Clinical Application of a Chromogenic Substrate Method for Determination of Factor VIII Activity

1985 ◽  
Vol 54 (04) ◽  
pp. 818-823 ◽  
Author(s):  
S Rosén ◽  
M Andersson ◽  
M Blombäck ◽  
U Hégglund ◽  
M J Larrieu ◽  
...  
Keyword(s):  
Factor Viii ◽  
Clinical Application ◽  
Healthy Subjects ◽  
Hemophilia A ◽  
Chromogenic Substrate ◽  
Factor Viii Activity ◽  
One Stage ◽  
The One ◽  
Good Agreement

SummaryA chromogenic substrate kit for the determination of factor VIII activity (COATEST® Factor VIII) has been evaluated in five different laboratories, one of them using a semi-automated procedure. This chromogenic method was compared to one-stage clotting assays for factor VIII determination in plasmas from healthy subjects, carriers of hemophilia A, severe, mild and moderate hemophilia A as well as von Willebrand’s patients. In all these cases, a high correlation between these two methods was obtained (r = 0.96-0.99, n = 385) with a good agreement of the assigned potencies at all levels of factor VIII. A good correlation (r = 0.94) was also obtained for the levels of factor VIII after infusion of concentrates in six severe hemophiliacs or after administration of DDAVP to von Willebrand’s patients.The chromogenic method is insensitive to preactivation of factor VIII by thrombin, thus yielding valid potency assignments also in these situations.The precision was higher with the chromogenic method than with the one-stage clotting assays (C.V. = 2-5% vs 4-15%). Altogether, the new chromogenic substrate method has proven itself suitable for determination of factor VIII in plasma and concentrates.

Issues with the Assay of Factor VIII Activity in Plasma and Factor VIII Concentrates

2000 ◽  
Vol 84 (12) ◽  
pp. 942-948 ◽  
Author(s):  
Henry Kingdon ◽  
Kenneth Mann ◽  
Gilbert White ◽  
Roger Lundblad
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factors ◽  
Chromogenic Substrate ◽  
In Vitro Measurements ◽  
Factor Viii Concentrates ◽  
The One

SummaryA review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VIII concentrates such as Hemofil ® M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements, it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor Villa during the assay process.

Factor VIII: C (FVIII: C) Recovery and Half-Life After Infusion of Steam-Treated High Purity Factor VIII Concentrate in Severe Hemophilia A - Comparison of One-Stage Assay, Two-Stage Assay and a Chromogenic Substrate Assay

1986 ◽  
Vol 56 (03) ◽  
pp. 353-359 ◽  
Author(s):  
Peter Hellstern ◽  
Ralf Kiehl ◽  
Chieko Miyashita ◽  
Holger Schwerdt ◽  
Gerhard von Blohn ◽  
...  
Keyword(s):  
Factor Viii ◽  
High Purity ◽  
Plasma Levels ◽  
Half Life ◽  
Hemophilia A ◽  
Least Square ◽  
Chromogenic Substrate ◽  
Two Stage ◽  
One Stage ◽  
The One

SummaryFactor VIII :C recovery and half-life was measured in 16 hemophilia A patients under comprehensively standardized conditions. Each patient received the same lot of a steam-treated high purity FVIII concentrate at a dose of 19-33 U/kg body weight. A comparison was made between the one-stage assay, the two-stage assay and a chromogenic substrate test for FVIII :C determination using a FXa-sensitive chromogenic substrate. Factor VIII :C potency of the administered FVIII concentrate was measured using calibration curves derived from a concentrate standard and FVIII: C plasma levels were read from calibration curves derived from a plasma standard. The chromogenic assay showed a good reproducibility at FVIII: C levels between 0.015 and 0.50 U/ml. The FVIII :C recoveries calculated from the results of the one-stage assay, the two-stage assay and the chromogenic substrate test were 109 ± 20, 92 ± 14 and 81 ± 11% (x ± SD), respectively. The elimination half-lives of FVIII :C were calculated by non-linear least square analysis using a modified computerized Gauss-Newton algorithm. The half-lives calculated from the FVIII: C plasma levels measured by the one-stage assay, the two-stage assay and the chromogenic test were 23.8 ± 6.4, 22.2 ± 5.7 and 17.1 ± 4.8 h (x ± SD), respectively. No previous study has reported such long half-life values. Our findings indicate that measurements of recoveries and half-lives by the chromogenic FVIII :C assay and by computerized nonlinear least square analysis allow the possibility of individualized FVIII replacement therapy.

scholarly journals Assessing the Performance of Extended Half-Life Coagulation Factor VIII, FC Fusion Protein by Using Chromogenic and One-Stage Assays in Saudi Hemophilia A Patients

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Tarek M. Owaidah ◽  
Hazzaa A. Alzahrani ◽  
Nouf S. Al-Numair ◽  
Abdulmjeed O. Alnosair ◽  
Amelita M. Aguilos ◽  
...  
Keyword(s):  
Factor Viii ◽  
Half Life ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Time Interval ◽  
One Stage ◽  
Chromogenic Assay ◽  
Significant Difference ◽  
Mean Differences ◽  
The One

Background. The one-stage assay is the most common method to measure factor VIII activity (FVIII : C) in hemophilia A patients. The chromogenic assay is another two-stage test involving purified coagulation factors followed by factor Xa-specific chromogenic substrate. Aim. This study aimed to assess the discrepancy and correlation between the chromogenic and one-stage assays in measuring FVIII : C levels in hemophilia patients receiving Extended Half-Life Elocta® as a recombinant extended half-life coagulation factor. Methods. We performed a study comparing the measurements of FVIII : C levels by the chromogenic versus the one-stage assays at different drug levels. Data of FVIII : C levels, dosage, and the time interval from administration to measurement were retrieved from the hospital records. The correlation, mean differences, and discrepancy between the two assays were calculated. The linear regression analysis was used to predict the time interval till reaching 1% FVIII : C. Results. Fourteen patients with 56 samples were included in the study. Of them, 13 patients were receiving Elocta® as a prophylactic, while one was receiving Elocta® on demand. One-third of these samples showed a discrepancy between the chromogenic and one-stage assays. The two assays were well correlated. Mean differences were significant at the individual and the time interval level. The time since the last Elocta® injection could significantly predict FVIII : C levels (β = 0.366, P<0.001). Conclusion. Our findings suggested a significant difference between both methods; the FVIII : C levels measured by the one-stage assay were less than those estimated by the chromogenic assay. However, the measurements of FVIII levels by the two assays were well correlated but discrepant in one-third of the samples. The levels of FVIII : C reach 1% after 5.4 days since the last Elocta® administration.

The Influence of Thrombin on the Clotting Activity of Factor VIII. A Study with Insolubilized Thrombin

1978 ◽  
Vol 39 (03) ◽  
pp. 600-606 ◽  
Author(s):  
Th Vukovich ◽  
E Koller ◽  
W Doleschel
Keyword(s):  
Factor Viii ◽  
Test Sample ◽  
Test System ◽  
Factor Viii Activity ◽  
Two Stage ◽  
One Stage ◽  
Analytical Procedures ◽  
The One

SummaryAn investigation of the influence of thrombin on the clotting activity of factor VIII was made. Purified factor VIII and different amounts of thrombin complexed to Sepharose 4 B were mixed and incubated for various periods of time. The factor VIII activities of these incubation mixtures were determined by the one- and two-stage analytical procedures in the presence of the thrombin-sepharose and in its absence following the latter removal from the test sample by filtration. The results so obtained confirm the view that thrombin inactivates factor VIII. Evidences for a thrombin-induced potentiation of the factor VIII activity, seen only in the thrombin-sepharose containing test samples analyzed by the one-stage method, are here interpreted as thrombin-effects peculiar to this factor VIII test system and not as potentiation by thrombin of the factor itself.

Pharmacokinetic In Vivo Comparison Using 1-Stage and Chromogenic Substrate Assays with Two Formulations of Hemofil-M

1996 ◽  
Vol 76 (06) ◽  
pp. 0950-0956 ◽  
Author(s):  
Christine Lee ◽  
Trevor Barrowcliffe ◽  
Gordon Bray ◽  
Ed Gomperts ◽  
Anthony Hubbard ◽  
...  
Keyword(s):  
Factor Viii ◽  
Pharmacokinetic Studies ◽  
Chromogenic Substrate ◽  
Single Centre ◽  
One Stage ◽  
Chromogenic Assay ◽  
The Usa ◽  
The Mean ◽  
The One

SummaryIn a study to demonstrate the safety and pharmacokinetics (half-life and recovery) of two different method M purified AHF (Hemofil-M) concentrates processed in the USA and Spain, two different methods of factor VIII assay (one-stage clotting and chromogenic) have been compared in vivo. The study was a single centre blinded, randomised, crossover study. Twelve patients with severe haemophilia A (VIII: C <2 u/dl) were divided into two subgroups of six. None had received factor VIII concentrate within 48 h preceding the study. Twenty-four pharmacokinetic studies were performed in the 12 patients. Each subgroup received two different lots of study material (US and Spanish) at a dose of 50 u/kg seven days apart. A second randomisation was nominal potency, high: 1000 u or mid: 500 u per vial. The potency label was a one-stage clotting assay using the mega I standard. A standard pharmacokinetic study was performed over 24 h and each blinded sample was analysed in duplicate by a one-stage clotting (aPTT) and a chromogenic (Chromogenix AB; CS) assay at the Royal Free and NIBSC. Pharmacokinetic modelling was performed. The mean label for Hemofil-M using the chromogenic substrate assay was 79% that using the one stage assay (Mega I standard). The recovery was 17-28% higher measured by chromogenic compared to the clotting assay. Since most clinicians use the clotting assay, potency labelling using the chromogenic assay, will overestimate predicted Hemofil-M recovery by as much as 25%.

A Collaborative Study to Establish the 6th International Standard for Factor VIII Concentrate

2001 ◽  
Vol 85 (06) ◽  
pp. 1071-1078 ◽  
Author(s):  
A. B. Heath ◽  
T. W. Barrowcliffe ◽  
S. Raut
Keyword(s):  
Factor Viii ◽  
Collaborative Study ◽  
World Health Organisation ◽  
World Health ◽  
Assay Method ◽  
Expert Committee ◽  
International Standard ◽  
One Stage ◽  
The One ◽  
Good Agreement

SummaryA study was carried out to replace the 5th WHO International Standard (IS) for factor VIII concentrate, because of depletion of stocks. Two candidate concentrates (X and Y) were assayed as potential replacements against the 5th IS for FVIII concentrate, in a collaborative study involving 33 laboratories. Collaborators were asked to use the ISTH/SSC recommendations, including pre-dilution of concentrates in FVIII deficient plasma in their assays. Several laboratories performed more than one assay method and altogether there were 21 sets of assays with the one-stage method, 6 with the two-stage method and 26 with the chromogenic method. There was good agreement between laboratories using each method for the comparison of concentrates X and Y against the 5th IS, but the overall potencies by one-stage and chromogenic methods each differed by approximately 5% from the overall mean, with the chromogenic potency approximately 10% higher than the one-stage. Inter-laboratory agreement was slightly better for concentrate Y than X, and stability studies indicated that Y was more stable than X. After considering all the information, together with comments from participants and from the FVIII/FIX Subcommittee of the ISTH/SSC, candidate Y (NIBSC code [97/616]), was proposed and accepted in October, 1998, by the Expert Committee on Biological Standardisation of the World Health Organisation to be the 6th International Standard for Factor VIII Concentrate with an assigned potency of 8.5 IU/ampoule.

Genetic analysis of non-severe hemophilia A phenotype with a discrepancy between one-stage and chromogenic factor VIII activity assays

2021 ◽  
pp. 103194
Author(s):  
Amir Valikhani ◽  
Mojgan Mirakhorly ◽  
Ali Namvar ◽  
Ghasem Rastegarlari ◽  
Gholamreza toogeh ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Factor Viii ◽  
Hemophilia A ◽  
Severe Hemophilia ◽  
Factor Viii Activity ◽  
One Stage

A Prospective, Multi-Center, Randomized Clinical Trial to Compare the Pharmacokinetic Properties of Human-Cl rhFVIII, a New Human-Cell Line Derived Recombinant Factor VIII, with Those of a Full-Length Recombinant Factor VIII Expressed in Baby Hamster Kidney Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3309-3309 ◽  
Author(s):  
Sigurd Knaub ◽  
Andreas Tiede ◽  
Toshko Lissitchkov ◽  
Leonard A. Valentino ◽  
Jerry Sherman Powell ◽  
...  
Keyword(s):  
Factor Viii ◽  
Cell Line ◽  
Human Cell ◽  
Hemophilia A ◽  
Human Cell Line ◽  
Recombinant Factor Viii ◽  
Baby Hamster Kidney ◽  
Efficacy And Safety ◽  
One Stage ◽  
The One

Abstract Abstract 3309 Introduction: human-cl rhFVIII is the first recombinant factor VIII (FVIII) concentrate expressed in a human cell line (Human Embryonic Kidney (HEK) 293F cells). The manufacturing process includes two steps for virus inactivation and removal (solvent/detergent treatment and a nanofiltration). No animal- or human-derived materials are added to the culture medium or during the purification and final formulation. Objective: The primary objective of this study is to compare the pharmacokinetics (PKs) of human-cl rhFVIII, a B-domain deleted rFVIII product expressed in a human cell line, with a commercially available full-length rFVIII product (Kogenate FS), which is expressed in Baby Hamster Kidney (BHK) cells. Methods: This study is an open, multinational, prospective, multicenter clinical phase-II trial. The PK properties of human-cl rhFVIII and Kogenate FS were determined using a randomized two-period, cross-over design. After a 96 hours or more wash-out phase of any previously administered FVIII product, subjects received a single intravenous dose of 50 IU/kg body weight of one of the products. Blood samples were collected over a 48-h period and FVIII coagulant activity (FVIII:C) was measured by validated chromogenic substrate and one-stage clotting assay in a central laboratory, which also assigned drug potency with the same assays. Following completion of the PK segment of the study, subjects will be followed for at least 50 Exposure Days and at least 6 months of treatment with human-cl rhFVIII to evaluate the efficacy and safety of on-demand treatment. The planned interim analysis stipulates the comparison of the complete PK profile of human-cl rhFVIII and Kogenate FS with the bioequivalence of the AUC as the primary endpoint. The full efficacy and safety profile will be reported when the study is completed. Results: Twenty-two adolescent and adult PTPs with severe hemophilia A (FVIII:C <1%) were enrolled at 10 study centers in the United States and Europe. The key PK parameters (AUC, elimination half-life, in-vivo recovery, clearance) are comparable between human-cl rhFVIII and Kogenate FS both for the one-stage and chromogenic assays. The two products were judged to be bioequivalent as the 90% confidence interval of the ratio of the geometric mean values of the AUC fell within 80–125%. human-cl rhFVIII has been well tolerated and no safety or efficacy issues have been reported so far, in particular no inhibitors or non inhibitory antibodies were detected or reported. Conclusion: The PK data from this study indicate that human-cl rh FVIII is bioequivalent to a licensed rFVIII concentrate measured by the one stage and the chromogenic assays. In this study as well as in the other ongoing clinical studies, which examine the efficacy and safety of human-cl rhFVIII during prophylactic treatment in children and adults with severe hemophilia A, the product has been safe and well tolerated so far. Disclosures: Knaub: Octapharma AG: Employment. Valentino:Baxter Bioscience: Consultancy; GTC Biotherapeutics: Consultancy; Bayer Healthcare: Consultancy; NovoNordisk: Consultancy; Inspiration Bioscience: Consultancy; Biogen: Consultancy. Manco-Johnson:Octapharma AG: Consultancy.

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