Summary of the WHO hearing on the development of product-specific reference materials for coagulation factor VIII and factor IX products.

In recent years, several modified recombinant factor (F) VIII and FIX therapeutics with extended half-life have been licensed internationally for the treatment of haemophilia. Safe and effective use of these products requires monitoring of factor activity in patient plasma. The potency of all FVIII and FIX products is currently assigned in International Units (IU) which anchors the relationship between potency labelling, dosing and clinical monitoring. However, varying degrees of discrepancies in factor activity assays are observed between and within the factor activity analytical methods (one-stage clotting and chromogenic), when measuring these modified products against plasma and plasma-derived (concentrate) International Standards (IS) or in-house reference standard traceable to the IS. Availability of product-specific reference reagents would mitigate assay discrepancies, facilitate independent testing of assay methods and reagents, and ensure long-term continuity of the IU related to each product. A hearing meeting was organised by the WHO to discuss the requirements for product-specific reference materials for these products and whether these reference materials should be produced by the WHO. Advantages and disadvantages of product-specific reference materials were identified and discussed.

Activity measurements of dalcinonacog alfa.

INTRODUCTION: Many recombinant and modified FIX products have been, and continue to be, developed with the aim of improving treatment for patients with haemophilia B. One such new product is dalcinonacog alfa, a recombinant FIX with modifications to provide improved features such as subcutaneous administration. AIM: In view of previously observed assay discrepancies with modified FIX therapeutics, the aim of this study was to assess potential discrepancies in potency measurement of dalcinonacog alfa between and within different assay methods. METHODS: Potency of dalcinonacog alfa was measured against the 5th International Standard (IS) for FIX Concentrate and the 4th IS for FIX Plasma by One-Stage Clotting Assay, using 9 different APTT reagents and 2 commercially available FIX chromogenic kits. Plasma-derived concentrate and recombinant FIX samples were also included for comparison in every assay. RESULTS: Substantial discrepancies were observed when assaying dalcinonacog alfa using the one-stage clotting assay against both standards. No statistically valid results were obtained when testing dalcinonacog alfa using either chromogenic kit. Increasing the incubation time with the activation reagent in both chromogenic kits resulted in valid assays and increased the potency to become more in line with potencies by one-stage clotting assays. Increasing the incubation time in the chromogenic kits had no effect on the potencies of the plasma-derived or recombinant samples. However, incubation time influenced in the one-stage clotting assay using Dapttin. CONCLUSIONS: Within and between assay method discrepancy was found when assaying dalcinonacog alfa. Methods for potency labelling and clinical monitoring should be given careful consideration.

The interplay between tissue plasminogen activator domains and fibrin structures in the regulation of fibrinolysis: kinetic and microscopic studies.

Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domain-agglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time.