Abstract
rFIX-Fc is an extended half-life (EHL) recombinant FIX concentrate approved for the prophylaxis and treatment of bleeding events in haemophilia B. A high laboratory and reagent-specific variability is described for rFIX-Fc activity measurements, overall in lower FIX concentrations.
The aim of this single-center study was to evaluate the intra-laboratory variability of 17 different combinations coagulometers/reagents including both one stage clotting (OSC) and chromogenic assays with a focus on lower concentrations.
Human FIX deficient plasma (FIX<1%, George King Bio-Medical) was spiked with rFIX-Fc at 150%, 100%, 80%, 20%, 5%, 2% and 1% based on label potency. We evaluated the spikes recovery with 3 coagulometers (STAR MAX (Stago), ACL TOP700 (Werfen) and Cs2100 (Siemens)) using for each instrument captive silica based aPTT reagents (PTT automate (Stago) and Pathromtin (Siemens)) and non-silica based (CK Prest (Stago), Synthafax (Werfen) and Actin FS (Siemens)). Two reagents (STA-Cephascreen (Stago) and Synthafax (Werfen)) were evaluated on all coagulometers because they potentially behave one the same way for EHL FIX products. The 2 available chromogenic assays (ROS FIXa (Rossix) and Biophen FIX:C were evaluated on all coagulometer. All experiments were performed by the same staff, in a single room, in triplicates with 3 dilutions. For a given combination, all the concentrations were evaluated on the same calibration curve. The spike recovery were calculated as the (mean of 3 experiments / targeted FIX value) x 100. The range values of 70% - 130% were considered as acceptable for spike recoveries'.
The summary of all spikes recoveries' are presented in table 1. We confirmed that kaolin based OSC assays underestimate rFIX-Fc including for low concentrations. Acceptable recoveries were found for PTT automate on Stago coagulometer from 150% to 5% and an underestimation was found for low concentrations (2% and 1 %). Pathromtin and Actin FS performed on Cs2100 display acceptable recoveries on all the range of target values. Synthasil performed on ACL TOP700 have acceptable recoveries from 150% to 20% and an overestimation was found for low concentrations (5% to 1%). STA-Cephascreen performed on the 3 coagulometers has acceptable recoveries, except for low concentrations (5% to 1%) on ACL TOP coagulometer, leading to an overestimation of rFIX-FC measurements. Synthafax performed on the 3 coagulometers has acceptable recoveries with ACL TOP700 coagulometer for all the range of concentrations. This reagent adapted on STAR MAX coagulometer shows an overall overestimation of rFIX-Fc measurements (except for the 150% target concentration) and when adapted on a Cs2100 coagulometer, it displays acceptable recoveries except for low concentrations (5% to 1%) that are overestimated. The two chromogenic assays have acceptable recoveries for rFIX-Fc on all coagulometers from 150% to 50% concentrations. ROS FIXa underestimated rFIX-Fc recovery for the lower concentrations (1% and 2%) on STAR MAX and ACL TOP700 coagulometers. Biophen FIX:C underestimated rFIX-Fc recoveries for concentrations below 50% with all coagulometers.
This study provides the pattern of several combinations coagulometers - reagents for rFIX-Fc measurements and confirms a high variability for several reagents for low concentrations.
Lebreton:Sobi: Consultancy, Research Funding.
