Elsevier

Biologicals

Volume 38, Issue 4, July 2010, Pages 430-436
Biologicals

Review
Thromboplastin standards

https://doi.org/10.1016/j.biologicals.2010.02.012Get rights and content

Abstract

The Prothrombin Time (PT) test is used for monitoring of treatment with Vitamin K-antagonists (VKA). The result of the PT test should be expressed as the International Normalized Ratio (INR). Calculation of INR is based on the availability of International Standards (IS) for thromboplastin and a calibration model. Calibration of a new PT test system is performed with the appropriate IS and fresh plasma samples of healthy (normal) volunteers and patients treated with VKA. The calibration model is based on the assumption of a linear relationship between the log(PT)'s obtained with the new PT system and the reference IS for both normal and patients' samples. Patients' samples for calibration should be selected by rejecting samples beyond the 1.5–4.5 INR range. Outliers should be rejected defined as points with a perpendicular distance greater than three residual standard deviations from the line of relationship. Selection of patients' samples and rejection of outliers result in a reduction of the between-laboratory variation of calibration. In addition to monitoring of VKA, the PT is used for management of patients with chronic liver disease. Likewise, INRliver should be based on calibration with an IS using samples from patients with chronic liver disease.

Introduction

Anti-vitamin K drugs (Vitamin K-antagonists, VKA) derived from coumarin are widely used in the treatment and prophylaxis of thrombotic disorders. These drugs inhibit the biosynthesis of vitamin K-dependent coagulation factors by the liver. For each patient, the dose of the coumarin drug must be adjusted periodically to ensure that an adequate but not excessive degree of anticoagulation is achieved. The adjustments are made on the basis of the results of a Tissue-Factor-induced coagulation time (Prothrombin Time) test on the patient's blood or plasma. When monitoring treatment with VKA drugs, it is common practice to require that the patient's prothrombin time (PT) be stabilized within an accepted therapeutic range. In many countries, the therapeutic range used to be specified in terms of the PT ratio, i.e., the patient's PT divided by the average PT for normal samples [1].

The Prothrombin Time (PT) is the primary measurement of monitoring of VKA treatment. The PT, originally described by Quick and co-workers [2], is based on the Tissue-Factor pathway of blood coagulation. Tissue-Factor is an integral transmembrane protein functioning as a cofactor enhancing the proteolytic activity of factor VIIa towards factor X and factor IX in the blood. Tissue-Factor needs to be associated with coagulant phospholipids for the full expression of its cofactor function. A reagent containing Tissue-Factor and coagulant phospholipids is also referred to as thromboplastin. Quick used an extract of rabbit brain as thromboplastin preparation, but others used human brain [3], or bovine brain [4]. The result of the PT test, expressed in seconds, is strongly dependent on the nature of the thromboplastin and the method used. Many modifications of the original Quick test have been applied for monitoring of VKA treatment. The quality of the thromboplastin is of decisive importance, and different thromboplastins may give widely differing results for the same test plasma.

The multiplicity of modifications of the PT test has contributed to the confusion about the optimum therapeutic target levels of VKA treatment [1], [5]. The confusion could only be resolved by the implementation of the principle of biological standardization. A reference method for the PT test and one or more reference thromboplastins had to be established, and a uniform reporting system had to be developed [6]. Finally, international agreement was reached on how to report PT or PT ratio in terms of the International Normalized Ratio, abbreviated as INR [7], [8].

Section snippets

WHO calibration model

To provide a means of standardizing PT tests, the World Health Organization (WHO) adopted in 1977 a scheme for calibrating thromboplastins in terms of an International Reference Preparation (IRP) [9]. Some time later it was recognized that the model on which this scheme was based had certain drawbacks [10]. Subsequently, a revised calibration model had been developed which was adopted by the WHO Expert Committee on Biological Standardization in 1982 [11]. In the revised calibration method, the

International Reference Preparations

The first (primary) International Reference Preparation (IRP) of Thromboplastin, Human, Combined (coded 67/40), was established by the WHO Expert Committee on Biological Standardization in 1976 [9]. It was a freeze-dried preparation, filled in sealed glass ampoules, and contained a human brain extract to which adsorbed bovine plasma had been added to optimize the content of non-vitamin-K-dependent coagulation factors (i.e. factor V and fibrinogen) [14]. Its ISI value was set at 1.0 by

Replacement of International Standards

The ISI for a new thromboplastin, ISIn, is obtained by multiplying the ISI for the reference preparation, ISIr, by the slope of the calibration line cnr. The statistical imprecision of ISIn is composed not only of statistical variation in the calibration line slope cnr, but also of any error in the calibration of the reference preparation. The calibration of a secondary reference thromboplastin must be carried out with extreme care, since any error in its ISI will be transmitted to all working

Selection of samples

The calibration procedure entails the determination of a series of prothrombin times, using normal and patients' samples, with both the reference and the test (new) thromboplastin. Blood samples from normal (healthy) subjects and patients who have been on VKA drugs for at least 6 weeks should be selected. Samples from patients treated with heparin should not be used. According to the established WHO Guidelines for thromboplastins and plasma used to control oral anticoagulant therapy [8], it is

Pre-analytical effects on the PT and ISI

The PT is sensitive to pre-analytical effects such as the sodium citrate concentration of the anticoagulant solution in which the blood is collected [22]. It is recommended to collect nine volumes of blood in one volume of 0.109 mol/L trisodium citrate solution [8]. There is slight shortening of the PT in the first hours after blood collection [23]. For the calibration of secondary standards with an IRP, individual fresh plasma samples should be prepared according to WHO guidelines [8]. The

Exclusion of outliers

The calibration of a new PT system involves the calculation of an orthogonal regression line relating log(PT) of the reference system to log(PT) of the new PT system. The orthogonal regression analysis provides an estimate of the standard deviation of data points about the regression line. According to the WHO Guidelines, any samples with a perpendicular distance greater than 3 residual standard deviations from the regression line should be excluded [8]. After removal of such samples, the final

Adequacy of the WHO calibration model

The WHO calibration model assumes a single linear relationship between the log(PT) of the reference PT system and the new PT system for both normal and patients' samples. In each case, care must be taken to ensure that the method of fitting the calibration line is statistically sound and the data should be checked for consistency with the assumed linear model. The latter is most easily done by plotting the data as in Fig. 1. The WHO calibration model requires that the overall regression line

Long-term stability of reference thromboplastins

The long-term stability of an established reference material is an essential requirement for its usefulness. As no biological material is absolutely stable, it has to be checked whether the real value after some time of storage still corresponds to the established value. For stability monitoring of a material, samples are measured at regular intervals and the measured values are to be compared to the established values. For stability monitoring of reference thromboplastins, different approaches

Other applications

The primary purpose of the International Standards for Thromboplastin is the ISI calibration of secondary thromboplastins. They may also be used to assign PT or INR values to designated freeze-dried reference plasmas [39]. Such certified plasmas may be used for simplified ISI calibration or direct INR determination [40]. It seems logical to use WHO International Standards or other standards because their ISI values are well-defined. Although thromboplastin standards should be used for the

The International Normalized Ratio calibrated for cirrhosis (INRliver)

The INR scale has been devised for patients on VKA and does not necessarily apply to conditions where the PT is prolonged because of other acquired coagulopathies. Unfortunately, this limitation has not been recognized and the INR has been extensively used to express results for patients with chronic liver disease (CLD) and to construct the model for end-stage-liver-disease (MELD) [45]. The MELD is a mathematical prognostic score used to prioritize patients for liver transplantation and

Conclusion

The INR system for laboratory monitoring of VKA treatment is not perfect. This is due to the fact that the PT test is rather non-specific, and sensitive to pre-analytical conditions. Nevertheless, the use of the INR enables comparisons to be made between results obtained with different thromboplastins and methods. It is a misconception, however, that for an individual patient's blood or plasma the INR will always be identical with different thromboplastins and methods. Different thromboplastins

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