Recombinant factor IX: discrepancies between one‐stage clotting and chromogenic assays

New and modified recombinant factor IX (rFIX) products are in development and accurate potency estimation is important to ensure the consistency of production and efficacy of these therapeutics. Collaborative study data obtained during the replacement of the 3rd International Standard (IS) for FIX concentrate suggested that there was a discrepancy between potency estimates for rFIX using clotting and chromogenic methods, when the rFIX candidate was measured against the plasma‐derived FIX (pdFIX) IS. This study explores potential chromogenic and one‐stage clotting method discrepancies in more detail. Five batches each of rFIX and pdFIX were assayed against the 4th IS FIX concentrate (a pdFIX) by activated partial thromboplastin time (APTT) one‐stage clotting assay and specific functional chromogenic assay. The potency of rFIX by chromogenic assay was consistently around 70% of the one‐stage clotting potency (average 78 and 108 IU mL^?1 respectively). These differences were not observed with pdFIX, which had similar potencies (average 96 IU mL^?1) by each assay method. In addition, different APTT reagents yielded different potency estimates for rFIX when assayed against the pdFIX IS, with a variation of up to 23%. In all cases, the differences were largely resolved when a rFIX reference was used as the standard. This study highlights some of the challenges associated with assay of rFIX products in the laboratory and that careful consideration needs to be given to the choice of reference material used. This is especially important with the imminent arrival of new and modified rFIX products.     

Practical and cost‐effective measurement of B‐domain deleted and full‐length recombinant FVIII in the routine haemostasis laboratory

The assessment of recombinant FVIII (rFVIII) activity (FVIII:C) in plasma of patients is dependent on the assay. Notably, a calibration with a product‐specific laboratory standard is recommended when measuring Refacto‐AF^R activity in plasma with a one‐stage assay. The objective of this study was to facilitate the measurement of rFVIII, taking into account the recent demonstration that a calibration curve does not have to be included in each run. FVIII:C was measured in patients' samples after infusion of different types of rFVIII with a one‐stage and a chromogenic assay calibrated either with pooled normal plasma or a product‐specific laboratory standard. Results obtained with the one‐stage coagulation assay were compared with these provided by a chromogenic assay. We confirmed that a calibration curve can be used for a prolonged period of time without loss of precision and accuracy. In such conditions, a stable relation between the calibration curves generated with a product‐specific laboratory standard and plasma can be established. In patients' plasma, Refacto‐AF levels measured with a one‐stage FVIII assay calibrated with plasma or a product‐specific laboratory standard diverged from ?58% to ?17% and from ?25% to +18%, respectively, from the activity determined with a chromogenic substrate assay. By comparison, FVIII:C levels of full‐length rFVIII measured with the one‐stage assay calibrated with plasma were 6–49% lower than with the chromogenic assay. In a monocentric setting, the long‐term stability of the calibration curves allows the implementation of a practical and cost‐effective approach to determine rFVIII:C levels.     

A blinded in vitro study with Refacto mock plasma samples: similar FVIII results between the chromogenic assay and a one-stage assay when using a higher cephalin dilution.

Assay of factor VIII (FVIII) in patient samples is routinely carried out using the one-stage assay rather than the chromogenic substrate assay. The introduction of new FVIII preparations for the treatment of haemophilia A, including immunopurified FVIII and particularly, recombinant FVIII (rFVIII) concentrates, has led to discrepancies between the results obtained with the two assays. In patients treated with rFVIII concentrates, FVIII levels measured with the one-stage assay can be 20-50% lower than those measured with the chromogenic assay. In this study, the one-stage assay was performed with cephalin dilutions higher than those recommended by the manufacturer. B-domain-deleted recombinant FVIII, Refacto, was diluted to eight different concentrations, ranging from 1-100 IU dL(-1), in FVIII-deficient plasma and the FVIII activity of the eight solutions was determined by the chromogenic method in a central laboratory. Aliquots were then assayed by the one-stage method in the four participating laboratories, using different dilutions of CK-Prest. When CK-Prest was reconstituted according to the manufacturer's recommendations (dilution 1 : 1), the difference between the one-stage and chromogenic methods was close to 30%. CK-Prest cephalin dilutions of 1 : 5 and 1 : 8 gave very similar results with the two methods, without increasing the interlaboratory coefficient of variation. These findings confirm the influence of phospholipids on the one-stage assay, particularly the importance of using a phospholipid concentration close to the physiological value in platelets. This modified one-stage method may therefore offer an alternative to the use of a concentrate-specific standard.     

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

Discrepancies exist for some of the modified coagulation factors when assayed with different one‐stage clotting and chromogenic substrate assay reagents. The aim of this study was to evaluate the performance of a recombinant factor VIII Fc fusion protein (rFVIIIFc), currently in clinical development for the treatment of severe haemophilia A, in a variety of one‐stage clotting and chromogenic substrate assays in clinical haemostasis laboratories. Haemophilic plasma samples spiked with rFVIIIFc or Advate^® at 0.05, 0.20 or 0.80 IU mL^?1 were tested by 30 laboratories using their routine procedures and plasma standards. Data were evaluated for intra‐ and inter‐laboratory variation, accuracy and possible rFVIIIFc‐specific assay discrepancies. For the one‐stage assay, mean recovery was 95% to 100% of expected for both Advate^® and rFVIIIFc at 0.8 IU mL^?1. Intra‐laboratory percent coefficient of variance (CV) ranged from 6.3% to 7.8% for Advate^®, and 6.0% to 10.3% for rFVIIIFc. Inter‐laboratory CV ranged from 10% for Advate^® and 16% for rFVIIIFc at 0.8 IU mL^?1, to over 30% at 0.05 IU mL^?1 for both products. For the chromogenic substrate assay, the average FVIII recovery was 107% ± 5% and 124% ± 8% of label potency across the three concentrations of Advate^® and rFVIIIFc, respectively. Plasma rFVIIIFc levels can be monitored by either the one‐stage or the chromogenic substrate assay routinely performed in clinical laboratories without the need for a product‐specific rFVIIIFc laboratory standard. Accuracy by the one‐stage assay was comparable to that of Advate^®, while marginally higher results may be observed for rFVIIIFc when using the chromogenic assay.     

International comparative field study of N8 evaluating factor VIII assay performance

Summary. Discrepancies between the one‐stage clotting assay and the chromogenic method, and also among different variations of each method, have been a significant challenge for one B‐domain deleted FVIII product. N8 is a B‐domain truncated FVIII product developed by Novo Nordisk. The comparison of N8 and Advate^® was performed in an international, multicentre, randomized and blinded field study of simulated postinfusion samples. Overall, Advate^® and N8 performed similarly in the one‐stage assay. In the one‐stage clotting assay, the measured mean FVIII levels of Advate^® vs. N8 were 0.046/0.047, 0.24/0.24, 0.58/0.60 and 0.82/0.83 IU mL^?1 for the target values of 0.03, 0.2, 0.6 and 0.9 IU mL^?1, respectively. In the chromogenic assays, the concentration estimates showed a tendency towards higher N8 values as compared with Advate^®; the measured FVIII levels of Advate^® vs. N8 were 0.030/0.032, 0.22/0.24, 0.65/0.74 and 0.98/1.08 IU mL^?1 for the target values of 0.03, 0.2, 0.6 and 0.9 IU mL^?1, respectively. In the one‐stage assays, the measured values were above 150% of target at the lowest concentration, decreasing to around 90% of target at the highest concentration. In contrast, the chromogenic assays were close to target at the lowest concentration and consistently above target at the three highest concentrations. Therefore, the ratio of chromogenic/one‐stage potencies was concentration dependent, ranging from 0.66 to 1.30. The SSC plasma standard was similar in both. Assay variability was similar for both compounds. The results show that N8 can be reliably measured in plasma without the need for a separate N8 standard.     

Measurement of factor VIII activity using one‐stage clotting assay: a calibration curve has not to be systematically included in each run

Summary. Coagulation factor VIII (FVIII) is usually evaluated using activated partial thromboplastin time‐based one‐stage clotting assays. Guidelines for clotting factor assays indicate that a calibration curve should be included each time the assay is performed. Therefore, FVIII measurement is expensive, reagent‐ and time‐consuming. The aim of this study was to compare FVIII activities obtained using the same fully automated assay that was calibrated once (stored calibration curve) or each time the assay was performed. Unique lots of reagents were used throughout the study. We analysed 255 frozen plasma samples from patients who were prescribed FVIII measurement including treated and untreated haemophilia A patients. Twenty‐six runs were performed on a 28‐week period, each including four lyophilized control and at most 10 patient plasma samples. In control samples, FVIII activities were not significantly different when the assay was performed using the stored calibration curve or was daily calibrated. The same applied to FVIII activities in patient plasma samples that were not significantly different throughout the measuring range of activities [68.3% (<1–179) vs. 67.6% (<1–177), P = 0.48] and no relevant bias could be demonstrated when data were compared according to Bland and Altman. These results suggest that in the studied technical conditions, performing the FVIII assay using a stored calibration curve is reliable, for at least 6 months. Therefore, as far as the same lots of reagents are used, it is not mandatory to include a calibration curve each time the FVIII assay was performed. However, this strategy has to be validated if the assay is performed in different technical conditions.     

Factor VIII activity of BAY 94‐9027 is accurately measured with most commonly used assays: Results from an international laboratory study

Introduction

Discrepancies in the measurement of modified factor VIII (FVIII) products have been recognized, highlighting the need for adjustments in clinical laboratory practices to ensure effective monitoring of patients treated with these products, particularly using the one‐stage (activated partial thromboplastin time [aPTT]) assay.

Aim

To assess the ability of clinical laboratories to measure the activity of BAY 94‐9027, a PEGylated extended half‐life FVIII product, using routine (predominantly one‐stage) assays in clinical laboratories

Methods

Blinded samples of FVIII‐deficient plasma spiked with defined levels of BAY 94‐9027 and a recombinant FVIII product comparator were provided to 52 clinical laboratories that routinely conduct FVIII testing. Samples were provided at 3 concentrations (low, medium and high), and laboratories analysed the samples using routine in‐house one‐stage and, when available, chromogenic assays. Acceptable spiked recovery (accuracy) of the local laboratory methods to measure BAY 94‐9027 was the primary endpoint of the study.

Results

Accurate FVIII measurements were obtained at all concentrations for both products using the chromogenic assay and most of the commonly used one‐stage reagents, both ellagic acid and silica based. Two specific silica‐based reagents, APTT‐SP and PTT‐A, underestimated BAY 94‐9027 levels at all concentrations, consistent with previous findings.

Conclusions

FVIII activity of BAY 94‐9027 was accurately measured with most commonly used one‐stage assays used in routine clinical practice. The chromogenic assay was also accurate. It is recommended that clinical laboratories identify and avoid specific inappropriate reagents, such as the APTT‐SP and PTT‐A, in their one‐stage assays for FVIII monitoring.