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%.

Pharmacokinetics of Recombinant Factor VIII (Recombinate) Using One-stage Clotting and Chromogenic Factor VIII Assay

1999 ◽  
Vol 82 (12) ◽  
pp. 1644-1647 ◽  
Author(s):  
D. Owens ◽  
G. Bray ◽  
P. Giangrande ◽  
P. Collins ◽  
C. Hay ◽  
...  
Keyword(s):  
Factor Viii ◽  
Clinical Situation ◽  
Recombinant Factor Viii ◽  
Pharmacokinetic Studies ◽  
Chromogenic Substrate ◽  
One Stage ◽  
Chromogenic Assay ◽  
Significant Difference ◽  
Post Infusion

SummaryIn a study designed to demonstrate the safety and pharmacokinetics of a recombinant factor VIII (Recombinate) manufactured in Andover, MA and Thousand Oaks, CA, two different methods of factor VIII assay (one-stage clotting and Chromogenic substrate) were compared in vivo. The study was performed in four centres in the UK: London, Oxford, Cardiff and Manchester. Two pharmacokinetic studies, at least one week apart, were performed in 30 patients with severe haemophilia A (VIII:C < 2 IU/dl). A dose of 50 IU/kg was administered with sampling pre-infusion, and +0.25, 0.5, 1, 3, 6, 9, 12 and 24 h post-infusion. The aggregate 60 pharmacokinetic study showed a half-life of 12.7 and 13.0 h (p = 0.28) and recovery of 127 and 161 IU/dl (p = 0.0001) using one-stage clotting or chromogenic substrate respectively. In a supplementary experiment, 20 post-infusion samples were re-assayed by 1-stage and chromogenic assay using two plasma (20th British plasma standard and an “in-house” pooled normal plasma) and two concentrate standards, derived from the same type, but different batch of infused concentrate (Recombinate) and pre-diluted in either individual pre-infusion sample or in pooled commercial haemophilic plasma. The use of the Recombinate concentrate standard overcame the significant difference in FVIII levels between 1-stage and chromogenic assay methods when a plasma standard was used (p <0.0001). It is concluded that where potency dosing designation is carried out by an assay system different to that used in the clinical situation, the use of the recombinant concentrate as a standard in post-infusion plasma samples is likely to give more reliable and reproducible results.

Validation of a Procedure for Potency Assessing of a High Purity Factor VIII Concentrate -Comparison of Different Factor VIII Coagulant Assays and Effect of Prediluent

1990 ◽  
Vol 64 (02) ◽  
pp. 251-255 ◽  
Author(s):  
Claudine Mazurier ◽  
Armelle Parquet-Gernez ◽  
Maurice Goudemand
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Assay Method ◽  
One Stage ◽  
Chromogenic Assay ◽  
Coagulant Activity ◽  
The One ◽  
In Vitro Data

SummaryThe assessment of factor VIII coagulant activity (FVTII: C) in recently available highly purified and concentrated FVTII therapeutic products calls for careful evaluation of assay methodologies. We assayed more than 130 batches of a concentrate with a specific activity of about 150 FVTII :C units/mg protein, using one-stage and two-stage clotting and chromogenic methods. There was good agreement between the potency estimates obtained with the different methods. We also compared the FVTII :C potencies obtained after predilution in buffer or FVIII-deficient plasma using either calibrated plasma or FVTII concentrate as references. With the one-stage assay we found a marked discrepancy between the potency values obtained with buffer and with FVTII-deficient plasma used as prediluents. In order to validate our “in vitro” data we performed 6 “in vivo” analyses in severe haemophilia A patients. On the basis of the overall data obtained we chose to label FVIII potency by using FVIII-deficient plasma as prediluent, reference plasma as standard and the chromogenic assay method.

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.

scholarly journals The one‐stage assay or chromogenic assay to monitor baseline factor VIII levels and desmopressin effect in non‐severe haemophilia A: Superiority or non‐inferiority?

Haemophilia ◽  
2020 ◽  
Vol 26 (5) ◽  
pp. 916-922
Author(s):  
Lisette M. Schütte ◽  
Luca S. Hodes ◽  
Iris Moort ◽  
Sara C. M. Stoof ◽  
Frank W. G. Leebeek ◽  
...  
Keyword(s):  
Factor Viii ◽  
Haemophilia A ◽  
Severe Haemophilia ◽  
One Stage ◽  
Chromogenic Assay ◽  
The One ◽  
Baseline Factor

A Methodological Investigation On The One-Stage Factor VIII Assay

1981 ◽  
Author(s):  
J Over ◽  
J A van Mourik ◽  
P van den Brink-Zantingh ◽  
R Smit-Jansen
Keyword(s):  
Factor Viii ◽  
Dose Response ◽  
Red Cross ◽  
Response Curves ◽  
One Stage ◽  
Factor Viii Concentrates ◽  
Dose Response Curves ◽  
The One ◽  
Protein Rich

Assay of Factor VIII coagulant activity (VIII: C) in Factor VIII concentrates has since long met difficulties, such as l) non-paralleility of dose-response curves of plasma standard and Factor VIII concentrate, 2) spuriously low values of VIII: C in concentrates as revealed by abnormally high in vivo recoveries after transfusion, and 3) large interlaboratory variation in assay results. In an attempt to analyze the cause of these problems several parameters of the one-stage assay system were varied systematically and their effect on the parallellity of dose-response curves and on the final VIII: C value was analyzed. Nonparallel1ity was partially corrected with a protein-rich dilution medium, and almost always completely with undiluted instead of 1:1 diluted hemophilic substrate plasma. In both conditions apparently higher VIII:C values were found.A number of assay systems used by different producers of Factor VIII concentrates were compared. The standard and, in some cases, the phospholipid reagent seemed to contribute for the largest past to the inter1aboratory variation, but also other, as yet unidentified, factors exerted some influence. These findings initiated a cooperative study by five Red Cross Blood Transfusion Services in Europe on standardization of the one-stage assay for VIII:C. This resulted in a better correspondence between these institutes (CV 13%) compared to the previous situation (CV 23%).It is concluded that 1) substrate plasma should not be diluted, especially when Factor VIII concentrate is to be tested against a plasma standard, 2) the standard should be of the same type as the testmaterial, and 3) this standard should be properly calibrated against the International Standard for Factor VIII.

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.

Decreased Potency Of Factor VIII Concentrates

1981 ◽  
Author(s):  
C K Kasper
Keyword(s):  
Factor Viii ◽  
In Vitro Assays ◽  
Assay Method ◽  
Two Stage ◽  
One Stage ◽  
Plasma Factor ◽  
Factor Viii Concentrates ◽  
The One

Plasma factor VIII recoveries after infusions of factor VIII concentrates into patients with classic hemophilia have been measured in this laboratory for 14 years. Recently, we observed a decline in the in vivo recovery of factor VIII per factor VIII unit infused. In 1980, plasma factor VIII levels were measured by a one-stage APTT-based assay before and 10 min after 150 infusions of 46 lots of 3 brands of factor VIII concentrate produced in the U.S.A. Our pooled normal plasma reference was calibrated against WHO International Standard 2 and results expressed in International factor VIII units. Observed in vivo factor VIII recovery was compared to the value expected from calculations based on the unitage stated on the label. The ratio of observed/expected recovery averaged 56% per lot for brand A, 60% per lot for brand B, and 103% per lot for brand C. In vitro assays were performed on 22 lots on 36 occasions, and the ratio of observed/labelled units average 46% per lot for brand A, 53% for brand B and 75% for brand C. The two-stage factor VIII assay method of Pool and Robinson was also used to assay plasma samples from 18 infusions, and results averaged 135% of the one-stage values for infusions of brand A, 160% for brand B, and 109% for brand C. (Brand A is assayed by the manufacturer by a two-stage method, brands B and C by one-stage methods.)Decreased clinical efficacy was observed when postinfusion plasma factor VIII levels were lower than customary. The decline in potency of brands A and B has necessitated more frequent assay of patients and use of larger amounts of concentrate, with greatly-increased expense. Investigation of the effect of different assay methods and different factor VIII standards and references on the apparent factor VIII content of concentrates has begun.

scholarly journals Comparative field study: impact of laboratory assay variability on the assessment of recombinant factor IX Fc fusion protein (rFIXFc) activity

2014 ◽  
Vol 112 (11) ◽  
pp. 932-940 ◽  
Author(s):  
Yang Buyue ◽  
Sara Bardan ◽  
Robert Peters ◽  
Haiyan Jiang ◽  
George Kamphaus ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Field Study ◽  
Factor Ix ◽  
One Stage ◽  
Chromogenic Assay ◽  
Fc Fusion Protein ◽  
Recombinant Factor Ix ◽  
Assay Methods ◽  
The Mean ◽  
The One

SummaryDue to variability in the one-stage clotting assay, the performance of new factor IX (FIX) products should be assessed in this assay. The objective of this field study was to evaluate the accuracy of measuring recombinant FIX Fc fusion protein (rFIXFc) activity in clinical haemostasis laboratories using the one-stage clotting assay. Human haemophilic donor plasma was spiked with rFIXFc or BeneFIX® at 0.80, 0.20, or 0.05 IU/ml based on label potency. Laboratories tested blinded samples using their routine one-stage assay and in-house FIX plasma standard. The mean spike recoveries for BeneFIX (n=30 laboratories) were 121 %, 144 %, and 168 % of expected at nominal 0.80, 0.20, and 0.05 IU/ml concentrations, respectively. Corresponding rFIXFc spike recoveries were 88 %, 107 %, and 132 % of expected, respectively. All BeneFIX concentrations were consistently overestimated by most laboratories. rFIXFc activity was reagent-dependent; ellagic acid and silica gave higher values than kaolin, which underestimated rFIXFc. BeneFIX demonstrated significantly reduced chromogenic assay activity relative to one-stage assay results and nominal activity, while rFIXFc activity was close to nominal activity at three concentrations with better dilution linearity than the typical one-stage assay. In conclusion, laboratory- and reagent-specific assay variabilities were revealed, with progressively higher variability at lower FIX concentrations. Non-parallelism against the FIX plasma standard was observed in all one-stage assays with rFIXFc and BeneFIX, leading to significant overestimation of FIX activity at lower levels and generally high inter-laboratory variability. Compared to the accuracy currently achieved in clinical laboratories when measuring other rFIX products, most laboratories measured rFIXFc activity with acceptable accuracy and reliability using routine one-stage assay methods and commercially available plasma standards.

BAY 81-8973, a Full-Length Recombinant Factor VIII: Results from Assay Field Study

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3539-3539
Author(s):  
Monika Maas Enriquez ◽  
Horst Beckmann ◽  
Yvonne Katterle ◽  
Stefan Bruns ◽  
Despina Tseneklidou-Stoeter ◽  
...  
Keyword(s):  
Factor Viii ◽  
Field Study ◽  
Full Length ◽  
Recombinant Factor Viii ◽  
One Stage ◽  
Chromogenic Assay ◽  
Clinical Laboratories ◽  
Interlaboratory Variability ◽  
The One ◽  
Plasma Control

Abstract Background: BAY 81-8973 is a full-length unmodified recombinant factor VIII (rFVIII) in development for the treatment of hemophilia A. BAY 81-8973 has the same amino acid sequence as Bayer's sucrose-formulated rFVIII but is manufactured using the latest technologies. Potency labeling is based on the chromogenic assay. BAY 81-8973 has demonstrated an excellent safety and efficacy profile in the clinical development program. This field study was conducted to evaluate assay variability in BAY 81-8973 measurements compared with a marketed rFVIII (Advate®, Baxter, Westlake Village, CA) using the methods, reference standards, and reagents typically used in clinical laboratories. Methods: Clinical laboratories in North America, Europe, Israel, and South Africa were invited to participate in the study. Each laboratory was provided with 21 blinded samples to analyze using their routine assay (one-stage, chromogenic, or both), reagents, and standards. The 21 samples consisted of 3 aliquots each of spiked hemophilia plasma containing normal von Willebrand factor levels with BAY 81-8973 at 3 different levels: <10 IU/dL (low), 10-50 IU/dL (medium), >50 IU/dL (high); 3 aliquots each of spiked hemophilia plasma with Advate at the same levels (low, medium, high); and 3 aliquots of commercially available positive control sample (normal human plasma). Samples were identified by unique numbers and by target FVIII levels (low, medium, high). The nominal spiked target levels of FVIII concentration were 0.043 IU/mL (low), 0.375 IU/mL (medium), and 0.865 IU/mL (high) for BAY 81-8973 and Advate and 0.960 IU/mL for the plasma control. Results were analyzed statistically for intra- and interlaboratory variability. Results: Of 82 laboratories contacted, 41 in 11 countries participated. Thirty-one laboratories used the one-stage assay only, 1 used the chromogenic assay only, and 9 used both assays. Intralaboratory variability was <11% for both assays at all FVIII levels and was similar for BAY 81-8973, Advate, and the plasma control. Interlaboratory variability was highest for the lowest concentration using the chromogenic assay (percent coefficient of variation: 60% for BAY 81-8973, 51% for Advate) and decreased to 14% for BAY 81-8973 (Advate, 12%; plasma control, 10%) with the one-stage assay and 5% (Advate, 6%; plasma control, 7%) with the chromogenic assay at the highest concentration. For the 9 laboratories that used both the one-stage and chromogenic assays, the chromogenic:one-stage ratio for mean values for BAY 81-8973 at low, medium, and high concentrations was 1.04, 1.04, and 1.14, respectively; for Advate, the ratios were 1.02, 1.07, and 1.21. Conclusions: The laboratories participating in this field study used a wide range of methods and reagents for FVIII measurements. The variability of the results was similar for both BAY 81-8973 and Advate and highest for low concentrations. There was no relevant difference in the results between the one-stage and chromogenic assays. Disclosures Maas Enriquez: Bayer Pharma AG: Employment. Beckmann:Bayer Pharma AG: Employment. Katterle:Bayer Pharma AG: Employment. Bruns:Winicker Norimed: Employment. Tseneklidou-Stoeter:Bayer Pharma AG: Employment. Kitchen:Bayer Pharma AG: Other: Advisory fees, Speakers Bureau.

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.

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