Comparison of Two Automated Coagulometers and the Manual Tilt-Tube Method for the Determination of Prothrombin Time

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1 Comparison of Two utomated Coagulometers and the Manual Tilt-Tube Method for the Determination of Prothrombin Time RMDO D'GELO, M.D., MRI POL SEVESO, M.D., SILV VIGO' D'GELO, M.D., FELICETT GILRDOI, B.SC, LESSDR MCGI, M.D., CESRE MOTTI, M.D., D PIERGELO BOII, M.D. Two automatic coagulometerscl 81 s1 (Instrumentation Laboratory), a laser-nephelometric centrifugal analyzer, and KoaguLab (Ortho Diagnostics), an optical automatic coagulometerwere compared with the manual tilt-tube method for the performance of prothrombin time (PT). Seven ISI- (International Sensitivity Index) calibrated commercial thromboplastin reagents were used for duplicate determinations in 3 normal subjects, 3 patients with liver disease, and 3 patients receiving stabilized oral anticoagulation. Clotting times were longer with the manual method than with CL 81 and, to a lesser extent, with KoaguLab. verage imprecision of duplicate determinations (CV) was less than 1% with CL 81; KoaguLab and the manual method had similarly higher imprecisions (.8% and.7%). Differences in origin and slope of the regression curves of clotting times obtained with the coagulometers over the tilt-tube method were observed with all the reagents tested. Transformation of clotting times to PT ratios did not eliminate the bias resulting from the different clot-detection methods. higher percentage of patients with liver disease had abnormal PT ratios when their plasma was tested with the coagulometers than with the manual method. Transformation of PT ratios to International ormalized Ratios effectively eliminated the bias resulting from the different thromboplastin reagents but had no effect on the bias resulting from the different clot-detection methods. significant proportion of patients appeared excessively anticoagulated (IR >.) with the coagulometers but not with the manual method. These results highlight the need for standardization of both instrumentations and reagents to improve monitoring of oral anticoagulant treatment. (Key words: Prothrombin time; Coagulometers; Oral anticoagulant treatment) m J Clin Pathol 1989;9:3-38 THE IM of oral anticoagulant therapy is to provide effective antithrombotic prophylaxis without exposing patients to an excessively increased risk of bleeding. It has been recently shown that different clinical conditions may benefit from different intensities of anticoagulant treatment ; thus, monitoring of oral anticoagulation is required to improve safety and efficacy. Despite the introduction of more sophisticated assays for evaluating the Received May 3, 1988; receivedrevisedmanuscript and accepted for publication March, Supported in part by a grant from "Programma azionale di Ricera nel Settore dei Farmaci, Decreto //89, Consorzio ntitrombotici," awarded to Dr.. D'ngelo. ddress reprint requests to Dr. rmando D'ngelo: Servizio di Coagulazione, Istituto Scientifico Ospedale S. Raffaele, Via Olgettina,3 Milano, Italy. Servizio di Coagulazione e Laboratories Centrals di nalisi, Istituto Scientifico, Ospedale S. Raffaele, Milano, Italy and Centro di Emostasi e Trombosi, Ospedale Regionale, Parma, Italy depression of vitamin K-dependent clotting factors, 7,8,1 ' the prothrombin time (PT) remains the most widely adopted assay for monitoring oral anticoagulant treatment. number of thromboplastins are commercially available that differ in composition and in the sensitivity to specific clotting factor deficiencies. 1 ' The different sensitivities of thromboplastins to the clotting defect induced by vitamin K antagonists hampers comparability of the results obtained in different laboratories, thus endangering safe and effective treatment in individual patients. s a result, considerable efforts have been made for reagent standardization, culminating with the introduction of the International Sensitivity Index (ISI) for thromboplastins. 8,3 In the last few years the increasing demand for coagulation testing in nonspecialized laboratories has led to the development of a series of semiautomated or fully automated coagulometers. In general, preference for one instrument over another has been given on the basis of cost-effectiveness analysis, so that the problems relating to comparability between instruments have been somehow neglected. In principle, the difference in the results obtained with the different instruments should depend on both the detection system and the reagents adopted. The few published comparative studies have provided conflicting results, indicating a need for the standardization of instruments as well as reagents.,,,9,13,1,3,7,9 ' 33 Recently, an automated microcentrifugal optical analyzer (utomated Coagulation Laboratory, CL 81, Instrumentation Laboratory, Milano, Italy) has been introduced. In evaluating this new coagulometer, we have compared the results obtained for PT determinations in clinical plasma samples with those obtained with another automated coagulometer (KoaguLab utomated Coagulation System, Ortho Diagnostic Systems, Raritan, J) and the tilt-tube technique. This comparison was done on May 8 3

2 3 D'GELO ET L..J.C.P. September 1989 over a wide range of clotting times and with the use of a series of commercially available reagents. Instrumentation Materials and Methods CL 81 (Instrumentation Laboratory, Milano, Italy) is a fully automated microcomputer-controlled microcentrifugal analyzer for prothrombin time (PT), activated partial thromboplastin time (PTT), fibrinogen, thrombin time, single factor determinations, and amydolitic assays (antithrombin HI, plasminogen, antiplasmin, and heparin). Plasma samples (. ml) are aspirated by a sampling arm from.-ml cuvettes with a dead volume of.1 ml (minimum plasma volume required,.1 ml) and dispensed in microcells positioned in disposable rotors. In the analytic system for clotting tests, a laser monochromatic light source (3 nm) is transmitted by an optic fiber. Readings, in light scattering, are obtained by a sensor mounted at 9 degrees at the rotor. For chromogenic tests, the light beam from a halogen lamp is transmitted by an optic fiber and the absorbance read at nm. The apparatus has temperature-controlled areas (1-1 C for reagent cooling and 37 C for the reaction chamber), a quality control program, computer interface (RS-3C), and automatic calibration for PT, fibrinogen, single factors, and amydolitic assays. For the PT assay, reagent volume is.1 ml/test, with an acquisition time of -11 seconds. KoaguLab utomated Coagulation System (Ortho Diagnostic System, Raritan, J) is a microprocessor-controlled, fully automated photooptical detector analyzer for PT, PTT, fibrinogen, thrombin time, and single factor determinations. The instrument aspirates plasma samples (. ml) from Vacutainer tubes or -ml cups with a dead volume greater than. ml (minimum plasma volume required, 1. ml) and dispenses.1 ml of sample into adjacent wells for duplicate tests. single incandescent light source is reflected through duplicate samples. Transmitted light beams are filtered and processed by two independent but identical photooptical detector systems. The apparatus has controlled temperature areas (1-38 C), a quality control program, computer interface (RS-3C), and extra program calibration for fibrinogen and single factors. For the PT assay, reagent volume is. ml/test, with an acquisition time of seconds. number of error messages are included in the software of the two instruments. In the oase of the PT test, if a sample coagulates in less than 3 seconds or the sample/ reagent mixture is very turbid, the CL 81 displays the message "coag error" instead of the result; if, on the contrary, the sample does not coagulate within the maximum acquisition time selected ( or 11 seconds) or the plasma is insufficient, the message "not coag" will be displayed. On KoaguLab, the message "no data" is printed when clotting time exceeds the maximum end point of seconds. Reagents The following commercially available thromboplastins were used throughout the study: PT-Fibrinogen (rabbit brain, Lot #719, International Sensitivity Index (ISI),.3, Instrumentation Laboratory, Milano, Italy); Calcium Thromboplastin (rabbit brain, Lot #L 1-979, ISI 1.7, Roche, Basel, Switzerland); Thromboplastin C (rabbit brain, Lot #TPCD 37 B, ISI.18, merican Dade, guada, Puerto Rico); Thrombomat (rabbit brain, Lot #L11, ISI., Bio Merieux, Marcy l'etoile, Charbonnieres les Bains, France); Simplastin Plus (rabbit brain and lung, Lot #R, ISI., General Diagnostics, Morris Plains, J); Calciplastina Set (Manchester Reagent, animal brain, Lot #89, ISI 1.17, Baldacci, Pisa, Italy); and Thrombotest (ox brain and bovine plasma, Lot #3, ISI 1., yegaard & Co., Oslo, orway). Reagents were handled according to the manufacturer's instructions and used within 1 hours. ll the reagents were calcium-containing thromboplastins with the exception of the Calciplastina Set, which was reconstituted with calcium chloride before use. Samples Replicate PT determinations were conducted on frozen plasma obtained from 3 apparently normal subjects; 3 patients with clinical, laboratory, or histologic evidence of chronic liver disease; and 3 patients receiving oral anticoagulation with warfarin or acenocoumalone for more than six months. The latter patients attended the outpatient clinic of the Center for Haemostasis and Thrombosis in Parma, Italy. Plasma pooled from normal donors ( ml) was used as normal reference standard. For the precision study, two additional plasma pools were used. The normal pool ( ml) was constituted of plasma obtained from ten healthy donors; the abnormal pool ( ml) consisted of a mixture of plasmas taken on a single day from patients with chronic liver disease or receiving oral anticoagulant therapy. Blood Sampling Venous blood samples ( ml) were collected in polystyrene tubes containing sodium citrate (.13 mol/l, ph., nine parts of blood to one part of anticoagulant). Within two hours after blood collection, tubes were centrifuged for 1 minutes at 1,8 X g at room temperature, on May 8

3 Vol. 9 o. 3 PROTHROMBI TIME METHODS Table 1. Precision Study 33 CL 81 KoaguLab Tilt-tube Thromboplastin PT-Fibrinogen Calcium Thromboplastin Thromboplastin C Thrombomat Simplastin Plus Calciplastina Set Thrombotest Sample* (/) X (sec) Total W-f B-* cv% X (sec) Total cv% W- cv% B X (sec) Total W- cv% B * ormal/abnormal plasma pool. t Within-assay coefficient of variation. X Between-assay coefficient of variation. and platelet-poor plasma was dispensed in.-ml aliquots into plastic tubes, snap-frozen in methanol and dry ice, and stored at -7 C. Prothrombin Time Determinations ll PT determinations were carried out within 1 days after the collection of blood. Replicate PT measurements were performed by three experienced operators on plasma samples thawed at 37 C, with the use of the two coagulometers and the manual tilt-tube technique. ll reagents were tested on the same day on plasma samples from five to eight patients. The order of reagent testing was assigned on a random basis each day. Because of the high plasma volume required by KoaguLab and the limited amount of plasma available from individual patients (1 ml), automated operation of this instrument proved unfeasible; therefore,.1-ml aliquots of plasma were manually dispensed in the adjacent disposable wells for duplicate determinations. Total, within- and between-run imprecisions were obtained on five replicates and six runs on both the normal and the abnormal pools. To minimize variability, a single lot of each reagent was used throughout the study. Statistical nalysis Data evaluation was conducted with the help of a statistical software package (Minitab, Minitab Inc., Boston, M) and an IBM T computer. Total imprecisions and between- and within-assay components of imprecision were calculated according to the method of Krouwer and Rabinowitz. 1 Imprecision of duplicate PT determinations in patients and controls were obtained as described by White and Fraser. 31 nalysis of the regression curves between clotting times observed with the different reagent-method combinations on the entire series of plasma samples (n = 9) was performed by Deming's regression. The ratio of measurement errors of x and y (Sex /Sey )as obtained by duplicate PT determinationswas used to compute slopes by Deming' formula. Unlike the least-squares method, Deming's method always results in one line, whether x or y was used as the independent variable. inety-five percent confidence limits of the origins and slopes of the regressions were computed. Slopes were compared with the use of the X ditribution as described by rmitage and Berry.' In the absence of significant difference between slopes, the origins of the regression curves were compared by the T ratio method. Prothrombin time ratios were log-transformed to obtain normal distributions. The differences in mean PT ratios observed with the different reagent-method combinations in the two groups of patients and in normal controls were tested by two-way analysis of variance. This allowed identification of the specific influences of methods and reagents on the differences observed, as well as of method-reagent interactions. Results Total imprecisions and within- and between-assay components of imprecisionexpressed as coefficient of variationobtained with the normal and the abnormal plasma pools are reported in Table 1. s expected, the higher imprecisions were observed with the tilt-tube method. Total imprecisions were similar with the two instruments; however, although most of the imprecision observed with CL 81 resulted from the between-assay on May 8

4 3 D'GELO ET L. Table. Imprecision of Duplicate PT Determinations in Patients and Controls (n = 9) JCP -'September 1989 CL 81 KoaguLab Tilt-tube Thromboplastin x (sec) CV x (sec) CV x (sec) CV PT-Fibrinogen Calcium Thromboplastin Thromboplastin C Thrombomat Simplastin Plus Calciplastina Set Thrombotest *.1% 1.%.3% 1.%.7% 1.3% * 3.% 1.93%.87%.99% 3.1%.89% f.%.8%.3%.3%.7%.%.9% Only patients with liver disease and controls (n = ). t Only patients taking oral anticoagulants and controls (n = ). component, with KoaguLab the within-assay component was often higher than the between-assay component, particularly when testing the abnormal plasma pool. Mean clotting times of normal and abnormal plasma pools were consistently longer with the manual method than with the instruments; in turn, CL 81 gave shorter clotting times than did KoaguLab (Table 1). s expected on the basis of the sensitivity of the different thromboplastins, the difference between the clotting times obtained with the normal and the abnormal plasma showed a large variability; however, different sensitivities were also observed, depending on the method used: the average PT ratios (abnormal/normal plasma) obtained with the different reagents were 1.7 with CL 81, 1. with KoaguLab, and 1.9 with the manual method. Thrombotest did not prove suitable for use with the coagulometers, most probably because of the high turbidity of the reagent; therefore, it was used with the tilt-tube method only. Because duplicate determinations were performed with all the reagent-method combinations, a comprehensive indication of within-assay imprecision could be obtained over the entire range of clotting times (Table ). In general, there was good agreement with the corresponding data obtained in the precision study. Coefficients of variation were lower with CL 81 than with KoaguLab and the manual method. In patients being treated with oral anticoagulants, Calciplastina Set did not prove suitable for use with the coagulometers because in many instances the messages "coag error" or "no data" were displayed on both instruments. In consideration of the different imprecision of the methods of clot detection, analysis of the regression curves between clotting times observed with the different reagent- Table 3. Regressions ccording to Deming of Clotting Times Obtained in Patients and Controls (n = 9) with the Different Reagent-Method Combinations Deming's Regression Thromboplastin y a b x r 9%CL*ofa 9%CLoffc PT-Fibrinogen Koagf = CL Koag = Tilt CL* = Tilt / / / /1.13.8/ /1. Calcium Thromboplastin Koag = CL Koag = Tilt CL = Tilt /.18.7/. -.7/ /1. 1.8/ /.777 Thromboplastin C Koag = CL Koag = Tilt CL = Tilt / /. -.33/ /1..733/.9.7/.793 Thrombomat Koag = CL Koag = Tilt CL = Tilt /..83/ /.3.9/ / /.783 Simplastin Plus Koag = Koag = CL = CL Tilt Tilt / /..378/.97.9/.973.8/.8./.91 * inety-five percent confidence limits. t KoaguLab. t CL 81. on May 8

5 Vol. 9 o. 3 PROTHROMBI TIME METHODS 3 FIG. 1. Geometric means and 9% confidence limits of the mean of PT ratios obtained with the different reagent-method combinations in controls D, patients with liver disease M, and patients receiving stabilized oral anticoagulant treatment. Dotted rectangles represent the normal range (geometric mean ± standard deviations). = CL 81;T = KoaguLab ; T = tilt-tube method; PT-Fibrin. = PT-Fibrinogen; Calcium Th. = Calcium Thromboplastin; Thromb.-C = Thromboplastin C; Thrombom - Thrombomat; Simpl. Plus = Simplastin Plus; Calcipl. Set = Calciplastina Set; Thrombot. = Thrombotest. The Calciplastina Set proved unsuitable for use with the coagulometers for PT determination in patients receiving oral anticoagulants; Thrombotest proved unsuitable for use with both coagulometers and was assayed with the manual method only in controls and patients taking oral anticoagulants. < * II]. IX) s B S BBB. B "S B B SL m HB HH Oft, B Sl MM ibi j l JL JL JL K T K T K T K T K T K T K T PT-Fibrin. Calcium Th. Thromb. C Thrombo. Simpl. Plus Calcipl.Set Thrombot. I I method combinations on the entire series of plasma samples (n = 9) was performed by Deming's regression (Table 3). Comparison of the regression curves of the two coagulometers over the tilt-tube method disclosed significant differences in slope with Calcium Thromboplastin (P <.) and Simplastin Plus (P <.1); differences in the origin of the regression curves were observed with the remaining thromboplastin reagents (PT-Fibrinogen, P <.; Thromboplastin C, P <.1; Thrombomat, P <.), consistent with the different mean values reported in Table. ccordingly, a significant difference (P <.) was also observed by comparison of slopes of the regression curves of clotting times obtained with the two coagulometers and the different reagents (Table 3). The use of ratios (PT of patient's plasma:pt of normal plasma) does not normalize variations resulting from the use of different thromboplastins 319 ' ; however it has been reported by several investigators"' 3 ' 7 ' 33 as a tool for eliminating the bias resulting from different instruments. Mean PT ratios and 9% confidence limits of the mean geometric values determined in patients and controls with the different reagent-method combinations are reported in Figure 1. To evaluate the specific influence of methods and reagents on the results observed, two-way analysis of variance was separately performed on PT ratios (log-transformed) of controls, patients with liver disease, and patients being treated with oral anticoagulants (Fig. and Table ). In normal controls, the differences observed in PT ratios were related to the different clot-detection methods used and did not depend on the reagents used; in addition, there was significant reagent-method interaction. In patients with liver disease and those taking oral anticoagulants, both methods and reagents significantly influenced the results; significant reagent-method interaction was only observed in patients with liver disease (Table ). In a document recently published by the World Health Organization (WHO), it is recommended that PTs of patients taking oral anticoagulants be expressed in International ormalized Ratios (IRs). 3 fter transformation of PT ratios to IRs, there was no significant influence of reagents on the difference in geometric mean IR values (Fig. and Table ). However, the influence of clot-detection methods was still highly significant and reagent-method interaction was present (Table ). Hence, transformation of PT ratios to IRs in patients taking oral anticoagulants did not reduce the bias resulting from the detection method, whereas it was effective in virtually abolishing the bias resulting from the reagent used. The clinical importance of the statistically significant differences observed was evaluated by analysis of the assignment of patients with the different reagent-method combinations. mong the 3 patients with liver disease, the number of patients with abnormal PT ratios was clearly higher with CL 81 and, to a lesser extent, with KoaguLab than with the manual method (Table ); moreover, the well-known difference in the sensitivity of thromboplastins to the coagulation defect induced by liver disease was clearly apparent when using the tilt-tube method but was much less apparent when using the coagulometers, and especially CL 81 (Table ). In patients taking oral anticoagulants, assignment to poorly anticoagulated (IR <.), properly anticoagulated (IR between. and.), or excessively anticoagulated patients (IR >.) was different with the manual technique, depending on the reagents used. With CL 81 and Koagulab, patients were similarly assigned, irrespective of the reagent adopted (Table ). Differences in the assignment of patients when using the same reagent with the different methods were also noted: for instance, with PT-Fibrinogen the proportion of patients poorly anticoagulated was three times higher with the tilt-tube method than with the coagulometers; moreover, although none on May 8

6 3 D'GELO ET L. J.CP. September LD ^ X a. a: z ' FIG.. Geometric means of PT ratios obtained with the different reagents and clot-detection methods in controls (C), patients with liver disease (LD), and patients receiving stabilized oral anticoagulant treatment (OT). Geometric means of IR values in the latter group of patients are also shown (OT). = CL 81; K = KoaguLab ; T = tilt-tube method; PT-Fibrinogen, O; Calcium Thromboplastin, ; Thromboplastin C, ; Thrombomat, ; Simplastin Plus, D; Calciplastina Set, ; Thrombotest, X. of the patients appeared excessively anticoagulated when the manual method was used, a significant proportion of patients appeared excessively anticoagulated according to the data obtained with the coagulometers and especially so with CL 81 (Table ). Discussion Introduction of automated coagulometers has led to improved efficiency of coagulation testing in nonspecialized laboratories of clinical chemistry. Instruments offer a number of advantages over the tilt-tube technique and result in improved precision, as confirmed by our study. Precision was much higher with the two instruments evaluated, CL 81 and KoaguLab, than with the manual method, with most of the difference resulting from the high between-assay component of imprecision observed with the tilt-tube technique. Within-assay components of imprecision were consistently lower with CL 81 than with KoaguLab and the manual technique. It has to be pointed out that plasma samples had to be manually dispensed with KoaguLab, possibly contributing to the higher imprecision; however, dispensing errors as high as % of the expected volume were not rehably distinguishable by replicate determinations in one study. Precision of duplicate PT determinations, also Table. Effects of Clot-Detection Methods, Reagents, and Reagent-Method Interactions (two-way analysis of variance, F-values) on the Difference in PT Ratios (log-transformed) in ormal Controls (n = 3), Patients with Liver Disease (n = 3), and Patients Taking Oral nticoagulants ormal Controls (PT ratio) Patients with Liver Disease (PT ratio) Patients Taking Oral nticoagulants PT Ratio IR F DF* P F DF P F DF P F P Methods Reagents Interaction <.1 S < <.1 <.1 < <.1 <.1 S 19.1 <.1.1 S.1 <. Error 3 * Degrees of freedom (reagents analyzed were PT-Fibrinogen, Calcium Thromboplastin, Thromboplastin C, Thrombomat, Simplastin Plus, Calciplastina Set in normal controls and in patients with liver disease; PT-Fibrinogen, Calcium Thromboplastin, Thromboplastin C, Thrombomat, Simplastin Plus in patients taking oral anticoagulants). on May 8

7 Vol. 9 o. 3 PROTHROMBI TIME METHODS 37 reflecting the within-assay component of imprecision, was higher with CL 81, with an average imprecision of less than 1%. With KoaguLab, imprecision of duplicate determinations was higher (.8%), mainly because of differences observed in the higher range of clotting times. The need for performing duplicate PT assays on all patients has been questioned recently. 18, ccording to generally accepted criteria of analytic precision, our data, and especially those obtained for CL 81, indicate that duplicates would serve little purpose; however, as already pointed out, elimination of this procedure cannot be recommended for all patients because there are no established clinical precision requirements that can be used to evaluate the clinical precision. In the absence of any generally accepted standard for normal control plasma, abnormality of PT can only be judged on the basis of normalrangesderived form a locally obtained control population. Our data confirm previous reports that both the reagents and the clot-detection methods adopted independently influence the results obtained. 9,3,7 ' 9 ' 33 With any reagent, shorter clotting times were observed with CL 81 than with Koagulab and the manual method. It has been claimed that the bias resulting from different instruments could be eliminated by the use of PT ratios."' 3 ' 7 ' 33 Our results do not support this conclusion: minor but statistically significant differences in normal ranges (PT ratios) were found with the different clot-detection methods. This observationcoupled with the higher sensitivity of CL 81 and, to a lesser extent, of KoaguLab over the tilt-tube method resulted in better discrimination of patients with liver disease when using the coagulometers, independent of the thromboplastin adopted. Prothrombin time ratios were also significantly different by both reagent and method comparisons in patients taking oral anticoagulants. Comparability of prothrombin assay results is of great importance for the safety of patients treated with oral anticoagulants. In an effort to achieve such comparability, recommendations have been pub- Table. Distribution of bnormal PT Values in the 3 Patients with Liver Disease with the Different Reagent-Method Combinations Thromboplastin CL 81 Koagulab Tilt-Tube PT-Fibrinogen 3 Calcium Thromboplastin Thromboplastin C 19 1 Thrombomat 19 Simplastin Plus Calciplastina Set 1 1 lished 1 ' 3 urging manufacturers of commercial thromboplastins to indicate the relation of their material to the WHO preparation by an International Sensitivity Index (ISI). Conversion of PT ratios to IRs would permit comparability of the results, 19 allowing for safe monitoring of anticoagulant treatment. In our study, transformation of PT ratios to IRs effectively reduced the bias resulting from the different reagents, but it did not affect the bias resulting from the different clot-detection methods. Major differences were found between IRs obtained with the two coagulometers or with the manual technique; however, depending on the reagent, differences in the determination of patients as being poorly, properly, or excessively anticoagulated were also found by comparison of the two coagulometers. Strictly speaking, the use of IR is valid only for fresh plasmas. The design of our study prevented determination of duplicate PT values on the same day with all reagent-method combinations on fresh samples; however, we believe that the results obtained are valid because care was taken to avoid cold-promoted contact activation of blood coagulation ' and because transformation of PT ratios to IRs effectively abolished the bias resulting from the different reagents. The influence of instrumentation on PT results has also been demonstrated with the use of fresh plasma samples ' 3 and with lyophilized plasmas. 9 Recently, manufacturers of instruments and throm- Table. Distribution of IR Values in the 3 Patients Taking Oral nticoagulants with the Different Reagent-Method Combinations Thromboplastin * CL 81 Bt c% KoaguLab B C Tilt-Tube B C PT-Fibrinogen Calcium Thromboplastin Thromboplastin C Thrombomat Simplastin Plus Calciplastina Set Thrombotest = Patients with IRs less than.. t C - Patients with IRs greater than.. t B = Patients with IRs between. and.. on May 8

8 38 D'GELO ET L..J.C.P. September 1989 boplastins have started to supply ISI values derived from calibration of their thromboplastins obtained with optical coagulometers in addition to ISI values traditionally obtained with the manual method or with mechanical coagulometers. For instance, General Diagnostic supplies an ISI value of. for the manual method and.3 for optical coagulometers for the thromboplastin lot used in this study; transformation of PT ratios to IRs with the use of the two different ISIs would have reduced the bias resulting from the clot-detection methods; on the other hand, Dade supplies ISI values of.18 for the manual method as opposed to. for optical coagulometers: in this case, use of the different ISI values would have increased rather than decreased the bias. different approach has been proposed recently, 9 based on the finding that a linear relationship exists between the mean PT and the effect on PT resulting from a particular thromboplastin or instrument. The additive, linear model proposed by van Besselaar and associates 9 could be complementary to standardization, based on IRs as proposed by WHO. Our data are not necessarily in contrast with this approach becausewith the exception of Simplastin Plus (Fig. )the instrument effect appeared independent of the thromboplastins adopted; however, it must be pointed out that such an approach would require that a particular instrument to thromboplastin combination be chosen as a reference method. 9 Standardization of PT is still far from being a solved question; more efforts are needed before safe monitoring of oral anticoagulant treatment is achieved. cknowledgments. The authors thank Drs. Laura Galli, Paola Garancini, and Giliola Calori from the Epidemiology Unit, IRCCS H. S. Raffaele, for their invaluable help in the statistical analysis of data. References 1. rmitage P, Berry G. Statistical methods in medical research. 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