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.     

Clinical utilization of the international normalized ratio (INR)

The prothrombin time (PT) is one of the most important laboratory tests to determine the functionality of the blood coagulation system. It is used in patient care to diagnose diseases of coagulation, assess the risk of bleeding in patients undergoing operative procedures, monitor patients being treated with oral anticoagulant (coumadin) therapy, and evaluate liver function. The PT is performed by measuring the clotting time of platelet-poor plasma after the addition of calcium and thromboplastin, a combination of tissue factor and phospholipid. Intra- and interlaboratory variation in the PT was a significant problem for clinical laboratories in the past, when crude extracts of rabbit brain or human placenta were the only source of thromboplastin. The international normalized ratio (INR), developed by the World Health Organization in the early 1980s, is designed to eliminate problems in oral anticoagulant therapy caused by variability in the sensitivity of different commercial sources and different lots of thromboplastin to blood coagulation factor VII. The INR is used worldwide by most laboratories performing oral anticoagulation monitoring, and is routinely incorporated into dosage planning for patients receiving warfarin. Although the recent availability of sensitive PT reagents prepared from recombinant human tissue factor (rHTF) and synthetic phospholipids eliminated many of the earlier problems associated with the use of crude thromboplastin preparations, local instrument variability in the INR still remains a problem. Presently, the use of plasma calibrants seems the best solution to this problem. Standardizing the point-of-care instruments for INR monitoring is another dilemma faced by the industry. Ultimately, new generations of anticoagulant drugs may eliminate the need for laboratory monitoring of anticoagulant therapy.     

Traces of factor VIIa modulate thromboplastin sensitivity to factors V, VII, X, and prothrombin

BACKGROUND: Thromboplastin reagents are used to conduct prothrombin time (PT) clotting tests to monitor oral anticoagulant therapy and screen for clotting factor deficiencies. Thromboplastins made from purified, recombinant tissue factor are generally more sensitive to changes in plasma factor (F) VII levels than are thromboplastins prepared from tissue extracts. This may be problematic as FVII's short plasma half-life can result in day-to-day fluctuation during oral anticoagulant therapy. We hypothesized that trace contamination of tissue-derived thromboplastins with FVII(a) blunts sensitivity to plasma FVII levels. METHODS: Traces of purified FVIIa were added to thromboplastin reagents prepared using recombinant human tissue factor and the effect on sensitivity to individual clotting factors was quantified in PT clotting assays. RESULTS AND CONCLUSIONS: Adding 5-100 pm FVIIa not only decreased thromboplastin sensitivity to plasma FVII, it surprisingly increased sensitivity to plasma levels of FV, FX and prothrombin. In addition, traces of FVIIa interacted with changes in the salt content and phospholipid composition of recombinant thromboplastins to further modulate their sensitivities to individual clotting factors. These results help explain how thromboplastin reagents of differing composition exhibit differing sensitivities to individual clotting factor levels. Implications of our results for monitoring oral anticoagulant therapy and other uses of the PT assay are discussed.     

乙型病毒性肝炎患者中末期凝血酶原时间标准化报告方式

目的 探讨乙型病毒性肝炎患者凝血酶原时间(PT)的标准化报告方式。方法 选择乙型病毒性肝炎患者61例,其中肝炎后肝硬化41例,慢性重型肝炎20例。20例口服华法令抗凝药患者作为对照组。采用来源不同、ISI值不同的6种凝血活酶试剂进行PT测定,以秒数、比率、活动度百分率以及国际正常化比率4种方式表示PT结果。结果 病毒性肝炎患者PT结果,当以活动度百分率和比率形式表示时,不同凝血活酶试剂之间差异没有显著性意义(F=1.289,P=0.268;F=I.992,,J=3.079),当以秒数和INR报告方式表示时,差异有显著意义(F=8.491,P=0.0001;F=2.497.P=0.031)。通过Neoplastin与其他5种试剂的PT结果作线性回归分析,当结果以活动度百分率表示时,Neoplastin与其他5种试剂之间存在高度一致性;而以秒数,比率和INR表示时,试剂之间不存在一致性。提示PT活动度百分率能使乙型病毒性肝炎中末期患者PT报告方式标准化。口服抗凝剂治疗的患者仅INR能使PT的报告方式标准化。结论 PT活动度百分率能使乙型病毒性肝炎中末期患者PT报告方式标准化,INR仅适用于抗凝治疗患者PT结果的报告。     

肝衰竭患者凝血酶原时间报告形式的探讨

目的 :探讨在肝衰竭病人的凝血酶原时间 (PT)以秒数、比率、活动百分率计 ,还是以国际正常化比率(INR)表示。方法 :采用 ISI 1.11,1.76和 2 .0 5的三种凝血活酶对 5 2例肝衰竭病人和 5 0例口服华法令的换瓣术后的病人进行 PT测定。以上述四种方式表示结果。结果 :肝衰竭病人 ,PT百分率能消除变异的可能性 (P<0 .0 5 ) ,而其他表示形式仍有明显的差异 (P>0 .0 1)。患者口服抗凝治疗后仅 INR能较准确地反映 PT的结果。结论 :INR不能用于表达非抗凝治疗患者的 PT结果。 PT活动的百分率是为肝衰竭病人的最好报告形式。     

International normalized ratio in the prothrombin test: clinical significance and use

The safety and efficiency of therapy by peroral anticoagulants (PA) depend on a laboratory monitoring based on the prothrombin test (PT). The test is distinguished through its variability conditioned by different means of results' presentation as well as through the sensitivity of thromboplastin and a type of a device used in coagulation detection. WHO recommended, 1983, to standardize the thromboplastin preparations through adjusting their sensitivity (the so-called International Sensitivity Index--ISI) to blood coagulation defects induced by PA versus the primary international reference thromboplastin. Thromboplastin ISI as well as the mean normal prothrombin time (MNPT) of blood plasma are used to calculate the international normalized ratio (INR). The presentation of PT results as INR is justified exclusively for the PA-therapy stabilized patients. The INR system makes it possible to optimize the PA therapy only if the laboratory expert and clinician can clearly understand the PT standardization essence and observe the key WHO recommendations, i.e. definition of a coagulometer-specific ISI by manufacturing companies, estimation of MNPT by laboratories and use of the correct anticoagulant concentration.     

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.     

Comparison of local International Sensitivity Index calibration and ''Direct INR'' methods in correction of locally reported International Normalized Ratios: an international study

BACKGROUND: It is no longer feasible to check local International Normalized Ratios (INR) by the World Health Organization International Sensitivity Index (ISI) calibrations because the necessary manual prothrombin time technique required has generally been discarded. OBJECTIVES: An international collaborative study at 77 centers has compared local INR correction using the two alternative methods recommended in the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis guidelines: local ISI calibration and 'Direct INR'. METHODS: Success of INR correction by local ISI calibration and with Direct INR was assessed with a set of 27 certified lyophilized plasmas (20 from patients on warfarin and seven from normals). RESULTS: At 49 centers using human thromboplastins, 3.0% initial average local INR deviation from certified INR was reduced by local ISI calibration to 0.7%, and at 25 centers using rabbit reagents, from 15.9% to 7.5%. With a minority of commercial thromboplastins, mainly 'combined' rabbit reagents, INR correction was not achieved by local ISI calibration. However, when rabbit combined reagents were excluded the overall mean INR deviation after correction was reduced further to 3.9%. In contrast, with Direct INR, mean deviation using human thromboplastins increased from 3.0% to 6.6%, but there was some reduction with rabbit reagents from 15.9% to 10% (12.3% with combined reagents excluded). CONCLUSIONS: Local ISI calibration gave INR correction for the majority of PT systems but failed at the small number using combined rabbit reagents suggesting a need for a combined reference thromboplastin. Direct INR correction was disappointing but better than local ISI calibration with combined rabbit reagents. Interlaboratory variability was improved by both procedures with human reagents only.     

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.     

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.     

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.     

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.     

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     

Comparison of quick and owren prothrombin time with regard to the harmonisation of the International Normalised Ratio (INR) system.

Prothrombin time (PT) is tested mostly to monitor patients on oral anticoagulant treatment. The International Normalised Ratio (INR) was introduced to improve and harmonise PT results and therapeutic range globally for patient care and the scientific literature. We studied the Quick PT in 179 patients and the Owren PT in 137 patients on oral anticoagulant therapy using two different reagents for the two methods of measuring PT. We assessed the clinical significance of the INR results obtained by each method using the two reagents and compared the Quick and Owren methods. We conclude that with the Quick method individual INR results differed from each other too much clinically, while using the Owren method individual INR results were clinically acceptable. Our opinion is that we should develop the INR system using the Owren PT method rather than the Quick to improve patient care.     

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.     

Poor agreement among prothrombin time international normalized ratio methods: comparison of seven commercial reagents

BACKGROUND: Prothrombin time (PT) has long been the most popular test for monitoring oral anticoagulation therapy. The International Normalized Ratio (INR) was introduced to overcome the problem of marked variation in PT results among laboratories and the various recommendations for patient care. According to this principle, all reagents should be calibrated to give identical results and the same patient care globally. This is necessary for monitoring of single patients and for application of the results of anticoagulation trials and guidelines to clinical practice. METHODS: We took blood samples from 150 patients for whom oral anticoagulation had been prescribed. Plasmas were separated and PTs determined by use of seven commercial reagents and four calibrator sets. The differences in results were assessed by plotting, for each possible pair of methods, the differences in INR values for each sample against the mean INR value (Bland-Altman difference plots). RESULTS: Mean results differed significantly (P <0.001) for 17 of 21 possible paired comparisons of methods. Only two pairs of methods produced very similar results when assessed for problems of substantial differences in INR values; a significant, systematic increase in the difference with INR; and a significant systematic increase in the variation in difference with increasing INR values. CONCLUSIONS: The agreement among several (and perhaps most) commercial INR methods is poor. The failure of current calibration strategies may severely compromise both the monitoring of individual patients and the application of oral anticoagulation guidelines and trial results to clinical practice.     

广东省凝血酶原时间测定的室间质量评价

目的 探讨广东省临床实验室监测凝血酶原时间（PT）的检测状况及影响PT测定室间可比性的因素.方法 通过每年两次定期向全省参评实验室寄发质控样品（每次5个样品）,然后对其回报的数据进行统计分析,作出实验室检验水平的评价.结果 近年来全省实验室间PT（INR）测定结果的CV值尽管有逐渐下降的趋势,但室间变异仍然相当高.同一凝血活酶试剂的测定结果,PT（INR）的CV明显大于PT（sec）的CV,特别是对异常水平质评物的测定.结论 凝血活酶试剂敏感度指数（ISI）值标定的不准确性和INR计算不正确的是引起广东省临床实验室间凝血酶原时间测定变异大的主要原因.     

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。     

Effect of lepirudin on the international normalized ratio

BACKGROUND: Many patients receiving direct thrombin inhibitor (DTI) therapy require transition to warfarin. This transition may be complicated by DTI-induced elevations in the international normalized ratio (INR). While the effect of argatroban on the INR has been characterized, data assessing the effect of lepirudin on the INR are limited. OBJECTIVE: To evaluate the effect of lepirudin on the INR. METHODS: Patients receiving lepirudin therapy between January 2000 and May 2001 were identified using the pharmacy database, and a retrospective chart review was conducted. Patients were included for analysis if they had paired activated partial thromboplastin time (aPTT) and INR data while receiving lepirudin monotherapy. RESULTS: Fifty-three paired aPTT and INR data points from 8 patients receiving lepirudin monotherapy were collected. The Organon MDA 180 instrument was used for aPTT and prothrombin time (PT) determination. Organon MDA Platelin L reagent was used for the aPTT and Organon Simplastin L reagent was used for the PT. The international sensitivity index (ISI) of the Simplastin L thromboplastin was 2.0. The mean +/- SD lepirudin dose was 0.05 +/- 0.04 mg/kg/h. Linear regression was used to identify the INRs that correspond to a therapeutic aPTT value of 45-75 seconds (1.5-2.5 times mean laboratory normal of 30 sec). The correlation between aPTT and INR was 0.77. An aPTT of 45-75 seconds with lepirudin correlated to an INR of 1.6-3.2. CONCLUSIONS: Based on laboratory results, when using a thromboplastin with an ISI of 2, lepirudin appears to elevate the INR in the absence of warfarin.     

仪器对凝血酶原时间国际标准化比值的影响及校正

为了评价血凝仪对国际标准化比值的影响。模拟ＷＨＯ标定凝血质的参比方法，用一种已 知国际敏感指标的凝血质对两种血凝仪的特异性ＩＳＩ进行标定，并以ＩＮＲ形式报告ＰＴ结果。