A critical appraisal of one‐stage and chromogenic assays of factor VIII activity

Peyvandi, Flora; Oldenburg, Johannes; Friedman, Kenneth D.

doi:10.1111/jth.13215

Cited by 130 publications

(201 citation statements)

References 69 publications

Supporting

Mentioning

191

Contrasting

Unclassified

Order By: Relevance

“…sensitive and have greater precision particularly in the low concentration range [24,[31][32][33]. One explanation of chromogenic assays appearing to be less precise in this study may be because the chromogenic assays were not the laboratories' routine assay and the imprecision could be due to inexperience.…”

Section: Discussionmentioning

confidence: 95%

A world‐wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE

Turecek¹,

Romeder‐Finger²,

Apostol³

et al. 2016

Haemophilia

View full text Add to dashboard Cite

Introduction: Discrepancies have been previously reported for one-stage clotting and chromogenic assays for FVIII activity analysis. Inter-laboratory variations in instruments, method of clot detection, assay set-up, reference standard calibration, reagent source and reagent composition all contribute to assay variability. Aim: To characterise multilaboratory assay variability in measuring ADYNOVATE, OBIZUR and ADVATE FVIII activity in human plasma and survey multinational FVIII activity assay preferences. Methods: As samples from patients treated with either of the FVIII products are not available in the quantities required for a systematic collaborative study, haemophilia A plasma was spiked in vitro with either ADYNOVATE (PEGylated rFVIII), OBIZUR [Porcine Sequence Antihaemophilic Factor (Recombinant)] or ADVATE at high (0.80 IU or U mL ) and low (0.05 IU or U mL À1 ) FVIII concentrations, based on labelled potencies. Clinical laboratories used their routine FVIII activity assay to determine FVIII activity of each product. Thirty-five data sets using one-stage clotting assay and 11 sets using chromogenic assay were obtained. Results: A vast majority of laboratories (98%) prefer and rely on the one-stage clotting assay. Mean recoveries across all concentrations were 113%, 120% and 127% for ADYNOVATE, OBIZUR and ADVATE respectively. Assay variation was comparable between ADVATE, ADYNOVATE and OBIZUR with inter-laboratory percent coefficients of variation (%CV) ranging from 11 to 22%. Mean chromogenic assay results were 116%, 51% and 113% for ADYNOVATE, OBIZUR and ADVATE respectively. Inter-laboratory CV's were similar for ADYNOVATE, OBIZUR and ADVATE. Conclusions: One-stage clotting assays can and will be used with sufficient accuracy and precision for the measurement of ADYNOVATE, OBIZUR and ADVATE in plasma samples from subjects with haemophilia A. Chromogenic assay underestimates OBIZUR potency, particularly at lower concentrations.

show abstract

Section: Discussionmentioning

confidence: 95%

A world‐wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE

Turecek¹,

Romeder‐Finger²,

Apostol³

et al. 2016

Haemophilia

View full text Add to dashboard Cite

show abstract

“…The same phenomenon is observed for patients harboring certain missense mutations with a higher value in the clotting assay compared to the chromogenic assay [27]. The observed discrepancy can be explained based on principal differences of the assays: 1) differences in the amount of thrombin (physiological amounts vs. excess of thrombin in chromogenic assay making it less sensitive to rate of activation), 2) different dilution factor of the samples (120x) for the chromogenic assay, 40x for the one-stage clotting assay assuring the activation of all FVIII molecules present in the chromogenic assay), 3) variations and differences in incubation and detection times (longer incubation with thrombin prior to initiation of FXa generation guaranteeing fully activated FVIII vs. the short time between the generation of catalytic amounts of thrombin and a measurable clot in one stage assays [24, 25]. …”

Section: Discussionmentioning

confidence: 99%

An in silico and in vitro approach to elucidate the impact of residues flanking the cleavage scissile bonds of FVIII

et al. 2017

View full text Add to dashboard Cite

Coagulation Factor VIII is activated by an ordered limited thrombin proteolysis with different catalytic efficiency at three P1 Arginine residues: Arg , indicating the flanking residues of the latter to be less optimal. This study aimed to investigate, in silico and in vitro, the impact of possessing hypothetically optimized residues at these three catalytic cleavage sites. The structural impact of the residues flanking Arginine cleavage sites was studied by in silico analysis through comparing the cleavage cleft of the native site with a hypothetically optimized sequence at each site. Moreover, recombinant FVIII proteins were prepared by replacing the sequences flanking native thrombin cleavage sites with the proposed cleavage-optimized sequence. FVIII specific activity was determined by assessing the FVIII activity levels in relation to FVIII antigen levels. We further investigated whether thrombin generation could reflect the haemostatic potential of the variants. Our in silico results show the impact of the residues directly in the cleavage bond, and their neighboring residues on the insertion efficiency of the loop into the thrombin cleavage cleft. Moreover, the in vitro analysis shows that the sequences flanking the Arg 1708 cleavage site seem to be the most close to optimal residues for achieving the maximal proteolytic activation and profactor activity of FVIII. The residues flanking the scissile bonds of FVIIII affect the cleavage rates and modulate the profactor activation. We were able to provide insights into the mechanisms of the specificity of thrombin for the P1 cleavage sites of FVIII. Thus, the P4-P2´resi-dues surrounding Arg 1708 of FVIII have the highest impact on rates of thrombin proteolysis which contributes to thrombin activation of the profactor and eventually to the thrombin generation potential.

show abstract

“…Because tests for FVIII using chromogenic substrates have a more specific endpoint, cleavage of factor Xa, their use in inhibitor testing was proposed by Blanco et al 58 Chromogenic factor assays also have the advantage of increased precision. 59 A chromogenic Bethesda assay (CBA), identical to the CDC-NBA except for use of a chromogenic factor assay to measure the FVIII endpoint, has been described. 60 When 1005 specimens were tested with both the CDC-NBA and the CBA, 0.3% of 883 NBA-negative specimens, 54% of 80 positive specimens with 0.5–1.9 Nijmegen-Bethesda units (NBU) and 100% of 42 specimens with ≥2.0 NBU were also positive with the CBA.…”

Section: | Inhibitor Measurement Methodsmentioning

confidence: 99%

“…60,71 Testing with the CBA confirms that the inhibition of clotting observed is specific to FVIII and not the result of a LA, UFH contamination or a non-specific inhibitor. 58,59 The absence of specific anti-FVIII or anti-FIX antibodies in immunologic tests makes a true inhibitor unlikely. Antibodies are present, however, in some patients who lack a functional inhibitor.…”

Section: | Recent Assay Modificationsmentioning

confidence: 99%

Laboratory testing for factor VIII and IX inhibitors in haemophilia: A review

Miller

2018

Haemophilia

View full text Add to dashboard Cite

Inhibitors are antibodies directed against haemophilia treatment products which interfere with their function. Factor VIII (FVIII) inhibitors in haemophilia A and factor IX (FIX) inhibitors in haemophilia B are significant clinically when they require a change in a patient’s treatment regimen. Their persistence may increase morbidity and mortality. Multiple laboratory tests are now available for detecting and understanding inhibitors in haemophilia. Inhibitors are traditionally measured by their interference in clotting or chromogenic factor assays. They may also be detected using immunologic assays, such as enzyme-linked immunosorbent assay or fluorescence immunoassay. Anti-FVIII or anti-FIX antibodies of IgG4 subclass best correlate with the presence of functional inhibitors. Improvements in inhibitor measurement have been recently introduced. Preanalytical heat treatment of patient specimens allows testing of patients without delaying treatment. Use of chromogenic and immunologic assays may aid in identification of false-positive results, which are frequent among low-titre inhibitors. Validated reagent substitutions can be used to reduce assay cost. New methods for defining assay positivity and reporting low-titre inhibitors have been suggested. Challenges remain in the areas of quality control, assay standardization, monitoring of patients undergoing immune tolerance induction therapy and testing in the presence of modified and novel treatment products.

show abstract

A critical appraisal of one‐stage and chromogenic assays of factor VIII activity

Cited by 130 publications

References 69 publications

A world‐wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE

A world‐wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE

An in silico and in vitro approach to elucidate the impact of residues flanking the cleavage scissile bonds of FVIII

Laboratory testing for factor VIII and IX inhibitors in haemophilia: A review

Contact Info

Product

Resources

About