Creating calibration curves via dilution in factor VIII (FVIII)-deficient plasma is critical for accurately measuring low levels of FVIII in patients with hemophilia, according to a recent article in Haemophilia. Additionally, analysis of serial dilutions of plasma samples requires predilution in FVIII-deficient plasma to align with requirements for parallelism, and these methods should be generally used for coagulation instruments.

FVIII is a beta globulin involved in clotting. Deficiency in FVIII causes one of the primary types of hemophilia. Monitoring replacement therapy for FVIII requires accurate measurement of FVIII over a wide range of concentrations, but unexplained overestimation of reduced amounts of FVIII can occur with extended half-life recombinant FVIIIs.

In this study, researchers created calibration curves for FVIII with FVIII-deficient plasma or with commercial diluent buffer. They then measured levels of FVIII in FVIII-deficient plasma treated with plasma FVIII, full-length recombinant FVIII (Advate®; Shire, Brussels, Belgium), or 1 of 2 extended half-life recombinant FVIIIs (Afstyla®, CSL Behring, Mechelen, Belgium; and Elocta® , Swedish Orphan Biovitrum, Woluwe Saint-Lambert, Belgium). Additionally, researchers assessed levels of FVIII in samples with added plasma FVIII that were prediluted 2- and 4-fold with either diluent buffer or FVIII-deficient plasma to analyze parallelism.

Coagulation times from calibration curves created with diluent buffer were higher than those created with FVIII-deficient plasmas. As a result, overestimation of FVIII levels at levels less than 25 IU/dL in samples with added FVIII and of FVIII activity detection (≥ 1 IU/dL) occurred.

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Of note, predilution of samples with diluent buffer instead of FVIII-deficient plasma yielded conflicting results.

The researchers noted that though dilution with the manufacturer-recommended buffer resulted in this overestimation, they did not test other diluent buffers and therefore cannot draw conclusions about how other buffers might affect estimation of FVIII levels.


1. Van den Bossche D, Toelen J, Schoeters J, et al. Accurate measurement of extended half-life and unmodified factor VIII low levels with one-stage FVIII assays is dependent on the matrix of calibration curves [published online December 27, 2018]. Haemophilia. doi: 10.1111/hae.13656