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Accurate measurement of extended half-life and unmodified factor VIII low levels with one-stage FVIII assays is dependent on the matrix of calibration curves.
Haemophilia. 2019 Jan; 25(1):e19-e26.H

Abstract

INTRODUCTION

The monitoring of factor VIII (FVIII) replacement therapy relies on the accurate measurement of FVIII activity over a large concentration range. However, unexplained overestimation of low FVIII levels has recently been reported with extended half-life recombinant FVIIIs.

AIM

The objective of this study was to confirm previous publications indicating that the reagents used to generate the calibration curves determine the accuracy of the measurement of low FVIII levels.

METHODS

We generated FVIII calibration curves with FVIII-deficient plasmas or a commercial diluent buffer. We then measured FVIII levels in FVIII-deficient plasma spiked with plasma FVIII, a full-length recombinant FVIII (Advate® , Shire, Brussels, Belgium) and two extended half-life recombinant FVIIIs (Elocta® , Swedish Orphan Biovitrum, Woluwe Saint-Lambert, Belgium and Afstyla® , CSL Behring, Mechelen, Belgium). FVIII levels were also analyzed in spiked samples prediluted two- and fourfold, either in diluent buffer or in FVIII-deficient plasma to evaluate parallelism.

RESULTS

Coagulation times of calibration curves generated with diluent buffer were longer than those with FVIII-deficient plasmas. This resulted in an overestimation of FVIII levels lower than 25 IU/dL in spiked samples and in detection of FVIII activity (≥1 IU/dL) in FVIII-deficient plasma. Predilution of samples with diluent buffer rather than with FVIII-deficient plasma also led to discordant results.

CONCLUSION

Our data confirm that the generation of calibration curves by dilution in FVIII-deficient plasma is crucial for the accurate measurement of low FVIII levels. When serial dilutions of samples are analyzed, predilution in FVIII-deficient plasma is required to respect the parallelism criteria. These methods should be more generally implemented for coagulation instruments.

Authors+Show Affiliations

Clinical Department of Laboratory Medicine, University Hospitals of Leuven, Leuven, Belgium.Clinical Department of Laboratory Medicine, University Hospitals of Leuven, Leuven, Belgium.Clinical Department of Laboratory Medicine, University Hospitals of Leuven, Leuven, Belgium.Clinical Department of Laboratory Medicine, University Hospitals of Leuven, Leuven, Belgium.Clinical Department of Laboratory Medicine, University Hospitals of Leuven, Leuven, Belgium.Clinical Department of Laboratory Medicine, University Hospitals of Leuven, Leuven, Belgium.Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium. Vascular Medicine and Hemostasis, University Hospitals of Leuven, Leuven, Belgium.Clinical Department of Laboratory Medicine, University Hospitals of Leuven, Leuven, Belgium. Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

30589148

Citation

Van den Bossche, Dorien, et al. "Accurate Measurement of Extended Half-life and Unmodified Factor VIII Low Levels With One-stage FVIII Assays Is Dependent On the Matrix of Calibration Curves." Haemophilia : the Official Journal of the World Federation of Hemophilia, vol. 25, no. 1, 2019, pp. e19-e26.
Van den Bossche D, Toelen J, Schoeters J, et al. Accurate measurement of extended half-life and unmodified factor VIII low levels with one-stage FVIII assays is dependent on the matrix of calibration curves. Haemophilia. 2019;25(1):e19-e26.
Van den Bossche, D., Toelen, J., Schoeters, J., Van Horenbeeck, I., Vanlinthout, I., Debasse, M., Peerlinck, K., & Jacquemin, M. (2019). Accurate measurement of extended half-life and unmodified factor VIII low levels with one-stage FVIII assays is dependent on the matrix of calibration curves. Haemophilia : the Official Journal of the World Federation of Hemophilia, 25(1), e19-e26. https://doi.org/10.1111/hae.13656
Van den Bossche D, et al. Accurate Measurement of Extended Half-life and Unmodified Factor VIII Low Levels With One-stage FVIII Assays Is Dependent On the Matrix of Calibration Curves. Haemophilia. 2019;25(1):e19-e26. PubMed PMID: 30589148.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Accurate measurement of extended half-life and unmodified factor VIII low levels with one-stage FVIII assays is dependent on the matrix of calibration curves. AU - Van den Bossche,Dorien, AU - Toelen,Jelle, AU - Schoeters,Joke, AU - Van Horenbeeck,Isa, AU - Vanlinthout,Ingrid, AU - Debasse,Mirjam, AU - Peerlinck,Kathelijne, AU - Jacquemin,Marc, Y1 - 2018/12/27/ PY - 2018/08/02/received PY - 2018/11/12/revised PY - 2018/11/12/accepted PY - 2018/12/28/pubmed PY - 2019/4/30/medline PY - 2018/12/28/entrez KW - FVIII one-stage FVIII assay KW - extended half-life FVIII KW - factor VIII KW - haemophilia A KW - parallelism SP - e19 EP - e26 JF - Haemophilia : the official journal of the World Federation of Hemophilia JO - Haemophilia VL - 25 IS - 1 N2 - INTRODUCTION: The monitoring of factor VIII (FVIII) replacement therapy relies on the accurate measurement of FVIII activity over a large concentration range. However, unexplained overestimation of low FVIII levels has recently been reported with extended half-life recombinant FVIIIs. AIM: The objective of this study was to confirm previous publications indicating that the reagents used to generate the calibration curves determine the accuracy of the measurement of low FVIII levels. METHODS: We generated FVIII calibration curves with FVIII-deficient plasmas or a commercial diluent buffer. We then measured FVIII levels in FVIII-deficient plasma spiked with plasma FVIII, a full-length recombinant FVIII (Advate® , Shire, Brussels, Belgium) and two extended half-life recombinant FVIIIs (Elocta® , Swedish Orphan Biovitrum, Woluwe Saint-Lambert, Belgium and Afstyla® , CSL Behring, Mechelen, Belgium). FVIII levels were also analyzed in spiked samples prediluted two- and fourfold, either in diluent buffer or in FVIII-deficient plasma to evaluate parallelism. RESULTS: Coagulation times of calibration curves generated with diluent buffer were longer than those with FVIII-deficient plasmas. This resulted in an overestimation of FVIII levels lower than 25 IU/dL in spiked samples and in detection of FVIII activity (≥1 IU/dL) in FVIII-deficient plasma. Predilution of samples with diluent buffer rather than with FVIII-deficient plasma also led to discordant results. CONCLUSION: Our data confirm that the generation of calibration curves by dilution in FVIII-deficient plasma is crucial for the accurate measurement of low FVIII levels. When serial dilutions of samples are analyzed, predilution in FVIII-deficient plasma is required to respect the parallelism criteria. These methods should be more generally implemented for coagulation instruments. SN - 1365-2516 UR - https://www.unboundmedicine.com/medline/citation/30589148/Accurate_measurement_of_extended_half_life_and_unmodified_factor_VIII_low_levels_with_one_stage_FVIII_assays_is_dependent_on_the_matrix_of_calibration_curves_ L2 - https://doi.org/10.1111/hae.13656 DB - PRIME DP - Unbound Medicine ER -