Applicability of the Thrombin Generation Test to Evaluate the Hemostatic Status of Hemophilia A Patients in Daily Clinical Practice

Abstract

Hemophilia A (HA) is a rare bleeding disorder caused by factor VIII (FVIII) deficiency due to various genetic mutations in the F8 gene. The disease severity inversely correlates with the plasma levels of functional FVIII. The treatment of HA patients is based on FVIII replacement therapy, either following a prophylactic or on-demand regime, depending on the severity of the disease at diagnosis and the patient's clinical manifestations. The hemorrhagic manifestations are widely variable amongst HA patients, who may require monitoring and treatment re-adjustment to minimize bleeding symptoms. Notably, laboratory monitoring of the FVIII activity is difficult due to a lack of sensitivity to various FVIII-related molecules, including non-factor replacement therapies. Hence, patient management is determined mainly based on clinical manifestations and patient-clinician history. Our goal was to validate the ST Genesia^® automated thrombin generation analyzer to quantify the relative hemostatic status in HA patients. We recruited a cohort of HA patients from the Principality of Asturias (Spain), following treatment and at a stable non-bleeding phase. The entire cohort (57 patients) had been comprehensively studied at diagnosis, including FVIII and VWF activity assays and F8 genetic screening, and then clinically monitored until the Thrombin Generation Test (TGT) was performed. All patients were recruited prior to treatment administration, at the maximum time-window following the previous dose. Interestingly, the severe/moderate patients had a similar TGT compared to the mild patients, reflecting the non-bleeding phase of our patient cohort, regardless of the initial diagnosis (i.e., the severity of the disease), treatment regime, and FVIII activity measured at the time of the TGT. Thus, TGT parameters, especially the peak height (Peak), may reflect the actual hemostatic status of a patient more accurately compared to FVIII activity assays, which may be compromised by non-factor replacement therapies. Furthermore, our data supports the utilization of combined TGT variables, together with the severity of patient symptoms, along with the F8 mutation type to augment the prognostic capacity of TGT. The results from this observational study suggest that TGT parameters measured with ST Genesia^® may represent a suitable tool to monitor the hemostatic status of patients requiring a closer follow-up and a tailored therapeutic adjustment, including other hemophilia subtypes or bleeding disorders.

Keywords: F8; FVIII; Hemophilia A; bleeding; mutation; thrombin generation test.

Figure 1

Schematic representation of TGT curves generated by the ST Genesia^®. Representative graphs of TGTs from samples obtained from mild HA patients, a newly diagnosed patient (left), and a previously diagnosed patient without current bleeding symptoms (right). The TGT variables are indicated.

Figure 2

Characterization of the Hemophilia A cohort. (A) Box plots depicting the FVIII activity levels at diagnosis, as measured by FVIII:Chr (left) and FVIII:C (right) assays in mild, moderate, and severe HA patients. (B) Correlation analysis of FVIII activity levels (measured using FVIII:Chr and FVIII:C assays), VWF:Ag (antigenic Von Willebrand Factor levels), VWF:RCo (Von Willebrand Factor ristocetin cofactor activity), severity, and annual bleeding rate (ABR preTGT) in HA patients. Grades of blue represent inverse correlation and grades of red represent direct correlation. The level of significance is indicated with asterisks (Spearman correlation, bilateral significance). (C) Schematic representation of FVIII mutations and variants across the FVIII precursor sequence, as identified in the entire HA cohort, separated by severity. The new variants are indicated in red. No mutations were identified in 10 mild HA patients. The number of patients bearing a given mutation is represented (scale n = 1–5). In, intron; ex, exon.

Figure 3

Box plots depicting TGT parameters and FVIII activity at the time of TGT. FVIII activity measured at the time of TGT and at the time of diagnosis (matched assay) in brown (upper left graph). In the other graphs, Endogenous Thrombin Potential (ETP); Peak; Time to Peak; Lag Time and Start Tail are depicted.

Figure 4

Correlation of TGT and clinical parameters. Correlation analysis of FVIII activity levels and TGT parameters in control subjects (left) and correlation analysis of FVIII activity, annual bleeding rates (ABR, preTGT, and postTGT), severity, and TGT parameters in HA patients (right). Grades of blue represent inverse correlation and grades of red represent direct correlation. The level of significance is indicated with asterisks (Spearman correlation, bilateral significance). Black squares indicate a change of significance (significant–not significant) as observed in control correlations, and the yellow squares indicate a change in the sign of the correlation (inverse–direct).