Calibration of a Lyophilized Pooled Plasma as Candidate Reference Plasma for Standardization of the Prothrombin Time Ratio
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Summary The prothrombin time (PT), obtained from a fresh normal plasma pool (FPP), is the basis both for the establishment of the 100% activity (normal plasma) and for the ratio calculation used in the International Normalized Ratio (INR) according to the recommendations of the ICSH/ICTH (6). Today the PT of lyophilized normal plasma pools are successfully used as reference for the assessment of samples in proficiency studies. However, a lack of comparability is to be recognized. Therefore the Committee of Hematology of the German Association of Diagnostics’ and Diagnostic Instruments’ Manufacturers (VDGH) decided to produce a candidate reference plasma (VDGH Reference Plasma) which was calibrated against fresh normal plasma pools in an international study. The basic calibration was performed by using the same certified BCR thromboplastin (BCT/099) by all participants. The endpoint was determined manually and by using the coagulometer Schnitger-Gross. In additional testings each participant used his own routine thromboplastins and methods. Calculating the ratio [PT VDGH Reference Plasma (sec)/PT fresh normal plasma pool (sec)] the VDGH Reference Plasma showed a deviation from the average fresh normal plasma pool of 1.05 both with the BCT/099 and with all thromboplastins. There were obtained some statistical differences between “plain” and “combined’’ (added factor V and fibrinogen) thromboplastins. No statistical difference was found between the different endpoint measurement methods (manual, mechanical, optical). In spite of these statistical deviations the VDGH Reference Plasma can be used for the standardization of the PT-normal (100%) value with different ratios for plain (1.06) and combined (1.02) thromboplastins. The manufacturers will use this VDGH Reference Plasma for the calibration of their commercially available calibration plasmas, which allows the user of such a material to calculate a calibrated 100% PT value.Keywords:
Prothrombin time
Reference values
Coefficient of variation
Thromboplastin
Thromboplastin
Prothrombin time
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Laboratory Wise Variations in Prothrombin Time - INR - A Cross Sectional Study in Trivandrum, Kerala
BACKGROUND Prothrombin time (PT) is routinely used as a test of coagulation. Thromboplastin is the key ingredient in the reagent for this test. Prothrombin time international normalized ratio (INR) readings can vary according to the thromboplastin used in the reagent. The composition of thromboplastin reagents can influence the sensitivity of each batch of reagents. Various thromboplastin reagents having different international sensitivity index (ISI) values are available now. This study was intended to evaluate the effect of different thromboplastins on INR reading for mitral valve replaced patients under stable oral anticoagulant therapy. METHODS The study was conducted on the citrated plasma received from the mitral valve replaced patients having stable international ratio between 2 to 3 for three months. 62 samples were collected from the clinical pathology laboratory, Govt. Medical College, Trivandrum. Each sample was tested with different thromboplastin reagents having international sensitivity index 1.0, 1.1 and 1.6 by measurement of the prothrombin time and conversion into international normalized ratio. The INR obtained from the thromboplastin with international sensitivity index 1.0 was considered as the standard. INR results obtained from samples tested with thromboplastin reagents with ISI 1.1 and 1.6 were compared with the standard by using analysis of variance (ANOVA) and Dunnett’s post hoc tests. RESULTS Sixty-two samples were tested with the thromboplastin reagent having ISI – 1.0, the mean INR is 2.42, for ISI – 1.1 mean INR value is 2.53 and for ISI 1.6, the mean INR value is 3.19. While comparing the mean value of INR for different reagents using ANOVA, the F value was 14.86, which was significant. P value less than 0.01. In the Dunnett post hoc test, the P value of difference between ISI 1.0/1.6 was < 0.01. Between ISI 1.1/1.6 also the P value is < 0.01. Both of these were significant. The P value of difference between the reagents having ISI 1.0 and 1.1 is 0.838 which denotes no significant difference. CONCLUSIONS In conclusion, the thromboplastin reagent with ISI 1.0 or nearest to 1.0 is most desirable for accurate INR report. KEYWORDS Prothrombin Time, International Sensitivity Index, International Normalized Ratio
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The prothrombin time is the coagulation time of citrated plasma in the presence of calcium and a tissue extract, thromboplastin, added in excess. The prothrombin time was historically the first method of evaluation and control of oral anticoagulation. Over the years, the different thromboplastins have changed, diversified, so affecting the result of the prothrombin ration established from the prothrombin time and a reference curve. In 1985, the International Committee on Thrombosis and Haemostasis requested that all the losts of thromboplastin have their international sensitivity index (ISI) indicated. This allowed uniformity of the results by the introduction of the INR (International Normalized Ratio) calculated by the formula: INR = (PTR)ISI, the PTR or prothrombin time ratio corresponding to the patients' prothrombin time divided by that of reference control plasma. It is, in fact, impossible to interpret the results of a prothrombin ration without knowing their expression in INR. The consequences of the absence of uniformity in the control of anticoagulant therapy are important and serious. The uncertainty concerning the degree of anticoagulation inherent in the use of a single prothrombin ratio may be the source of bleeding or thromboembolic complications. Curiously, the system based on the INR is neither generalised, nearly 10 years after its recommendation, nor adopted by the majority of practitioners. However, the stakes are high because the principal complication of oral anticoagulants remains bleeding, including the dramatic strokes. Moreover, the global mortality due to haemorrhagic complications is about 0.1 to 0.5% for treatments of short duration and much higher in prolonged therapy.(ABSTRACT TRUNCATED AT 250 WORDS)
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Summary Tissue thromboplastin is the key reagent in the one-stage prothrombin time test. To obtain reliable results, a potent thromboplastin with constant activity and stability is required. This need is met by acetone-dehydrated rabbit brain. This reagent, when protected against oxidation by sealing in an evacuated tube, retains its full activity indefinitely. The basic prothrombin time serves as a screening test for depletion of prothrombin, factors V, VII and X. Thromboplastin is slowly inactivated by oxidation and also by bacterial action. Phenol prevents the latter reaction and therefore a phenolized extract of either human brain or acetone-dehydrated rabbit brain serves as a satisfactory reagent for the prothrombin time when employed to control oral anticoagulant therapy. The constant 12 sec prothrombin time of fresh normal human plasma is fixed by the concentration of active prothrombin. By modifying the basic prothrombin time by adding excess of factors V, VII or X, the test is made a specific quantitative measure of each of these factors.
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The objective of this report is to compare the prothrombin time performed with thromboplastin of different tissues and species in patients under oral anticoagulant therapy as well as the way of expressing the results. The results showed that the ISI of the thromboplastin of human and rabbit brain are very close to the IRP BCT/253 (1.2 vs 1.1) and RBT/79 (1.3 vs 1.4), respectively. In contrast, the rabbit lung thromboplastin showed the greatest differences in the ISI values (1.6 vs 1.4) and in the CV (6.1%). The authors found significant statistical differences with the results of the prothrombin time as expressed in activity percentage (p less than 0.001) in three plasma pools of patients under different oral anticoagulant level for all thromboplastins studied. However, if the results are expressed in terms of INR, the values obtained are almost the same. The results here reported would demonstrate that the prothrombin time as INR allows the use of only one scheme for oral anticoagulant control when the thromboplastin reagent is calibrated according to the recommendations of the WHO.
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For determination of the international normalized ratio (INR), it has been suggested that “highly sensitive” thromboplastin reagents (International Sensitivity Index [ISI] ≤ 1.2) provide the most consistent performance and minimize interlaboratory variability. We compared the INR values obtained from 69 specimens drawn from patients receiving long-term oral anticoagulant therapy, using four thromboplastin preparations (manufacturerassigned ISI range of 0.96–1.10) and two automated photo-optical analyzers. Multivariate analysis of the INR response matrix (552 INR values) indicated that the eight reagent-coagulometer combinations did not produce equivalent INR values. Similar analysis indicated that INR values were not normalized when uncorrected prothrombin ratios or INR values, calculated after assignment of “local ISI values” to each thromboplastin reagent, were compared. The INR differences also seemed to be clinically significant because 17% to 29% of paired thromboplastin values were discordant when all INR values were assigned to one of four therapeutic categories used in oral anticoagulant therapy (< 2.0; 2.0–3.0; 3.0–4.5; or > 4.5). These differences in INR values obtained with two photo-optical coagulometers and four highly sensitive thromboplastin reagents suggest that the existing INR system has not achieved the goal of standardized prothrombin time values and does not support the recommendation to use only highly sensitive reagents for the regulation of oral anticoagulant therapy.
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Prospective population studies have established that fibrinogen is an independent predictor for ischemic heart disease and stroke. These study conclusions have prompted recommendations that fibrinogen determinations be included in the cardiovascular risk profile. The routine availability of fibrinogen measurements may result in widespread screening prior to establishing the validity of a single fibrinogen level as an accurate descriptor for individual subjects. The objectives of this study were to describe the methodological and intraindividual components of variability in fibrinogen measurements determined by using the Clauss method; to establish the usefulness of a single fibrinogen measurement on risk stratification and retest reproducibility; and to determine the influence of intraindividual fibrinogen variability on sample size estimates. Fibrinogen levels were measured by a modification of the Clauss method. Three cohorts of apparently healthy, nonsmoking volunteers were recruited. The single-day intra-individual component of fibrinogen variability was determined in 39 subjects. For the 5-day intraindividual component of fibrinogen variability, 32 subjects were recruited, and in the 6-week intraindividual study, 28 subjects were included. The coefficient of variation for the methodological component of fibrinogen variability was 5.8% as determined from batch analyses, but the intraindividual coefficient of variation for replicate measures on a single day was 10.7%. The 5-day intraindividual coefficient of variation was 14.2%, and for the 6-week period it was 17.8%. Based on the 6-week data, an average of four fibrinogen measures is required to reduce misclassification error to less than 10%. Sample size estimates were made based on predetermined levels of statistical power and the 6-week intraindividual and interindividual variability estimates.(ABSTRACT TRUNCATED AT 250 WORDS)
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In the UK, rabbit brain thromboplastin has recently replaced human thromboplastin. Since the sensitivity of thromboplastin varies according to species of origin, and the calibration of thromboplastins is based entirely on samples from normal subjects and patients on oral anticoagulants, a separate assessment is required in patients with liver disease. We have compared prothrombin times and specific one stage assays of factors V, VII and X in plasma from 19 patients with establishe < liver disease using rabbit thromboplastin (Manchester reagent, MR) and human thromboplastin (Manchester comparative reagent, MCR). Both materials were kindly provided by the National (UK) Reference Laboratory for Anticoagulant Control. Three separate analyses were performed on the prothrombin time data viz clotting time, prolongation of prothrombin time compared with control and prothrombin ratio. All were significantly longer with MR (p 0.001, paired ‘t’ test) although correlation was goo< (r=0.95 in all instances). In the assay of factors V, VII and X no significant differences were obtained with the two thromboplastins and correlation was good over a range of abnormality (Ranges for MCR and MR respectively were Factor V:0.31-1.23u/ml and 0.32-1.I6u/ml, r=0.96; Factor VII:0.07-1.22u/ml and 0.07-1.17u/ml, r=0.97; Factor X;0.18-1.Olu/ml and 0.17-1.03u/ml, r=0.96. We conclude that in the investigation of the haemostatic defect associated with liver disease rabbit brain thromboplastin is a suitable alternative to human material.
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Commercially available thromboplastin reagents and two human brain preparations have been compared using the one-stage prothrombin time and plasma samples from patients receiving long-term oral anticoagulant therapy. Considerable variation is noted between various thromboplastins using the same plasma sample. The commercially available thromboplastins give shorter prothrombin times than do human brain preparations. With the latter, the "therapeutic range" is represented by a prothrombin time of about 1.8 to 3.0 times the normal control value, whereas with commercial preparations the "therapeutic range" is about 1.25 to 1.75 times normal. The implications of these observations are discussed; the desirability of standardization of the one-stage prothrombin time is emphasized.
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Prothrombin time
Oral anticoagulant
Therapeutic index
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The response of the prothrombin time to oral anticoagulant treatment depends on the thromboplastin reagent and method of testing. A prothrombin time assay system may be characterized by its international sensitivity index (ISI) with which a coumarin-treated patient's international normalized ratio can be calculated. Both the anticoagulant (sodium oxalate or sodium citrate) and calcium chloride concentrations influenced the ISI. An ISI of 2.0 was obtained for Quick's prothrombin time assay using rabbit brain thromboplastin. Replacing rabbit brain by human brain thromboplastin (Aggeler's method) resulted in an ISI of 1.3. The results described herein are mainly of historical interest and may assist in the interpretation of anticoagulation intensity in early American recommendations.
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Oral anticoagulant
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