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

Hubbard, A. R.; Bevan, Sally A.; Weller, Lynne J.

doi:10.1046/j.1365-2141.2001.02761.x

Cited by 31 publications

(26 citation statements)

References 10 publications

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“…Differences can be observed of up to 40% for full‐length recombinant products . This can be abolished by use of a concentrate standard for assay calibration . In earlier studies, results of chromogenic assays have been up to 50% higher than those obtained by one‐stage assay for some B‐domain deleted (BDD) FVIII products with standard half‐lives .…”

Section: Introductionmentioning

confidence: 99%

Clotting and chromogenic factor VIII assay variability in post‐infusion and spiked samples containing full‐length recombinant FVIII or recombinant factor VIII Fc fusion protein (rFVIIIFc)

Kitchen¹,

Jennings²,

Makris

et al. 2018

Int J Lab Hematology

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Introduction Variability in FVIII measurement is a recognized problem. There are limited data for samples containing recombinant Factor VIII Fc fusion protein (rFVIIIFc). Many studies use samples for which factor concentrate has been spiked into FVIII deficient plasma in vitro. This approach requires validation. Aim/Methods Four samples were distributed in a UK National External Quality Assessment Scheme for Blood Coagulation (NEQAS BC) survey. One contained Advate (full‐length recombinant FVIII) (rFVIII) added to FVIII deficient plasma, one was from a severe haemophilia A patient after infusion of Advate, one was prepared by addition of rFVIIIFc (marketed as Elocta/Eloctate) to FVIII deficient plasma and the fourth was collected from a severe haemophilia A patient following rFVIIIFc (Eloctate) infusion. Fifty‐three haemophilia centres (UK and Scandinavia) performed one‐stage FVIII assays and 27 performed chromogenic FVIII assays. Results/Conclusions One‐stage assays gave significantly lower results than chromogenic assays by 7% (P < 0.01) and 13%(P < 0.001) for post‐Advate and Advate spiked samples, and by 22% (P < 0.001) and 23% (P < 0.001) for post‐rFVIIIFc and rFVIIIFc spiked samples. The interlaboratory variation was similar for all samples, with CVs of 12%‐16% (chromogenic) and 10%‐13% (one stage). The data indicate that either product can be safely monitored by one‐stage or chromogenic assay. Spiked samples behaved in a similar way to post‐infusion samples for both products and could be substituted for post‐infusion samples for use in proficiency testing exercises (ie, samples were commutable).

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Section: Introductionmentioning

confidence: 99%

Clotting and chromogenic factor VIII assay variability in post‐infusion and spiked samples containing full‐length recombinant FVIII or recombinant factor VIII Fc fusion protein (rFVIIIFc)

Kitchen¹,

Jennings²,

Makris

et al. 2018

Int J Lab Hematology

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“…These discrepancies are dependent upon the concentration of FVIII in test plasmas [6], standards used [7, 8] and incubation times in the chromogenic assay [3, 9]. The nature, purity and structure of natural and recombinant FVIII proteins are also confounders for these two assays as well [8, 10, 11].…”

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The “normal” factor VIII concentration in plasma

Butenas

Parhami-Seren

Undas

et al. 2010

Thrombosis Research

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Introduction The quantitation of factor (F)VIII by activity-based assays is influenced by the method, procedure, the quality and properties of reagents used and concentrations of other plasma proteins, including von Willebrand factor (VWF). Objective To compare FVIII concentrations measured by activity-based assays with those obtained by an immunoassay and to establish the influence of plasma dilution on the FVIII clotting activity (FVIIIc). Methods The APTT, a chromogenic assay (Coatest) and two in-house immunoassays were used. Albumin-free recombinant FVIII was used as the calibrator in all assays. Results For a group of 44 healthy individuals (HI), the mean value observed for FVIII antigen (FVIIIag; 1.22±0.56 nM; S.D.) is substantially higher than that for FVIIIc (0.65±0.29 nM) and the chromogenic assay (FVIIIch; 0.50±0.23 nM). A positive correlation between FVIIIag and VWFag with R2=0.20 was observed. Since plasma VWF has an inhibitory effect on FVIIIc, we evaluated the influence of plasma dilutions on FVIIIc in HI (n=105). At a 4-fold dilution, estimates of FVIIIc by clotting assay were much lower than FVIIIag (0.77±0.31 vs. 1.14±0.48 nM). At 10- and 25-fold dilutions, the estimated FVIIIc increased to 0.87±0.36 and 0.94±0.44 nM, respectively. Conclusions 1) In plasma, FVIIIag is higher than FVIIIc and FVIIIch; and 2) Real FVIII concentrations in plasma can be estimated by measuring FVIIIag.

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“…For example, Hubbard et al . recently found the 20th British Plasma Standard produced less absorbance than the fifth IS for FVIII Concentrate in the chromogenic assay at an equivalent assigned potency [9]. The consequence of this behavior is that the potencies of recombinant FVIII test substances were significantly higher in the chromogenic assay using the Plasma Standard compared with the Concentrate Standard.…”

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confidence: 99%