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.     

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.     

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。     

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.     

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     

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.     

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.     

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。     

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.     

初诊2型糖尿病患者胰岛素强化治疗对凝血功能的影响

目的观察初诊2型糖尿病患者经胰岛素强化治疗对凝血功能的影响。方法将新诊断的2型糖尿病患者使用胰岛素强化治疗前及治疗后3个月。分别抽取肘静脉血,进行凝血功能测定,取毛细血管血做糖化血红蛋白测定。结果初诊2型糖尿痛患者经胰岛素强化治疗后糖化血红蛋白（HbAlc）明显下降,凝血酶原时间（PT）延长、部分活化凝血活酶时间（删）延长、国际标准化比值（矾R）延长、凝血活酶时间（TT）无明显改变。结论初诊2型糖尿病患者短期胰岛素强化治疗有利于改善凝血功能。     

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。     

A review of variables affecting PTs/INRs.

The prothrombin time is a common method of monitoring patients undergoing oral anticoagulant therapy. The proliferation of commercial thromboplastin brands with different international sensitivity indices (ISI) in conjunction with wider availability of automated coagulation analyzers has elevated the need for standardization in monitoring therapy.     

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。     

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.     

Reference intervals for coagulation tests in adults with different ABO blood types

IntroductionCoagulation tests are affected by many factors, such as age, race, and gestation. Although coagulation test results vary by ABO blood type, reference intervals of different ABO blood groups remain to be determined. This study aims to investigate the reference ranges of coagulation tests for different ABO blood groups in the Han population in South China.MethodsA retrospective study was conducted in the First Affiliated Hospital of Shantou University Medical College. In all, 9600 individuals aged between 20 and 79 years were included. Coagulation tests, including prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), thrombin time, and fibrinogen, were performed.ResultsThere was a significant difference in PT, INR, and aPTT among ABO blood groups. PT and INR varied slightly between ABO blood groups. There was a higher aPTT value in individuals in the O blood group than in those in non‐O blood groups, in both males and females across the included age range. No differences were found in thrombin time and fibrinogen between the ABO blood groups.ConclusionThe study provides reference data on coagulation tests from ABO blood groups in South China. The established reference intervals specific to ABO blood type, sex, and age may improve clinical decisions based on coagulation tests.     

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.     

Variability of prothrombin time and activated partial thromboplastin time in the diagnosis of increased surgical bleeding

BACKGROUND: Prothrombin time (PT) and activated partial thromboplastin time (APTT) are used to diagnose causes of increased surgical bleeding and to guide treatment of acquired coagulation factor deficiency. This study compared the sensitivity of various commercial PT and APTT tests in patients with dilutional coagulopathy. STUDY DESIGN AND METHODS: A prospective study was used to identify patients who experienced increased surgical bleeding during elective extensive (>10 spinal segments) spinal fusion and instrumentation. In patients with clinical signs of increased bleeding, blood was obtained to compare the sensitivity of various commercial PT and APTT tests. PT, PT ratio, the International Normalized Ratio (INR), APTT, and APTT ratio were compared for their sensitivity in the diagnosis of a dilutional coagulopathy. RESULTS: Sixteen patients experienced increased bleeding during surgery. Mean estimated blood volume lost exceeded 1 blood volume (1.14 +/− 0.28). PT and APTT test results varied markedly. In the most sensitive PT and APTT tests, the results were 1.5 times the mean reference range values in all but on of the patients. The least sensitive combination of tests had results that were 1.5 times the mean reference range values in only 2 of 16 patients. Variability among tests was not reduced by the use of the PT or the APTT ratio, by the use of INR, or by incorporation of a measure of PT or APTT test sensitivity to factor-deficient serum. CONCLUSION: In surgical patients with dilutional coagulopathy, diagnostic and treatment decisions could depend on which PT and APTT test was used to determine the etiology of increased bleeding. This study indicates that the relationship between increased bleeding and an increased PT and APTT may be more difficult to define than is suggested by current practice guidelines. Each laboratory must establish guidelines based on reagent and instrument sensitivity to coagulation factor dilution.     

Laboratory control over warfarin therapy in patients with acute coronary syndrome

AIM: To elicit the most sensitive and reliable method of control over administration of indirect anticoagulants. MATERIAL AND METHODS: Sixty patients with acute coronary syndrome were studied. They received varfarin under the control of international normalized ratio (INR) calculated by the tables in cases of prothrombin time (PT) estimation on a stationary turbidynamic hemocoagulometer from venous and capillary blood by thromboplastins. In parallel, PT was estimated from whole capillary blood on individual portable coagulometer "Coagu Check C" by means of standard test-strip (prothrombin test--PT) on the study day 1, 3, 5, 7, 14 and 21. The results were assessed on the study day 21. All the patients were treated in hospital then outpatiently. RESULTS: The assessment of PT results implied calculation of variation coefficient (VC) and error (m) by each index obtained with thromboplastins. VS and m were the lowest when INR was made from whole capillary blood on coagulometer "Coagu Check C". CONCLUSION: An optimal method of control over efficacy of varfarin treatment is calculation of INR from capillary blood on coagulometer "Coagu Check C" as calculation of INR from capillary and venous blood on stationary coagulometer is complicated and reliability depends on thromboplastin used.