Report of the Factor XIII Standard Working Party session

49 th Meeting of the German Society on Thrombosis and Haemostasis, Mannheim , Wednesday, February 23, 2005.
Chair persons: Dr A Ichinose ( Yamagata University , Japan ), Dr R Seitz ( Paul-Ehrlich-Institute , Germany )

Dr Laszlo Muszbek ( Debrecen University , Hungary ) discussed issues related to standardisation of FXIII antigen assays. The physiology of FXIII is complex. In plasma it circulates as an A2B2 tetramer, much of which is in complex with fibrinogen gamma prime splice variant, with 50% free B-subunit. In other body fluids FXIII is present as an A2 dimer and A2B2 tetramer. Cellular FXIII consists of an A2 dimer only. A decision is to be made with regards to which modality of FXIII antigen measurement needs standardisation: A-subunit, B-subunit, or A2B2 complex antigen. Other issues that require attention include the unity of measurement to be chosen for standardisation, g/L or percentage of pooled normal plasma. The point was also raised whether or not we need to establish inclusion or exclusion criteria for laboratories involved in the standardisation study. Methodologies for antigen measurements were discussed: RIA, competitive or sandwich ELISA. It was suggested that for a sandwich A-subunit ELISA, a capture monoclonal antibody followed by polyclonal developing antibody, both against FXIIIA, or vice versa, as opposed to a sandwich based on two polyclonal antibodies may be preferable to avoid epitope overlap. It was also suggested that for a sandwich A2B2 ELISA, a capture antibody against the B-subunit would be preferable, followed by a developing antibody against FXIIIA. Issues of specificity (cross-reactivity with fibrinogen) and sensitivity were discussed. Drs Kohler ( Bern University , Switzerland ) and Ariëns ( Leeds University , United Kingdom ) raised the point that for measurement of A-subunit in plasma, an a A2B2/ a A sandwich may be as effective as a a A/ a A sandwich, since all A-subunit in plasma is in complex with B.

Dr Sanj Raut (National Institute for Biological Standards and Control , United Kingdom ) presented data from the collaborative international study that has lead to the development of the 1 st International Standard for FXIII activity. Antigen measurement was performed in a limited number of laboratories with different methodologies. There were three major antigen protocol groups: a A2B2/ a A, a A(Monoclonal)/ a A(Polyclonal) and Laurell/RIA. There were no clear differences in variability if the data were re-analysed by method group. Two possibilities were proposed: 1) the current antigen estimate of 0.93 u/ml with 14.8% GCV from a limited number of laboratories is accepted, or 2) further studies are performed to compare the methods.

Dr Rainer Seitz ( Paul-Ehrlich-Institute , Germany ) presented data on measurement of FXIII in concentrates and fibrin sealants. It was observed that concentrates are slower to reach Vmax and that this delay was corrected by dilution in FXIII deficient plasma. A preliminary study on plasma (1 u/ml), FXIII concentrates of 30 and 50 u/ml and fibrin sealants (2-10 u/ml) with two commercially available FXIII activity assays showed that dilution in FXIII deficient plasma improved consistency for the FXIII concentrates but not fibrin sealants. It was concluded that FXIII concentrates behave differently from fibrin sealants, the latter of which contain a variable concentration of FXIII.

Dr Metzner (Behring, Marburg , Germany ) discussed production issues for FXIII concentrates and fibrin sealants. In-vivo halflife of FXIII from concentrate is 9.2 days for both activity and antigen. Pre-dilution of concentrate in FXIII depleted plasma rather than buffer increases consistency in its measurement, but it was noted that currently the commercial supplies for FXIII deficient plasma are limited. FXIII content in fibrin sealants is a complex issue as this is highly variable. Discrepancies between antigen and activity levels suggest that FXIII in fibrin sealants may be partly inactivated.

In a round-table discussion, Drs Ichinose (chair of FXIII SWP), Muszbek (FXIII SWP), Seitz (FXIII SWP), Barrowcliffe (FXIII SWP), Raut (FXIII SWP) and Ariëns (chair of FXIII SSC) planned a pilot study to compare FXIII A-subunit antigen measurement in plasma with that of A2B2 (provided by Dr Muszbek to the laboratories). Future agenda items were discussed.