Hemostasis is a physiological process that keeps the blood in a liquid state and stops bleeding after vascular injury. It consists of primary hemostasis, involving platelet activation and plug formation, and secondary hemostasis, based on the activation of the coagulation cascade, divided into the intrinsic and extrinsic pathway. In the latter, coagulation factor VIII (FVIII) plays a key role; it circulates in plasma bound to von Willebrand factor (vWF). Deficiency of either protein leads to bleeding disorders treated with plasma-derived FVIII and vWF concentrates obtained by isolation from plasma via precipitation and chromatography. During chromatographic purification, monitoring protein concentration and activity is crucial for ensuring the appropriate therapeutic effect of the concentrates. Therefore we determined FVIII using chromogenic and coagulation methods, and vWF using an immunoturbidimetric and method ELISA. We compared methods using standards of both proteins in terms of analytical characteristics such as repeatability and accuracy. The results showed that both methods for determining FVIII achieved adequate intra- and inter-day repeatability, with the coagulation method showing better repeatability and accuracy at higher FVIII levels, that are typically found in real process samples, which is likely due to greater automation. Post purification we obtained fractions containing FVIII in process media of varying compositions, mostly in terms of salt concentration. Since the composition of these media can affect measurements, we also compared the methods in terms of the matrix effect on the determination of both proteins. The results showed that the composition of the process media affects the analytical response. In most methods, we determined lower activities or concentrations of analytes in samples prepared by diluting standards in the process medium with the highest salt concentration, with the exception of the method ELISA, which showed the opposite trend. We also evaluated the possibility of reducing the influence of process media by additionaly diluting and desalting the samples. Additional dilution did not improve the analytical response of the methods, which is likely due to errors in preparing higher dilutions and reduced sensitivity of the methods at low analyte concentrations. Desalting, which we intended to use to remove low-molecular-weight components, was also inadequate, as the loss of analytes during the process caused major deviations in the measured values.
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