Influence of phospholipids on the assessment of factor VIII activity

Abstract

In view of reported discrepancies between different factor VIII assays, the influence of phospholipids on the performance of one-stage clotting (OS) and chromogenic substrate (CS) assays was evaluated. The B domain deleted recombinant factor VIII, rVIII SQ, two full-length recombinant products and a plasma derived factor VIII concentrate were each diluted into severe haemophilia A plasma and assayed against a plasma standard. The one-stage activity was 50, 80, 75 and 106%, respectively, of the chromogenic result. Variations in the phospholipid concentration did not affect the chromogenic assay, except at very low levels where the apparent activity increased. In contrast, dilution of the phospholipid reagent had a substantial influence on the activity measured by OS assays, especially in the case of rVIII SQ. At low levels of phospholipid, the one-stage activity of rVIII SQ exceeded the chromogenic result. When mixtures of phosphatidylserine (PS) and phosphatidyl-choline (PC) were used as a source of phospholipid, the OS results for rVIII SQ agreed well with the CS activity as long as the content of PS was below 10%, i.e., closer to the physiological level. At higher levels of PS, as in most commercial APTT reagents, the OS activity decreased. When the APTT reagent was replaced by platelets in the OS assay, the results compared well with those obtained by the CS assay for both t-VIII SQ and full-length factor VIII products.

Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories

“…Substantial variability has been observed with the assay of high-purity factor products 2. For full-length products, rFVIII activity has been reported to be 20% lower with the one-stage assay compared with the chromogenic substrate assay; however, this difference can increase to up to 50% for the BDD-rFVIII ReFacto® (Pfizer Inc, New York, NY, USA) 6,7. The increased discrepancy with ReFacto® may be related to the phospholipid composition of the reagents in the one-stage assay 7.…”

“…For full-length products, rFVIII activity has been reported to be 20% lower with the one-stage assay compared with the chromogenic substrate assay; however, this difference can increase to up to 50% for the BDD-rFVIII ReFacto® (Pfizer Inc, New York, NY, USA) 6,7. The increased discrepancy with ReFacto® may be related to the phospholipid composition of the reagents in the one-stage assay 7. The development of a product-specific reference standard was introduced to enable more accurate assessment of ReFacto® activity 4,8–10.…”

Determination of APTT factor sensitivity – the misguiding guideline

“…Consequently, the major role of the APTT at many centres is now detection of clotting factor deficiencies either in the form of a preoperative clotting screen or in patients who present with a history of a haemorrhagic diathesis. However, APTT reagents show different sensitivities to deficiencies of factors VIII, IX, XI and XII; this is thought to be because of differences in the activator or phospholipids used in the reagent 1,2.…”

Considerations on activity assay discrepancies in factor VIII and factor IX products

“…Historically, reports of discrepant CS and OC assay results have been known since purified FVIII concentrates became available, 27–30 although assay issues may have resulted in few dosing or efficacy concerns. One notable incident was reported for a B‐domain‐deleted (BDD) recombinant FVIII product, which rendered about a 50% higher potency value by a CS assay than the OC assay 31–34 . The clinical trials that provided the data to support this product's safety and efficacy were performed with subjects being dosed and monitored based on CS‐derived FVIII values.…”

“…One notable incident was reported for a B-domaindeleted (BDD) recombinant FVIII product, which rendered about a 50% higher potency value by a CS assay than the OC assay. [31][32][33][34] The clinical trials that provided the data to support this product's safety and efficacy were performed with subjects being dosed and monitored based on CS-derived FVIII values. The product was licensed using the CS assay for potency assignment.…”