Development and validation of a novel qualitative test for plasma fibrinogen utilizing clot waveform analysis

Abstract

Plasma fibrinogen is commonly examined by Clauss fibrinogen assay, which cannot distinguish between quantitative and qualitative fibrinogen anomalies. However, our previously reported Clauss fibrinogen assay utilizing clot waveform analysis (Clauss-CWA) provides additional information that contributes to the classification of fibrinogen anomalies. In this study, we adopted the Clauss-CWA method for an autoanalyzer to automatically measure the antigenic estimate (eAg) of fibrinogen in addition to the functional amount (Ac), and to thus provide the Ac/eAg ratio as a qualitative indicator. Performance was validated by receiver operating characteristics (ROC) and precision recall (PR) curve analyses using a patient cohort, consisting of a training cohort (n = 519) and a validation cohort (n = 523), both of which contained cases of congenital (hypo)dysfibrinogenemia as qualitative defects. We obtained an optimal cutoff of 0.65 for Ac/eAg by ROC curve analysis of the training cohort, offering superior sensitivity (> 0.9661) and specificity (1.000). This cutoff was validated in the validation cohort, providing positive predictive value > 0.933 and negative predictive value > 0.998. PR curve analysis also showed that Clauss-CWA provided excellent performance for detecting qualitative fibrinogen anomalies. The Clauss-CWA method may represent a useful approach for detecting qualitative fibrinogen abnormalities in routine laboratory testing.

International council for standardisation in haematology recommendations on fibrinogen assays, thrombin clotting time and related tests in the investigation of bleeding disorders

“…particularly for differential diagnosis of FBG anomalies. 49,[54][55][56][57] A clot waveform in optical changes on a coagulometer indicates the generation of fibrin clot, thus CWA can be considered as an investigation of fibrin clot formation and fibrin(ogen) itself. The Min1 value of CWA in Clauss assay or TT strongly correlates with the antigenic FBG amount rather than the functional amount and can be a surrogate for FBG antigen value.…”

“…54,55 Recent studies demonstrated that the ratio of functional FBG to Min1-derived antigenic FBG estimates in the Clauss assay could be a novel index for differential diagnosis of FBG anomalies 56,57 and showed comparable performance for detecting qualitative FBG abnormalities to the standard method (the combination of Clauss and immunological assays). 49 This novel approach is available on certain analysers, but may not have regulatory approval for clinical use in all regions. where practicable, statistical techniques shall be applied to review the results.…”

“…Immunological assays determine the amount of antigenic rather than functional protein. Although immunoassay values generally show good agreement with the Clauss or PT-Fg values in normal subjects, 33,[49][50][51] depending on the reagents and assay types, degraded fibrinogens (if present) may be included, resulting in false estimation of FBG antigen levels. Immunological assays generally provide commutable FBG values when using an appropriate reference plasma.…”

Diagnosis and classification of hereditary fibrinogen disorders

“…A ratio fibrinogen activity / antigen with a cut-off of 0.7 has historically be proposed to distinguish between quantitative and qualitative fibrinogen disorders, but not fully validated 12 . Recently, a cut-off of 0.55 has also been tested 13 . If fibrinogen activity and antigen are disproportionally decreased, a hypodysfibrinogenemia should be considered.…”

“…In addition, recent data promotes novel approaches for diagnosis of HFDs. Clot wave form from Clauss could have a similar performance to detect dysfibrinogenemia 13 .…”