Tripodi et al. (Thromb Haemost 1993;70:921-4) has been shown to be dependable but is too complex and demanding. The use of plasma would simplify calibration of whole-blood POCT PT monitors, but important differences may exist between the ISI for whole blood and plasma calibrations. METHODS: In a 10-center calibration study of two POCT whole-blood monitoring systems (CoaguChek Mini and TAS PT-NC), we characterized the relationship between the log PT for whole blood and fresh plasma with use of single lots of test strips/cards. This relationship (linear) was used to correct the difference between the whole-blood and plasma ISI. The reliability of the correction with different lots of test strips/cards was assessed at three centers. The linear relationship was used to correct the difference in the whole-blood and plasma ISI with four other lots of TAS PT-NC cards and with two additional lots of CoaguChek Mini test strips. RESULTS: The correction decreased the ISI difference from 13.3% to 0.9% for the TAS PT-NC and from 5.7% to 0.6% for the CoaguChek Mini. In assessments at three centers, which included different lots of test strips/cards, the mean ISI difference was markedly decreased with the TAS PT-NC but not with the CoaguChek Mini, for which the mean ISI difference increased slightly. CONCLUSIONS: The proposed correction resolves the discrepancy between whole-blood and fresh plasma ISI calibrations with TAS PT-NC test cards. The CoaguChek Mini systems could be calibrated without this correction.     

European Concerted Action on Anticoagulation (ECAA): an assessment of a method for ISI calibration of two whole blood point-of-care PT monitor systems based on lyophilized plasmas using whole blood equivalent PT

Summary.  Previously, the attempt to simplify the International Sensitivity Index (ISI) calibration of the CoaguChek Mini whole blood point-of-care test prothrombin time (PT) monitor system was successful using lyophilized plasmas from coumarin-treated patients but not with lyophilized artificially depleted plasmas. With the TAS PT-NC monitor system, both types of plasma failed to provide reliable calibrations. The present study assesses a procedure for the ISI calibration of a TAS PT-NC and CoaguChek Mini whole blood point-of-care test PT monitor systems using lyophilized plasmas. Using lyophilized artificially depleted and coumarin plasma calibrations, we have evaluated a correction for the monitor displayed PT. This was based on a 'line of equivalence' derived from the relationship between whole blood and fresh plasma PT with both types of monitor system. With the TAS PT-NC, the use of this 'line of equivalence' resulted in reliable ISI with both lyophilized coumarin and artificially depleted plasmas. There was no significant difference between mean monitor and mean reference International Normalized Ratio (INR) with the artificially depleted plasmas. With the lyophilized coumarin plasma calibrations there was only a small INR difference. Correction with the 'line of equivalence' therefore facilitates calibration of the TAS PT-NC with lyophilized plasmas. With the CoaguChek Mini, the correction based on the 'line of equivalence' did not improve results but was not required with this system.     

A comparison of INRs after local calibration of thromboplastin international sensitivity indexes.

There are approximately 300 reagent/instrument combinations for performing prothrombin times/international normalized ratios (PT/INR) in the United States. Manufacturers and laboratories continually struggle to ensure that the International Sensitivity Index (ISI) of their thromboplastin is accurate for assaying PT/INR. OBJECTIVE: This study reports the feasibility of a new method to locally calibrate ISI of thromboplastin using the mechanical STA automated coagulation analyzer (Diagnostica-Stago Inc.) and two photo-optic coagulation analyzers, the BCS (Dade-Behring) and CA-540 (Sysmex). DESIGN: Neoplastine CI+ (CI+) (Diagnostica-Stago Inc); Thromboplastin C+ (TC+); Thromborel S (TRS); and Innovin (I) (Dade-Behring) were used in this study. A mean normal PT (MNPT) was determined for each reagent/instrument combination using samples from 25 normal individuals. Manufacturer instrument specific ISI values were not available for the STA with TC+, TRS and I. The CA540 had no ISI value for CI+ and the BCS system had no manufacturer assigned ISI values for TC+ and I; generic photo-optic and mechanical ISI manufacturer values were used for these two systems. Local on-site calibration was performed using frozen plasma calibrators to determine ISI values for each thromboplastin. Post-calibration, 95 patient samples were assayed for each reagent/instrument system combination using the manufacturer ISI and the local calibrated ISI to determine the INR result. PATIENTS: Patients from whom samples were obtained included five with a lupus anticoagulant, 30 on heparin therapy, and 60 on coumadin therapy. RESULTS: Differences between manufacturer versus local calibrated ISI ranged from 0.9% to 18.9% for normal sample INRs and from 0.8% to 16.4% for patient sample INRs. The number (or proportion) of patient specimens with clinically significantly different INR values (>10.0% difference) ranged from zero for several reagent combinations to more than half (or >50.0%) of those tested for several other combinations. CONCLUSION: Our results indicated that by locally calibrating ISI values, each laboratory may eliminate variability and guesswork between different reagent/instrument systems for ISI values when performing PT/INR assays and potentially improve the clinical accuracy of their patients' PT/INR results.     

凝血酶原时间ISI/INR系统测定中的一些问题及改进意见

目的对凝血酶原时间(PT)测定ISI/INR系统出现的一些问题提出相应的改进建议。方法对上海市12家医院在用的仪器和匹配的PT试剂,对日常使用的正常血浆平均凝血酶原时间(MNPT)作调研实测,将结果进行分析;调查试剂的仪器特定(spec ific)国际敏感度指数(ISI)值与世界卫生组织(WHO)的手工法ISI定标值之间的差异;用2种已知国际标准化比值(INR)的异常参比血浆代替WHO的ISI系统作质控并行比较。结果12家中有4家日常使用的平均正常凝血酶原时间(MNPT)明显偏离实测值,分别为0.8、0.9、1.0和1.8 s。用WHO CRM149R参比,用手工法标定的凝血活酶和109 mmol枸橼酸钠抗凝的不同PT值血标本,在Sysm ex1500型、C.2000型仪器上测定试剂的仪器特定ISI,其结果比手工法分别减少4.1%和4.7%,但采用HEPES-枸橼酸钠抗凝剂标本时,2种型号仪器的特定ISI比手工法分别减少16.7%及7.7%。用已知INR异常参比血浆,国产品与进口品对照的结果良好。结论受调研12家中,有4家血凝分析仪器调研时实测的MNPT明显偏离日常使用值。有几家医院试剂的仪器特定ISI值也存在问题,建议纠正。用已知INR异常参比血浆代替WHO手工法标定凝血活酶ISI法作质控,使用简便,又不需MNPT参数,值得推广。     

凝血质的标定及凝血酶原时间标准化的临床应用

本文根据ＷＨＯ参比方法用已知国际敏感度指数的凝血质对两种未知ＩＳＩ的凝血质进行了标定，并以标准形式报告ＰＴ结果。     

International multicenter international sensitivity index (ISI) calibration of a new human tissue factor thromboplastin reagent derived from cultured human cells

Summary.  The international sensitivity index (ISI) of the first working standard of Simplastin HTF, a new human tissue factor thromboplastin derived from cultured human cells, has been assessed in a calibration exercise in two Canadian and five European laboratories. Calibrations against international reference preparations (IRP) were performed for the manual method and six types of automated coagulometers that cover the majority of clotting endpoint principles in routine use. The ISI was method-dependent and varied between 1.03 and 1.29 when calibrated against rTF/95 (human IRP). The ISI was also dependent on the route of calibration. Compared with calibration against rTF/95, the ISIs obtained by calibration against RBT/90 (rabbit IRP) were on average 4.4% higher ( P  < 0.005). Considering the principle of 'like vs. like', the ISIs obtained by calibration against rTF/95 should be preferred.     

凝血活酶敏感指数对凝血酶原时间测定的影响

本文观察了四种不同国际敏感指数的凝血活酶试剂，在相同条件下，对２５例正常人和５０例口服华法令病人进行了ＰＴ测定，结果显示正常参考值随所用凝血活酶的ＩＳＩ不同而异。建议各实验室应依其所用试剂建立自己实验室的正常参考值。对口服抗凝药的５０例患者测得ＰＴｓ（秒）和ＰＴＲ（比率）经方差分析，四组结果有显著性差异（Ｐ〈０．０１），而按ＩＮＲ＝ＰＴＲ＾ＩＳＩ换算成国际标准化比率（ＩＮＲ）后，经方差分析，四组Ｉ     

The effect of instruments for prothrombin time testing on the international normalized ratio

Prothrombin time (PT) testing is used for monitoring oral anticoagulant therapy, its result being usually expressed as international normalized ratio (INR). This is done using the international sensitivity index (ISI) specific of thromboplastin employed to carry out the test. In this way a good PT standardization may be achieved although the instruments used to calibrate the thromboplastins might influence the ISI value. Presented at the ‘2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implications for the Clinical Laboratory’, Rome, September 28–29, 1989.     

Quality assessment of CoaguChek point-of-care prothrombin time monitors: comparison of the European community-approved procedure and conventional external quality assessment

BACKGROUND: There is a need for dependable quality assessment (QA) of the widely used CoaguChek point-of-care testing prothrombin time monitor. By use of the prescribed set of 5 CoaguChek certified international normalized ratio (INR) QA plasmas, we compared the reliability of the immediate QA of individual monitors described in the European Community-recommended Technology Implementation Plan with conventional external QA analysis. METHODS: Experienced staff tested CoaguChek point-of-care monitors in routine use for controlling oral anticoagulant dosage at 9 Netherlands Thrombosis Service Centres. Testing was performed with both the certified CoaguChek INR for a set of 5 QA individual plasmas from the Eur Con Action on Anticoag (ECAA) and conventional external QA analysis. RESULTS: Patients brought 523 CoaguChek monitors to our service centers for assessment. The proportion with unsatisfactory performance indicated by a 15% deviation from the ECAA set was compared with 15% deviation from overall median INR of all CoaguChek monitors in the survey, as in conventional QA analysis. The results were similar (20.3% and 18.5%, respectively). Interlot differences of CoaguChek test strips were detected, but the incidence of unsatisfactory performance was similar with both analyses, from 6.5% to 37.5% with the certified INR method and from 5.9% to 33.3% with the overall median analysis. CONCLUSIONS: The results validate the use of the European Action on Anticoagulation rapid single-instrument QA-specific procedure for CoaguChek users compared with the nonspecific conventional QA analysis that relies on deviation from the overall median INR.     

肝病患者凝血酶原时间报告方式的评价

目的 评价凝血酶原时间（PT）作为判断肝病患者细胞损伤程度实验指标的两种报告方式。方法 选择肝病患者104例,其中肝炎肝硬化37例,慢性肝炎32例,肝癌36例,急性肝炎35例。30例口服华法林病人做对照组。采用ISI值不同的2种凝血活酶试剂进行PT测定。结果 急性黄疸性肝炎患者,二种凝血活酶试剂测定的INR结果有显著性差异;慢性活动性肝炎、肝硬化、肝癌患者,二种凝血活酶试剂测定的国际标准化比值（INR）结果无明显差异。急性黄疸性肝炎、肝癌患者,二种凝血活酶试剂测定的活动度（PTA）结果有显著性差异。结论 目前尚不能否定INR可作为肝病病情估计时的门标准化报告方式;PT并不是评价肝病病情的最佳指标;PTA仍为目前门作为肝病病情估计时的首选报告方式。     

血浆凝血酶原时间测定中建立仪器区域国际敏感指数的实验探讨

目的　探讨临床口服抗凝治疗时血浆凝血酶原时间 (PT)监测的标准化 ,为临床提供比较准确的PT 国际标准化比值 (PT INR)结果。方法　使用PT INR校准血浆建立PT测定试剂在不同仪器上的区域性国际敏感度指数 (LocalISI) ,以此对新鲜血浆进行PT测定。结果　未经LocalISI校准的PT试剂测定血浆的PT时 ,INR结果差异较大 (P <0 .0 1)。试剂经LocalISI校准后 ,测定血浆PT时 ,INR结果良好 (P >0 .0 5及Kap pa >0 .75 )。 结论　在PT测定时 ,只要建立仪器和测定试剂的LocalISI ,INR结果就具有较好的一致性。     

Use of lyophilized calibrant plasmas for simplified international normalized ratio determination with a human tissue factor thromboplastin reagent derived from cultured human cells

BACKGROUND: For monitoring of treatment with oral anticoagulants, the clotting time obtained in the prothrombin time (PT) test is transformed to the International Normalized Ratio (INR) with use of a system-specific International Sensitivity Index (ISI). The calibrant plasma procedure (CPP) is an alternative approach to INR calculation based on the use of a set of lyophilized plasmas with assigned INRs. METHODS: With the CPP, a linear relationship is established between log(PT) and log(INR), using orthogonal regression. CPP was validated for Simplastin HTF, a new human tissue factor reagent derived from cultured human cells. CPP precision was assessed as the CV of the slope of the regression line. The accuracy of the CPP was determined by comparing the INR obtained with the CPP with that obtained with the established ISI-based reference method. INRs of the calibrants were assigned by different routes: by manufacturer (consensus labeling) or by use of Simplastin HTF or International Reference Preparations (IRPs; rTF/95 or RBT/90). RESULTS: The mean CV of the CPP regression slope ranged from 1.0% (Simplastin HTF reagent-specific INR) to 2.4% (INR assigned with rTF/95). INRs calculated with the CPP were similar to those obtained with the reference method, but when the routes for assigning INRs to the calibrant plasmas were compared, the mean difference in INR between CPP and the reference method was smaller with Simplastin HTF reagent-specific values. In several (but not all) cases, this difference was significant (P <0.05, t-test). CONCLUSION: CPP can be used for local INR determination, but better precision and accuracy are obtained with reagent-specific INRs compared with INR assignment by consensus labeling or IRP.