The International Normalized Ratio (INR) for Monitoring Warfarin Therapy: Reliability and Relation to Other Monitoring Methods

Reporting prothrombin times used to monitor warfarin therapy as an international normalized ratio (INR) has been proposed as a way to eliminate interlaboratory differences in test results caused by the use of thromboplastins with different sensitivities [1-4]. The INR is calculated by raising the prothrombin time ratio (PTR; the patient's prothrombin time divided by a reference normal prothrombin time) to the power of a coefficient known as the international sensitivity index (ISI). This coefficient relates the sensitivity for monitoring warfarin therapy of a given thromboplastin to the sensitivity of the World Health Organization's first primary international reference preparation of thromboplastin, which was assigned an ISI of 1.0 [5]. Thromboplastins less sensitive than this international reference preparation of thromboplastin to decreased levels of the vitamin K-dependent clotting factors have proportionately higher ISI values. In 1992, a prothrombin time ratio was still used to monitor warfarin therapy at our medical center. The ratio was determined for inpatients from a plasma prothrombin time and for outpatients from a capillary whole-blood prothrombin time measurement system (the Coumatrak portable monitor [Du Pont Pharmaceuticals; Wilmington, Delaware]). To prepare for reporting prothrombin time results as INRs, we compared the INRs as routinely determined by the capillary monitor in a large group of outpatients on maintenance warfarin therapy with the INRs as determined by plasma prothrombin times done with six commonly used thromboplastins. We also measured the prothrombin time by the so-called prothrombin-proconvertin (P&P) method [6], residual plasma native prothrombin antigen [7], and specific prothrombin (factor II) activity [8]. Methods One hundred plasma samples were collected from 79 patients between 8 April and 20 May 1992 at seven weekly sessions of the outpatient anticoagulant clinic of the University of California, San Diego, Medical Center. All samples except one were obtained from patients who had taken warfarin for longer than 14 days. All patients gave informed consent. After the Coumatrak prothrombin time was measured, 4.5 mL of venous blood was collected in a siliconized BD Vacutainer tube (Becton-Dickinson; Rutherford, New Jersey) containing 0.5 mL of 3.2% sodium citrate and sent to the medical center's Special Coagulation Laboratory. All subsequent handling and testing were done by the same experienced medical technologist who followed the routine techniques used in this laboratory. Platelet-poor plasma was prepared within 2 hours of the time of sampling by centrifugation at 3000 rpm for 15 minutes at room temperature. In the early afternoon, when all samples for a given day had been processed, prothrombin times were done on each sample with six thromboplastin reagents. The plasma samples were then stored at 70C for subsequent measurement of the P&P percent activity, specific prothrombin (factor II) activity, and native prothrombin antigen. This protocol was adopted after a preliminary experiment was done in which blood samples from five patients were divided into two aliquots and allowed to stand for either 2 hours or 5 hours before separating the plasma and measuring the prothrombin time. The prothrombin time values, as measured with three different thromboplastins, did not differ significantly for the paired samples (data not shown). In addition, virtually identical P&P test results were obtained on fresh plasma samples from five patients and on these samples after they had been stored frozen for 1 week. Control Pooled Plasma The normal plasma used to obtain the prothrombin time ratio for plasma prothrombin times was the routine control pooled plasma of the Special Coagulation Laboratory. This plasma is prepared by pooling plasma obtained by the technique described above from blood collected in 1/10 volume of a buffered citrate anticoagulant (0.06 mol/L sodium citrate plus 0.04 mol/L citric acid) from between 15 and 20 healthy donors. It is stored in small aliquots at 70C. The prothrombin time and activated partial thromboplastin time of the plasma from each donor had to be within 2 standard deviations (SD) of the mean of the individual plasma samples from the donors that were included in the laboratory's previous batch of control pooled plasma. Coagulation Assays Capillary Blood Prothrombin Time (Coumatrak) This test was done by two experienced practitioners on finger-stick blood. The thromboplastin used in the cartridge for the Coumatrak instrument had an ISI of 2.04. The value for a normal prothrombin time was set by the instrument at 12 s, and the prothrombin time ratio and INR were provided by the instrument as a direct readout. Prothrombin Time Assays The prothrombin times were done on an Electra 800 (Medical Laboratory Automation; Pleasantville, New York). The six thromboplastins, made available by sales representatives of the manufacturers, were Thromboplastin C (Dade C; ISI, 2.88), Thromboplastin C-plus (Dade C-plus; ISI, 2.03), and Thromboplastin IS (Dade IS; ISI, 1.27) from Dade division, Baxter Scientific Products, Miami, Florida; Simplastin Excel (Excel; ISI, 1.99 or 2.04 for two lots used) and Simplastin Excel-S (Excel-S; ISI, 1.34) from Organon Teknica, Durham, North Carolina; and Ortho Brain Thromboplastin (Ortho; ISI, 2.30) from Ortho, Raritan, New Jersey. Prothrombin time ratios for assays done with each thromboplastin were calculated using a normal value obtained with each thromboplastin for each run with the control pooled plasma. The mean clotting times ( SD, n = 7) of the control pooled plasma with the different thromboplastins were as follows: Dade C, 11.8 0.2 s; Dade C-plus, 12.2 0.2 s; Dade IS, 13.7 0.2 s; Excel, 11.7 0.1 s; Excel-S, 13.4 0.2 s; and Ortho, 12.7 0.3 s. The INR was calculated as INR = PTRISI with the manufacturer's value for the photo-optical ISI as given in the package insert. Prothrombin-Proconvertin Test This test was done according to the manufacturer's instructions with Simplastin A (Organon Teknika), which is the source not only of the thromboplastin (ISI, 1.70) and CaCl2 but of an optimal concentration of factor V and fibrinogen. The test plasma was diluted in saline 1:10, which sensitized the assay to small changes in the prothrombin, factor VII, and factor X activity of the sample. Clotting times were determined with an ST-4 semi-automated coagulometer (Diagnostica Stago; Parsippany, New Jersey). A log-log plot of clotting times against dilutions of control pooled plasma in saline between 1:10 and 1:80 yielded linear standard reference curves. The clotting time of the 1:10 dilution of the control pooled plasma was assigned a value of 100% P&P activity, and clotting times of test samples were converted into percent of the control pooled plasma activity. Specific Prothrombin Assay (Factor II) Prothrombin activity was assayed by a one-stage assay in which a mixture of 100 L of a prothrombin-depleted human serum/barium-adsorbed bovine plasma reagent [8] and 100 L of a 1:10 to 1:40 dilution of test plasma were clotted by the addition of 200 L of a thromboplastin C reagent containing CaCl2. Clotting times were converted to percent normal plasma prothrombin activity from a log-log standard curve prepared with dilutions of control pooled plasma. Native Prothrombin Antigen Assay Plasma native prothrombin antigen concentration was measured with native prothrombin antigen enzyme immunoassay kits provided by Organon Teknika. Color was measured at 450 nm with a Thermomax enzyme-linked immunosorbent assay reader (Molecular Devices; Menlo Park, California). Results Relations between the Prothrombin Time Ratio and International Normalized Ratio Values Obtained with Six Thromboplastins and with the Coumatrak Monitor The thromboplastins used in this study had ISI values ranging from 1.27 to 2.88 (see Methods). Dade Thromboplastin IS (Dade IS; ISI, 1.27) was the most sensitive of the thromboplastins and was therefore selected as the standard against which we compared data obtained with the other thromboplastins. In an initial analysis, the prothrombin time values obtained with the Coumatrak capillary monitor and with Dade IS were plotted both as prothrombin time ratios Figure 1, A) and as INR values (Figure 1, B). Because of the different ISI values for the Coumatrak monitor and Dade IS, the slope of the linear regression line for the data plotted as prothrombin time ratios, 0.36 (r = 0.87, n = 100), clearly differed from 1. The slope of the regression line of the data plotted as INRs, 0.84 (r = 0.90, n = 100), was closer to 1, which is the theoretically expected slope when plotting INR values against each other. Figure 1. Plots of Coumatrak monitor data as a function of plasma prothrombin time data. A. r n B. INRs r n The linear regression lines obtained by plotting the prothrombin time ratios obtained from all of the other thromboplastins and from the Coumatrak monitor as a function of the prothrombin time ratios obtained with Dade IS are shown in Figure 2 A. The slopes of the linear regression lines varied between 0.38 to 0.88. The individual prothrombin time ratios obtained for a given sample varied with the sensitivity of the thromboplastin. Thus, a sample that gave a prothrombin time ratio with Dade C (ISI, 2.88) of 1.5 gave a prothrombin time ratio with Dade IS (ISI, 1.27) of 2.3. Figure 2. Linear regression lines derived from prothrombin time ratios and from international normalized ratios. A. B. The prothrombin time ratios that yielded the regression lines of Figure 2A were then converted into INRs and plotted against the INRs obtained with Dade IS Figure 2B. The slopes of the resultant linear regression lines varied between 0.96 to 1.97, with a correlation coefficient range of 0.96 to 0.98. Thus, converting prothrombin time ratios to INRsby use of the ISI given in the thromboplastin package inserts and clotting times obtained on the sam