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.

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.

Ektachem Slides for Direct Potentiometric Determination of Sodium in Plasma: Effect of Natremia, Blood pH, and Type of Electrolyte Reference Fluid on Concordance with Flame Photometry and Other Potentiometric Methods

1992 ◽  
Vol 38 (1) ◽  
pp. 114-118 ◽  
Author(s):  
Ann Boeyckens ◽  
Jan Schots ◽  
Hugo Vandenplas ◽  
Freedy Senesael ◽  
Wim Goedhuys ◽  
...  
Keyword(s):  
Gastrointestinal Disease ◽  
Flame Photometry ◽  
Direct Potentiometry ◽  
Blood Ph ◽  
Ph Dependency ◽  
Analytical Recovery ◽  
The One

Abstract With electrolyte reference fluid (ERF)00, results from Kodak Ektachem slides for the direct potentiometric assay of sodium in plasma were significantly correlated with results from flame photometry, but also appeared to be systematically higher, especially in hypernatremic patients. Indirect potentiometry with the Technicon RA-1000 yielded intermediate values. In 23 hypernatremic patients with greater than or equal to 6 mmol/L difference in sodium between Ektachem ERF00 and flame photometry, a clinical survey disclosed the frequent association of large between-method differences with renal failure, diabetes mellitus, and gastrointestinal disease. However, there was no correlation between differences in sodium on the one hand and anion gaps or (lipo)protein concentrations on the other, nor did in vitro addition studies implicate metabolites that often accumulate in the above-mentioned disorders. Unlike indirect methods, sodium measurements by direct potentiometry on Ektachem and Corning were influenced by in vitro changes of pH between 7.0 and 7.9. However, in a group of patients that included many acidotic individuals, between-method differences in sodium appeared not significantly correlated with in vivo blood pH. Use of the equitransferant ERF04 on Ektachem strongly diminishes the systematic differences with flame photometry, reduces the pH-dependency of the results to that of the direct Corning method, and brings the mean analytical recovery of sodium to below 95% (instead of 115% previously) without affecting the ability of Ektachem slides to avoid spuriously low results in the presence of increased (monoclonal) protein concentrations.

Comparative Investigations on Factor VIII Assays

1975 ◽  
Author(s):  
R. Pflugshaupt ◽  
S. Moser ◽  
K. Züger ◽  
R. Bütler
Keyword(s):  
Factor Viii ◽  
High Activity ◽  
Hemophilia A ◽  
Statistical Evaluation ◽  
Two Stage ◽  
One Stage ◽  
Normal Plasma ◽  
Calibration Curves ◽  
Factor Viii Concentrates

Six one stage methods and one two stage method were tested for precision and reproducibility. With each method twenty calibration curves of normal plasma and two lots of Factor VIII concentrates were established. Statistical evaluation revealed only minor differences. Neither one of the methods was optimal for both the physiological-pathological region and the region of high activity preparations.Three selected methods were tested in vivo for accuracy: nine patients with hemophilia A were treated with equal amounts of Factor VIII concentrates or kryoprecipitates respectively. The methods showed different activities for preparations as well as for patient’s plasma. The discrepancy between measured and expected recovery differed for each method.

scholarly journals Alteration of Coagulation in Intensively Transfused Hemophilic Patients

1977 ◽  
Author(s):  
Helen S. Hathaway ◽  
Roger D. Hamstra ◽  
Linda Jacobson ◽  
William E. Hathaway
Keyword(s):  
Factor Viii ◽  
Degradation Products ◽  
Bleeding Tendency ◽  
Transfusion Therapy ◽  
Coagulation Tests ◽  
Factor Viii Concentrates ◽  
The One ◽  
Altered Proteins ◽  
Dose Factor

Bleeding may occasionally occur in adequately transfused hemophilic patients. To investigate this phenomenon, 11 patients with classical hemophilia had serial coagulation studies performed during intensive transfusion therapy with factor VIII concentrates given for surgical procedures. The studies included kaolin partial thromboplastin times (KPTT), fibrinogen, monomer, and fibrin split products (FSP) levels, and assays for factor VIII by the one-stage PTT method (PTT-VIII), thromboplastin generation time method (TGT-VIII), and immunologic method (VIII Ag). After an initial correcting dose, factor VIII concentrates were administered once to twice daily in a dose to keep the minimal level above 20 percent. Alterations of coagulation assays were most pronounced 7–10 days postoperatively. These included (1) KPTT values at least 20 seconds longer than expected for the percent factor VIII;(2) TGT-VIII levels consistently higher than PTT-VIII levels (mean difference was 20 percent);(3) VIII Ag values from 216–660 percent of normal. Fibrin monomer and FSP tests were frequently positive and fibrinogen levels ranged from 300–700 mgm percent. Four patients exhibited spontaneous wound bleeding on postoperative days 7, 7, 9, and 12 in spite of adequate factor VIII levels. These studies and the results of in vitro experiments with factor VIII concentrates suggest that altered proteins or degradation products of fibrinogen or factor VIII may produce spurious values for coagulation tests and may be associated with an increased bleeding tendency and/or abnormal wound healing.

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

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.

In Vivo Recovery and Survival of Monoclonal-Antibody-Purified Factor VIII Concentrates

1991 ◽  
Vol 66 (06) ◽  
pp. 730-733 ◽  
Author(s):  
Carol K Kasper ◽  
Hugh C Kim ◽  
Edward D Gomperts ◽  
Kenneth J Smith ◽  
Phyllis M Salzman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
In Vitro Assays ◽  
Two Stage ◽  
Double Blind ◽  
One Stage ◽  
Factor Viii Concentrates ◽  
In Vivo Recovery

SummaryIn response to reports of discrepant in vitro assays of high-purity concentrates, a double-blind crossover study of in vivo recovery and half-life of two brands of monoclonal-antibody-purified factor VIII concentrates (Monoclate and Hemofil-M) was performed in 23 patients with hemophilia A. In vivo recoveries were close to values predicted from the labelled unitage when plasma samples were assayed by a one-stage method. When a two-stage assay was used, lower recoveries were calculated and the recovery with Hemofil-M was slightly but significantly lower than that with Monoclate. The concentrates were re-assayed in vitro by the two-stage method. Monoclate (which is assayed by the manufacturer using a two-stage method) contained 97% of the labelled potency and Hemofil-M (which is assayed by the manufacturer using a one-stage method) contained 81% of the labelled potency. Differences in in vitro and in vivo assay methods contribute to disparities between expected and observed factor VIII recovery. Clearance of Hemofil-M was significantly faster than that of Monoclate, but volume of distribution at the steady state, mean residence time, and plasma half-disappearance times of the two concentrates were not significantly different.

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%.

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