Assay Discrepancies with Highly Purified Factor VIII Concentrates

Dawson, N J; Kemball‐Cook, Geoffrey; Barrowcliffe, T W

doi:10.1159/000215905

Cited by 28 publications

(27 citation statements)

References 9 publications

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“…2b). Wide variations between the results obtained by chromogenic and onestage FVIII clotting assays were described previously [5,13,26]. These variations are supposed to be caused by interlaboratory variations and by the use of different standards.…”

Section: Discussionmentioning

confidence: 92%

Analysis of expression kinetics and activity of a new B-domain truncated and full-length FVIII protein in three different cell lines

Haack

Schmitt

Poller

et al. 1999

Annals of Hematology

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Transient expression of full-length wild-type (wt) and a new B-domain truncated (deltaB) FVIII has been investigated in three eukaryotic cell lines (HEK-293, COS, CHO). When expressed in CHO cells, both FVIII proteins reached the same peak antigen levels, whereas in HEK-293 and COS cells those of FVIII/deltaB were up to sixfold those of FVIII/wt. Investigation of specific activity of the recombinant FVIII proteins using a chromogenic and a one-stage assay in addition to the FVIII-antigen ELISA revealed large variations: In HEK-293 cells specific activity of FVIII/deltaB measured with both assays was higher than that of FVIII/wt. In COS cells specific activity of both FVIII proteins was higher measured in the one-stage assay than in the chromogenic assay. In CHO cells both FVIII proteins had similar specific activity in each assay. In summary, expression kinetics and specific activity of conditioned medium vary depending on cell type used.

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Section: Discussionmentioning

confidence: 92%

Analysis of expression kinetics and activity of a new B-domain truncated and full-length FVIII protein in three different cell lines

Haack

Schmitt

Poller

et al. 1999

Annals of Hematology

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“…Bei dem Einsatz von Faktor VIII-Mangelplasmen mit oder ohne von Willebrandfaktor waren jedoch bei Patienten mit von Willebrand-Syndrom keine Unterschiede in den Faktor VIII-Aktivitäten feststellbar [13]. Dagegen wurden niedrige Aktivitäten in einigen Faktor VIII-Konzentrattypen und bei Patienten, die mit bestimmten Konzentraten behandelt worden waren, dann beobachtet, wenn mit immundepletierten Mangelplasmen ohne von Willebrandfaktor gearbeitet wurde [5,14]. Schließlich liegen Beobachtungen darüber vor, daß eine korrekte quantitative Bestimmung der Wirkung humaner Faktor VHI-Inhibitoren auf tierischen Faktor VIII nur durch Verdünnung der tierischen Plasmen mit Faktor VIII-Mangelplasmen, die ausreichende Mengen an von Willebrandfaktor enthielten, möglich war [15].…”

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Gerinnungsfaktorenanalysen in diagnostisch angewendeten kommerziellen Faktor-VIII- und Faktor-IX-Mangelplasmen. Commercial factor VIII and factor IX depleted plasmas: analysis of clotting factors

Lutze¹,

Kunstmann²

2001

LaboratoriumsMedizin

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Zusammenfassung: Native und immunadsorbierte Faktor VIII-und Faktor IX-Mangelplasmen von 4 Herstellern, die zu diagnostischen Zwecken für koagulometrische Faktor VIII-und Faktor IX-Aktivitätsbe-stimmungen eingesetzt werden, wurden gerinnungsanalytisch untersucht. Bei allen Mangelplasmen lagen die Restaktivitäten des depletierten Faktors unter l % der Norm. Die für koagulometrische Untersuchungen erforderlichen Begleitfaktoren hatten überwiegend Aktivitäten innerhalb der Referenzbereiche, mindestens jedoch 50 % der Norm. Wesentliche Unterschiede zwischen den Mangelplasmen bestanden lediglich bei den Faktor VIII-Mangelplasmen im Vorhandensein oder Fehlen des von Willebrandfaktors. Schlüsselwörter: Gerinnungsuntersuchungen; Mangelplasma; Faktor VIII; Faktor IX; Restaktivität; von Willebrandfaktor.Summary: Native and immunodepleted factor VIII or IX deficient plasmas from 4 different commercial suppliers were examined in clotting factor analysis. Compared to standard plasma, the residual activity of the depleted factor was below 1 % in all examined plasmas. The activity of the additional factors needed for successful coagulation was mainly in normal range, but at least 50 % of normal. Only the factor VIII deficient plasmas differed from each other due to presence or absence of von Willebrand factor.

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“…Activation has been shown to occur with other coagulant enzymes including factor IXa and factor Xa [13,14], It is assumed that the actiThe determination of the quantity and quality of factor VIII in therapeutic preparations is of concern since this will impact on the therapeutic effectiveness of the factor VIII infused. There are a variety of known variables leading to skewed assay results [1][2][3][4][5][6][7]. These include degree of purity, type of test system, and the amount of von Willebrand factor (vWf) present.…”

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confidence: 99%

Activated Factor VIII in Therapeutic Preparations: Analysis by Ultracentrifugation

Aronson¹,

Chang²

1996

Vox Sanguinis

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Activation of factor VIII is signaled by an increase in the 1-stage factor VIII activity and release from von Willebrand factor. Ultracentrifugation in 10—40% sucrose gradients was used to identify such activation in therapeutic concentrates. Plasma-derived factor VIII lots were examined and factor VIII sedimenting independently of von Willebrand factor was identified in some of the preparations. In addition there was slower sedimentation of factor VIII by the 1-stage assay than by the chromogenic assay. These results are consistent with a factor VIII cleaved at residue 1689, a site important for von Willebrand binding. This activated form leads to some of the assay discrepancies between the 1-stage assay and the chromogenic or the 2-stage assay. There is more rapid sedimentation of the factor VIII measured by the chromogenic assay than the von Willebrand factor in some manufacturers’ samples indicating that the method of fractionation may select a low molecular weight von Willebrand factor which does not bind factor VIII. Routine comparison between the 1-stage and chromogenic assays during fractionation may be able to identify such activated preparations. Other assay discrepancies may be due to structural differences between the standards and the tested product.

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Assay Discrepancies with Highly Purified Factor VIII Concentrates

Cited by 28 publications

References 9 publications

Analysis of expression kinetics and activity of a new B-domain truncated and full-length FVIII protein in three different cell lines

Analysis of expression kinetics and activity of a new B-domain truncated and full-length FVIII protein in three different cell lines

Gerinnungsfaktorenanalysen in diagnostisch angewendeten kommerziellen Faktor-VIII- und Faktor-IX-Mangelplasmen. Commercial factor VIII and factor IX depleted plasmas: analysis of clotting factors

Activated Factor VIII in Therapeutic Preparations: Analysis by Ultracentrifugation

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