Potency estimation of recombinant factor VIII: effect of assay method and standard.

Large potency discrepancies between the chromogenic and one-stage clotting methods have been reported for patients' plasma samples following the infusion of recombinant factor VIII (rFVIII) concentrates. We have investigated the potency estimation of two different full-length rFVIII concentrates using both assay methods relative to both plasma and concentrate standards. Potencies by the chromogenic method were significantly higher (53% and 45%) than potencies by the one-stage clotting method when a plasma standard was used. In contrast, there was no significant potency difference between methods when a concentrate standard was used. Time-course studies into thrombin and activated factor X (FXa) generation, in modified clotting and chromogenic methods, respectively, revealed that the two rFVIII concentrates behaved very similarly to the concentrate standard, whereas the plasma standard showed slightly more rapid thrombin generation and markedly slower FXa generation. The different behaviour of rFVIII and plasma FVIII in the chromogenic method is proposed as the main cause of the methods-based potency discrepancy. The results support the use of a concentrate standard to measure rFVIII in post-infusion plasma.

An international collaborative study on the INR calibration of freeze-dried reference plasmas.

A study was carried out to calibrate potential European Reference Plasmas for prothrombin time (PT) standardization. The International Normalized Ratio (INR) values of three freeze-dried candidate plasmas (one pooled normal and two pools from anticoagulated patients) were determined in 20 laboratories using six thromboplastin reagents comprising three International Reference Thromboplastins (human, rabbit and bovine), two recombinant human reagents and one placental human reagent. Interlaboratory variability of INR estimation was low with geometric coefficients of variation (gcv) <10% except in one case. Significant differences in mean INR were found between the different thromboplastins with lowest INR values found with the bovine reagent. INR values from the International rabbit and human reagents differed by <6% and were combined to give proposed assigned INR values. Significant differences in INR estimates from four thromboplastins of human origin may indicate that single assigned INR values are not applicable for use with all thromboplastin reagents. Field trials to assess the validity of single assigned INR values in clinical practice are required.

Measurement of tissue factor pathway inhibitor in normal and post-heparin plasma.

Assay methods for the detection of both tissue factor pathway inhibitor (TFPI) function (two-stage chromogenic assay) and for TFPI antigen levels (competitive ELISA) have been developed and applied to the measurement of TFPI in normal plasma, in post-heparin plasma and to recombinant TFPI. There was good correlation in TFPI levels, measured using the two methods (r = 0.848; P < 0.001) in the normal plasma samples (n = 21) with the values ranging from 0.6 to 1.4 units per ml relative to a normal reference plasma pool (assigned 1.0 unit per ml). The post-heparin plasma samples were associated with increased levels of both TFPI functional activity and antigen. However, there was poor correlation between the two methods, with an increase in antigen levels greatly exceeding the increase in functional activity. This discrepancy was also found with recombinant TFPI and may reflect the different responses of the two assay methods to lipoprotein-bound TFPI (in the normal plasma reference) and the 'free' TFPI in post-heparin plasma and recombinant TFPI. These findings have implications in the choice of suitable reference materials for the assay of TFPI.