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.

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 Physiologically based kinetic modelling based prediction of in vivo rat and human acetylcholinesterase (AChE) inhibition upon exposure to diazinon

2021 ◽  
Author(s):  
Shensheng Zhao ◽  
Sebastiaan Wesseling ◽  
Bert Spenkelink ◽  
Ivonne M. C. M. Rietjens
Keyword(s):  
Dose Response ◽  
Kinetic Modelling ◽  
Ache Inhibition ◽  
Response Curves ◽  
Alternative Testing ◽  
Dose Response Curves ◽  
Physiologically Based Kinetic Modelling ◽  
Point Of Departure

AbstractThe present study predicts in vivo human and rat red blood cell (RBC) acetylcholinesterase (AChE) inhibition upon diazinon (DZN) exposure using physiological based kinetic (PBK) modelling-facilitated reverse dosimetry. Due to the fact that both DZN and its oxon metabolite diazoxon (DZO) can inhibit AChE, a toxic equivalency factor (TEF) was included in the PBK model to combine the effect of DZN and DZO when predicting in vivo AChE inhibition. The PBK models were defined based on kinetic constants derived from in vitro incubations with liver fractions or plasma of rat and human, and were used to translate in vitro concentration–response curves for AChE inhibition obtained in the current study to predicted in vivo dose–response curves. The predicted dose–response curves for rat matched available in vivo data on AChE inhibition, and the benchmark dose lower confidence limits for 10% inhibition (BMDL10 values) were in line with the reported BMDL10 values. Humans were predicted to be 6-fold more sensitive than rats in terms of AChE inhibition, mainly because of inter-species differences in toxicokinetics. It is concluded that the TEF-coded DZN PBK model combined with quantitative in vitro to in vivo extrapolation (QIVIVE) provides an adequate approach to predict RBC AChE inhibition upon acute oral DZN exposure, and can provide an alternative testing strategy for derivation of a point of departure (POD) in risk assessment.

scholarly journals Characteristics of Factor VIII Related Protein in Different Types of von Willebrand’s Disease

1977 ◽  
Author(s):  
A. L. Bloom ◽  
I. R. Peake
Keyword(s):  
Factor Viii ◽  
Dose Response ◽  
Related Protein ◽  
Con A ◽  
Response Curves ◽  
Von Willebrand's Disease ◽  
Normal Plasma ◽  
Different Types ◽  
Dose Response Curves ◽  
Antigenic Reactivity

The antigenic, biochemical and biological reactions of factor VIII related protein were studied in normal plasma and in the plasma of patients with different types of von Willebrand’s disease (vWd). Antigenic reactivity was compared using the Laurell electroimmunoassay (LA) and an immunoradiometric assay (IRMA). Biochemical characteristics of factor VIII related antigen (FVIIIRAG) were compared by examining its electrophoretic mobility (EM) on two dimensional crossed Immunoelectrophoresis (2DCIE) and by determining its precipitation properties with the glycoprotein precipitant concanavalin A (Con A),Biological reactions were compared using the ristocetin cofactor (RiCoF) assay on fixed platelets and by determining procoagulant factor VIII (FVIIIC). In normal plasma the levels of FVIIIRAG measured by LA correlated with the IRMA and dose response curves were parallel. At a concentration of lmg/ml Con A completely precipitated FVIIIRAG. The biological activities, RiCoF anú FVIIIC, were normal and correlated with those of FVIIIRAG. In patients with vWd in whom levels of FVIIIRAG by LA were normal the EM by 2DCIE was increased. In these patients the dose-response curves of the IRMA were not parallel to normal, FVIIIRAG was not precipitated normally by Con A and the RiCoF activity was reduced. Similar findings were observed in some patients with “typical” intermediate vWd in whom the plasma levels of FVIIIRAG were too low to determine EM, In other patients with vWd the dose response curves of the IRMA were parallel. The results suggest that the non-parallel dose response curves of the IRMA were due to the presence of abnormal FVIIIRAG and were consistent with variations of antigenic reactivity or binding sites in these patients.

Histamine H2-receptor of human and rabbit parietal cells

1987 ◽  
Vol 253 (4) ◽  
pp. G497-G501 ◽  
Author(s):  
R. Leth ◽  
B. Elander ◽  
U. Haglund ◽  
L. Olbe ◽  
E. Fellenius
Keyword(s):  
Dose Response ◽  
In Vivo Model ◽  
Response Curves ◽  
Gastric Glands ◽  
Histamine H2 Receptor ◽  
Receptor Affinity ◽  
H2 Receptor ◽  
Dose Response Curves

The histamine H2-receptor on the human parietal cell has been characterized by using dose-response curves and the negative logarithm of the molar concentration of an antagonist (pA2) analyses of cimetidine antagonism of betazole, histamine, and impromidine stimulation in isolated human and rabbit gastric glands. To evaluate the in vitro results, betazole-stimulated gastric acid secretion with and without cimetidine was also studied in healthy subjects. In the in vivo model, individual dose-response curves were shifted to the right with increasing cimetidine concentrations, but this was counteracted by increasing betazole doses, indicating competitive, reversible antagonism. The pA2 values ranged from 6.1 to 6.3. In isolated human gastric glands, impromidine was shown to be eight times more potent than histamine, indicating higher receptor affinity, but the maximally stimulated aminopyrine accumulation was the same as for histamine, and the pA2 values for cimetidine antagonism did not differ significantly, i.e., 5.7 (histamine) and 6.1 (impromidine). In isolated rabbit gastric glands, cimetidine inhibited the histamine- and impromidine-stimulated response with pA2 values of 6.0 and 7.3, respectively. Impromidine was shown to be approximately 100 times more potent than in human gastric glands, whereas histamine had the same potency. This confirms the role of the histamine H2-receptor and suggests a difference between the species concerning receptor affinity.

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 Pharmacological curve fitting to analyze cutaneous adrenergic responses

2011 ◽  
Vol 111 (6) ◽  
pp. 1703-1709 ◽  
Author(s):  
Megan M. Wenner ◽  
Thad E. Wilson ◽  
Scott L. Davis ◽  
Nina S. Stachenfeld
Keyword(s):  
Dose Response ◽  
Curve Fitting ◽  
Repeated Measures ◽  
Nonlinear Modeling ◽  
Response Curves ◽  
Data Set ◽  
Sigmoidal Model ◽  
Dose Response Curves ◽  
Cutaneous Vasoconstriction

Although dose-response curves are commonly used to describe in vivo cutaneous α-adrenergic responses, modeling parameters and analyses methods are not consistent across studies. The goal of the present investigation was to compare three analysis methods for in vivo cutaneous vasoconstriction studies using one reference data set. Eight women (22 ± 1 yr, 24 ± 1 kg/m2) were instrumented with three cutaneous microdialysis probes for progressive norepinephrine (NE) infusions (1 × 10−8, 1 × 10−6, 1 × 10−5, 1 × 10−4, and 1 × 10−3 logM). NE was infused alone, co-infused with NG-monomethyl-l-arginine (l-NMMA, 10 mM) or Ketorolac tromethamine (KETO, 10 mM). For each probe, dose-response curves were generated using three commonly reported analyses methods: 1) nonlinear modeling without data manipulation, 2) nonlinear modeling with data normalization and constraints, and 3) percent change from baseline without modeling. Not all data conformed to sigmoidal dose-response curves using analysis 1, whereas all subjects' curves were modeled using analysis 2. When analyzing only curves that fit the sigmoidal model, NE + KETO induced a leftward shift in ED50 compared with NE alone with analyses 1 and 2 ( F test, P < 0.05) but only tended to shift the response leftward with analysis 3 (repeated-measures ANOVA, P = 0.08). Neither maximal vasoconstrictor capacity (Emax) in analysis 1 nor %change CVC change from baseline in analysis 3 were altered by blocking agents. In conclusion, although the overall detection of curve shifts and interpretation was similar between the two modeling methods of curve fitting, analysis 2 produced more sigmoidal curves.

In vivo evaluation of the effectiveness of bronchial thermoplasty with computed tomography

2005 ◽  
Vol 98 (5) ◽  
pp. 1603-1606 ◽  
Author(s):  
Robert H. Brown ◽  
William Wizeman ◽  
Christopher Danek ◽  
Wayne Mitzner
Keyword(s):  
Computed Tomography ◽  
Smooth Muscle ◽  
Dose Response ◽  
Response Curves ◽  
High Resolution Computed Tomography ◽  
In Vivo Evaluation ◽  
Airway Narrowing ◽  
Bronchial Thermoplasty ◽  
Dose Response Curves

A recent study has reported that the application of thermal energy delivered through a bronchoscope (bronchial thermoplasty) impairs the ability of airway smooth muscle to shorten in response to methacholine (MCh)(Danek CJ, Lombard CM, Dungworth DL, Cox PG, Miller JD, Biggs MJ, Keast TM, Loomas BE, Wizeman WJ, Hogg JC, and Leff AR. J Appl Physiol 97: 1946–1953, 2004). If such a technique is successful, it has the potential to serve as a therapy to attenuate airway narrowing in asthmatic subjects regardless of the initiating cause that stimulates the smooth muscle. In the present study, we have applied high-resolution computed tomography to accurately quantify the changes in airway area before and after a standard MCh aerosol challenge in airways treated with bronchial thermoplasty. We studied a total of 193 airways ranging from 2 to 15 mm in six dogs. These were divided into treated and control populations. The MCh dose-response curves in untreated airways and soon-to-be-treated airways were superimposable. In contrast, the dose-response curves in treated airways were shifted upward at all points, showing a significantly decreased sensitivity to MCh at both 2 and 4 wk posttreatment. These results thus show that treated airways have significantly increased luminal area at any dose of inhaled MCh compared with untreated airways. The work in this study thus supports the underlying concept that impairing the smooth muscle may be an effective treatment for asthma.

scholarly journals A Model Mechanism Based Explanation of an In Vitro-In Vivo Disconnect for Improving Extrapolation and Translation

2017 ◽  
Author(s):  
Andrew K. Smith ◽  
Yanli Xu ◽  
Glen E.P. Ropella ◽  
C. Anthony Hunt
Keyword(s):  
Dose Response ◽  
Mitochondrial Damage ◽  
System Level ◽  
Central Vein ◽  
Response Curves ◽  
Causal Explanations ◽  
Dose Response Curves ◽  
Virtual Culture

AbstractAn improved understanding of in vivo-to-in vitro hepatocyte changes is crucial to interpreting in vitro data correctly and further improving hepatocyte-based in vitro-to-in vivo extrapolations to human targets. We demonstrate using virtual experiments as a means to help untangle plausible causes of inaccurate extrapolations. We start with virtual mice that have biomimetic software livers. Earlier, using those mice, we discovered model mechanisms that enabled achieving quantitative validation targets while also providing plausible causal explanations for temporal characteristics of acetaminophen hepatotoxicity. We isolated virtual hepatocytes, created a virtual culture, and then conducted dose-response experiments in both culture and mice. We expected the two dose-response curves to be displaced. We were surprised that they crossed because it evidenced that simulated acetaminophen metabolism and toxicity are different for virtual culture and mouse contexts even though individual hepatocyte mechanisms were unchanged. Crossing dose-response curves is a virtual example of an in vivo-to-in vitro disconnect. We use detailed results of experiments to explain the disconnect. Individual hepatocytes contribute differently to system level phenomena. In liver, hepatocytes are exposed to acetaminophen sequentially. Relative production of the reactive acetaminophen metabolite is largest (smallest) in pericentral (periportal) hepatocytes. Because that sequential exposure is absent in culture, hepatocytes from different lobular locations do not respond the same. A virtual Culture-to-Mouse translation can stand as a scientifically challengeable theory explaining an in vitro-in vivo disconnect. It provides a framework to develop more reliable interpretations of in vitro observations, which then may be used to improve extrapolations.AbbreviationsaHPCanalog hepatocyteAPAPacetaminophenCVCentral VeinSSsinusoidal segmentNAPQIN-acetyl-p-benzoquinone iminemitoDmitochondrial damage productsnonMDnon-mitochondrial damage products

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.

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