A Standardized Rotation Thromboelastometry (ROTEM) Method for Evaluation of FVIII and FIX Products in a Multi-National Clinical Trial

Abstract 1199

Measurement of FVIII or FIX activity in clinical samples by the one-stage clotting assay is the standard method for estimating the in vivo activity of replacement factor products in the treatment of hemophilia. Global hemostasis assays, such as thromboelastography (TEG) and rotation thromboelastometry (ROTEM) could provide additional information about the in vivo function of FVIII and FIX products since these whole blood assays are thought to more closely reflect in vivo coagulation. However, the TEG and ROTEM assays have not been routinely used in hemophilia care, mainly due to the lack of assay standardization and the absence of reference materials appropriate for hemophilic patient samples. We show here that standardization of the ROTEM assay is possible across multiple sites in a clinical trial. Reproducibility of this method would not only allow ex vivo confirmation of the potency of a novel product in comparison to current replacement factors, but may also reveal patient-specific clotting parameters related to their individual bleeding tendency.

We evaluated the effectiveness of a standardized ROTEM procedure in support of Biogen Idec's phase 3 trial of a long-lasting clotting factor IX Fc fusion protein (rFIXFc). Prior to evaluating clinical specimens by ROTEM, each site performed a set of quality control (QC) assays using the ROTROLN reagents provided by TEM Innovations GmbH and a set of frozen plasma controls that were prepared by Biogen Idec by spiking hemophilic plasma with 4 different levels of FIX drug product (1 IU/dL, 5 IU/dL, 15 IU/dL and 30 IU/dL). A detailed protocol and all reagents needed to perform the assay, including custom diluents, were provided to the sites by Biogen Idec in order to ensure a consistent method and reagent lot uniformity across all centers participating in this exploratory study. In addition, local operators at each site participated in hands-on training provided by Biogen Idec.

An interim analysis was performed on the QC data gathered from 6 clinical sites that performed a total of 44 QC runs for FIX on 8 ROTEM instruments (7 model Gamma and 1 model Delta). Among the 4 ROTEM parameters evaluated (clotting time, CT; clot formation rate, CFR; maximum clot firmness, MCF and alpha angle), CT was most reproducible, with relatively small variation among the 6 sites, ranging from 8% to 24% CV across the 4 concentration levels of FIX (Table 1).

A mixed effect model was fitted for the ROTEM global QC data (the response variable) with ‘centers’ and ‘plasma FIX levels’ as covariates. The center had no statistically significant effect on clotting time (p-value = 0.57) and alpha angle (p-value = 0.85), but a mild impact on MCF (p = 0.12) and CFR (p-value = 0.07). As expected, an increase in FIX concentration level was shown to significantly reduce the clotting times (p-value = 0.001). To further examine our standardization approach in controlling inter-site variability of ROTEM data, two parameters, operator and instrument, were also evaluated. Comparison of the results obtained at the 6 clinical sites to a replicate analysis at a single site (19 runs at each FIX level performed at Biogen Idec by a single operator on 6 model Delta instruments) indicated equivalent variability with an overall ANOVA p-value of 0.85 for CT (accounting for the correlation between ROTEM data and FIX level) and a similar comparability for CFR, MCF and alpha angle.

All instruments were maintained within the manufacturer's specification (per ROTROL N data), and there was no instrument-related clustering of results when samples were assayed on multiple instruments at a single site by one operator. We thus conclude that the inter-site variability (i.e. operator and instrument variability) is not a significant factor in our ROTEM study. Assuming the preanalytical variables for the blood collection are also minimized in our standardized procedures, we expect that major differences in a subject's ROTEM parameters will be a meaningful indicator or their hemostatic potential rather than assay variability between individual test sites.