Measurement of heparin, direct oral anti‐coagulants and other non‐coumarin anti‐coagulants and their effects on haemostasis assays: A British Society for Haematology Guideline

Abstract

This guideline was compiled according to the BSH process at [https://b-s-h.org.uk/media/16732/bsh-guidance-development-process-dec-5-18.pdf]. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) nomenclature was used to evaluate levels of evidence and assess the strength of recommendations. The GRADE criteria can be found at http://www.gradeworkinggroup.org. Literature search criteria can be found in Appendix A. Review of the manuscript was performed by the British Society for Haematology (BSH) Haemostasis and Thrombosis Task Force and the BSH Guidelines Committee. It was also placed on the members section of the BSH website for comment. This guideline aims to update healthcare professionals working in the UK on the measurement of anti-coagulants (other than coumarins) currently licensed for use in the UK, and their effects on laboratory assays (Table 1). It provides recommendations based on the body of literature produced since the previous guidance published in 2014.1 Direct factor (F)XIa- and direct FXIIa-inhibiting anti-coagulants are at various stages of development but not yet licensed, so are not discussed in this guideline.2 The recent guidelines from the International Society of Thrombosis and Haemostasis Scientific Standardization Committee (ISTH/SSC) on the nomenclature to be used when describing non-vitamin K anti-coagulation3 are followed. Hepatobiliary (20) Renal (80) Dabigatran dTT Dabigatran ECT/ECA Dabigatran anti-FIIa Hepatobiliary (66) Renal (33) Hepatobiliary (75) Renal (25) Hepatobiliary (50) Renal (50) Low dose—Reticuloendothelial system High dose—Renal APTT UFH anti-FXa Combined UFH/LMWH anti-FXab UFH anti-FIIa APTT Argatroban dTT Argatroban ECT/ECA Argatroban anti-FIIa LMWH anti-FXaa Combined UFH/LMWH anti-FXab Some anti-coagulants, such as unfractionated heparin (UFH) and argatroban, have been in clinical use for decades. Laboratory monitoring to guide dose adjustments has been with the widely available activated partial thromboplastin time (APTT). However, the COVID-19 pandemic highlighted the wide variability in the sensitivity of different APTT reagents in patients with acute illness, emphasising the need for accessible and cost-effective anti-FIIa and anti-FXa assays for routine monitoring. The introduction of specific anti-FIIa or anti-FXa-based assays has provided a means to quantitate plasma drug concentrations of the newer fixed-dose FIIa inhibitors (FIIaI) and FXa inhibitors (FXaI). When used according to licence, monitoring these direct oral anti-coagulants (DOACs) is not required, but measuring drug concentration can add value in some circumstances (Table 2). As neither drug concentration nor dose-adjustment based on the measured concentration has yet been shown to affect efficacy or safety, it is incorrect to refer to a therapeutic range. In this manuscript, the term ‘expected range’ is used to acknowledge this limitation. There are many reports in the literature about the effects of anti-coagulants on measurable parameters of haemostasis. Lack of awareness of these effects, which are variable depending on anti-coagulant, timing of sampling and reagents, can cause confusion and delay diagnosis and care. Table 3 gives a broad overview of the types of impact on laboratory assays that may be seen. All assays described must be used in accordance with requirements of ISO15189. Activated charcoal products (ACP) (tablets or filters) that adsorb some anti-coagulant activities from plasma samples have been suggested as a way of undertaking haemostatic tests while continuing anti-coagulant therapy.4-6 The process appears to remove not only the effects of rivaroxaban, apixaban edoxaban, dabigatran, argatroban, protamine, aprotinin and polymyxin but also leaves heparin-like and coumarin anti-coagulant activity intact.7, 8 Many haemostatic parameters have been reported to be unaffected by ACP, including those associated with haemophilia, thrombophilia (including lupus anti-coagulant assays) and thrombin generation assays (TGA), making the process attractive for many diagnostic algorithms.9-11 ACP do not remove the effects of heparin-based or coumarin anti-coagulation, and caution is required if there are high levels of FXaI present, as these may be incompletely removed.11, 12 If laboratories wish to use this approach, the impact on local assays and reagents needs to be assessed in-house and documented in accordance with the requirements of ISO15189. In general, delaying testing until off anti-coagulation is the preferred option whenever possible. Heparin is a naturally occurring glycosaminoglycan polymer that has a physiological anti-coagulant function. It exists naturally as polymers of varying sizes (20 000–50 000 Daltons), and all pharmaceutical-grade UFH is derived from porcine or bovine intestinal mucosa. Fractionation of primary polymers produces smaller molecules of varying sizes referred to as low-molecular-weight heparin (LMWH). UFH produces its anti-coagulant effect mainly through inactivation of FIIa and FXa (as well as FIXa, FXIa and FXIIa to a lesser extent) through an anti-thrombin-dependent mechanism.13 UFH is a highly negatively charged molecule with a propensity for reversibly binding to proteins and surfaces. Pharmacokinetic limitations are caused by anti-thrombin-independent binding of heparin to plasma proteins released from platelets and endothelial cells, resulting in a variable anti-coagulant response leading not just to large interindividual variability but also to intraindividual variability influenced by the patient's inflammatory response.14 One effect of this is to release tissue factor pathway inhibitor (TFPI), inhibiting thrombin generation in vivo, which is likely to contribute to the anti-coagulant action.15 Therefore, the anti-coagulant effect is not directly proportional to the dose of UFH, which requires routine monitoring to optimise the balance between the required anti-thrombotic effect and excessive bleeding risk. Tests suitable for monitoring UFH are APTT, activated clotting time (ACT) and heparin anti-FXa activity assay. None of these assess all the anti-thrombotic effects of UFH and all have limitations. Furthermore, evidence to support the widely used expected ranges by either APTT or heparin anti-FXa assay is weak. When using tri-sodium citrate blood collection tubes for UFH monitoring, there should be only a small residual air space in the tube once blood is added, achieved mainly with a predetermined vacuum.16, 17 Citrated samples containing UFH destined for APTT must be centrifuged within 1 h of collection and analysed within 4 h to avoid leakage of platelet factor-4 (PF4), leading to neutralisation of heparin.18-20 Dextran sulphate releases heparin from its complex with PF4 so when samples are analysed by anti-FXa assay using dextran sulphate-containing reagents, centrifugation can be delayed for up to 4 h since there is only minor or no loss of heparin anti-FXa activity and little clinically relevant impact on management decisions.20, 21 Samples collected into citrate–theophylline–adenosine–dipyridamole (CTAD) are stable for at least 4 h, even when used for an APTT.21-23 One major limitation of the APTT for monitoring UFH is the lack of specificity. For example, a lupus anti-coagulant or deficiency of one or more clotting factors, such as FXII, may prolong the APTT. This may falsely raise the APTT into the target range despite suboptimal heparin levels. Conversely, the APTT may not be within the target range even if the heparin is at the therapeutic concentration in the of levels of such as and are to the response that is in patients deficiency can be in to a UFH Furthermore, may be in patients with acute of the and This the APTT to UFH, leading to the incorrect that heparin levels are The target APTT range for UFH for was in a in which only patients and with there was a of when using an APTT of a APTT, the evidence to support this as a target range was weak. The APTT is no in use and is not to The target range is not since APTT reagents in their to the for using UFH for anti-coagulation have and the used for to The target APTT was shown to to heparin when measured using assay or heparin anti-FXa of Laboratory of clinical have shown that a target range for APTT by to or heparin for the variable response of APTT to heparin using samples from to The limitations of the APTT for UFH monitoring are and even use of an APTT target range heparin anti-FXa to as to of therapeutic or when different APTT reagents used in UFH patients to APTT APTT is for UFH monitoring should have a APTT performed to of UFH If the APTT is or the APTT is for monitoring UFH for that In these a heparin anti-FXa assay is a option to drug UFH anti-FXa of is as the UFH therapeutic range for of and a as to of This was derived from a in which patients UFH 000 to monitoring with APTT or UFH anti-FXa using therapeutic ranges to by The UFH anti-FXa range was to using an assay dextran sulphate The APTT of UFH than the UFH anti-FXa within the 12 of in and in the heparin anti-FXa and APTT There bleeding in the APTT and one in the by UFH of the UFH anti-FXa assay should be with a UFH to have to a anti-FXa assay. There is literature to and these should be if Dextran sulphate is to some reagents used for heparin anti-FXa assays to binding of UFH to plasma which may in or in containing dextran sulphate may than those at least when some of the heparin is to proteins in the This can to of heparin anti-FXa activity in patients heparin by the of dextran sulphate in anti-FXa reagents of the heparin available in of in binding of heparin to recent using samples by UFH into plasma suggested using heparin anti-FXa assays provided blood collection is performed and the tube of blood is to the of PF4 produced there is currently no on heparin anti-FXa assays with or dextran sulphate should be for monitoring of the or of dextran sulphate of heparin anti-FXa assays as in a UK the of for of different heparin anti-FXa assays with and some dextran was for UFH samples in the therapeutic range to for APTT with reagents, making the heparin anti-FXa assay a more attractive Furthermore, use of heparin anti-FXa assays to UFH achieved a time to therapeutic range and dose adjustments to use of APTT in of 000 patients that by heparin anti-FXa likely to have a than patients by APTT for and these and reported of a heparin anti-FXa assay APTT, a of with patients found that use of APTT to heparin anti-FXa was not associated with of bleeding or an of There no in in the not suitable for it has been reported that the heparin assay was the preferred for monitoring UFH used to a in a of of or there are to a heparin anti-FXa assay to APTT for monitoring UFH, and it is a of a patient's response to However, there is no evidence that clinical if heparin anti-FXa is used of APTT for monitoring Lack of of the heparin anti-FXa assay a at some the from monitoring UFH with the APTT. provided by UK UK for heparin monitoring using the APTT, with for anti-FXa measurement of However, the heparin anti-FXa assay should not need to be performed as as the APTT as it is to in stable patients on UFH, testing should be The of LMWH that when according to their licence, plasma concentration monitoring is not LMWH has anti-FIIa activity to anti-FXa and LMWH anti-FXa activity is preferred when monitoring is LMWH anti-FXa activity is not well with the of bleeding or and routine monitoring not clinical The of LMWH is through a monitoring LMWH anti-FXa activity for the of bleeding in patients with LMWH is the used anti-coagulant to physiological such as levels of clotting and As these be expected to affect and efficacy of anti-coagulation, many have assessed LMWH anti-FXa monitoring clinical in when LMWH is used at either or are in a recent which that LMWH based on anti-FXa activity not the of bleeding or with There are some when LMWH anti-FXa monitoring is such as in with In and the of LMWH is different from there are on LMWH anti-FXa monitoring LMWH and guidelines support monitoring with and different to those for As monitoring is clinical the of monitoring are to be The effect of LMWH h which an is The time a dose when samples for LMWH assay are as it is to the this If bleeding is a sampling at the time just the is the as this provides an of LMWH This is of with when may be LMWH concentration monitoring may be more in there be about for example, while on but there are no to support an in and resulting in in some minor anti-coagulant such as anti-FIIa However, there is no evidence that these are clinically As the anti-FXa is by the that is to all anti-FXa concentrations do not need to be specific for different recent and LMWH and patients in the LMWH anti-FXa and patients in the There was no in the of bleeding between the but the LMWH anti-FXa a of that the of in the LMWH anti-FXa monitoring was than that in the when the was while there was no between the when the was this that monitoring of levels can be with have not range for LMWH anti-FXa activity that with bleeding or has not been as ‘expected range’ is a more When LMWH anti-FXa activity is it should be using a with an LMWH a UFH/LMWH has been for use as described LMWH anti-FXa activity levels have not been clinically and there is no for based on LMWH anti-FXa Furthermore, depending on reagents and as by in target ranges have been suggested by BSH the evidence in specific clinical is and these should be as for laboratories and LMWH has effect on the time to the of heparin to high levels of LMWH may cause The APTT can be with LMWH in a depending on the LMWH and the therapeutic LMWH not be expected to prolong the thrombin time depending on the LMWH and but can impact lupus anti-coagulant in lupus anti-coagulant testing should be even when using reagents with heparin (Table should be off anti-coagulation or just the dose of LMWH to these effects, if clinically thrombin in a to of LMWH with a that between LMWH haemostatic assays be by LMWH in a with the that has more impact at heparin anti-FXa activity to As LMWH monitoring is not required, the clinical of and are for the The is as an when LMWH is not or is It and means that monitoring is not required, in those discussed (Table 2). When anti-FXa activity is it should be using a with and reported in or The use of LMWH as the for the measured anti-FXa activity of is and should not be therapeutic range has not been but when as a the plasma concentration is 3 h For patients of body the plasma concentration is 3 h In a of laboratories in the of samples with different concentrations of and or dose the by 1 and the APTT by 4 to and measured levels. concentrations the expected range measured from to The APTT was in and of laboratories with and levels and assays not clinically generation assays be by in a in the impact is than with in no impact of on in the expected but with effects at concentrations the expected as an anti-coagulant by anti-FXa activity in an anti-thrombin-dependent It is for patients have or are to LMWH for Renal is the of of for to of its plasma There is no evidence of by the on anti-FXa it has a of of anti-FXa activity is not When anti-FXa activity is it should be using a with The expected anti-FXa activity for patients by is h for patients to an of or is not expected to prolong the but in that it prolong the APTT and this may the of APTT assays for lupus anti-coagulant at anti-FXa activity assays are at levels the expected and ACP not anti-FXa activity in If samples for testing should be just the dose of to of is expected to impact the parameters of in a Dabigatran levels can be measured using a thrombin time clotting time assay or anti-FIIa monitoring of is not required, as there are or high levels on clinical and expected concentrations are described with concentrations are for and may be in patients with or with or a may that an anti-coagulant be In patients for an acute or with an may be may be used to the use of a in patients with and a of is for those or If a assay be tests can be The sensitivity of the to by the with a from to at a concentration of The was a of in of patients on in samples collected to 3 h The APTT in the of dabigatran, but the is not in or the APTT is to in concentration within the expected range and at In the of patients with by a APTT, but all a The is to dabigatran, up to within the expected and even levels to (Table with and APTT may but may be h Argatroban is a direct thrombin inhibitor no to reversibly to and The of for a target of the APTT, not This was based on a clinical which with in of the APTT in this is by the that patients with at be their APTT was This that an APTT within at which is a limitation of using the APTT for monitoring APTT reagents and different also varying sensitivity to and to an APTT of if the APTT is of the The APTT at high concentrations of and is influenced by high factor and lupus anti-coagulants, leading many to the of using the or APTT for evidence to support use of an APTT with a target of to is weak. measurement of levels using a or assay many of the limitations of the APTT. However, there is the therapeutic since use the APTT as a Some evidence to support use of concentration APTT for monitoring from a of patients which assay with APTT The a in by when concentration was used than an APTT, an in and no in the of Argatroban the and this needs to be for when anti-coagulation to (Table target is described in the which that can be when the on this that the is a of the anti-coagulants, so the should be h is to that the is within the therapeutic target for ACP may be of use in these of may not be in all is required this can be is a binding to the thrombin and its with high The for that the should be used to a response to but no laboratory monitoring is the and this needs to be for when anti-coagulation to (Table target is described in the but monitoring should be once with has in the of with function. There is no clinical for the measurement of but a or assay be In patients on thrombin some assays if reagents levels of thrombin or plasma is may cause a in anti-coagulation or to be laboratories should their of reagents for their of in patients on oral or thrombin assays should not be as an the guideline in has been to and apixaban as a licensed direct FXaI in the In a was but was not a in the UK, and has been the therapeutic of the monitoring of their effects and dose is not However, to not in the previous BSH guidelines recommendations for testing in specific As of the to in plasma concentration was measured 1 into the in a of patients and the followed. The was based on and suggested a between concentration and and bleeding of patients in the measured apixaban concentration 3 of The concentration for those patients was while the was The was not to an between apixaban concentration and to However, that patients in the of levels a of than those with between bleeding and apixaban was also The of the anti-FXa assay is in some clinical (Table 2). There are and FXaI concentrations and and these do not evidence for the of monitoring. The laboratory on patients with of this a between and FXaI concentrations and patients a and these associated with high in patients with acute or on or apixaban for between and and levels measured h of and patients for a of with bleeding on anti-FXa levels than those levels within the expected range. with bleeding on apixaban and levels than those In of patients on and levels measured in the with bleeding or concentrations in the expected range. concentrations in those with or body The that there was no from and FXaI this was in The International for in Haematology published guidance on the laboratory of and FXaI which a of expected and concentrations in and in Table of the and APTT can be with the different to be associated with the of the reagents and and (Table In general, FXaI have a impact on assays than on APTT some reagents are to apixaban even at levels the expected range. In the UK, many routine laboratories have and the between and FXaI activity has been as mainly monitoring using the and APTT is not as can be within ranges even when FXaI can be by When FXaI anti-FXa activity is it should be using a with The of for measurement of FXaI by anti-FXa assay by and and should be assays are for therapeutic fixed-dose ranges for all the FXaI and can be used as a guide when for if the is can be achieved if and are Some reports have suggested caution when between FXaI and For a there may be a these it has been to more should be if between with as no assays can be used to between complex has been the in the UK for the of major bleeding associated with FXaI despite not licensed for this Laboratory of its use are likely to be of APTT and assays with its of and FXaI anti-FXa activity may not be The FXa is an option in the UK for of apixaban and not in and The of and published a of in the lack of an for the of when bleeding have shown that the anti-FXa assays can be used as of the process to However, FXaI activity can the of these and may not be available in an Furthermore, may for FXaI anti-FXa activity to be leading to of the are in some of which are based on molecules to in a to have a anti-FIIa anti-FXa effect a have been widely reported for use in measuring the impact of recent on the use of in the monitoring of in clinical FXaI concentrations with and parameters some not between FXaI levels and have been reported in to be in the of the specific its parameters including have been shown to to levels about a effect caused by binding to to be the for measuring the effect of and its but it is of a is of bleeding in all with is an to of complex of and their It is to anti-coagulants in the assay a wide range of It is as the for FXaI anti-FXa but its in laboratories it for routine have been reported to and FXaI activity with specific and there are on the impact of FXaI on newer and are The use of to the of and has been described using the and the assays limitations in as laboratory assays to FXaI and should be levels can also be measured using a 1 h plasma to FXaI anti-FXa This provides option for or between and FXaI in an have suggested that the can levels of and can be used with a to FXaI can have an impact on specific factor assays using APTT or assays to varying This can be by assays at if is testing is also (Table with and assays levels and activated of the assays used for lupus anti-coagulant testing is likely even with levels of be in assays based on platelet are also in the of all However, appears more complex with reports as to the impact of FXaI to the impact of Laboratory and should have a of the effects of anti-coagulants on their tests so that may tests and of This and avoid tests or For tests in a a should not be as a lack of anti-coagulant effect and may not need if with For example, inhibitors of or the pathway may the concentration of while can have the Some of the anti-coagulants discussed have therapeutic As are used in the for through or is laboratory are those that are and The tests used for monitoring anti-coagulation associated with anti-coagulation are monitoring or to on a laboratory is cause of including when using and in have been as of in by to When monitoring an anti-coagulant, it is that the anti-coagulant is in the to the This the to be used and the reported in a way that it that it is specific to that in the laboratory do not the drug or that the is on an the of this occurring by making of the anti-coagulant a in samples are for healthcare between As this is a of varying it is by local of clinical and laboratory All to the of this The wish to and for in undertaking the literature The members of the BSH Haemostasis and Thrombosis Task Force at the time of this guideline as and The to the BSH and the BSH guidelines for their support in this for this manuscript was by the British Society for The BSH the the of this All have a of to the BSH and Task Force which may be on the and in this guidance are to be and at the time of to the the BSH or the for the of this of the the if evidence available that the strength of the recommendations in this or it The be by the relevant Task Force and the literature search be 3 to search for evidence that may have been The be and from the BSH guidelines website if it If recommendations are an be published on the BSH guidelines website for Table clinical thrombin inhibitors Direct inhibitors Argatroban Dabigatran Low heparin LMWH heparin assays Haemostasis assays

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