Laboratory Control of Anticoagulant, Thrombolytic and Antiplatelet Therapy

Anticoagulant and antithrombotic therapy is given to prevent formation or propagation of thrombi. Anticoagulant drugs, unlike fibrinolytic agents, have little if any effect on an already-formed thrombus. There are five main classes of drugs that require consideration:

1.
Coumarins and indanediones, which are orally active and act by interfering with the γ-carboxylation step in the synthesis of the vitamin K-dependent factors (see p. 371).
2.
Heparin, heparinoids (low molecular weight and synthetic compounds) and the heparin pentasaccharide (fondaparinux), which have a complex action on haemostasis, the main effect being the potentiation and acceleration of the effect of antithrombin.
3.
Direct thrombin and Xa inhibitors. These include derivatives of hirudin (natural or recombinant), and a number of orally active synthetic compounds which are now entering clinical use.
4.
Antiplatelet drugs such as aspirin, nonsteroidal anti- inflammatory drugs, dipyridamole, inhibitors of the P2Y ₁₂ adenosine diphosphate (ADP) receptor and inhibitors of glycoprotein IIb/IIIa function, some of which are antibodies. Novel agents targeting other platelet receptors and functions are also in development.
5.
Thrombolytic agents including streptokinase, urokinase and tPA as well as a number of recombinant tPA derivatives with modified properties.

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 minimises the combined risks of haemorrhage and thrombosis: the therapeutic range. This range will be different for different patients and individual responses to oral anticoagulant treatment with vitamin K antagonists are extremely variable, so must be regularly and frequently controlled by using 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) 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 standardisation 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 Normalised Ratio (INR), to report the results, has minimised 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. ^,

The Thrombotest of Owren and the prothrombin and proconvertin (P&P) method of Owren and Aas were used in the past, but they are no longer recommended for oral anticoagulant control. Historically, standardisation of oral anticoagulant therapy was carried out following the procedure described below. It is now possible to simplify this by using local procedures which are discussed afterwards.

Standardisation of oral anticoagulant treatment

1.
A thromboplastin is chosen, and its ISI is determined by comparison with a reference thromboplastin.
2.
The log mean normal PT is determined for that thromboplastin.
3.
PTs are performed on patient samples, and the results are converted to an INR.

World Health Organisation (WHO) Reference Preparations are available from the National Institute for Biological Standards and Control (NIBSC) ( www.nibsc.org ). Certified preparations calibrated against the WHO material are available from the Institute for Reference Materials and Measurements (IRMM) ( https://ec.europa.eu/jrc/institutes/irmm/ ) and from commercial suppliers (see p. 535). All the reference preparations have been calibrated, now sometimes indirectly, against a primary WHO reference of human brain thromboplastin, which was established in 1967. ^,

The following terms are used in the calibration procedure described below:

International Sensitivity Index (ISI). This is the slope of the calibration line obtained when the PTs obtained with the reference preparation are plotted on the vertical axis of log-log paper and the PTs obtained by the test thromboplastin are plotted on the horizontal axis. The same normal and anticoagulated patient plasma samples are used for both sets of results.
International Normalised Ratio (INR). This is the PT ratio for a sample, which, by calculation, would have been obtained had the original primary, human reference thromboplastin been used to perform the PT. Its calculation is shown below.

Calibration of thromboplastins

Principle

The test thromboplastin should be calibrated against a reference thromboplastin of the same species (rabbit vs. rabbit, bovine vs. bovine) although reference plasmas from different species must at some stage be compared with each other. All reference preparations are calibrated in terms of the primary material of human origin and have an ISI which is assigned after a collaborative study involving many laboratories from different countries.

Reagents

Normal citrated plasma. From 20 healthy donors.
Anticoagulated plasma. From 60 patients stabilised on oral anticoagulant treatment for at least 6 weeks.

The tests need not all be done at the same time; they may be carried out on freshly collected samples on successive days.
Reference and test thromboplastins.
CaCl ₂ . 0.025 mol/l.

Method

Carry out PT tests as described on p. 380. 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 PT 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 ). If using arithmetical paper, plot the logarithms of the PTs ( Fig. 20-2 ). The relationship between the two thromboplastins is determined by the slope of the line (b).

An estimate of the slope can be obtained as shown in Figures 20-1 and 20-2 ; this can then be used to obtain the ISI of the test thromboplastin. Alternatively the slope can be calculated using a computer or online system to apply simple linear regression. This has been shown to be sufficiently accurate compared to the orthogonal regression that is theoretically preferable. A procedure for manual calculation of the slope is given in previous editions of this book.

Figure 20-2, Calibration of thromboplastin.

Whenever possible, however, to obtain a reliable measurement, the following more complicated calculation should be used instead.

Calculation of international sensitivity index

The natural logarithms of the PTs obtained using the reference thromboplastin and the test thromboplastin are called y _i and x _i , respectively, where i = 1,2,3…N for N pairs of results.

The following designations are then made:

x ₀ and y ₀ are the arithmetical means of the N values of x _i and y _i , respectively.
Q ₁ and Q ₂ are the sums of the squares of ( x _i − x ₀ ) and ( y _i − y ₀ ), respectively.
P is the sum of their products Σ( x _i − x ₀ )( y _i – y ₀ )

E = {(Q_{2} - Q_{1})}^{2} + 4 P^{2}

$E = {(Q_{2} - Q_{1})}^{2} + 4 P^{2}$

And b = \frac{Q_{2} - Q_{1} + E^{1 / 2}}{2 P}

$And b = \frac{Q_{2} - Q_{1} + E^{1 / 2}}{2 P}$

where b is the slope of the graph. The ISI of the preparation under test (ISI _t ) is then given by the following:

{ISI}_{t} = {ISI}_{IRP} \times b

${ISI}_{t} = {ISI}_{IRP} \times b$

where IRP stands for International Reference Preparation.

Local calibration of thromboplastins and direct INR determination

Although the ISI system has been very effective in standardising 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 using plasmas with a certified INR is now recommended.

In the method called ‘local calibration’ the manufacturer supplies a set of plasmas representing a range of INRs with an assigned PT using a reference thromboplastin. The laboratory then measures the PTs of these samples locally and they are plotted against the assigned PT on a log/log plot and an orthogonal regression line is calculated to give the ISI as with the standard method above. It has been shown that use of a linear regression, which is easier to calculate, is also acceptable.

A simpler method is direct INR determination. In this system, a set of plasmas with an assigned INR are tested with the local thromboplastin-machine combination. These results are then 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. Thus the PT is converted directly into an INR without the need for measurement of the ISI: this has been described as ‘direct INR determination’. The accuracy of the system relies on the ability of the supplier to validate the INR for the (usually) lyophilised plasma on the system to be used by the laboratory. For this method it is recommended that a minimum set of one normal and three abnormal plasmas are used to reduce imprecision. The plasmas may be lyophilised or frozen and from anticoagulated patients or artificially factor-depleted but should span a range of INRs from 1.5 to 4.5.

Geometric mean normal prothrombin time

If the INR is to be calculated using the PT and the determined ISI then the geometric mean normal PT (GMNPT) for each batch of thromboplastin must be determined by testing 20 normal samples or blood donors. An equal number of males and females should be tested. The GMNPT is the logarithmic mean normal PT (i.e. e ^(ΣlnPT)/N ).

Calibration audits

External quality-assurance surveys (e.g. United Kingdom National Quality Assessment Service, UK NEQAS, see p. 539) will reflect differences relating to 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 internal 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.

Determination of the international normalised ratio

If a local calibration scheme is not used then it is essential to use a thromboplastin the ISI of which 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. The INR is calculated as follows:

INR = {(PT patient / GMNPT)}^{ISI}

$INR = {(PT patient / GMNPT)}^{ISI}$

For example, a PT ratio of 2.5 using a thromboplastin with ISI of 1.4 can be calculated from the formula to be 2.5 ^1.4 = 3.61, which is either read from a logarithmic table or determined on an electronic calculator.

Capillary reagents

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.

Point-of-care testing

There are now schemes for monitoring INR using point-of- care devices outside the hospital clinic. These require selection and standardisation of appropriate analysers and a quality-control programme that includes participation in an external quality assessment scheme. There should be an established procedure for checking any problems of instrument performance and for referring to the specialist centre patients who are difficult to control. ^,

Self-management of warfarin treatment may also be an effective point-of-care procedure for selected patients. They should first attend two or more training sessions on the use and quality control of the appropriate analyser, interpretation of INR, adjustment of warfarin dosage and guidance on when it is necessary to be seen at the specialist clinic. ^,

Therapeutic range and choice of thromboplastin

Several authorities have now published recommended therapeutic targets denoting the appropriate degree of anticoagulation in different clinical circumstances. ^, 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 . 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.

Management of overanticoagulation

The approach to management of a patient whose INR exceeds the therapeutic range with or without bleeding is shown in Table 20-1 and guidelines have been published. ^,

Table 20-1

Recommendations for management of bleeding and excessive anticoagulation

INR 3.0–6.0 (target INR 2.5) INR 4.0–6.0 (target INR 3.5)	1. Reduce warfarin dose or stop 2. Restart warfarin when INR < 5.0
INR > 6.0–8.0 (No bleeding or minor bleeding)	1. Stop warfarin 2. Restart when INR < 5.0
INR > 8.0 (No bleeding or minor bleeding)	1. Stop warfarin 2. Restart warfarin when INR < 5.0 3. If other risk factors for bleeding give 0.5–2.5 mg of vitamin K (oral or IV)
Major bleeding	1. Stop warfarin
	2. Give PCC 25-50 u/kg or FFP 15 ml/kg only if PCC not available
	3. Give 5 mg of vitamin K (oral or IV)

FFP, fresh-frozen plasma; INR, International Normalised Ratio; IV, intravenous; PCC, prothrombin complex concentrate.

Heparin treatment

The anticoagulant action of heparin is primarily a result of its ability to bind to antithrombin (AT), thereby accelerating and enhancing the latter’s rate of inhibition of the major coagulation enzymes (i.e., factors IIa and Xa and to lesser extents IXa, XIa and XIIa). The two main effects of heparin, the antithrombin and the anti-Xa effects, are differentially dependent on the size of the heparin molecule. The basic minimum sequence needed to promote anticoagulant activity has been identified as a pentasaccharide unit. Of the molecules containing this pentasaccharide, those comprising fewer than 18 saccharide units and of molecular weight less than 5000 Da can only augment the inhibitory activity of AT against Xa. In contrast, longer chains can augment anti-IIa activity as well by formation of a tertiary complex bridging AT and thrombin molecules.

Hence low molecular weight heparins (LMWH), which have an average molecular mass of 5000 Da, have a ratio of anti-Xa to antithrombin effect of 2–5 compared to that of unfractionated heparin (UFH), which is defined as having a ratio of 1. However, all heparin preparations are heterogeneous mixtures of molecules with different molecular weight, many of which do not contain the crucial pentasaccharide sequence. Heparin also produces some anticoagulant effect by promoting the release of tissue factor pathway inhibitor (TFPI) from the endothelium (see p. 372) which will not be present in plasma ‘spiked’ with heparin.

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