PO060 | DISCREPANCY IN FACTOR VIII LEVELS AMONG HEMOPHILIA A CARRIERS: ONE-STAGE VS. CHROMOGENIC ASSAY
Abstract
Background. Hemophilia A (HA) is an X-linked bleeding disorder caused by mutations in the F8 gene. While clinical focus is often on affected males, obligate and symptomatic carriers frequently exhibit reduced Factor VIII (FVIII) activity due to lyonization (X-chromosome inactivation). A significant diagnostic challenge has emerged regarding the "assay discrepancy," where FVIII:C values differ significantly between the One-Stage Clotting Assay (OSA) and the Chromogenic Substrate Assay (CSA). This study investigates the prevalence of this discrepancy in HA carriers and its implications for clinical risk stratification. Methods. A comparative analysis was performed on a cohort of 16 confirmed F8 mutation carriers. FVIII activity was measured using both OSA (based on the activated partial thromboplastin time - aPTT) and CSA (measuring the rate of Factor Xa generation). Discrepancy was defined as a ratio of CSA/OSA < 0.6 or > 1.6. Genetic sequencing was integrated to correlate specific missense mutations with the observed biochemical phenotypes. Results. The results demonstrate that 50% of HA carriers manifest a significant discrepancy between the two assays (Table 1). Analysis of FVIII activity levels revealed a unidirectional discrepancy, characterized exclusively by One-Stage Assay (OSA) values significantly exceeding Chromogenic Substrate Assay (CSA) results (OSA > CSA). No instances of the inverse relationship (CSA > OSA) were observed. This systematic overestimation by the OSA potentially obscures diminished biological clotting activity, posing a significant diagnostic risk. Patients harboring a mild bleeding phenotype may be inaccurately classified as having "hemostatically secure" FVIII levels by traditional clotting assays, despite manifesting a functional deficiency in chromogenic quantification. These findings indicate that OSA-only protocols are insufficient for detecting sub-optimal FVIII function in carriers of "discrepancy-prone" mutations. In such cohorts, clinical bleeding scores (ISTH-BAT) often correlate poorly with OSA-derived FVIII levels. Consequently, the CSA appears to more accurately reflect in vivo hemostatic capacity and the stability of the tenase complex. Conclusions. Relying exclusively on the One-Stage Assay limits the scope of a comprehensive assessment for Hemophilia A carriers, as this methodology may not fully capture the complexities of an individual’s hemostatic profile. The discrepancy between the OSA and the Chromogenic Substrate Assay (CSA) is a tangible biological phenomenon rooted in the molecular stability of Factor VIII and the kinetic integrity of the tenase complex. Consequently, a dual-testing approach is essential for ensuring clinical safety, particularly for symptomatic carriers or those undergoing surgical procedures. This integrated strategy provides a more reliable foundation for personalized prophylactic or on-demand treatment plans, ensuring that clinical decisions are based on the most accurate reflection of in vivo clotting activity.
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BibTeX
@article{strangio2026po060,
title = {{PO060 | DISCREPANCY IN FACTOR VIII LEVELS AMONG HEMOPHILIA A CARRIERS: ONE-STAGE VS. CHROMOGENIC ASSAY}},
author = {Alessandra Strangio},
journal = {Bleeding, Thrombosis and Vascular Biology},
year = {2026},
month = oct,
url = {https://www.btvb.org/btvb/article/view/876},
note = {CC BY-NC},
}
RIS
TY - JOUR
TI - PO060 | DISCREPANCY IN FACTOR VIII LEVELS AMONG HEMOPHILIA A CARRIERS: ONE-STAGE VS. CHROMOGENIC ASSAY
AU - Alessandra Strangio
PY - 2026
DA - 2026/10/26
JO - Bleeding, Thrombosis and Vascular Biology
UR - https://www.btvb.org/btvb/article/view/876
AB - Background. Hemophilia A (HA) is an X-linked bleeding disorder caused by mutations in the F8 gene. While clinical focus is often on affected males, obligate and symptomatic carriers frequently exhibit reduced Factor VIII (FVIII) activity due to lyonization (X-chromosome inactivation). A significant diagnostic challenge has emerged regarding the "assay discrepancy," where FVIII:C values differ significantly between the One-Stage Clotting Assay (OSA) and the Chromogenic Substrate Assay (CSA). This study investigates the prevalence of this discrepancy in HA carriers and its implications for clinical risk stratification. Methods. A comparative analysis was performed on a cohort of 16 confirmed F8 mutation carriers. FVIII activity was measured using both OSA (based on the activated partial thromboplastin time - aPTT) and CSA (measuring the rate of Factor Xa generation). Discrepancy was defined as a ratio of CSA/OSA < 0.6 or > 1.6. Genetic sequencing was integrated to correlate specific missense mutations with the observed biochemical phenotypes. Results. The results demonstrate that 50% of HA carriers manifest a significant discrepancy between the two assays (Table 1). Analysis of FVIII activity levels revealed a unidirectional discrepancy, characterized exclusively by One-Stage Assay (OSA) values significantly exceeding Chromogenic Substrate Assay (CSA) results (OSA > CSA). No instances of the inverse relationship (CSA > OSA) were observed. This systematic overestimation by the OSA potentially obscures diminished biological clotting activity, posing a significant diagnostic risk. Patients harboring a mild bleeding phenotype may be inaccurately classified as having "hemostatically secure" FVIII levels by traditional clotting assays, despite manifesting a functional deficiency in chromogenic quantification. These findings indicate that OSA-only protocols are insufficient for detecting sub-optimal FVIII function in carriers of "discrepancy-prone" mutations. In such cohorts, clinical bleeding scores (ISTH-BAT) often correlate poorly with OSA-derived FVIII levels. Consequently, the CSA appears to more accurately reflect in vivo hemostatic capacity and the stability of the tenase complex. Conclusions. Relying exclusively on the One-Stage Assay limits the scope of a comprehensive assessment for Hemophilia A carriers, as this methodology may not fully capture the complexities of an individual’s hemostatic profile. The discrepancy between the OSA and the Chromogenic Substrate Assay (CSA) is a tangible biological phenomenon rooted in the molecular stability of Factor VIII and the kinetic integrity of the tenase complex. Consequently, a dual-testing approach is essential for ensuring clinical safety, particularly for symptomatic carriers or those undergoing surgical procedures. This integrated strategy provides a more reliable foundation for personalized prophylactic or on-demand treatment plans, ensuring that clinical decisions are based on the most accurate reflection of in vivo clotting activity.
SN - 2785-5309
ER -