Effect of Direct Oral Anticoagulants on Fibrinogen Pseudoparaproteinemia

Background:

High-resolution serum protein electrophoresis (SPEP) is the initial test for detecting monoclonal paraproteins (M-protein), however the specificity is as low as 43% (Katzmann et al, 2010). Residual fibrinogen due to incomplete clotting of sample during preparation can create a band at the β/γ SPEP junction (Qiu et al, 2003). Excess fibrinogen occurs in patients on systemic anticoagulation (warfarin, heparin) or when there is heparin contamination of the sample (Qiu et al, 2003, Hryszko et al, 2009). Fibrinogen pseudoparaproteinemia has been reported in up to 3% of SPEPs in the pre-direct oral anticoagulant (DOAC) use era (Strobel, 2004). The optimal technique for removing fibrinogen in cases of a suspected pseudoparaprotein is unclear. Methods include pre-treatment with thrombin or reptilase to complete specimen clotting, or fibrinogen precipitation with ethanol (Qui et al, 2003; Ibrahim et al, 2005). Moreover, ethanol sample-treatment itself can produce an artificial band at the α2/β junction (Zhu et al, 2006). The misinterpretation of this fibrinogen band as a potential M-protein leads to costly, resource intensive downstream testing as well as unnecessary hematology referrals. To date, there are no data on direct oral anticoagulant (DOAC) induced fibrinogen pseudoparaprotein detection. This is of particular interest given the widespread and increasing use of DOACs.

Objective:

The primary objective was to evaluate the effect of DOACs on fibrinogen induced pseudoparaproteinemia in healthy adult serum anticoagulated ex-vivo. The secondary objective was to determine the optimal technique for fibrinogen removal in this context.

Methods:

Blood samples collected in biochemistry tubes (with and without pro-clotting agent) from a single human subject with a previously normal SPEP were pre-treated with commercially available calibrators for apixaban, rivaroxaban and dabigatran. SPEP was used to assess for a band near the β/γ junction. To determine the optimal method for fibrinogen removal, blood from a single healthy subject was collected in heparin tubes and treated with either reptilase, ethanol, or thrombin according to previously published protocols (Qui et al, 2003; Ibrahim et al, 2004). DOAC-spiked samples were then treated with reptilase and retested by SPEP.

Results:

Plasma collected in heparin tubes showed a discrete fibrinogen band measuring 3.29 g/L on SPEP. Treatment with thrombin or ethanol failed to eradicate the fibrinogen band (2.52 g/L and 1.89 g/L, respectively), while treatment with reptilase abolished the fibrinogen band.

Among DOAC-spiked serum samples analyzed by SPEP, no quantifiable fibrinogen band was identified with any of the four apixaban calibrators pre- or post-reptilase, although faint bands were suspected with calibrators 3 and 4 pre-reptilase. Treatment with each of the three rivaroxaban calibrators resulted in discrete fibrinogen bands (1.96, 3.57 and 3.22 g/L, respectively) that disappeared with reptilase treatment. Treatment with each of the three dabigatran calibrators resulted in discrete fibrinogen bands (3.08, 3.57 and 4.27 g/L, respectively) that disappeared with reptilase (Figure 1). Findings were consistent among samples collected in tubes both with and without pro-clotting agent.

Conclusion:

Our data suggest that DOACs induce fibrinogen pseudoparaproteinemia that is effectively circumvented with reptilase. To our knowledge, this is the first time that the effect of DOACs on SPEP has been explored. Future directions include determination of ex vivo DOAC calibrator concentrations that best represent in vivo expected "therapeutic" levels and expansion to a larger number of human subjects.

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