Usefulness of Global Coagulation Tests, Thrombin Generation and Viscoelastic Tests for Assessing the Bleeding Phenotype in Rare Coagulation Factor Deficiencies

Abstract

Inherited deficiencies of coagulation factors are rare and exhibit variable associations with severe bleeding phenotypes. Although conventional hemostatic assays serve as useful screening tools, they often fail to accurately predict clinical bleeding severity. Disease management is further complicated by poor correlation between residual factor levels and the overall symptom severity in affected patients and limited clinical experience. In this review, we evaluate the utility of global coagulation tests, such as the thrombin generation assay, plasmin generation assay (PGA), and rotational thromboelastometry (ROTEM), in assessing the severity of rare coagulation factor deficiencies and their clinical manifestations.

Overall, thrombin generation, plasmin generation, and ROTEM were impaired in most rare coagulation factor deficiencies. Furthermore, significantly reduced coagulation factor activity and consequently decreased thrombin generation potential correlated with the clinical bleeding severity in deficiencies of prothrombin and factors V, VII, X, and XI. PGA was significantly impaired in fibrinogen and prothrombin deficiency and variably reduced in FV- and FX-deficient patients, but did not correlate with the presence or severity of bleeding manifestations. Lastly, ROTEM parameters were able to discriminate between asymptomatic FX-, FXI-, and fibrinogen-deficient patients and those with a history of bleeding.

Although these studies are mostly limited to small sample sizes and prospective data are lacking, the available literature suggests that TGA, PGA, and ROTEM may be useful in stratifying patients according to their overall bleeding severity, as well as their risk of major bleeding complications in some of the rare coagulation factor deficiencies.

Keywords

inherited bleeding disorders - coagulation factor deficiency - thrombin generation assay - rotational thromboelastometry - routine hemostatic tests

Introduction

Rare coagulation factor deficiencies (CFDs) typically include deficiencies of fibrinogen, prothrombin, and factors (F) V, VII, IX, X, XI, and XIII. These deficiencies are rare, but their diagnosis and treatment are essential, as they can be associated with a bleeding phenotype ranging from mild to severe and, in some cases, even life-threatening.[1] In clinical practice, the management of patients with CFD is often challenging. Widely available routine hemostatic tests, such as the activated partial thromboplastin time (aPTT), thrombin time (TT), and prothrombin time (PT), as well as the residual factor activity correlate poorly with the clinical bleeding severity and symptom frequency in some rare CFD.[2] [3] Furthermore, due to the rarity of these conditions, clinical expertise and access to specialized hemostatic assays are often limited. In centers without a specialized hemostasis department, this lack of experience and resources can further complicate the management of CFD patients.

To address current challenges in the diagnosis and management of rare CFD, several studies have evaluated the clinical utility of global hemostatic assays, including the thrombin generation assay (TGA), plasmin generation assay (PGA), viscoelastic assays such as rotational thromboelastometry (ROTEM) and thromboelastography (TEG), and the optical thrombodynamics assay (TD) as tools to predict the bleeding phenotype in rare CFD. TGA and PGA measure thrombin and plasmin generation primarily from plasma over time, reflecting the overall coagulation and fibrinolytic potential, while viscoelastic and optical tests provide insight into clot formation kinetics, strength, and susceptibility to lysis in whole blood.[4] [5]

By providing a more comprehensive assessment of hemostasis and fibrinolysis, these assays may enhance phenotypic characterization of patients with CFD and facilitate the identification of those at high risk for bleeding.[6]

In more common CFD, such as hemophilia A and B, both TGA and PGA parameters differed between patients with severe and mild bleeding manifestations.[7] [8] Similarly, ROTEM has shown the ability to differentiate hemophilia patients according to their bleeding severity.[9] In contrast, TD parameters were not able to distinguish between mild and severe hemophilia A.[10]

This review examines the utility of global coagulation assays in the evaluation of rare CFD and their associated bleeding phenotypes. We report on the ability of established global assays to identify patients at high risk for severe bleeding and discuss their potential role as screening tools.

Diagnostic Workup and Clinical Presentation of Rare Coagulation Factor Deficiencies

Routine screening for rare CFD is mostly based on the routinely performed coagulation tests aPTT, PT, and TT, followed by the measurement of respective factor activity levels. As illustrated in [Fig. 1], each of these routine coagulation tests assesses a specific part of the coagulation cascade. In CFD, an isolated aPTT prolongation is associated with deficiencies of FXII (without clinical relevance) or FXI, as well as with the more common deficiencies of FVIII and FIX, while a prolonged PT may indicate FVII deficiency. Deficiencies affecting the common segment of the coagulation cascade, downstream of both the intrinsic and extrinsic pathways, usually prolong both aPTT and PT, particularly deficiencies in fibrinogen, prothrombin, FV, and FX. However, FXIII deficiency, another exceedingly rare CFD, has no effect on these routinely performed tests and therefore no screening method is available.[11] Furthermore, routine hemostasis tests may be within normal ranges in mild CFD, such as FXI deficiency and hemophilia A and B.[12] [13] In these cases, patients may show normal results on routine hemostatic investigations while still experiencing a clinically relevant bleeding tendency. Hence, when a CFD is suspected—whether based on prolonged routine coagulation tests or a history of bleeding symptoms—the diagnosis has to be confirmed by measuring the activity of the respective coagulation factor.

Fig. 1 Overview of the coagulation cascade and routine hemostatic tests. *FVIII and FIX deficiency are not discussed in this review. aPTT, activated partial thromboplastin time; F, factor; PT, prothrombin time; ROTEM, rotational thromboelastometry; TGA, thrombin generation assay; TT, thrombin time. (Created in BioRender. Dreier, T. (2025) https://BioRender.com/d8fpk2f.)

Management of patients with rare CFD is further complicated by the limited correlation between reduced coagulation factor activities and the clinical severity in some CFD. Although residual activities of at least 10% (for prothrombin and FV), 20% (for FVII and FXI), 30% (for FXIII), and 40% (for FX) appear to be sufficient for physiological hemostasis in most patients,[3] [14] deficiencies of some factors, particularly prothrombin, FV, FVII, and FXI, show a poor correlation with the observed bleeding severity.

The clinically relevant cutoff of each coagulation factor, the correlation between the clinical bleeding severity in affected patients and the residual factor activity, as well as commonly reported symptoms are outlined in [Table 1]. The outlined cutoffs were recommended by the European Network of Rare Bleeding Disorders (EN-RBD) as the target trough level for an asymptomatic state in patients with the respective CFD.[3] [14]

Table 1 Overview of the coagulation factor deficiencies included in this review, and their clinically relevant cutoffs, clinical presentation, and the correlation between residual factor activities and the clinical bleeding severity

Diagnosis	Clinically relevant cutoff for bleeding manifestations	Correlation between residual factor activity and bleeding	Common clinical presentation
Fibrinogen deficiency	Asymptomatic/mild: 150–100 mg/dL Moderate: 100–50 mg/dL Severe: <50 mg/dL[64]	Strong	Hypermenorrhagia, post-surgical and postpartum bleeding complications[65]
Afibrinogenemia	Complete lack of fibrinogen[64]	No residual fibrinogen	Umbilical cord bleeding, joint bleeding, mucocutaneous bleeding, intracranial bleeding, hypermenorrhagia, post-surgical and postpartum bleeding complications[16] [66]
Prothrombin deficiency	Asymptomatic/mild: >10% activity Mild/moderate: ≤10% activity[2] [3] Moderate/severe: ≤5% activity[67]	Poor, very limited data[67] [68]	Hypermenorrhagia, post-surgical and postpartum bleeding, bleeding after tooth extractions, intramuscular bleeding, hemarthrosis[69]
FV deficiency	Asymptomatic/mild: ≥10% activity[3] [70] Variable: 1–10% activity[71] Severe: <1% activity[72]	Poor[22] [73]	Variable, mostly post-surgical bleeding and mucosal bleeding[71]
FVII deficiency	Asymptomatic/mild: >20% activity[24] Mild/variable: 10–20% activity[3] Severe: <10% activity[74]	Poor[2] [75]	Post-surgical bleeding and mucosal bleeding, intracranial and umbilical cord bleeding reported in a few severe cases[76]
FX deficiency	Asymptomatic/mild: >40% activity[2] [3] Mild: 10–40% activity[77] Severe: <10% activity[78] [79]	Strong[2]	Gastro-intestinal and intracranial bleeding in severe cases[78] [79]
FXI deficiency	Asymptomatic/mild: ≤20% activity[80] Mild: ≤1% activity[81]	None[82]	Overall mild bleeding severity regardless of activity[2] [83] Mostly provoked bleeding symptoms (post-surgical bleeding),[80] oral bleeding and hypermenorrhagia[3]
FXIII deficiency	Mostly asymptomatic: >30% activity[2] [14] Mild to severe: <30% activity[2] Severe: <5% activity	Strong[77] [84]	Severe cases: Intracranial and post-surgical bleeding,[85] miscarriages[2] [86] [87] [88]

Abbreviation: F, factor.

Clinical Presentation of Rare Coagulation Factor Deficiencies

The prevalence of fibrinogen deficiency, defined by plasma fibrinogen levels below 150 mg/dL (mild), 100 mg/dL (moderate), or 50 mg/dL (severe), is estimated at 13 to 18 cases per 1,000,000 individuals in European countries such as Ireland, the United Kingdom, and Slovakia.[15] In fibrinogen deficiency, clinical severity of the disease and the prolongation of aPTT and PT is mainly dependent on the residual fibrinogen level, with TT being the most sensitive parameter.[17] Afibrinogenemia, a complete lack of fibrinogen, is a rare disease with an estimated global prevalence of 1 to 2 per 1,000,000,[18] and is associated with a severe bleeding tendency often diagnosed early in life using routine coagulation tests, such as aPTT, PT, and TT, which are extremely prolonged or not measurable.[15]

Dysfibrinogenemia, characterized by normal antigen levels but dysfunctional fibrinogen, and hypodysfibrinogenemia, which involves reduced levels of dysfunctional fibrinogen, results from a spectrum of mutations affecting fibrinogen structure and function.[19] [20] Given the considerable clinical heterogeneity and the potential for thrombotic complications in dysfibrinogenemia, this review deliberately focuses on hypofibrinogenemia, where diagnostic challenges are closely linked to a quantitative deficiency of functional fibrinogen. A more detailed discussion of dysfibrinogenemia is beyond the scope of this review.

With an estimated prevalence of about 1 in 2,000,000 individuals, inherited prothrombin (FII) deficiency represents an extremely rare coagulation disorder. Prothrombin levels <10% are typically associated with a severe bleeding tendency,[2] [3] although with a limited correlation between residual prothrombin activity and the individual bleeding severity. However, the residual prothrombin activity has shown a weak, but significant, correlation with the age at first bleed, and an inverse correlation with lifetime number of both trauma-related and musculoskeletal bleedings.[2] Similarly to deficiencies in fibrinogen, prothrombin deficiency leads to variable prolongations of aPTT and PT.[21]

A deficiency in FV is a rare bleeding disorder with an estimated prevalence of 1 in 1,000,000, with an FV activity of >10% required for normal hemostasis.[2] Residual FV activity levels have been shown to correlate poorly with the observed clinical bleeding manifestations.[22] In routine laboratory investigations, FV deficiency is associated with a prolonged aPTT and PT, but with normal TT.[23]

With an estimated prevalence of 1 in 500,000, the clinically relevant deficiency of FVII (<20% residual activity) is the most common among the rare CFD.[24] Residual FVII activity levels in affected patients have been shown to only poorly correlate with the individual clinical severity of the disease.[25] [26] However, clinically relevant FVII deficiencies, with a residual FVII activity of <2%, can be accompanied by life-threatening spontaneous bleeding.[27] Lastly, patients with quantitative FVII deficiencies present with a prolonged PT, but normal aPTT values, reflective of the isolated impairment of the extrinsic pathway.[28]

Deficiencies of coagulation FX are estimated to affect comparably fewer individuals, with an estimated global prevalence of 1 in 1,000,000.[29] In FX deficiencies with FX activity <40%, the clinical severity of the bleeding disorder is strongly dependent on the residual FX levels, with low FX levels (<10%) generally leading to a severe and spontaneous bleeding tendency.[2] Initial suspicion of an FX deficiency affecting the common pathway of coagulation is often based on a prolongation of both aPTT and PT.[30]

Factor XI deficiency, defined by residual activity below 20%, occurs with an estimated prevalence ranging from 1 to 250 cases per 1,000,000 individuals, depending on sociodemographic and geographic variables.[31] Importantly, the individual severity and frequency of the bleeding symptoms are not dependent on FXI activity levels.[2] In laboratory investigations, a decrease of FXI activity to <30% is associated with an increase in aPTT, while the PT remains within the normal range.[32] As patients with higher FXI activity of between 30 and 60% can still experience bleeding symptoms (mostly mild or only after hemostatic challenges),[2] FXI-deficient patients with normal aPTT might still be at risk of bleeding.[12]

FXIII deficiency (activity <30%) is an extremely rare disease, with an estimated prevalence of 1 in 2,000,000 to 5,000,000 persons according to data from the United Kingdom.[33] In FXIII deficiency residual factor activity correlated strongly with the severity of the bleeding tendency. In FXIII deficiency, routine hemostatic tests such as aPTT, PT, and TT are within normal ranges; thus, FXIII activity needs to be determined in case of high clinical suspicion.

Global Hemostatic Tests in Rare Coagulation Factor Deficiencies

As outlined, routine hemostatic tests such as aPTT and PT represent robust and cheap screening tools for the majority of rare CFD. However, some, and especially mild, CFDs do not impair these tests, which may be partially explained by the fact that these routine assays terminate upon initial fibrin formation, an event that occurs when less than 5% of the total thrombin has been generated.[34] As a result, these tests fail to capture the extent of in vivo thrombin generation. Furthermore, routine hemostatic assays and residual factor activity levels often fail to correlate with clinical severity in rare CFD, limiting their utility in predicting bleeding risk. As mentioned, global hemostasis assays may offer a more comprehensive assessment of bleeding tendency in these patients.

TGA, PGA, and ROTEM, performed in plasma or whole blood, have been developed to provide a more complete assessment of hemostasis and fibrinolysis and might therefore be able to assist in estimating the individual prospective probability and severity of bleeding symptoms. In the following sections, currently available clinical and experimental data on these global hemostatic tests in cohorts of patients with rare CFD will be summarized and discussed.

Thrombin Generation Assay

TGA enables the time-dependent quantitative measurement of thrombin generation. As outlined in [Fig. 2], a TGA curve has five important parameters: lag time (the time from addition of assay activators to initial thrombin formation), time to peak thrombin (time from the start of the test to the maximum thrombin concentration), peak thrombin concentration (amount of maximum thrombin generated), the velocity index (the maximum slope steepness), and AUC/ETP (the area under the curve, also called the endogenous thrombin potential).[35]

Fig. 2 Representative thrombin generation assay (TGA) curve. AUC, area under the curve; ETP, endogenous thrombin potential; min, minutes; nM, nano molar. (Created by the authors.)

Importantly, the results of TGA measurements are highly dependent not only on the preanalytical variables, such as the type of sample used (e.g., platelet-poor plasma or platelet-rich plasma), but also on the concentration of the tissue factor used to trigger the in vitro thrombin generation. To address this issue, standardized TGA kits have been developed (e.g., the novel hemostasis assay, “NHA,” the calibrated automated thrombogram, “CAT,” and established protocols for other automated TGA), while in-house assays with custom concentrations of triggers to initiate thrombin generation, such as phospholipids and tissue factor, are also in use. Different preanalytical setups used in studies investigating thrombin generation in cohorts of patients with rare CFDs will be highlighted in this review, where applicable.

A study assessing thrombin generation using the NHA in a pooled cohort of 39 patients with various CFDs—including deficiencies of fibrinogen, prothrombin, and FV, FVII, FX, and FXIII—evaluated TGA parameters for their ability to differentiate between patients with overall minor bleeding symptoms and those with major bleeding manifestations. Although differences in lag time, time to peak thrombin, and the AUC were not statistically significant between patients with minor and major bleeding symptoms, patients classified as having major manifestations had significantly lower peak thrombin concentrations.[5] Furthermore, in a similar study using a custom chromogenic TGA assay with phospholipids and tissue factor or phospholipids and actin FS as triggers, a total of 88 patients with deficiencies of prothrombin, FV, FVII, FX, and FXII, all patients with severe bleeding symptoms had an ETP of <20% of healthy controls, while mild or asymptomatic patients had an ETP of more than 30% of healthy controls.[36]

However, the results of TGA in individual CFDs showed considerable variability:

TGA data from four patients with inherited afibrinogenemia showed normal results in the clotting time in seconds (CT) using platelet-poor plasma (PPP), with no changes after the infusion of fibrinogen concentrates.[37] In line with this, five patients with fibrinogen deficiency (<100 mg/dL) assessed using the NHA showed similar AUC and non-significantly reduced thrombin peak height compared with normal pooled plasma (NPP).[5] However, one of these hypofibrinogenemic patients had no detectable thrombin generation, indicating a significant variability in the TGA in hypofibrinogenemia. Unfortunately, neither study investigated the correlation between TGA parameters and the severity of the reported bleeding manifestations.

In three patients with prothrombin deficiency (residual activity 7–10%), all parameters in the NHA were reduced or non-measurable. Here, one patient showed a significantly impaired overall thrombin generation potential with an AUC <5% of NPP, while the other two patients could not produce any thrombin.[5] In a separate study using a custom TGA setup conducted on a cohort of 21 patients with prothrombin deficiency including their relatives carrying prothrombin mutations (residual activity 1–85%), the ETP strongly correlated with the residual prothrombin activity.[36] Although this study did not provide specific details on the individual bleeding phenotypes of the analyzed patients, all patients with an (extrinsic or intrinsic) ETP of >20% of normal were reported as asymptomatic. Of nine patients with an ETP ≤20%, two were reported as having mild symptoms, and the bleeding phenotype of the remaining seven patients (with an ETP ranging from 18 to <5%) was classified as severe.

Plasma from three patients with FV activity levels <1% showed no capability to produce thrombin in the CT.[38] Although this study reported no details on the bleeding severity of FV-deficient patients, another study evaluating 22 FV-deficient patients and their affected relatives using a custom TGA found that 6 patients with FV activity <1% had negligible ETP and severe bleeding phenotypes.[36] However, in all other patients, an FV activity of ≥2% was sufficient for physiological thrombin generation, as indicated by an ETP ranging from 79 to 100% independent of individual FV activity. Two patients with FV activity of 2% reported mild bleeding symptoms, while all others with FV activity ≥4% were asymptomatic.

Even trace amounts of FVII in patients with <1% remaining FVII activity was enough to produce normal amounts of thrombin in the NHA (normal peak thrombin concentration and ETP), although with a prolonged time to peak thrombin (TTP)[39] and a high variability between patients.[5] In an analysis using an established protocol[40] for an automated, calibrated TGA, peak thrombin concentration and TTP, peak thrombin concentration and TTP have been proposed as the most sensitive parameters for FVII activity.[6] Clinically, lag time and TTP have been positively correlated with disease severity in FVII-deficient patients.[6] Although TGA did not directly correlate with bleeding scores, parameters such as lag time, TTP, and peak thrombin concentration distinguished patients with normal ISTH-BAT (Bleeding Assessment Tool by the International Society on Thrombosis and Hemostasis) scores from those with abnormal scores.[6] Similar results were reported in a study including 22 FVII-deficient patients also using a custom TGA: An FVII activity <2% led to a significantly impaired TTP and peak thrombin concentration (although with a normal ETP and a variably severe bleeding tendency), while patients with ≥2% activity showed lower impairments of these parameters and an overall “mild” bleeding phenotype or were asymptomatic.[36]

In two separate studies using the NHA and an established protocol for automated TGA,[40] TGA data on a total of nine patients with severe FX deficiency (FX activity <1%) have been reported.[5] [6] Here, one patient showed significantly impaired thrombin generation potential (<5% AUC compared with NPP) while all other patients had non-measurable thrombin generation. In a separate cohort of 10 FX-deficient patients assessed using a custom TGA setup, a residual activity of <10% significantly impaired the TTP and peak thrombin concentration.[36] Importantly, the resulting ETP correlated with the overall bleeding severity of the patients. FX activity ≤2% was associated with non-measurable ETP and severe bleeding, while three patients with FX activity levels of 3, 6, and 10% (ETP: 38, 59, and 72% of normal, respectively) reported only mild symptoms. All patients with FX activity ≥25% (ETP ≥80%) were asymptomatic.

TGA was also performed in a study assessing 39 FXI-deficient patients in an established[40] automated TGA assay.[41] Importantly, when TGA was performed with recalcification as a trigger only, patients with a history of bleeding had significantly lower ETP and peak height than non-bleeding patients. In the tissue factor–activated assay, bleeding patients had significantly impaired peak height, but similar ETP than non-bleeding patients. Furthermore, in a cohort of seven FXI-deficient patients and relatives carrying FXI mutations (with FXI activities ranging from <1 to 54%), mild bleeding symptoms were reported only by patients with <1% residual FXI activity with a consequent ETP reduction of 55 to 65% of normal, while all other patients were asymptomatic and had normal ETP in a custom TGA.[36]

Lastly, in a cohort of eight patients with FXIII activity between <3 and 11%, NHA results varied significantly.[5] In these patients, the AUC ranged from 120% of NPP to non-measurable thrombin generation. However, the overall mean peak thrombin was significantly reduced in FXIII-deficient patients compared with NPP. Interestingly, three patients with FXIII deficiency on prophylaxis (activity at baseline without substitution: 15–24%) showed a 1.6- to 1.8-fold increase in peak thrombin generation compared with normal control platelet-poor plasma, while ETP, TTP, lag time, and the α-angle were similar. The difference in peak thrombin generation in TGA was attenuated with FXIII substitution. The authors of this study argue that increased plasma levels of prothrombin fragments in these patients might have led to increase in peak thrombin generation. Another possible explanation for an increase of thrombin generation in FXIII-deficient patients is the accelerated degradation of formed fibrin due to the lack of crosslinking by FXIII, and hence a delayed termination of the assay.[42] No data on the individual bleeding severity of these patients were reported.

Plasmin Generation Assay

The PGA measures plasmin production upon hemostatic activation in plasma using fluorogenic substrates. These assays provide a dynamic assessment of fibrinolytic activity and offer valuable insight into a patient's overall hemostatic capacity.

Overall, data on PG in patients with rare CFD are scarce. A total of 41 patients with different CFDs (5 cases of fibrinogen deficiency, 3 cases of prothrombin deficiency, 6 cases of FV deficiency, 11 cases of FVII deficiency, 8 cases of FX deficiency, and 8 cases of FXIII deficiency) were investigated using PGA in a study by Van Geffen et al.[5] In this study, the “novel hemostasis assay” (NHA) was used to measure PGA in parallel to TGA (data reported earlier in this review). Unfortunately, this study did not provide details on the bleeding phenotype of individual patients, or the correlations between PGA parameters and bleeding severity. Plasmin generation potential in patients was described using three parameters, as illustrated in [Fig. 3]: the fibrin lysis time (the time until the formed clot is lysed by plasmin, FLT), the peak plasmin concentration in nanomolar, and the plasmin potential (which corresponds to the AUC/ETP in TGA).[43] In this pooled cohort of all 41 patients, the plasmin potential was able to distinguish between patients with mild and major bleeding symptoms, while the fibrin lysis time was similar and the peak plasmin concentration was non-significantly reduced in patients with a history of major bleeding symptoms.[5] As with the TGA, the detailed results in this study varied widely between deficiencies in different factors.

Fig. 3 Representative plasmin generation assay (PGA) curve. FLT, fibrin lysis time; Min, minutes; nmol, nanomolar; TGA, thrombin generation assay. (Created by the authors.)

As expected, in five cases of fibrinogen deficiency with residual fibrinogen levels <100 mg/dL, PGA was significantly impaired. The FLT was unmeasurable in all cases, while the peak plasmin concentration only reached 2 to 29% of the normal pooled plasma.[5] In contrast, three patients with prothrombin deficiency showed only a delayed onset of PG, but with normal or only slightly reduced FLT. Similarly to the fibrinogen-deficient patients, peak plasmin concentration as well as plasmin potential was reduced in comparison to NPP, however.[5]

In five patients with FV deficiency (all of whom with residual activity <5%), results of plasmin generation varied widely, with the peak plasmin concentration ranging from non-detectable to 478% of NPP. However, in all cases where plasmin generation was measurable, FLT was reduced in comparison to NPP.[5]

Even though FVII-deficient patients showed slight but clinically relevant abnormalities in TGA, all patients were able to produce normal amounts of plasmin. Mean FLT, peak plasmin concentration, and the overall plasmin potential were comparable to NPP in this cohort.[5]

As outlined previously, only one of the eight patients with FX deficiency (<1% residual activity in all patients) was able to produce a miniscule amount of thrombin in TGA. In the simultaneous PGA, this low thrombin level was sufficient to trigger normal plasmin production, while no plasmin generation was observed in the other patients.[5]

Unfortunately, no PGA data on patients with inherited FXI deficiency have been published to date. However, a study conducted on 71 FXI-deficient patients (mean FXI activity 37% in non-bleeding and 27% in bleeding patients) showed impaired clot formation and an increased fibrinolytic susceptibility in a clot formation and fibrinolysis turbidity assay in FXI-deficient patients with a positive history of bleeding compared with asymptomatic patients.[44]

Lastly, in FXIII deficiency there was no significant impact on the mean FLT, peak plasmin concentration, or overall plasmin potential in eight affected patients (FXIII activity ranging from <3 to 11%) compared with NPP.[5]

Viscoelastic and Optical Tests

Viscoelastic tests are based on the increasing viscosity of clotting blood, quantified by mechanical or resonant resistance. Although routine hemostatic tests, TGA and PGA, are often limited to citrated plasma and therefore omit central contributors of in vivo hemostasis, such as platelets and erythrocytes, viscoelastic tests can be performed using whole blood. Hence, viscoelastic tests provide a holistic overview of hemostasis—from initial clot formation to fibrinolysis in whole blood.[45]

Different platforms have been developed, such as TEG, rotational thromboelastography (ROTEG), and ROTEM. Briefly, all these assays measure real-time coagulation by detecting the resistance of clotting blood against an oscillating pin with varying mechanical differences. As the clot forms, resistance increases, reflecting thrombus formation and strength. Once fibrinolysis begins, resistance decreases. A key advantage of ROTEM is its ability to assess specific parts of the hemostatic system using targeted activators: INTEM (intrinsic pathway), EXTEM (extrinsic pathway), FIBTEM (fibrinogen versus platelet contribution), and NATEM (native hemostasis).[46]

The increase in rotational resistance is used to chart the ROTEM output as shown in [Fig. 4]. Key parameters include CT, clot formation time in seconds (CFT), the slope steepness (α-angle), A10 in mm (amplitude at 10 minutes), maximum clot firmness in mm (MCF), maximum lysis (ML), and clot lysis indices (CLI) at set time points.[46]

Fig. 4 Representative rotational thromboelastometry (ROTEM) curve. A10, amplitude at 10 minutes in mm; CFT, clot formation time; CLI, clot lysis index; CT, clotting time; MCF, maximum clot firmness; min, minutes; ML, maximum lysis; mm, millimeters. (Created by the authors.)

In contrast, TD enables real-time visualization of initiation and propagation of spatial thrombus formation, and thrombin generation.[4] By adding plasma to a chamber with immobilized tissue factor, the generation of thrombin and the formation of thrombi can be visually captured and measured. Clot formation is monitored via parameters including lag time (minutes), initial and stationary clot growth rates (µM/min), clot size after 30 minutes (µm), maximum clot density (arbitrary units), and time to spontaneous clot appearance (minutes). Thrombin generation metrics in TD are analogous to those in standard TGA.

As ROTEM covers most of secondary hemostasis—from the initiation of clot formation to fibrinolysis—it has been used routinely in emergency departments and intensive care units for early detection of abnormalities in coagulation and fibrinolysis.[47] [48] Furthermore, ROTEM has been shown to be impaired in patients with severe hemophilia A, with the extent of the impairment strongly related to the clinical severity and residual factor activity.[49] Hence, ROTEM may also correlate with the clinical severity in affected patients with other CFDs.

A case study on a patient with inherited afibrinogenemia showed an undetectable MCF and CT, alongside barely measurable A10 in both FIBTEM and EXTEM.[50] Similarly, TEG was not measurable in four patients with afibrinogenemia, but this was attenuated with the infusion of human fibrinogen concentrate.[37] FIBTEM CT and MCF have been shown to be impaired in patients with fibrinogen deficiency in comparison to patients with dysfibrinogenemia,[51] although not to the extent seen in afibrinogenemia. In a cohort of seven hypofibrinogenemia and five dysfibrinogenemia patients, FIBTEM CT was significantly more prolonged in patients with bleeding symptoms in comparison with asymptomatic patients or patients with thrombotic complications.[51] In the same study, FIBTEM MCF moderately correlated with the residual fibrinogen antigen levels, but not with fibrinogen activity.

Unfortunately, an extensive literature review found no data on ROTEM in patients with inherited prothrombin deficiency. However, Yamada et al published two cases of acquired lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS)[52] where no clot formation was detectable in ROTEM, which improved with treatment of the underlying systemic lupus erythematosus.

In nine FV-deficient patients, ROTEM analyses with INTEM, EXTEM, NATEM, and FIBTEM showed significantly prolonged CT when compared with healthy controls, with variable differences in other parameters.[53] However, ROTEM parameters did not correlate with the individual clinical severity in the patient cohort.

A study on 27 children with FVII activity levels <35% showed no correlation between TEG parameters and residual FVII activity levels. Similar results were also shown in clinical case presentations of patients with FVII deficiency, where pre- and perioperative NATEM and EXTEM showed near-normal or normal parameters that did not significantly change upon the perioperative infusion of recombinant activated FVII (rFVIIa).[54] [55] Similarly, in a case of a patient with 4% remaining FVII activity, ROTEM parameters were within normal ranges before and after rFVIIa infusion.[56] This discrepancy between residual FVII levels and ROTEM results can be in part explained by the high concentrations of tissue factor used in ROTEM, and particularly in EXTEM, to initiate coagulation.[57] This high concentration of tissue factor might “overwhelm” the extrinsic pathway and therefore compensate for lower levels of FVII. Currently, no data are available to reliably explain the discrepancy between NATEM, which does not use tissue factor, and the severity of FVII deficiency. However, published data show a higher susceptibility for pre-analytical variability in NATEM, which might contribute to the outlined results.[58]

ROTEM data in patients with FX deficiency are scarce. However, a case report of a 17-year-old pregnant patient with inherited FX deficiency (residual activity 2%) undergoing antepartum checkups reported EXTEM CT parameters.[59] At baseline in the 3rd trimester, this patient had significantly prolonged EXTEM CT of 244 seconds (normal range 43–82 seconds[60]). In this case, the antepartum transfusion of an FX concentrate normalized the EXTEM CT to 77 seconds.

As mentioned before, residual FXI activity levels do not correlate with the presence or severity of bleeding manifestations in patients with FXI deficiency. To address this issue, a few studies have investigated ROTEM as an alternative method to stratify bleeding risk in FXI-deficient patients. In a study assessing 25 patients with FXI deficiency using NATEM and INTEM, patients with severe FXI deficiency (FXI <1 IU/dL) had significantly impaired results in NATEM CT, α-angle, MCF, and a non-measurable fibrinolysis when compared with healthy controls. NATEM was also able to distinguish between different underlying FXI genotypes. Although most NATEM parameters did not significantly correlate with the bleeding severity or symptom presence, CT was slightly but significantly longer in patients with bleeding manifestations when compared with non-bleeding patients. Similarly, INTEM parameters were impaired in FX-deficient patients but did not correlate with the clinical bleeding severity.[41]

In a separate study on 68 FXI-deficient patients investigating ROTEM using platelet-rich plasma (PRP), patients with a positive bleeding history had a significantly longer CFT and a significantly lower α-angle when compared with non-bleeding patients. However, these results were heavily influenced by additional parameters, as a ROC/AUC analysis only resulted in a specificity of 38.5% (sensitivity 100%) for the bleeding phenotype.[61]

To date, TD was only assessed in FXI-deficient patients in a single study. Calderara et al demonstrated that peak thrombin concentration and clot growth parameters, particularly velocity and clot size in the stationary tissue factor–independent propagation phase, differentiated bleeding FXI-deficient patients from non-bleeding FXI-deficient patients and healthy controls.[62] In receiver operating characteristic analysis, clot growth rate emerged as the strongest predictor of bleeding phenotype, with a sensitivity of 92.3% and a specificity of 81.8%.

Lastly, no ROTEM data in patients with inherited FXIII deficiency are available in published literature to date. However, ROTEM has been measured in patients with liver cirrhosis and acquired FXIII deficiency, where a decrease in FXIII activity was associated with a decrease in MCF in FIBTEM and EXTEM, indicating an impaired clot stability.[63]

Summary

The impact of different CFDs on routine hemostatic tests, TGA, PGA, and ROTEM is summarized in [Fig. 5].

Fig. 5 Impact of different coagulation factor deficiencies on routine hemostatic tests, thrombin generation assay (TGA), plasmin generation assay (PGA), and rotational thromboelastometry (ROTEM). aPTT, activated partial thromboplastin time; CT, clotting time; ETP, endogenous thrombin potential; MCF, maximum clot firmness; PPP, platelet poor plasma; PRP, platelet rich plasma; PT, prothrombin time; TT, thrombin time.

Conclusions

Inherited rare CFD comprise a heterogeneous group of bleeding disorders with diverse clinical phenotypes, ranging from asymptomatic or mild bleeding, as in FXI deficiency, to severe, life-threatening hemorrhage, as observed in FXIII deficiency or afibrinogenemia. Although most CFDs prolong one or more routine hemostatic test (aPTT, PT, or TT), these changes are often nonspecific. Moreover, some CFDs, particularly mild deficiencies, do not affect these tests at all. Importantly, the prolongation of routine assays does not reliably correlate with bleeding risk or severity in affected individuals. This is because such tests only reflect the initiation phase of coagulation and do not assess the full thrombin generation potential, clot stability, or susceptibility to fibrinolysis.

We therefore aimed to examine global hemostatic tests, such as TGA, PGA, and viscoelastic tests, like TEG and ROTEM, as additions to routine hemostatic investigations for their ability to screen for CFD and to predict the individual clinical bleeding phenotype in rare CFD.

Disruptions in the coagulation cascade, which ultimately culminates in thrombin and fibrin production can significantly affect TGA results. In line with this, TGA was impaired in all rare CFDs except for afibrinogenemia and, except for one patient, in fibrinogen deficiency. In deficiencies of prothrombin, FV, FX, and FXI, low residual coagulation factor activities resulted in significantly reduced ETP. In these disorders, the bleeding severity was also associated with the extent of TGA impairment. In FVII deficiency, TGA correlated with a pathologic bleeding score (assessed by the ISTH-BAT). Clinical PGA data in cohorts of CFDs are scarce. However, plasmin generation, indicating higher fibrinolytic activity, was impaired in patients with fibrinogen and prothrombin deficiency. Factor V–deficient patients showed a high variability in peak plasmin generation. In FX-deficient patients, PGA was normal, except in patients with no thrombin production in the simultaneously measured TGA. In these patients, PGA was also not measurable. TEG and ROTEM, with their variants INTEM, EXTEM, and NATEM, have been shown to be impaired in the majority of CFDs, with FVII-deficient patients being the only cohort with normal ROTEM results. Importantly, ROTEM was able to discriminate between asymptomatic patients with fibrinogen, FX, or FXI deficiency and those with a history of clinically relevant bleeding.

To summarize, TGA, PGA, and ROTEM were affected in the majority of CFDs. An impaired overall thrombin generation potential was most commonly observed in patients with very low residual factor activities and was associated with a severe bleeding phenotype. ROTEM was able to distinguish symptomatic from asymptomatic patients with certain rare CFD. However, due to the limited clinical data on rare CFD and significant inter-patient variability, recommendations for the introduction of ROTEM and TGA into routine clinical practice cannot be made at this time, although both TGA and ROTEM parameters show impairment in several different CFDs and correlate with the disease severity. Nevertheless, global tests of hemostasis, such as ROTEM and TGA, may serve as valuable tools in the future to assess the impact of rare CFD on the individual patients' hemostatic capacity and the extent of their overall bleeding propensity.

Conflict of Interest

The authors declare that they have no conflict of interest.

Support for Attending Meetings and/or Travel

Novo Nordisk, travel support to GTH 2023.

Roche, travel support to ASH 2023.

Sobi, travel support to EHA 2023.

Pfizer, travel support to ISTH 2023.

Consulting Fees

Novartis, SOBI, Amgen, Sanofi, CSL Behring.

Grant

Prof. Heimburger Award, research grant (to institution).

EHA Research Mobility Grant, research grant (to institution).

Medical-scientific fund of the mayor of the federal capital Vienna, research grant (to institution).

Research support from Novartis, SOBI, Amgen, Sanofi, and CSL Behring.

Invited Review for the “Diagnosis and treatment of rare congenital bleeding disorders” themed issue.

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Publication History

Received: 27 March 2025

Accepted: 18 August 2025

Article published online:
15 October 2025