Management of the thrombotic risk associated with COVID-19: guidance for the hemostasis laboratory

Even if the usefulness of D-dimer testing in the management of COVID-19 patients remains uncertain, some expert groups suggested the use of this parameter together with clinical variables to stratify patients according to their thrombotic risk and determine thromboprophylaxis intensity accordingly [15‐19]. This implies that test results shall be timely and reliable, and accurate threshold values need to be defined according to the specific clinical context and the immunoassay used.

Soluble fibrin degradation products containing the D-dimer motif constitute a heterogeneous set of molecules produced during degradation of polymerized fibrin network, which has been previously covalently cross-linked by activated factor XIII. This explains differences observed between fibrinogen and fibrin degradation products (FDP), the former being generated by degradation of fibrinogen from the fibrinolytic system (Fig. 1) [58]. A large inter-laboratory variability has been reported for the measurement of the plasma D-dimers concentration, which mostly reflects the different assay methodologies, the different mix of antibodies with variable antigenic specificity, the individual calibration and the variation of measuring units [64‐66]. The lack of internationally certified calibrators and quality controls also challenges to achieve better degree of universal harmonization. The large inter-individual variability (depending among others on renal function), is a further source of uncertainty in test results interpretation. A diagnostic threshold that has been validated within a specific clinical setting, using a certain assay, cannot be translated to different analytical conditions and different clinical settings [67]. The diagnostic thresholds shall hence be validated according to the method used and for the intended diagnostic purpose. Most of the available D-dimers assays have also been developed to have the best reproducibility around the threshold value used for excluding deep vein thrombosis and/or pulmonary embolism, which is usually around 500 ng/mL. Therefore, their performance at higher values, such as those proposed for initiating high dose anticoagulation in COVID-19 patients (i.e., over 3000 ng/mL), is probably suboptimal and would need to be assessed in order to avoid the use of inaccurate results in these patients. For example, external quality controls performed with moderately elevated D-dimers samples (target value 4000 ng/mL FEU) identified method-specific D-dimers means ranging from 470 to 10,150 ng/mL FEU (all methods coefficient of variation: 23%) [68]. As a possible solution, the threshold values of plasma D-dimers could be adjusted based on assay methodology. For example, for DIC diagnosis according to the ISTH definition, the appropriate D-dimer cut-off value for 2 points ranged from 3500 ng/mL to 6500 ng/mL, depending on the reagents used [69].

The potential impact of many preanalytical variables on measurement of plasma D-dimers should also be considered, as this test is vulnerable to the excessive presence of free hemoglobin in plasma, and becomes virtually uninterpretable when the plasma free hemoglobin concentration is over 30 g/L [70]. Hyperlipemia, hyperbilirubinemia or a high concentration of immunoglobulins can also generate a bias in the measurement, though the influence of these conditions has been less studied [70]. However, it has to be considered in the context of COVID-19 due to the high concentration of immunoglobulins measured in some of these patients 2 weeks after the symptom onset [71].

Many of the methods available for the study of fibrinolysis have significant drawbacks. Fibrinolysis assays are technically challenging, time consuming and cannot be easily automated, thus explaining their underuse in clinical laboratories. Under physiological circumstances, fibrinolysis takes many hours or days to develop in healthy blood after clotting, and this is indeed a major limitation for rapidly assessing the ‘global fibrinolysis capacity’ (GFC). The accelerated (albeit remaining long) assessment of fibrinolysis needs prior removal of inhibitors or the addition of tPA to initiate plasmin generation, complicating our appraisal of what is going on in vivo. Plasma-based systems, for example, where clotting and lysis may be easily monitored with turbidimetric assessment, need tPA to accelerate the reaction [72]. Alternatively, euglobulin may be prepared from plasma, thus lowering the concentration of fibrinolytic inhibitors (including PAI-1 and antiplasmin) [73, 74].

Individual components of the fibrinolytic system can also be measured. Although interesting evidence is emerging especially for PAI-1 in COVID-19 [30], its measurement is limited by large inter-laboratory variability, which is attributable to the different assay methodology, antigenic specificity of the antibodies, the lack of internationally certified calibrators and quality controls, as well as to the many measuring units that can be used for reporting data.

Interestingly, PAI-1 follows a circadian rhythm in humans, with a morning peak (around 8 AM) independent from the sleep pattern [75, 76]. The time-interval between blood collection and analysis, the anticoagulant used in sample tubes and storage temperature are other variables that are known to have an impact on plasma PAI-1 measurement [77‐79].

A weak stability at room temperature of the plasma used for assessing fibrinolysis (and especially PAI-1) has been occasionally described, whilst hemolysis (with release of cell free hemoglobin and other intracellular components) may contribute to produce an inhibitory effect on some fibrinolysis assays [80]. The contact phase (i.e., activated factor XII) also stimulates fibrinolysis through conversion of single chain urokinase-type plasminogen activator (scu-PA) in uPA [81]. Therefore, standardization of preanalytical and analytical steps is of utmost importance for obtaining accurate and reliable results of most fibrinolysis assays.

Regarding viscoelastic tests, ROTEM and TEG currently lack sensitivity to disordered fibrinolysis but there is still room for improvement [82].

As some extracellular vesicles are able to facilitate plasmin generation, it would be interesting to assess the contribution of such vesicle-dependent fibrinolytic activity in COVID-19 [83, 84].

Viscoelastic tests (VETs) are “global” hemostasis tests evaluating mechanical properties of the clot as it forms and lyses. The most frequent used VETs comprise thromboelastography (TEG) and rotational thromboelastometry (ROTEM). These commercial assays are embedded in the care for critically ill patients in many institutions, in particular for guiding transfusion management in patients at high risk of bleeding. In general, these assays are sensitive to detect hypocoagulability related to consumption of coagulation factors like fibrinogen, but hypercoagulability can also be detected. Indeed, increased clot firmness has been associated with occurrence of venous thromboembolism (VTE) in various clinical settings [85‐91], and has hence been proposed for evaluating the thrombotic risk associated with SARS-CoV-2 infection.

In COVID-19 patients, whole blood rotational thromboelastometry (ROTEM) has been able to detect accelerated clot formation and increased clot strength, persisting for at least 5 days [92]. Similar findings were reported by other groups using ROTEM and Quantra instruments, with both elevated platelets and fibrinogen contribution to clot strength [93, 94]. Increased heparin prophylaxis dose was associated with attenuation of fibrinogen contribution to clot strength and fibrinogen level, although lacking control conditions, this may have been a chance finding. Data regarding fibrinolysis evaluation in COVID-19 using VETs is sparser; hypofibrinolysis has been identified in one study [95], although another could not confirm this result [92]. Only one study evaluated the association between clinical outcomes and VETs parameters in COVID-19. The authors identified that the lack of clot lysis at 30 min on citrated kaolin TEG with heparinase was associated with VTE, whereas increased clot strength was not [95]. The combination of absence of lysis at 30 min with D-dimers levels > 2600 ng/mL was also strongly associated with VTE and with the need for dialysis [95]. Nonetheless, the place of VETs in the management of COVID-19 deserves further evaluation.

When using VETs, some drawbacks have to be considered. First, these tests are generally poorly sensitive to platelet function and mild fibrinolysis disorders [96, 97], and their sensitivity to fibrinogen levels is quite variable depending on the test methodology [98]. Second, even if considered as being global tests, they do not evaluate the contribution of endothelium, whose dysfunction likely contributes to COVID-19 associated hemostasis disturbances [34]. Moreover, similarly to D-dimers and SFC, correlation between methods is moderate, and inter-laboratory variation is high [99], but probably improving with introduction of new methods (ROTEM Sigma®, TEG6S® and Quantra®). Regarding preanalytical conditions, the time-interval between blood collection and VET [100, 101], anticoagulants and/or additives used in sample tubes [102, 103], over or underfilling of blood tubes [104], hemolysis [105] and hematocrit [106‐108] can influence the test results. Pneumatic tubes transport systems (PTS) may also exert little influence on test results, depending on acceleration forces of the local system [109‐112]. Therefore, this effect should be evaluated locally when utilization of PTS is considered for blood sampled for VETs. Finally, the accuracy of these tests for bleeding management or thrombotic risk stratification has not been validated in any hyperinflammatory context. Caution is therefore required when using VETs in COVID-19 patients, and further studies are needed to precise their added value in the management of these patients.

Thrombin generation assays (TGA) enable an integrative approach of coagulation. Commercial assays include Technothrombin® TGA kit from Technoclone and Calibrated Automated Thrombogram® (CAT) and ST-Genesia® from Stago Diagnostica [113, 114]. While TGA may be used to detect hypercoagulability, the frequent use of heparin/low molecular weight heparin (LMWH) in hospitalized COVID-19 patients could make this test relatively unsuitable for evaluating hypercoagulability. Modification of the TGA conditions, i.e. by adding polybrene or heparinase as neutralizing agent [115], could be of interest to eliminate heparin. However, data has not been reported yet in COVID-19 patients. Of note, pre-analytical variables and choice of the reference plasma to normalize the result also have important impact on TGA measurement and must be controlled in order to reduce the variability of measurement [116].

LMWH is recommended as first-line therapy for the prophylaxis of VTE in hospitalized COVID-19 patients [15, 20, 117, 118]. Direct oral anticoagulants (DOACs) or vitamin K antagonists, are not recommended because of the risk of drug interactions, among others with some antiviral drugs, the expected broad fluctuation in plasma concentrations (for DOACs), especially in patients at higher risk of rapid clinical deterioration, and because of the late onset of anticoagulation and higher risk of bleeding with VKA [119]. Heparin, and especially the long heparin chains, was reported to also exhibit anti-inflammatory activity in addition to the anticoagulant effect, by binding and neutralizing inflammatory cytokines and acute phase proteins, while potentially exerting a protective effect on endothelium [120]. It also interferes with neutrophils recruitment into tissue and impairs neutrophil function by inhibiting the activity of the neutrophil protease’s human leukocyte elastase and cathepsin G, which can promote inflammation [121, 122]. It has also been hypothesized that heparin may hinder the interaction between the virus and the host cell through non-specific ionic bond, and thus may contribute to decrease the rate of infected cells [120, 123]. However, we do not yet know precisely to what extent heparin is clinically effective in this infection.

Compared to UFH, LMWH has better bioavailability after subcutaneous administration and longer duration of action, allowing daily administration in one or two injections. No regular laboratory monitoring is necessary for treatment with LMWH because of the predictable anticoagulant activity after administration of doses adjusted to body weight on the one hand, and the lack of formal association demonstrated between laboratory tests results and clinical efficacy or complications on the other [124]. However, a single anti-Xa activity measurement can be proposed in case of administration of high or intermediate doses in a patient with moderate renal impairment, with a BMI < 18 kg/m² or > 30 kg/m², or during pregnancy, mainly to rule out drug accumulation [125]. In the event of increased doses in patients at enhanced thrombotic risk, measurement of anti-Xa activity is also recommended 4 h after the third dose to exclude accumulation. The anti-Xa value must be based on a calibration curve with the specific heparin type and interpretation of test result must also be made for the same compound; e.g. with therapeutic doses of enoxaparin, the mean peak concentration observed is 1.2 IU/mL. This measurement can be repeated for example in case of renal function impairment.

UFH is only recommended in case of severe renal failure (creatinine clearance according to Cockcroft-Gault equation < 30 mL/min), extracorporeal membrane oxygenation (ECMO) [15] or significant bleeding risk (shorter half-life than LMWHs, easier neutralization by protamine) [126]. The risk of heparin-induced thrombocytopenia (HIT) is also much higher with UFH [127]. Regular laboratory monitoring during anticoagulation is necessary because of the high inter- and intra-individual variability in the anticoagulant response [125].

Adjustment is thought to be required because of high inter and intra-individual variability. Historically, adjustment of UFH dosage was based on APTT. The measurement is performed 4 to 6 h after initiation and any dose change, and once a day at least, to reach a target APTT ratio between 1.5 and 2.5. This therapeutic target dates from work in 1972 and has never been confirmed in large clinical studies [128]. Since then, the number of reagents used for the APTT has exponentially increased, the sensitivity of which is very different both to heparin and biological interference (including several proteins of the acute phase) [129‐131]. Therefore, calculation of the APTT ratio corresponding to an anti-Xa activity between 0.3 and 0.7 IU/mL would be recommended for each analyzer and each new batch of reagents. This calculation should best be performed with plasma samples from patients treated with UFH because the use of plasma spiked with UFH gives less relevant results due to the influence of the metabolism of heparin in vivo and its bioavailability [132]. For some reagents, the relationship between heparin levels and APTT is linear, but this can change in case of significant inflammation [133]. Most importantly, APTT is very dependent on pre-analytical conditions. Among others, platelet factor 4 (PF4) released by activated platelets during inadequate sampling procedure or prolonged delay before centrifugation can neutralize part of the heparin, leading to a risk of underestimating its activity [134].

Several biological parameters can cause a prolongation of the APTT (e.g. high CRP, presence of lupus anticoagulants, coagulation factors deficiency, high plasma concentration in FDPs which oppose the polymerization of fibrin) or its shortening (e.g. increased plasma concentration of acute phase proteins such as FVIII and fibrinogen) [63, 135, 136]. The influence of these parameters will also depend on assay methodology, the type of reagents, and has a high inter-individual and intra-individual (i.e., during hospitalization) variability [137, 138]. For these reasons, the GFHT advises against the use of APTT for monitoring treatment with UFH [133]. Moreover, the use of APTT is problematic when this test is prolonged prior to initiation of UFH treatment (for example, in case of lupus anticoagulants or coagulation factors deficiency); anti-Xa target should also consider the etiology of this prolongation when clinically relevant (i.e. defects with a bleeding risk).

In COVID-19, the increased plasma concentrations of fibrinogen and FVIII can cause a shortening of APTT (observed in 16% of affected patients) [55], and this may lead to underestimating the anticoagulant effect of heparin. This situation can contribute to excess heparin dosing, enhancing the bleeding risk, and underlines the importance of collecting a basal APTT value before anticoagulation. This can be problematic in some settings, e.g. in the ICU, where patients are frequently transferred with anticoagulation already started at intermediate or therapeutic doses [13]. Conversely, APTT prolongation may be related to the transient increased levels of antiphospholipid antibodies, a situation encountered during viral infections [139], including COVID-19 [10, 140‐143], or when CRP is high [138], thus leading to a risk of heparin underdosage. Lupus anticoagulant screening can also be falsely positive in the presence of variables prolonging clotting time of tests used for its screening (for example, elevated CRP or presence of anticoagulants, among which heparin) [143‐146]. The APTT may also be prolonged in the presence of DIC (which is relatively rare in COVID-19). In this situation, its measurement by means of an optical system may become uninterpretable: an immediate and gradual decrease in light transmittance can be observed even before clot formation due to the presence of a complex between CRP and very low density lipoproteins in the presence of calcium [147], thus rendering the measurement unreliable [148]. Therefore, mechanical methods are advisable in this circumstance, where measurement anti-Xa activity may even be the best choice.

Thus, heparin monitoring with aPTT may be challenging in COVID-19 patients due to the hyper-inflammatory status of the patient. Indeed, the high fibrinogen and factor VIII levels, the interference of CRP (depending on the reagents used) and also the potential presence of antiphospholipid antibodies may affect the aPTT. Therefore, anti-Xa activity seems more suitable to monitor UFH treatment in these patients and more generally in ICU patients for the very same reasons. However, there are several caveats here as well. First, FXa is not the essential target of UFH. Its inhibition is studied under very artificial conditions: in the fluid phase (and not within the prothrombinase complex formed on a phospholipid surface) and in a calcium-depleted medium. The in vivo inhibitory activity of UFH is indeed three times stronger towards FIIa than FXa [149]. This difference is further artificially increased in vitro by use of low calcium concentrations in the assay mixture: the anti-Xa activity was halved under these conditions, compared to physiological concentrations of calcium, but the effect of low calcium on anti-IIa activity is more limited [149]. Having mentioned that, a good correlation exists in vitro between anti-Xa and anti-IIa activities, thus enabling the use of the former test to assess the effect of heparin therapy. The anti-Xa assay consists of measuring in vitro the residual activity on a chromogenic substrate specific for FXa added to citrated plasma. Compared to APTT, this test has the advantage of being less vulnerable to biological interference (possible interference of free hemoglobin and bilirubin in case of significant elevation [150]) and less dependent on pre-analytical conditions - with the notable exception of PF4 released in vitro by platelets. Even if the validity of anti-Xa activity of UFH in the presence of an important inflammatory syndrome has not been formally established (or for that matter under any circumstances), this measure will be less impacted in this context, particularly if the reagents contain antithrombin (AT). However, it is not advisable to use kits with exogenous AT to avoid overestimation of anticoagulant activity in case of AT deficiency, with risk of heparin underdosing. The plasma concentration of AT can indeed decrease in case of sepsis [151]. Only few data are available on the sensitivity of kits without exogenous AT to changes in plasma AT concentration [152]. Some reagents also contain dextran sulfate, which will displace heparin from its non-specific binding (including PF4). Particularly, the influence of PF4 released by activated platelets is therefore minimized, which is favorable for limiting the impact of pre-analytical conditions on test result, but problematic when the concentration of PF4 is actually high in vivo. Unlike a ‘global’ test like APTT, the measurement of anti-Xa activity is insensitive to fluctuations in the underlying hemostatic state (for example, coagulation factor defect following hemorrhage or DIC), which should prompt an adjustment in therapeutic targets to the clinical context and the possible identification of such defects. Finally, significant variability in heparin sensitivity has been reported between the different commercial kits [153‐156].

The therapeutic range in terms of anti-Xa activity is considered between 0.3 and 0.7 IU/mL [125]. These values ​​are derived from the work of 1972 that was previously mentioned. It was hence inferred from the APTT target to be reached for secondary prevention of VTE. The bottom line is that it lacks validation for the same reasons [157]. The application and interpretation of these tests in a hyperinflammatory context also raises important questions. Given the very high thrombotic risk described or suspected in some subgroups of patients, the GIHP suggested narrowing the target of anti-Xa activity in the upper zone, to 0.5–0.7 IU/mL, for those at the highest thromboembolic risk [15]. This increase in dosage, not supported by objective data, remains debated [20‐23], whilst ongoing prospective, multicenter clinical trials aim at addressing this question. In addition to the selection of patients who could potentially benefit from increased doses of anticoagulants, the question of treatment duration is a major one. The resolution of the inflammatory syndrome should be accompanied by a reduction in the thrombotic risk, thus exposing the patient to excessive anticoagulation and risk of bleeding when higher doses of heparin are maintained. However, no firm recommendation on duration and intensity of COVID-19 anticoagulation can be made at this time.

In patients receiving UFH, a laboratory resistance to the anticoagulant effect of heparin, arbitrarily defined by failure to reach the therapeutic target despite the administration of doses > 1.5 times usual doses, which are about 400 to 600 U/kg/24 h [158, 159], is frequently observed in COVID-19 [13, 160], and adds to the clinical resistance previously outlined (occurrence of thrombotic events under well-conducted drug thromboprophylaxis). The hyperinflammatory context could also explain part of this observation. Indeed, UFH is able to non-specifically bind several acute phase proteins as well as activated endothelial cells and platelets, thus limiting its anticoagulant activity [161, 162]. The administration of an initial bolus of UFH is therefore needed to saturate non-specific fixation [163]. The increased plasma concentration of fibrinogen and FVIII will also contribute to generate heparin resistance when the effect is monitored with the APTT, which is less likely to be observed when anti-Xa activity is used. Acquired AT deficiency by consumption or production defects (negative protein of the acute phase) could also contribute to the heparin resistance observed in some COVID-19 patients [151], especially those most seriously affected [10, 49], but in most patients it does not justify the prescription of AT concentrates. To the best of our knowledge there is one single prospective interventional study on UFH monitoring in case of laboratory heparin resistance. In this study, the utilization of anti-Xa activity instead of aPTT permitted to avoid UFH dosage escalation with similar clinical outcomes [164]. Whether this holds true for COVID-19 patients remains to be established. When heparin resistance is suspected based on APTT values, UFH shall be administered according to the anti-Xa activity [165]. The advantages and limitations of APTT vs anti-Xa for UFH monitoring are summarized in Table 3.

A final aspect of heparin monitoring is screening for HIT. A platelet count should be performed before administering the first injection of heparin, if possible, or as soon as possible thereafter. In the COVID-19 setting, it is reasonable to monitor the platelet count regularly between the 4th and the 14th day following the initiation of heparin therapy (once or twice a week in case of LMWH treatment, two to three times a week during UFH treatment), then once a week until the end of the first month of therapy. The development of thrombocytopenia (< 100 × 10⁹/L) or the rapid decrease in platelet count (especially if ≥50% in less than 24 h) should then suggest the diagnosis of HIT [166]. However, especially in the presence of acute inflammation and infection, other etiologies may explain a decrease in platelet count. Therefore, a systematic evaluation of clinical probability of diagnosis allows better identification of patients in whom the occurrence of this complication must be suspected, and for whom laboratory work-up for HIT antibodies is indicated. This evaluation is generally performed with the 4Ts’ score, much studied [167, 168] [159], despite its limitations, particularly in more complex situations such as those encountered in ICU (no consensus on the drugs responsible for thrombocytopenia, many other causes of thrombocytopenia, very high negative predictive value but not absolute (e.g. thrombosis in the absence of thrombocytopenia), insufficient data on platelet count, weak agreement in the determination of the 4th criteria (other causes of thrombocytopenia)) [166, 169, 170].

In case of strong suspicion, or as soon as the antibodies are identified, treatment with heparin should be stopped and replaced by a direct thrombin inhibitor (DTI; argatroban, bivalirudin) or by danaparoid sodium. Of note, the presence of a DTI can lead to underestimation of plasma fibrinogen concentrations by inhibition of the thrombin present in Clauss’ reagent [171]. This interference will vary depending on the thrombin concentration used in the reagent [172, 173]. To a lesser extent, interference may also exist in the presence of high concentration of UFH (0.6 to 2.0 IU/mL, depending on the reagent), which would exceed the neutralization capacities of the reagent used, or in the presence of high concentration of FDPs (> 100–130 μg/mL) [173].