Problems In Laboratory Monitoring Of Heparin Dosage (2000)

Heparin is a sulphated polysaccharide widely used as an anticoagulant for the treatment and prevention of thromboembolic disease (Hirsh, 1991; Hyers et al, 1992; Hirsh & Fuster, 1994). For many years, clinical preparations have been unfractionated, containing a mixture of polysaccharide chains of different lengths made up largely of alternating residues of uronic acid and glucosamine (Casu, 1990). Unfractionated heparin (UFH) contains material with a molecular weight range of 3000–35 000 Da with a mean of 12–15 000 (Andersson et al, 1979, Fareed et al, 1996). Unfractionated heparin can be administered by the intravenous or the subcutaneous route (Hull et al, 1986; Hirsh & Fuster, 1994), both of which are safe and effective. These studies have demonstrated that the risk of bleeding probably increases with increasing doses of heparin and that the frequency of recurrent venous thromboembolism is low provided that adequate heparin doses are employed. Laboratory monitoring of full-dose UFH is required, irrespective of the route of administration. For heparin to exert its anticoagulant activity, the cofactor anti-thrombin (AT) is required (Brinkhous et al, 1939; Abildgaard, 1968). After heparin binding, AT undergoes a conformational change and is converted into a very rapid inhibitor with the active centre arginine residue in AT much more reactive against the active centre serine of several coagulation enzymes (Lindahl et al, 1979; Rosenberg & Lam, 1979). With respect to the saccharide polymers, there is a requirement for a pentasaccharide to potentiate the action of AT. Polymers less than five units in length have no anticoagulant activity. For inhibition of thrombin, heparin must bind to both the enzyme and AT. Fractions with fewer than 18 saccharide units are unable to bind thrombin and AT simultaneously (Casu et al, 1981) and therefore do not catalyse inhibition of thrombin. Simultaneous binding of enzyme and heparin is not required for neutralization of activated factor X (Casu et al, 1981). Fragments with 5–17 (or a greater number) of saccharides are therefore able to catalyse inhibition of activated factor X (Lane et al, 1984). Low molecular weight heparins (LMWHs) that contain a majority of polymers less than 18 units in length are manufactured from UFH by controlled depolymerization employing chemical, enzymatic or irradiation techniques (Fareed et al, 1996). These processes yield products that differ in final composition and which may have different properties in clinical usage. The complex of heparin with AT is able to inactivate a number of coagulation enzymes including factor IIa (thrombin), factor VIIa, factor IXa, factor Xa, factor XIa and factor XIIa. The relative importance of inhibition of different enzymes remains controversial although the most important effects may relate to inhibition of IIa or Xa. As many LMWH preparations contain few heparin fragments sufficiently large enough to inhibit thrombin, the anticoagulant actions of LMWHs are mainly through neutralization of factor Xa. Heparins interact with other inhibitors of coagulation, including a second plasma cofactor, named heparin cofactor II (HCII), that can bind thrombin and has some anticoagulant action (Tollefsen et al, 1982). Tissue factor pathway inhibitor (TFPI) may also be involved in the anticoagulant and anti-thrombotic actions of heparin. The action of this inhibitor of factor Xa is enhanced by heparin, at least in purified systems (Broze et al, 1988), and injection of heparin results in a several-fold increase in TFPI activity in the blood (Sandset et al, 1988). Several studies have subsequently concluded that TFPI contributes to the anti-thrombotic activity of UFH and LMWH (Abildgaard et al, 1991, Lindahl et al, 1991; Kijowski et al, 1994). A number of different blood sample collection systems have been used for collection of blood samples for heparin monitoring that vary in respect of tube composition (plastic or glass), and siliconization and anticoagulant. There is evidence that use of 0·109 mol/l trisodium citrate (3·2%) or 0·129 mol/l trisodium citrate (3·8%) can influence the activated partial thromboplastin time (APTT) determinations in samples from patients receiving intravenous UFH, results being on average 10% greater in samples collected into 0·129 mol/l citrate (Adcock et al, 1997). The lower strength is increasingly used as it is recommended for samples collected for monitoring oral anticoagulant therapy (WHO, 1999). It is now well known that, after blood has been collected, any heparin present may be partially neutralized during any delay before analysis. This is caused by release of platelet factor 4 (PF4) from platelets within the sample (Rucinski et al, 1979). The PF4 binds to heparin and neutralizes it. In order to combat this problem, samples from heparinized patients can be collected into an anticoagulant cocktail that inhibits platelet release, thereby avoiding PF4-mediated neutralization of heparin. It has been demonstrated that collection of blood into a citrate, theophylline, adenosine, dypyridamole mixture (CTAD) can greatly reduce the neutralization of heparin activity in blood (Contant et al, 1983; Van den Besselaar et al, 1987). In the study of Van den Besselaar et al (1987), APTTs of blood samples collected into CTAD were associated with less than 11% shortening depending on APTT reagent, compared with a 15–29% shortening of citrated APTTs between 1 h and 5 h after collection. Based on their results, the authors recommended the use of CTAD for heparin monitoring samples. The same study concluded that room temperature was the most appropriate storage temperature and also recommended minimum centrifugation at 940 g for 30 min or 2200 g for 10 min. These studies suggest that if the APTT is used for heparin monitoring, it is important to perform the measurement within 5–6 h if CTAD is used as the anticoagulant. If citrate is used, testing of samples more than 2 h after collection may lead to important underestimation of in vivo heparin effects. Freezing samples for later analysis should be avoided as this can lead to shorter APTTs unless great care is taken to remove the maximum possible number of platelets (Van den Besselaar et al, 1990). There is evidence that the volume of blood within a particular vial size can influence the APTT as determined for UFH control, even when the ratio of anticoagulant to blood (1:9) is maintained (Ray, 1991; Ray et al, 1993). The mean APTTs of 5 ml samples from heparinized patients was 120 s compared with a mean of 79 s for 2 ml samples (Ray, 1991) when both were collected into containers with a capacity of 6·7 ml. The author speculated that the air bubble of 4·7 ml within the sample containing 2 ml of blood caused greater mixing, release of PF4 and neutralization of heparin than the 1·7 ml air bubble in the samples containing 5 ml of blood. In a subsequent study, increased release of PF4 produced similar effects (i.e. significantly shorter APTTs in the container with a smaller volume of blood) if citrate was used, but not if CTAD was used, as the anticoagulant (Ray et al, 1993). For heparin monitoring it is therefore inappropriate to use sample containers that contain a large air volume unless CTAD is used as the anticoagulant. The timing of blood sample collection for heparin monitoring should depend on the administration of the drug. If heparin is given by intravenous infusion, the sample should be collected after equilibrium has been reached (Samama, 1995a), normally 4–6 h after initiation of the infusion. A similar lag phase should pass prior to sampling following a dose adjustment. When heparin is given by subcutaneous injection, the maximal response of the APTT occurs after 4–6 h (Griffith & Boggs, 1964, Hull et al, 1982). Samples should therefore be collected at the mid-interval between injections if given twice daily. These blood collection times have been successfully used in a number of clinical trials (Hull et al, 1986,1990; Turpie et al, 1989). There are conflicting reports in relation to possible diurnal changes in the anticoagulant effect of infused heparin. In studies of heparinized patients (Decousus et al, 1985) or healthy volunteers (Krulder et al, 1992) there was considerable diurnal variation, with significantly greater APTT prolongation in the night (3·30–8·30am) than in the day (Krulder et al, 1992) and up to 60% difference in individual results between night and day (Decousus et al, 1985). Such a diurnal effect was not detected in two further studies (Toulon et al, 1987; Fagrell et al, 1989). On occasion, it may be necessary to eliminate heparin in a plasma sample. Several methods for removal of heparin from plasma have been described that involve absorption of heparin by cellulose resin (Cumming et al, 1986; Wenz & Burns, 1991), affinity filtration (Wenz & Burns, 1991) or enzymatic elimination (Hutt & Kingdom, 1972). Some heparin elimination techniques can lead to reduction of clotting factors, particularly factor IX, which was reduced to about 60% of initial levels by cellulose resin adsorption and to 70% by affinity filtration in one study (Wenz & Burns, 1991). The most widely used test for heparin monitoring in the USA (Banez et al, 1980), the Netherlands (Van den Besselaar et al, 1990) and the UK (Kitchen et al, 1996) is the APTT. In a study of thrombo-prophylaxis, Basu et al (1972) concluded that the risk of venous thromboembolism was reduced when APTT ratios (patient–control) were greater than 1·5 and they suggested a range of The same range was to be in the study of Hull et al and this range remains in There is evidence that the between heparin and APTT is if a ratio is used than the APTT in (Van den Besselaar et al, 1990). In this study, the ratio was by the APTT by the same determined after the heparin been with Such an may not be for in of the time and A widely used is to the APTT by the mean although this not of the APTT in the of heparin. A number of studies have in the of different APTT to heparin (Banez et al, & et al, den Besselaar et al, Ray et al, 1992; et al, & although is required in the of some studies as the response to heparin to plasma may be different from the response to vivo samples from patients receiving heparin et al, et al, & 1996). In one study, the mean APTT ratio of patients receiving UFH from to depending on the and used, and the mean heparin associated with an APTT ratio of from to for different APTT techniques (Kitchen & 1996). As a the range of not to There is also evidence of between APTT in respect of to the levels of clotting & et al, 1979; et al, particularly factor As factor is an levels probably vary in a of patients with thromboembolic disease and this may have to the difference in of described in some of the There are a number of that to between in respect of heparin including the use of different and In there is of the of APTT used in the et al, and in the (Kitchen et al, The of APTT a range from (Kitchen et al, was from a from material and from in containing (Kitchen et al, It is therefore not that as influence heparin et al, 1986; Van den Besselaar et al, 1993). In study (Kitchen et al, there was a between a lower of present as or and increasing heparin There is also evidence that different with the same are associated with in heparin & 1981). there are a number of APTT have different heparin The recommended ratio range of is largely on studies in which the heparin was by the (Hirsh, 1991). The that a heparin of is an appropriate lower for therapy is by a number of studies (Hull et al, 1986,1990; Turpie et al, et al, 1994). The evidence for an increased risk of bleeding when a heparin of is is less well (Hull et al, although there are reports of increased bleeding at levels of or greater by et al, to the of between in respect of heparin is for a to a range for the APTT in use that to a heparin of by or by (Hirsh, 1991). As the between heparin and APTT is different to heparin is to plasma in compared with this must be samples from patients receiving heparin. This has been used in at least studies different APTT et al, & et al, In two of et al, & the between APTT ratios and heparin was analysis and important were when the APTT ratios to by were for This range was for one APTT and for (Kitchen & 1996). It should be that the between heparin and APTT is the influence of other effects on particularly Samples with the same APTT ratio can have different heparin and samples with the same heparin have very different APTT ratios (Kitchen & 1996). When a a change of APTT the to heparin can be used to the range for the as described the of this use of that to their which may be to is to a with similar heparin to the used In this a of a sufficiently similar for clinical has been described et al, A further is the of oral anticoagulant and heparin of and X APTTs irrespective of the heparin is therefore necessary when a range by APTT ratio from patients an increase in is to patients in the is If an APTT ratio that the heparin effect is required, the ratio of the APTT before and after heparin elimination can be It should also be that between heparin of different or of produced by the same can In one study on of heparinized of one APTT not of that a sample with heparin APTT ratios between and with different et al, 1988). in heparin of the same was in on samples from patients et al, 1993). important when the range by APTT for a different or different of the same with heparin is the number of samples required to in the 30 and samples have been used in some studies employing analysis et al, & 1996). analysis on a number of samples not yield the same results as a large sample as in a study APTT and activity in more than samples 1997). This study suggested that at least several samples may be on the and of between clinical has been suggested The results of APTTs determined for heparin monitoring can be by the of et al, 1990). have demonstrated that use of one particular with one was not associated with between APTT ratio when compared to (Kitchen & with the same a increase in APTT ratios was mean APTT ratio of patients compared with mean A range for heparin should therefore of both and APTTs determined for monitoring heparin that an to is required in order to the patients being within the range for of treatment in one study et al, 1985). A of dose changes to of for one particular APTT has been et al, 1988). It should be that this may well to of the in There has been some about the importance of adequate initial doses of UFH, as by APTT within h and the risk of recurrent et al a analysis of several studies and no evidence that the risk of was to APTT results within In a analysis of studies by Hull et al suggested that venous thromboembolism was times more in patients for APTT was not at h compared with APTT the by The of different in respect of the by oral have been a recommended by Several studies have the possible use of a similar to of heparin therapy by APTT et al, Van & In one study, use of a for the same used in different was not (Van & In study, within and between after of a was to be et al, and the authors concluded that the of a similar to the be These studies that the is an inappropriate for of monitoring the APTT. the APTT remains the most used test for monitoring heparin the between APTT and heparin is This is a of the of of APTT for heparin There are a number of when the APTT may be inappropriate for heparin monitoring of this of in which APTT may be within the for heparin therapy in the of heparin the the of and heparin therapy in which reduction of and X to prolongation of heparin and and or of clotting APTT IX, II or In there are other in which doses of heparin may be required to APTT into the This can when the APTT in the of heparin is reduced as a of levels of clotting factors, particularly or during the phase levels of or reduction of anti-thrombin can similar effects. In some of a heparin is more appropriate than APTT as an of the anti-thrombotic effects of heparin. important study by et al compared heparin with APTT for monitoring in patients required a large 000 of heparin. were into two one of which was by APTT with a range of s and the other by a with a of The were to be to of heparin by to be the in the required significantly more heparin units compared with in the day after The APTTs of the were shorter than the and were the smaller dose of heparin given to the to be adequate as the of of thromboembolism was similar in the two of the compared to in the APTT within There were bleeding in patients by APTT more and one in the monitoring with was safe and in patients APTT large doses of heparin. study that patients receiving heparin may to be by APTT the of adequate heparin levels et al, 1994). studies which the of clinical are required to that can be successfully for monitoring of heparinized The for heparin is not widely and is not other of heparin have been described employing a range of different have been widely as they are more for heparin than APTT. These the of anti-thrombin to inhibit activated factor X to plasma or of enzyme is by its clotting activity & or by of a et al, et al, et al, 1994). in the are by factor Xa the from to a to is associated with an increase in at In this the strength of is to heparin activity. In the clotting a of factor Xa is neutralized by The plasma is with a mixture containing clotting factors, and the clotting time being to heparin heparin to lower Xa and subsequently clotting of heparin use et al, neutralization of thrombin (Van den Besselaar et al, 1990) or et al, 1993). of heparin may differ to the and it has been that results are greater than heparin levels by In one study, by to by et al, 1994). In study, the mean in samples from patients receiving UFH by was depending on compared with a mean of by (Kitchen et al, and by to by Some techniques involve of anti-thrombin to the some this to be and that results may be less as they do not the anticoagulant effect in which is through plasma anti-thrombin et al, et al, 1987). This is of importance as heparin therapy a in anti-thrombin that is of the initial is after 1 at after and to after of therapy & et al, 1985). The reduction is a of et al, and may lead to therapy at plasma levels of AT 1984). As in the of it is important to perform determinations as as possible after sample collection to PF4-mediated neutralization of heparin. Samples that are successfully of platelets can be important of activity et al, Van den Besselaar et al, 1990). Heparin are by a by of heparin to plasma at a range of The heparin being used for treatment has been used in this et al, although is by a heparin that has been against a for heparin et al, 1984). test that has been used for heparin monitoring is the thrombin time which was to be more to plasma heparin than one APTT et al, 1980), a that was in a later study a thrombin (Ray et al, 1996). with this test is that the of the thrombin time to increasing of heparin is not (Ray et al, 1996). In the test may at doses of heparin not the between and The evidence that low molecular heparins (LMWHs) are at least as as heparin in and has been the of (Hull & 1994). In there are reports to use of LMWH in in et al, and it has been suggested that LMWH be increasingly used in The or for monitoring of LMWH is not 1994). as the in is much lower for LMWH et al, 1990) than for heparin and as the of LMWH is of dose et al, it is that monitoring of LMWH to be as as for This is by a in which a dose was compared to a by activity to a of et al, 1994). The authors concluded that the two were with respect to and that the of treatment not differ between the two there was no evidence for a requirement for A on of the of et al, also that patients receiving LMWH or for of venous or patients for venous thromboembolism by a do not There a that monitoring is necessary in and the same monitoring in patients and that monitoring may be of in patients with receiving therapy including at risk of bleeding or and patients with or low Low molecular weight heparins have less effect on inhibition of thrombin than UFH as they contain fewer of the large chains necessary to catalyse this For this many LMWHs APTT (or thrombin compared with UFH, an effect which is to their ratio et al, 1991). after administration of the chains inhibition of IIa are twice as as the shorter chains activity et al, 1994). The test of if monitoring is necessary is therefore the even the between and or risk of is not 1994). In one suggested as for a risk for a risk for In the on of et al, a activity of was recommended for samples collected 4 h after subcutaneous injection from patients receiving twice As for of UFH, both clotting and have been used for monitoring There is evidence that some may activity relative to et al, 1991; et al, et al, have compared the results of five and clotting in samples from patients receiving or and between the results of different techniques (Kitchen et al, of the clotting were significantly lower than results by some other clotting and by in therapy and in a which was by the of with different methods of (Kitchen et al, It has been suggested that the should be the of et al, of the influence of activity on some clotting although not clotting techniques are in this et al, et al, There is evidence that the of results in different samples from patients receiving different LMWH preparations be reduced if LMWH was used in the of in of heparin et al, and there is evidence that the for heparin is as a for LMWH et al, 1985). The of for of for monitoring LMWH therapy should therefore be a LMWH that has been against the for LMWH et al, 1988). A number of have now been that heparin monitoring at the Some of are for during or et al, have been for monitoring patients with heparin for thromboembolic disease et al, et al, et al, 1996). have the of results from the requirement for centrifugation of samples. blood collected, for by required blood. UK for testing in including monitoring of heparin have been et al, and testing of samples to the range of heparin levels in the being compared to the APTT in use and to activity. These that the of the should for of the with of results, and that should be by an appropriate of the may also for The and of or testing in coagulation have been of the most important of heparin therapy is in platelet in as many as 10% of patients receiving heparin and to during therapy The reduction in platelet to as may be associated with and between the and day after initiation of therapy in patients should be if to of the initial platelet after several of heparin therapy As UFH therapy is if occurs and can be successfully used in some et al, patients receiving heparin platelet to be in to monitoring, particularly therapy is It has been suggested that patients receiving heparin for the time should have platelet at and on after day monitoring is required from the in patients with to heparin et al, The anticoagulant of for monitoring UFH therapy is CTAD (Van den Besselaar et al, 1987). Samples should be collected 4–6 h after initiation on infusion, injection or dose et al, When employing APTT for monitoring UFH, the of the monitoring must be taken into when a range et al, & and should to by or by (Hirsh, 1991). The heparin can be used for monitoring UFH, particularly when APTT is inappropriate et al, 1994). patients receiving LMWH as or patients for venous thromboembolism by a do not monitoring et al, LMWH may monitoring in as therapy including at risk of bleeding or and patients with or low et al, A range of has been recommended for patients receiving twice LMWH therapy et al, for LMWH should be a material against the for of may vary to the (Kitchen et al,