Laboratory Monitoring in Patients Receiving Emicizumab

Abstract

Emicizumab is a bispecific monoclonal antibody that mimics the cofactor function of activated factor VIII (FVIIIa). It is approved for routine prophylaxis in patients with severe or moderate congenital hemophilia A (HA), both with and without FVIII inhibitors, and is increasingly used as a first-line treatment in acquired HA (AHA). Owing to its predictable pharmacokinetic profile, emicizumab monitoring is generally limited to cases with suspected reduced efficacy, such as due to poor adherence or the development of anti-drug antibodies (ADAs). However, emicizumab interferes with standard clotting assays, particularly by shortening activated partial thromboplastin times (APTT). To address this, modified FVIII one-stage clotting assays (mOSA), which use higher sample pre-dilution and emicizumab-specific calibration, are commonly employed to estimate plasma levels, although other assay formats like emicizumab-calibrated chromogenic substrate assays based on human factors or liquid chromatography tandem mass spectrometry are also available. Additionally, global assays such as in vitro thrombin generation testing are being explored to better reflect clinical hemostatic efficacy. This review summarizes current knowledge on assay interferences caused by emicizumab, challenges in functional measurement of plasma levels, and strategies to ensure reliable laboratory assessment. We also discuss the relevance and methods for ADA detection and provide an overview of current and emerging strategies for thrombin generation measurement as a global indicator of treatment effectiveness.

 

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Keywords

hemophilia A - blood coagulation tests - emicizumab - factor VIII - drug monitoring

Introduction

Congenital hemophilia A (CHA) is an X-linked inherited bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII).[1] Affected individuals have a lifelong tendency to bleed, often beginning in early childhood. Bleeding typically involves large joints—such as ankles, knees, and elbows—and may lead to hemophilic arthropathy.[2] Other common bleeding sites include muscles, the gastrointestinal and urogenital tracts, and the central nervous system. For decades, the standard of care has been regular prophylactic infusion of FVIII concentrates.[3] However, approximately one-third of patients develop neutralizing alloantibodies against FVIII, known as inhibitors, which render FVIII replacement therapy ineffective.[4]

Acquired hemophilia A (AHA) is an autoimmune bleeding disorder that affects previously healthy men and women of all ages. It is caused by the development of neutralizing autoantibodies, also referred to as inhibitors, against FVIII, which reduces endogenous FVIII activity to varying degrees, from slightly decreased to undetectable levels. Common bleeding sites include the skin (usually presenting as subcutaneous hematomas), the muscles, the gastrointestinal and urogenital tracts, and the central nervous system. In contrast to CHA, joint bleeding is less common in AHA.[5]

Emicizumab is a monoclonal, humanized, bispecific antibody that binds to factor IX/IXa and factor X, thereby facilitating the proteolytic activation of FX by FIXa on the phospholipid membrane ([Fig. 1]). Because of this function, it is often referred to as an FVIIIa-mimetic antibody.[6] Emicizumab is clinically effective in CHA, both in patients with and without inhibitors, and has also shown efficacy in AHA ([Table 1]).[5] [7] [8] [9] Although its mechanism of action functionally mimics FVIIIa, important differences exist: (1) emicizumab does not require activation by thrombin (“always on”); (2) it is not subject to spontaneous decay or inactivation by activated protein C (“never off”); and (3) it binds FIX/IXa and FX with significantly lower affinity than FVIIIa, resulting in slower generation of FXa.[10] [11] Because of these differences in activation, activity, and inactivation, assigning a direct FVIII-equivalent activity to emicizumab is challenging.[12] [13] Clinical trial data suggest that it converts severe HA to a milder phenotype, indicating a partial correction of FVIII deficiency. However, in cases of breakthrough bleeding or surgery, additional treatment with FVIII concentrates may still be necessary, as emicizumab does not fully restore hemostasis.[14] [15]

Fig. 1 Mechanism of action of emicizumab. Emicizumab is a bispecific antibody that binds activated factor IX (FIXa) and factor X (FX), mimicking the cofactor activity of factor VIIIa. This facilitates the colocalization of FIXa and FX on phospholipid surfaces, enhancing FX activation to FXa. (Figure made using Biorender.)

Table 1 Indications for the use of emicizumab

Indication	Loading dose	Maintenance dose	EMA license	Pivotal studies
Severe CHA (FVIII <1%)	3 mg/kg Q1W for 4 weeks	1.5 mg/kg Q1W or 3 mg/kg Q2W or 6 mg/kg Q4W	Yes	HAVEN 3[8]
Moderate CHA (FVIII 1–5%) with severe bleeding phenotype	3 mg/kg Q1W for 4 weeks	1.5 mg/kg Q1W or 3 mg/kg Q2W or 6 mg/kg Q4W	Yes	HAVEN 6[88]
CHA of any severity with inhibitors against FVIII	3 mg/kg Q1W for 4 weeks	1.5 mg/kg Q1W or 3 mg/kg Q2W or 6 mg/kg Q4W	Yes	Adults: HAVEN 1[9] Children: HAVEN 2[89]
AHA	6 mg/kg day 1 and 3 mg/kg day 2	1.5 mg/kg Q1W starting day 8	No (off-label use)	AGEHA[29] GTH-AHA-EMI[7]

Abbreviations: AHA, acquired hemophilia A; CHA, congenital hemophilia A; FVIII, factor VIII.

Although emicizumab only partially replaces FVIII functionally, it fully corrects (or even over-corrects) the activated partial thromboplastin time (APTT). As a result, APTT-based factor assays may report falsely elevated factor activity levels.[16] [17] [18] This may lead to misinterpretation of coagulation results in clinical practice and underscores the importance of clearly documenting when a sample is obtained from a patient receiving emicizumab.[19]

This article reviews recent literature and expert insights on the impact of emicizumab on standard laboratory assays, offering solutions through modified assay setups or emicizumab neutralization. It also addresses the monitoring of FVIIIa-like activity and the screening for anti-drug antibodies (ADAs). Additionally, we explore the potential use of thrombin generation assays (TGAs) as a more complete reflection of emicizumab's pharmacodynamic action and their possible correlation with clinical efficacy.

 

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Influence of Emicizumab on Laboratory Assay

[Table 2] summarizes the influence of emicizumab on standard coagulation tests.

Table 2 Influence of emicizumab on standard coagulation tests

Assay	Influence of emicizumab	Solution
APTT	Artificially short	Neutralize emicizumabRUO When monitoring heparin: consider anti-Xa or thrombin time
APTT-based assays for factors VIII, IX, XI, XII, etc.	Artificially high	Factors VIII and IX: use CSA with bovine components Other factors: antigen assaysRUO
APTT-based Bethesda inhibitor assay	Artificially low (false negative)	Use modified assay with chromogenic with bovine components
Activated clotting time	Artificially short	Neutralize emicizumabRUO When monitoring heparin: consider alternative assays (see above)
Prothrombin time	Little effect (not clinically relevant)	Not applicable
Thrombin time, lupus anticoagulant (if based on diluted Russel viper venom time), VWF:Act, immunoassays	Not affected	Not applicable

Abbreviations: APTT, activated partial thromboplastin time; CSA, chromogenic substrate assay; RUO, research use only.

APTT and APTT-based Factor Assays

Among the most affected laboratory parameters are APTT-based clotting assays. Emicizumab markedly shortens the APTT, even at sub-therapeutic plasma concentrations, and this effect is independent of the specific APTT reagent used.[20] As a result, one-stage clotting assays (OSAs) routinely used to assess FVIII levels are no longer reliable, as they produce falsely elevated values under emicizumab prophylaxis. These assays are therefore unsuitable for assessing endogenous FVIII levels or monitoring additional FVIII administration during emicizumab treatment. Furthermore, other factor activity assays based on the APTT principle, such as OSA for factors IX, XI, and XII, are similarly affected, yielding falsely high results.[16]

To overcome this diagnostic challenge posed by emicizumab, laboratories and clinicians must employ suitable alternatives. For FVIII activity testing, chromogenic substrate assays (CSAs) using bovine factors IXa and/or X are the preferred option.[16] [20] A FIX CSA is also available to reliably measure FIX activity in patients on emicizumab.[17] In certain situations, emicizumab can be neutralized in vitro using anti-idiotype monoclonal antibodies, enabling the use of otherwise affected assays.[21]

 

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Inhibitor Assays

The detection of FVIII inhibitors is also compromised in the presence of emicizumab. In the Nijmegen-Bethesda assay (NBA), residual FVIII activity is detected with APTT-based OSA, yielding false-negative results in patients on emicizumab.[20] Replacing residual FVIII detection by CSA with bovine reagents is a reliable solution.[22] The CSA-based NBA has already been implemented in clinical laboratories as it can be routinely used for all patients, and knowledge about the use of emicizumab is not required. As an alternative, quantification of anti-FVIII antibodies by ELISA can be considered.[23] However, the latter does not differentiate between inhibitory and non-inhibitory antibodies.[16]

 

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Other APTT-based Coagulation Assays

Emicizumab also interferes with other APTT-based diagnostics. For example, tests for activated protein C (APC) resistance and lupus anticoagulant detection that rely on the APTT become invalid.[16] In cases where thrombophilia screening is indicated, clinicians should switch to non-APTT-based assays, such as genetic testing for factor V Leiden in the case of APC resistance or use assays like the diluted Russell's viper venom time (DRVVT) or antiphospholipid antibody ELISAs for suspected antiphospholipid syndrome. The activated clotting time (ACT), used intraoperatively to monitor unfractionated heparin (UFH), is shortened in the presence of emicizumab. Although some authors recommend switching to anti-Xa, thrombin time, or protamine titration assays for UFH monitoring,[24] others maintain that ACT should still be used, as heparin continues to cause a concentration-dependent prolongation of ACT under emicizumab.[25]

 

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Assays not Affected by Emicizumab

A range of assays remain unaffected by emicizumab and can be reliably used in clinical practice. Chromogenic substrate assays for antithrombin, protein C, and anti-Xa activity, as well as ELISA-based methods for plasminogen and von Willebrand factor (vWF) antigen, have shown no interference.[17] Likewise, latex-enhanced turbidimetric assays for markers such as free protein S, D-dimer, or FXIII antigen yield accurate results. The effect of emicizumab on the prothrombin time (PT/INR) is negligible and clinically irrelevant. Similarly, coagulation tests triggered by FXa or thrombin, such as thrombin time or fibrinogen assays, are not influenced by the drug.[17]

 

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Conclusions

Emicizumab introduces substantial interference with some standard coagulation assays, especially those based on the APTT. Clinicians and laboratories must be aware of these effects and adapt diagnostic strategies accordingly. The implementation of emicizumab-insensitive assays is crucial to ensure accurate monitoring, guide therapeutic interventions, and maintain patient safety.

 

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Monitoring of Emicizumab

The standard subcutaneous treatment regimen for emicizumab in CHA includes a loading dose of 3 mg/kg body weight (BW) once weekly for 4 weeks, followed by maintenance dosing of 1.5 mg/kg once weekly, 3 mg/kg every 2 weeks, or 6 mg/kg once per month ([Table 1]).[26] With its established clinical efficacy at steady-state plasma concentrations, typically ranging from 30 to 80 µg/mL, routine monitoring of emicizumab levels is not necessary during the uneventful course of treatment.[27] [28]

Potential Indications for Monitoring

Nevertheless, the availability of appropriate analytical methods for determining emicizumab activity is crucial for measuring in specific clinical scenarios, such as unexplained bleeding episodes. Potential causes for such episodes may include unfavorable patient-specific pharmacodynamic characteristics, development of ADAs, or poor patient adherence to therapy.[26] Additional indications for plasma emicizumab measurements include transitions to alternative treatments or participation in research or clinical studies, especially when applying alternative dosing regimens.[7] [29]

 

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Qualitative Assessment of Emicizumab Effects

If a dedicated assay for emicizumab quantification is not available, surrogate laboratory parameters can indicate the presence or absence of the drug. For example, shortened APTT and unexpectedly high FVIII OSA activity levels suggest the presence of circulating emicizumab. Conversely, prolonged APTT and reduced FVIII activity measured by OSA may indicate insufficient emicizumab levels or activity.[16] [30] [31] [32]

 

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Calibrated Functional Emicizumab Assays

Although liquid chromatography–tandem mass spectrometry (LC-MS/MS) techniques have been developed for direct quantification of emicizumab, routine analysis is usually performed by assessing the FVIIIa-mimetic cofactor activity of emicizumab using a modified FVIII one-stage clotting assay (mOSA) calibrated against emicizumab.[16] [18] [33] [34] Given the artificially shortened APTT observed in the presence of emicizumab, mOSA involves a higher pre-dilution of the plasma sample to meet the dynamic range of the assay.[35] Commercial calibrators and controls are available, with calibration ranges typically spanning 10 to 100 µg/mL, which is considered sufficient for evaluation of therapeutic efficacy ([Table 3]).[18]

Table 3 Assays for monitoring of emicizumab

	Modified one-stage clot assay (mOSA)	Human component chromogenic substrate assay (hCSA)
Assay principle	FVIIIa-mimetic cofactor activity	FVIIIa-mimetic cofactor activity
Assay principle	Higher dilution (1:80) compared to conventional FVIII OSA (1:5 or 1:10)	Chromogenic FVIII assay with human FIX and FX components
Commercial calibrators and controls available	Yes	No (can use components from mOSA)
Range of quantification	10–100 µg/mL	10–100 µg/mL
Influence of residual or infused FVIII on assay resulta	Moderate	Strong

Note: ^aConsider neutralization with commercial, inhibitory anti-FVIII antibody.

FVIII CSA can also be used to measure emicizumab activity, if these utilize human FIX and FX components (hCSA) ([Table 3]). In contrast to APTT-based OSAs, emicizumab acts within the dynamic range of hCSA and yield reliable results if calibrated for emicizumab.[16] [36] Notably, recent field data suggest that mOSA using various APTT reagents and hCSA yields comparable results when calibrated against emicizumab.[37]

 

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Interference of FVIII with Emicizumab Measurement

Both mOSA and hCSA detect the functional FVIII-mimetic activity of emicizumab. Additional FVIII in the sample (e.g., in moderate or mild CHA, after replacement of FVIII concentrate, or in patients with AHA) may lead to falsely elevated functional emicizumab levels.[38] [39] [40] This confounding effect is less pronounced with mOSA due to the higher pre-dilution of the sample compared to the inverse scenario, where emicizumab interferes with OSA-based FVIII measurement.[40] In hCSA, due to non-adjusted sample pre-dilution, overestimation of emicizumab activity by additional FVIII may be even more significant.[16]

Heat inactivation of FVIII, a standard preanalytical procedure applied in routine FVIII inhibitor quantification by NBA, appears to be a logical approach to mitigate FVIII interference in functional emicizumab assays.[41] However, emicizumab exhibits considerable heat sensitivity in clinical samples.[39] Interestingly, studies have shown that circulating emicizumab is notably less heat-stable than emicizumab spiked into FVIII-deficient plasma (dp).[40] In general, the reduced heat stability is likely attributable to the engineered nature of emicizumab, with its biochemical properties differing from that of native antibodies.[42] Further modifications of emicizumab in vivo as well as stabilizing excipients present in commercial plasma preparations may explain the observed differences in heat stability between endogenous emicizumab and emicizumab spiked into FVIII-dp.[43] [44] [45]

To selectively neutralize interfering FVIII activity without compromising the function of emicizumab, FVIII-specific inhibitory antibodies have been successfully employed. In a recent study, an equal volume of a commercially available, high inhibitor titer plasma of approximately 20 Bethesda Units/mL was added and found to allow for precise emicizumab measurement. This approach achieved complete and reliable suppression of FVIII activity from various sources. However, it is anticipated that future applications of this FVIII neutralizing strategy will be based on more standardized sources of FVIII inhibitors to ensure reproducible and cost-effective implementation in the coagulation laboratory.[40]

A visual overview on when to measure emicizumab plasma levels and corresponding conclusions to be drawn is given in [Fig. 2].

Fig. 2 A virtual patient on a virtual timeline. Schematic overview of when to measure emicizumab plasma levels and corresponding conclusions/consequences.

 

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Conclusions

Monitoring of the functional activity of emicizumab can be done using mOSA or hCSA calibrated against emicizumab. Residual FVIII in the sample can falsely increase emicizumab activity results, in particular with hCSA. Inactivation of residual FVIII should be attempted using standardized FVIII inhibitor reagents rather than heat inactivation.

 

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Screening for Anti-drug Antibodies

As with all protein-based biological drugs, ADAs against emicizumab, although humanized, can develop or even preexist in patients. ADA can be neutralizing (e.g., interfering directly with emicizumab function) or non-neutralizing (i.e., binding to emicizumab without compromising its function but potentially affecting pharmacokinetic behavior or causing hypersensitivity reactions). These mechanisms are summarized in [Fig. 3].

Fig. 3 Proposed mechanisms of action of anti-drug antibody (ADA) against emicizumab. Non-neutralizing ADAs may promote immune complex formation and clearance of emicizumab. Neutralizing ADAs interfere with emicizumab's ability to bind FIXa and FX, preventing FXa production. (Figure made using Biorender.)

Reported Frequency of ADA Against Emicizumab

Reports on ADA against emicizumab remain rare. Emicizumab was associated with the development of ADA in 34 (5.1%) of 688 patients who participated in the HAVEN 1-5, HOHOEMI, and STASEY clinical trials.[46] These were found to be non-neutralizing in 16 out of 34 cases (47%) and neutralizing in 18 participants (53%). Of the latter, four showed decreased emicizumab levels while one discontinued emicizumab therapy due to loss of efficacy. As of April 2025, nine additional cases of neutralizing ADA outside clinical trials have been reported, representing only a small fraction (<1%) of the more than 20,000 patients treated worldwide so far.[47] [48] [49] [50] [51] [52] [53]

Non-neutralizing ADAs have been proposed to enhance emicizumab clearance by immune complex formation.[47] [48] [49] Of note, this was not seen in the 16 patients with non-neutralizing ADA in the HAVEN trials.[46]

Since the complementarity-determining regions of emicizumab are derived from non-human species, the possibility of pre-existing ADA cannot be entirely excluded. These ADAs were detectable at baseline in one patient, with levels decreasing during treatment but remaining measurable.[54]

 

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Indications for ADA Testing

Given the low frequency of ADA, the question arises whether routine screening is justified. Current consensus guidelines do not recommend routine screening for all patients on emicizumab.[55] Instead, a targeted testing approach is favored. This strategy is supported by observations that ADA-related insufficient treatment response commonly manifests with clinical worsening and discordant laboratory results.[56] Indications for ADA testing can include unexpected clinical events, such as spontaneous or recurrent bleeding despite adequate emicizumab administration. Furthermore, incongruous laboratory findings—such as persistently prolonged APTT without a clear explanation, a decline in pharmacodynamic markers, or unexpectedly low emicizumab levels—may also indicate the presence of ADA.

 

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Neutralizing ADA (Inhibitors)

Neutralizing ADAs, although rare, represent a particularly concerning challenge. These antibodies can directly inhibit the activity of emicizumab, likely by steric hindrance that interferes with its intended bridging function between FIXa and FX. Clinically, such cases are typically characterized by breakthrough bleeding episodes accompanied by a prolonged APTT.[32]

To evaluate neutralizing ADA, Bethesda-like assays—originally developed for quantifying FVIII inhibitors in HA—have been adapted for emicizumab.[47] Although preliminary studies suggest that such approaches can provide a quantitative readout of inhibitor titers, the establishment of a standardized protocol remains challenging due to the distinct mechanism of action of emicizumab.

Given the analytical challenges inherent to direct detection using a Bethesda-like assay, some investigators have proposed TGA as a functional surrogate marker for ADA activity.[52] A reduction in TGA peak despite adequate dosing may raise suspicion of ADA interference with emicizumab's function. However, standardization of TGA-based methodologies for ADA screening remains pending.

 

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Screening Assays for ADA

Several screening methodologies have been employed including: (i) Western blotting of purified IgG from patient plasma followed by detection of ADA with biotin-labeled emicizumab,[50] (ii) enzyme-linked immunosorbent assay (ELISA) bridging assay,[47] [56] [57] and (iii) bead-based Luminex assay using the F(ab́)2 binding regions of emicizumab to improve specificity ([Fig. 4]).[58]

Fig. 4 Schematic representation of immunoassay for the detection of anti-drug antibodies (ADAs) against emicizumab. In a fluorescent bead-based assay, emicizumab-F(ab')₂ fragments are immobilized on beads to capture ADA from a sample. Bound ADAs are then detected using a PE-labeled anti-human IgG Fc antibody (right panel). In an ELISA assay, ADAs are detected via bridging between biotinylated emicizumab (immobilized via, e.g., streptavidin) and labeled emicizumab (e.g., labeled with horseradish peroxidase). Detection is achieved through enzymatic conversion of a substrate (left panel). (Figure made using Biorender.)

These methods can serve as screening assays for both neutralizing and non-neutralizing antibodies. Two main limitations exist: first, these methods cannot distinguish between neutralizing and non-neutralizing ADA; second, assay sensitivity is influenced by the amount of emicizumab in patient plasma and due to structural similarities between ADA against emicizumab and other endogenous immunoglobulins in the plasma. Therefore, detection of ADAs should prompt additional testing to determine their specificity. One common method involves spiking patient's samples with emicizumab prior to immunoassay analysis. Specific ADA should yield a decreased signal upon spiking with excess emicizumab.[57] [58] Alternatively, anti-idiotypic antibodies targeting distinct antigenic epitopes of emicizumab can be used. This approach not only confirms ADA specificity but also facilitates precise epitope mapping of the antibody response.[47]

 

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Conclusions

Although anti-emicizumab ADAs are rare, their potential to neutralize drug activity or alter pharmacokinetics warrants targeted screening as part of a broader diagnostic workup in clinically suspected cases. Routine testing of all patients is not required. Instead, clinical awareness and access to reliable, validated assays for patients with suspected ADAs are essential. In the absence of standardized commercial assays, ADA testing remains restricted to reference laboratories with in-house protocols. As emicizumab use continues to expand, the development and harmonization of diagnostic assays will be crucial for identifying immune-mediated treatment impairment.

 

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Thrombin Generation Assays

Indications for Using TGA in Patients Receiving Emicizumab

In recent years, TGA has received considerable attention as a global assay that provides an integrated assessment of coagulation. In patients with CHA or AHA, TGA may offer valuable insights into hemostatic potential beyond what single-factor assays such as mOSA or hCSA can provide. Unlike these assays, TGA can partially reflect individual variations in both procoagulant (e.g., FIX, FX) and anticoagulant (e.g., antithrombin) pathways. Furthermore, TGA has the capacity to capture the net effects of complex therapeutic interventions, such as the combination of emicizumab with FVIII or bypassing agents—situations in which conventional coagulation assays may be unreliable.[59] Integrating TGA into clinical practice could therefore aid in identifying patients at increased risk of bleeding and supports personalized treatment strategies in challenging clinical scenarios.

 

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TGA for Predicting Treatment Efficacy and Thromboembolic Safety

Heterogeneous data exist regarding the association between TGA and clinical phenotype. TGA has been shown to correlate with the bleeding phenotype in HA patients,[60] [61] and is increasingly recognized as a valuable tool to facilitate a personalized approach to hemophilia management.[62] A recent observational study reported reduced thrombin generation capacity in CHA patients with breakthrough bleeds during prophylaxis with emicizumab.[63]

TGA has also been used to evaluate the necessity of additional treatment in cases of surgery or breakthrough bleeding and to guide the choice of replacement therapy. Importantly, the synergistic effects of emicizumab with activated prothrombin complex concentrate that caused thromboembolic events were mirrored by excessive thrombin generation in TGA.[62] [64] However, discrepant results were obtained in larger patient cohorts under real-world conditions. Recent data did not confirm correlation of TGA parameters with emicizumab plasma levels, and TGA failed to predict traumatic bleeding events.[65] Another study did in fact observe correlation between TGA parameters and emicizumab concentrations but no association with spontaneous bleeding episodes.[66] Both the studies highlighted considerable interpatient variability and therefore did not recommend the routine use of TGA as a monitoring tool in patients receiving emicizumab.

Discrepancies in published data are perhaps attributable to the lack of standardization, which remains one of the primary challenges in implementing TGA in clinical practice ([Fig. 5]). Several efforts have been made to standardize the assay. Although some consensus has been reached regarding pre-analytical conditions, the performance parameters of TGA still vary considerably across laboratories.[67] [68]

Fig. 5 Challenges in standardizing thrombin generation assay (TGA) in patients receiving emicizumab. Key variables influencing TGA results include the choice of sample matrix (platelet-poor plasma [PPP] vs. platelet-rich plasma [PRP]), the triggering reagent, and the analytical platform. The lack of consensus on these parameters limits comparability and hinders clinical interpretation in the management of hemophilia A patients. CTI, corn trypsin inhibitor. (Figure made using Biorender.)

 

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Current Approaches to TGA Protocols

Consideration of Choice of Trigger Reagent

One key aspect to consider is the selection of appropriate triggers for initiating thrombin generation. There are two primary pathways for tenase complex formation: the extrinsic pathway, which is typically triggered in TGA by tissue factor (TF), and the intrinsic pathway, which can be activated using a broader range of reagents, including contact activators like kaolin or silica, or FXIa.

Thrombin generation via extrinsic pathway stimulation is commonly used; however, the concentration of TF and phospholipids in the triggering agent may vary. For assessing bleeding tendencies, a low TF concentration (approximately 1 pM or even less) has been shown to be preferable.[67] [69] An international reference standard for TF is lacking, and manufacturers of commercially available triggers do not consistently report the TF concentration used.

Although TF-triggered TGA remains the standard method for assessing thrombin generation capacity, its utility is limited in certain contexts. For instance, it failed to show a correlation with emicizumab activity in spiking experiments or with the combination of emicizumab and bypassing agents.[70] This could be explained by the fact that the TF/FVIIa complex activates both FIX and FX, whereas emicizumab activity depends on the activation of FIX, which in turn requires FXI activation.[71] [72] FXI can be activated by thrombin, particularly on the surface of activated platelets, which provide a procoagulant environment. Kaolin and silica, which activate FXII and subsequently FXI, can also be used. Using kaolin as a trigger has been reported to increase the sensitivity of TGA in hemophilia patients,[73] but could also contribute to heterogeneity in thrombin generation results.[74] Therefore, FXIa might be preferred over classical contact factor activators for monitoring emicizumab.

Some studies have indeed demonstrated that FXIa-triggered TGA is more reliable for evaluating thrombin generation in HA patients.[72] [75] At present, FXIa is not available as a ready-to-use reagent for TGA and must therefore be prepared in-house by laboratories, typically in combination with phospholipids. Nonetheless, since FXIa can be precisely dosed and calibrated, FXIa-triggered TGA offers greater potential for standardization. However, the exclusive use of FXIa as a trigger in TGA activates only the intrinsic pathway. A combination of TF and activated FXI as a trigger has been proposed to better mimic physiological conditions. Notably, this approach has been shown to correlate with clinical outcomes in patients receiving emicizumab.[63]

 

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Use of Corn Trypsin Inhibitor

Some studies suggest that performing TGA with TF concentrations of 1 pM or lower may benefit from collecting blood into tubes prefilled with corn trypsin inhibitor (CTI).[76] [77] The addition of CTI has been shown to decrease TGA parameters under low TF conditions, yet strong correlations between TGA results obtained with and without CTI have been reported. Furthermore, no consistent influence on the coagulation proteins that determine TGA parameters was observed.[78] [79] [80] In patients with severe hemophilia A, CTI did not affect TGA results, regardless of the presence of inhibitors.[81] Current guidelines emphasize that the use of CTI remains debatable and is not recommended for routine practice.[67] [82]

 

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Consideration of Choice of Matrix

Another important consideration in TGA is the choice of matrix—either platelet-poor plasma (PPP) or platelet-rich plasma (PRP). Platelets play a crucial role in hemostasis by providing an activated surface for coagulation factor interactions; however, working with fresh platelets can be challenging in a routine clinical laboratory.

PPP has the advantage of being storable, which allows for flexibility in assay timing. However, it is important to note that PPP may underestimate thrombin generation when evaluating therapies that rely on platelet activity, such as rFVIIa. As mentioned earlier, in the context of emicizumab therapy, using FXIa as a trigger can help mitigate this limitation by enhancing the assay's sensitivity in the absence of platelets.[63]

 

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Available TGA Platforms

Calibrated Automated Thrombogram (CAT Assay)

A key factor in selecting the appropriate equipment for TGA is the method used to measure thrombin generation. The most widely used method has been the CAT assay by Diagnostica Stago (Düsseldorf, Germany).[68] Developed in 2003 by Hemker et al., this semi-automated system has been a standard tool for thrombin generation measurements.[83] However, it is expected to be withdrawn from the market due to several limitations, including its lack of standardization and the requirement for specially trained personnel, which restrict its implementation in routine laboratory settings.

 

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Modern Platforms for TGA Measurement

In recent years, fully automated TGA platforms employing fluorogenic methods have been developed, including ST Genesia (Diagnostica Stago) and Ceveron TGA (Technoclone, Vienna, Austria).[84]

The ST Genesia platform is noted for being easy to implement in routine laboratory practice, supporting continuous sample accessioning.[85] An advantage of this system is the sample-specific, internal (background fluorescence) calibration as well as the availability of an included reference plasma for results normalization, which helps reduce inter-laboratory variation.[86] However, the platform appears to be less flexible regarding customization of assays.

On the other hand, the Ceveron system is a more open platform. Currently, however, only an external thrombin calibration curve is used, and a dedicated reference plasma is not available, which may reduce inter-laboratory comparability. Despite this, the Ceveron system is an option for broader hemostasis testing in routine practice due to its flexibility to perform additional coagulation tests.[87]

 

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Conclusions

In summary, TGAs have the potential to play a significant role in the monitoring of patients with CHA. Although study results exhibit variability, TGA appears to offer valuable insights into predicting bleeding risk and guiding replacement therapy during surgery or for managing bleeding events. However, for TGA to be fully effective in clinical practice, standardization remains a key challenge, necessitating unified pre-analytical and analytical procedures. Current recommendations from the International Society on Thrombosis and Haemostasis (ISTH)[67] [82] provide a robust framework for improving standardization, though updates are needed as new data and methodologies—such as FXIa activation—emerge. The use of automated platforms could further enhance the reliability of TGA, helping to reduce inter-laboratory variation and increase its utility in routine clinical practice.

 

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Overall Conclusions

The application and monitoring of emicizumab require a comprehensive understanding of the underlying molecular mechanisms, as well as awareness of the limitations of routine coagulation assays. It is well-established that standard APTT-based assays do not yield reliable results in patients undergoing emicizumab therapy. To determine FVIII and FIX activity in these patients, bovine factor-based CSA can be used instead. These assays also allow for reliable measurement of FVIII inhibitors in the modified NBA.

Although the functional measurement of emicizumab in patient plasma using mOSA has become an established standard, residual FVIII activity levels may lead to falsely elevated results. Since heat inactivation of residual FVIII can reduce emicizumab activity, it is suggested to neutralize this residual FVIII with inhibitory antibodies instead. With more standardized adaptations of this method, it is expected to gain broader acceptance in the future.

Although the formation of ADA against emicizumab is rare, assays for screening and specificity confirmation have been proposed and validated. Due to the complexity of these assays and the infrequent ordering, these assays are likely to be offered by specialized laboratories only. ADA-induced reductions in circulating functional emicizumab plasma levels can be detected by mOSA.

Although fully automated analyzers for TGAs are already in use, the development of appropriate reagents for monitoring FVIII or FVIII-like activity is ongoing. Current data suggest that FXIa may be a relevant reaction trigger, perhaps in addition to TF, which has been the trigger in most commercial reagents to date.

In summary, clinicians should be aware about the challenges that emicizumab poses in the diagnostic laboratory to avoid potentially harmful misinterpretation of standard laboratory results. Specialized laboratories have amended their diagnostic portfolio to offer precise and valid monitoring for patients using emicizumab. This portfolio continues to evolve while emicizumab and second-generation FVIIIa mimetics are or will be being used by a growing number of patients.

 

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Conflicts of Interest

Jens Müller: Institutional grants for research from Novo Nordisk, honoraria for lectures and consultancy from Octapharma, Siemens Healtineers, and Pfizer.

Martin Büchsel: Institutional grants for research and studies from Bayer, CSL Behring, Roche, SOBI, and Takeda and honoraria for lectures from Bayer, Chugai, and Roche.

Olga Oleshko: Institutional grants for research and studies from Biotest, CSL Behring, and Octapharma; honoraria for lectures from CSL Behring, Chugai, and Roche.

Behnaz Pezeshkpoor: Institutional grants for research from Biotest, Octapharma, and NovoNordisk as well as honoraria for lectures or consultancy from NovoNordisk and Octapharma.

Ulrich Sachs: Institutional grants for research from Bayer, CSL Behring, Johnson & Johnson, Leo Pharma, Novo Nordisk, Octapharma, Pfizer, Siemens Healthineers and Sobi, as well as personal fees for advisory board meetings, consulting, and/or travel support from Bayer, Biomarin, Biotest, CSL Behring, Johnson & Johnson, Novo Nordisk, Octapharma, Pfizer, Siemens Healthineers, Sobi, and Werfen.

Ute Scholz: Honoraria for consultancy from Pfizer,

Andreas Tiede: Institutional grants for research and studies from Bayer, Biotest, Chugai, Novo Nordisk, Octapharma, Pfizer, Roche, SOBI, and Takeda, and honoraria for lectures or consultancy from Bayer, Biomarin, Biotest, Chugai, CSL Behring, Novo Nordisk, Octapharma, Pfizer, Roche, SOBI, and Takeda.

Authors' Contributions

All authors participated in writing the manuscript and approved the final version.

Standing Commission Labor (STAEKOLA) of the Society of Thrombosis and Haemostasis Research (GTH).

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Publikationsverlauf

Eingereicht: 01. Juli 2025

Angenommen: 19. August 2025

Artikel online veröffentlicht:
09. Oktober 2025