Chapter 20 Laboratory control of anticoagulant, thrombolytic and antiplatelet therapy
Oral anticoagulant treatment using vitamin K antagonists
It has not yet proved possible to produce a therapeutic reduction in thrombotic tendency without increasing the risk of haemorrhage. The purpose of laboratory control is to maintain a level of hypocoagulability that effectively minimizes the combined risks of haemorrhage and thrombosis: the therapeutic range. Individual responses to oral anticoagulant treatment with vitamin K antagonists1 are extremely variable and so must be regularly and frequently controlled by laboratory tests to ensure that the anticoagulant effect remains within the therapeutic range.
Selection of Patients
Before starting oral anticoagulant treatment it is advisable to perform the first-line coagulation screen – a prothrombin time (PT), an activated partial thromboplastin time (APTT), a thrombin time (TT) and a platelet count. Any abnormality of these tests must be investigated because a contraindication to the use of oral anticoagulants may be revealed and an abnormality will confound their use for controlling anticoagulant effect. History and clinical examination should be assessed to ensure that no local or general haemorrhagic diathesis exists.
Methods Used for the Laboratory Control of Oral Anticoagulant Treatment
The one-stage PT of Quick is the most commonly used test. Originally, lack of standardization of the thromboplastin preparations and methods of expressing the PT results led to great discrepancies in the reported results and hence also in anticoagulant dosage. The use of the International Sensitivity Index (ISI), to assess the sensitivity of any given thromboplastin and the International Normalized Ratio (INR), to report the results, has minimized these difficulties and greatly improved uniformity of anticoagulation throughout the world.
Chromogenic substrate assays of factors X, VII or II have been used for the control of anticoagulant treatment and might be necessary when baseline tests are abnormal. Although it is possible to use such a single factor measurement, it must be remembered that the PT measures the effect of three vitamin K-dependent factors (factors VII, X and II) and is also affected by the presence of PIVKAs (proteins induced by vitamin K absence or antagonism), which are the acarboxy forms of vitamin K-dependent factors. It thus gives a better assessment of the situation in vivo: in addition, data on the appropriate individual factor levels corresponding to a given INR are limited.2,3
Standardization of Oral Anticoagulant Treatment
Standardization of oral anticoagulant therapy comprises the following steps:
Reference thromboplastins (rabbit and bovine) are available as World Health Organization (WHO) Reference Preparations via the National Institute for Biological Standards and Control (NIBSC) (www.nibsc.ac.uk), the Institute for Reference Materials and Measurements (IRMM) (Irc-irmm-rm-sales@ec.europa.eu) or certified reference materials from commercial suppliers (see p. 588). All the reference preparations have been calibrated, now sometimes indirectly, against a primary WHO reference of human brain thromboplastin, which was established in 1967.4,5
The following terms are used in the calibration procedure described below:
Calibration of Thromboplastins
Principle
The test thromboplastin should be calibrated against a reference thromboplastin of the same species (rabbit versus rabbit, bovine versus bovine) although reference plasmas from different species must at some stage be compared with each other.3 All reference preparations are calibrated in terms of the primary material of human origin and have an ISI, which is assigned after a collaborative trial involving many laboratories from different countries.
Reagents
Method
Carry out PT tests as described on p. 409. Allow the plasma and thromboplastin to warm up to 37°C for at least 2 min before mixing or adding CaCl2. Test each plasma in duplicate with each of the two thromboplastins in the following order with minimum delay between tests.
Reference Thromboplastin | Test Thromboplastin | |
---|---|---|
Plasma 1 | Test 1 | Test 2 |
Test 4 | Test 3 | |
Plasma 2 | Test 5 | Test 6 |
Test 8 | Test 7, etc. |
Record the mean time for each plasma. If there is a discrepancy of more than 10% in the clotting times between duplicates, repeat the test on that plasma.
Calibration
Plot the PTs on log-log graph paper, with results using the reference preparation (y) on the vertical axis and results with the test thromboplastin (x) on the horizontal axis (Fig. 20.1). On arithmetic paper, it is necessary to plot the logarithms of the PTs (Fig. 20.2). The relationship between the two thromboplastins is determined by the slope of the line (b).

Figure 20.1 Calibration of thromboplastin. The PTs (in seconds) with the test thromboplastin are plotted on the horizontal axis (x) and with the reference thromboplastin on the vertical axis (y) on double-log graph paper. The best-fit line is drawn by eye and the slope is obtained as follows: Points (A) and (B) are marked on the line just below the lowest recorded PT and just above the longest recorded PT, respectively, (C) and a vertical through (B) meet. The distance between (B) and (C) is measured accurately in mm. The slope b = (B to C)/(A to C). In this example B to C = 55 mm, A to C = 35 mm, b = 55/35 = 1.57. The ISI of the reference thromboplastin was 1.11. Therefore, the ISI of the test thromboplastin = 1.11 × 1.57 = 1.74.

Figure 20.2 Calibration of thromboplastin. The PTs (in seconds) are converted to their logarithms, which are plotted on arithmetic graph paper. The slope is calculated as in Figure 20.1. In this example, A to C = 42 mm, B to C = 65 mm, b = 65/42 = 1.54. Therefore, ISI = 1.11 × 1.54 = 1.71.
An estimate of the slope can be obtained as shown in Figures 20.1 and 20.2; this can then be used to obtain an approximation of the ISI of the test thromboplastin.
Calculation of International Sensitivity Index
The natural logarithms of the PTs obtained using the reference thromboplastin and the test thromboplastin are called yi and xi, respectively, where i = 1,2,3… N for N pairs of results.
The following designations are then made:
x0 and y0 are the arithmetic means of the N values of xi and yi, respectively;
Q1 and Q2 are the sums of the squares of (xi–x0) and (yi–y0), respectively;
P is the sum of their products Σ(xi–x0)(yi–y0) E = (Q2–Q1)2 + 4P2
where b is the slope of the graph. The ISI of the preparation under test (ISIt) is then given by the following:
where IRP stands for International Reference Preparation.
Local Calibration of Thromboplastins
Although the ISI system has been very effective in standardizing anticoagulant control and improving agreement between laboratories, it is not perfect. One reason is that the ISI of a thromboplastin may vary according to the technique or coagulometer used and even with different models of the same instrument. To circumvent this, a system of local calibration has been suggested. In this system, a set of plasmas with an assigned INR are tested with the local thromboplastin–machine combination. These results are plotted on log-log paper against the assigned INR. The INR for subsequent patient samples can then be read off the graph using the locally measured PT.3,4 Thus, the PT is converted directly into an INR without the need for measurement of the ISI.
Geometric Mean Normal Prothrombin Time
The geometric mean normal PT (GMNPT) for each batch of thromboplastin should be determined by testing 20 normal samples or blood donors. An equal number of males and females should be tested.
Calibration Audits
External quality-assurance surveys (e.g. UKNEQAS, see p. 594) will reflect differences regarding thromboplastin–machine combinations but not differences in blood sampling techniques (i.e. capillary and venous blood sampling). This can be a problem when capillary blood sampling is used in an outpatient setting, whereas venous samples are taken for inpatient anticoagulant monitoring. Regular audits comparing results from a range of patients whose blood has been sampled by both capillary and venous techniques will provide information not provided by NEQAS surveys.4,5
Determination of the International Normalized Ratio
If a local calibration scheme is not used, then it is essential to use a thromboplastin whose ISI has been determined either by the commercial supplier or (preferably) according to a local, regional or national procedure. The PT result can then be expressed as an INR. Using the INR/ISI system, the patient’s INR should be the same in any laboratory in the world. To ensure safety and uniformity of anticoagulation, the results should be reported as an INR, either alone or in parallel with the locally accepted method of reporting.
INR = prothrombin time ratio obtained using the test thromboplastin to the power of the ISI of the test reagent. The PT ratio is calculated using the patient’s test result and the geometric mean normal prothrombin time (GMNPT) from 20 normal donors: INR = (PT patient/GMNPT)ISI.
For example, a ratio of 2.5 using a thromboplastin with ISI of 1.4 can be calculated from the formula to be 2.51.4 = 3.61, which is either read from a logarithmic table or calculated on an electronic calculator.
The GMNPT is the logarithmic mean normal PT (i.e. e(ΣlnPT)/N). In this way, the level of anticoagulation in all plasma samples can be compared and a meaningful therapeutic range can be established regardless of the thromboplastin used.
Capillary Reagent
Reagents are commercially available for monitoring the INR using samples of capillary blood. These are usually a mixture of thromboplastin, calcium and adsorbed plasma so that when whole blood is added the reagent measures the overall clotting activity; it is sensitive to deficiency of factors II, VII and X. The reagents have an ISI assigned to them in the same way as individual thromboplastins and the INR is calculated from the PT ratio. These reagents are frequently used in anticoagulant clinics, when a large number of INRs need to be performed rapidly, and in point-of-care testing (see p. 471).
Therapeutic Range and Choice of Thromboplastin
Several authorities have now published recommended therapeutic ranges denoting the appropriate degree of anticoagulation in different clinical circumstances.7,8 These are largely based on controlled clinical trials but to some extent also represent a consensus on practice that has emerged over many years.
The choice of thromboplastin largely determines the accuracy with which anticoagulant control can be maintained. If the ISI of the thromboplastin is high, then a small change in PT represents a large change in the degree of anticoagulation. This affects the precision of the analysis and the coefficient of variation for the test increases with the ISI. Moreover, the target prothrombin ratio range becomes very small for any given range of INR. This is illustrated in Figure 20.3 and Table 20.1. For these reasons, it is strongly recommended that a thromboplastin with a low ISI (i.e. close to 1) is used.

Figure 20.3 The ratios obtained with thromboplastins with given ISI values equivalent to INR therapeutic range of 2.0–4.5.
(Slightly modified, from Poller L 1987 Oral anticoagulant therapy. In: Bloom AL, Thomas DP (eds) Haemostasis and Thrombosis, 2nd edition. Churchill Livingstone, Edinburgh, with permission.)
Table 20.1 Therapeutic ranges equivalent to an INR of 2.0–4.0 using different commercial thromboplastins
Thromboplastin | ISI | Ratios equivalent to INR 2.0–4.0 |
---|---|---|
Thrombotest | 1.03 | 2.0–3.8 |
Thromborel | 1.23 | 1.7–3.1 |
Dade FS | 1.35 | 1.65–2.8 |
Simplastin | 2.0 | 1.3–2.0 |
Boehringer | 2.1 | 1.35–1.9 |
Ortho | 2.3 | 1.3–1.8 |
ISI, International Sensitivity Index; INR, International Normalized Ratio.
(Modified from Poller L 1987 Oral anticoagulant therapy. In: Bloom AL, Thomas DP (eds) Haemostasis and Thrombosis, p. 870, 2nd edition. Churchill Livingstone, Edinburgh.)
Management of Overanticoagulation
The approach to management of a patient whose INR exceeds the therapeutic range with or without bleeding is shown in Table 20.2.7
Table 20.2 Recommendations for management of bleeding and excessive anticoagulation7
3.0<INR<6.0 (target INR 2.5) | 1. Reduce warfarin dose or stop |
4.0<INR<6.0 (target INR 3.5) | 2. Restart warfarin when INR <5.0 |
6.0<INR<8.0No bleeding or minor bleeding | 1. Stop warfarin2. Restart when INR <5.0 |
INR <8.0No bleeding or minor bleeding | 1. Stop warfarin2. Restart warfarin when INR <5.03. If other risk factors for bleeding give 0.5–2.5 mg of vitamin K (oral or i.v.) |
Major bleeding | 1. Stop warfarin2. Give PCC30–50 u/kg or FFP 15 ml/kg if PCC not available3. Give 5 mg of vitamin K (i.v.) |
FFP, fresh-frozen plasma; INR, International Normalized Ratio; PCC, prothrombin complex concentrate.

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