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Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers
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| Zeitschriftentitel: | Blood |
|---|---|
| Personen und Körperschaften: | , , , , , , |
| In: | Blood, 134, 2019, Supplement_1, S. 1153-1153 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Society of Hematology
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| Schlagwörter: |
| author_facet |
Khan, Anuja Shishko, Krisjela Arcidiacono, Sam Bui, Khanh Lu, Genmin Conley, Pamela B. Winkler, Anne Khan, Anuja Shishko, Krisjela Arcidiacono, Sam Bui, Khanh Lu, Genmin Conley, Pamela B. Winkler, Anne |
|---|---|
| author |
Khan, Anuja Shishko, Krisjela Arcidiacono, Sam Bui, Khanh Lu, Genmin Conley, Pamela B. Winkler, Anne |
| spellingShingle |
Khan, Anuja Shishko, Krisjela Arcidiacono, Sam Bui, Khanh Lu, Genmin Conley, Pamela B. Winkler, Anne Blood Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers Cell Biology Hematology Immunology Biochemistry |
| author_sort |
khan, anuja |
| spelling |
Khan, Anuja Shishko, Krisjela Arcidiacono, Sam Bui, Khanh Lu, Genmin Conley, Pamela B. Winkler, Anne 0006-4971 1528-0020 American Society of Hematology Cell Biology Hematology Immunology Biochemistry http://dx.doi.org/10.1182/blood-2019-126254 <jats:p>Background: Andexanet alfa (AnXa) is a modified human FXa protein approved in the US and EU for reversal of direct factor Xa (FXa) inhibitors (apixaban, rivaroxaban) due to life-threatening or uncontrolled bleeding. AnXa reverses the anticoagulant effect of FXa inhibitors by binding and sequestering FXa inhibitor. Commercially available anti-FXa assays measure FXa inhibitors using drug specific calibrators and controls. However, there are limitations when these assays are used for measuring AnXa-containing patient samples. One of the limitations is the large sample dilution in the assay setup, which causes dissociation of the inhibitor from the AnXa-inhibitor complex (due to the reversible binding equilibrium of the AnXa-inhibitor), resulting in an erroneous elevation of the anti-FXa activity following AnXa administration. We have developed modified assay parameters for the drug-specific anti-FXa activity assays, apixaban and rivaroxaban, on the ACL TOP Family instruments, which minimizes the effect of sample dilution suitable for analyzing AnXa-containing samples.</jats:p> <jats:p>Methods: Pooled normal human plasma was spiked with different concentrations of apixaban (230, 460, 920 ng/mL, 0.5 - 2.0 μM) or rivaroxaban (218, 436, 872 ng/mL, 0.5 - 2.0 μM) and AnXa at different AnXa:inhibitor molar ratios (0, 0.5,1.0, 2.0). The reversal activity of AnXa for apixaban and rivaroxaban was measured by a modified anti-FXa assay in a 96-well format previously used in the AnXa preclinical and clinical studies. The same set of contrived samples was assessed using the apixaban and rivaroxaban assays on the ACL TOP 700 with HemosIL Anti-Xa with HemosIL Apixaban or HemosIL Rivaroxaban Calibrators and Controls according to the manufacturer's recommendations. The ACL TOP 700 assay parameters were modified with an optimized plasma to reagent volume ratio to reduce the overall sample dilution. The calibration curves for both assays were optimized to report in the 0-100 ng/mL range.</jats:p> <jats:p>Results: When assessed by modified anti-FXa assays in the 96-well format (with an overall 2-fold sample dilution), AnXa dose-dependently reversed the anti-FXa activity of both apixaban and rivaroxaban spiked in plasma. % Reversal increased as a function of the molar ratios (AnXa:inhibitor) with ~50% (1:2), > 95% (1:1), and >99% reversal (2:1) respectively. Conversely, the Anti-FXa setup with the HemosIL Anti-Xa reagents and calibrators showed <75% reversal even with a molar excess of AnXa over the inhibitors (2:1) due to the large sample dilution in the overall reaction mixture. The calibrations of the new modified assays, in the range of 0-100 ng/mL, reported %CV <6% across all levels on the ACL TOP 700. The apixaban and rivaroxaban low controls recovered within the corresponding product insert range. The low controls were diluted to three additional levels and recovered in the modified assays as expected. Preliminary data on the AnXa-containing samples with the modified Anti-Xa assays on the ACL TOP 700 instrument showed similar or slightly lower percent reversal compared to the 96-well format manual method, warranting further verification of the modified test on the ACL TOP Family instruments.</jats:p> <jats:p>Conclusions: The modified parameters for apixaban and rivaroxaban on the ACL TOP Family instruments minimized the dilution effect seen in the commercially released assays for the AnXa-containing samples and the data showed a similar trend in % reversal compared to the 96-well plate manual assays.</jats:p> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>Bui: Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Lu:Portola Pharmaceuticals: Employment, Equity Ownership. Conley:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership.</jats:p> </jats:sec> Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers Blood |
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American Society of Hematology, 2019 |
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| title |
Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers |
| title_unstemmed |
Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers |
| title_full |
Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers |
| title_fullStr |
Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers |
| title_full_unstemmed |
Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers |
| title_short |
Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers |
| title_sort |
modified anti-fxa assays for measuring the residual activity of apixaban and rivaroxaban in andexanet alfa-containing samples on the acl top family® coagulation analyzers |
| topic |
Cell Biology Hematology Immunology Biochemistry |
| url |
http://dx.doi.org/10.1182/blood-2019-126254 |
| publishDate |
2019 |
| physical |
1153-1153 |
| description |
<jats:p>Background: Andexanet alfa (AnXa) is a modified human FXa protein approved in the US and EU for reversal of direct factor Xa (FXa) inhibitors (apixaban, rivaroxaban) due to life-threatening or uncontrolled bleeding. AnXa reverses the anticoagulant effect of FXa inhibitors by binding and sequestering FXa inhibitor. Commercially available anti-FXa assays measure FXa inhibitors using drug specific calibrators and controls. However, there are limitations when these assays are used for measuring AnXa-containing patient samples. One of the limitations is the large sample dilution in the assay setup, which causes dissociation of the inhibitor from the AnXa-inhibitor complex (due to the reversible binding equilibrium of the AnXa-inhibitor), resulting in an erroneous elevation of the anti-FXa activity following AnXa administration. We have developed modified assay parameters for the drug-specific anti-FXa activity assays, apixaban and rivaroxaban, on the ACL TOP Family instruments, which minimizes the effect of sample dilution suitable for analyzing AnXa-containing samples.</jats:p>
<jats:p>Methods: Pooled normal human plasma was spiked with different concentrations of apixaban (230, 460, 920 ng/mL, 0.5 - 2.0 μM) or rivaroxaban (218, 436, 872 ng/mL, 0.5 - 2.0 μM) and AnXa at different AnXa:inhibitor molar ratios (0, 0.5,1.0, 2.0). The reversal activity of AnXa for apixaban and rivaroxaban was measured by a modified anti-FXa assay in a 96-well format previously used in the AnXa preclinical and clinical studies. The same set of contrived samples was assessed using the apixaban and rivaroxaban assays on the ACL TOP 700 with HemosIL Anti-Xa with HemosIL Apixaban or HemosIL Rivaroxaban Calibrators and Controls according to the manufacturer's recommendations. The ACL TOP 700 assay parameters were modified with an optimized plasma to reagent volume ratio to reduce the overall sample dilution. The calibration curves for both assays were optimized to report in the 0-100 ng/mL range.</jats:p>
<jats:p>Results: When assessed by modified anti-FXa assays in the 96-well format (with an overall 2-fold sample dilution), AnXa dose-dependently reversed the anti-FXa activity of both apixaban and rivaroxaban spiked in plasma. % Reversal increased as a function of the molar ratios (AnXa:inhibitor) with ~50% (1:2), > 95% (1:1), and >99% reversal (2:1) respectively. Conversely, the Anti-FXa setup with the HemosIL Anti-Xa reagents and calibrators showed <75% reversal even with a molar excess of AnXa over the inhibitors (2:1) due to the large sample dilution in the overall reaction mixture. The calibrations of the new modified assays, in the range of 0-100 ng/mL, reported %CV <6% across all levels on the ACL TOP 700. The apixaban and rivaroxaban low controls recovered within the corresponding product insert range. The low controls were diluted to three additional levels and recovered in the modified assays as expected. Preliminary data on the AnXa-containing samples with the modified Anti-Xa assays on the ACL TOP 700 instrument showed similar or slightly lower percent reversal compared to the 96-well format manual method, warranting further verification of the modified test on the ACL TOP Family instruments.</jats:p>
<jats:p>Conclusions: The modified parameters for apixaban and rivaroxaban on the ACL TOP Family instruments minimized the dilution effect seen in the commercially released assays for the AnXa-containing samples and the data showed a similar trend in % reversal compared to the 96-well plate manual assays.</jats:p>
<jats:sec>
<jats:title>Disclosures</jats:title>
<jats:p>Bui: Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Lu:Portola Pharmaceuticals: Employment, Equity Ownership. Conley:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership.</jats:p>
</jats:sec> |
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| description | <jats:p>Background: Andexanet alfa (AnXa) is a modified human FXa protein approved in the US and EU for reversal of direct factor Xa (FXa) inhibitors (apixaban, rivaroxaban) due to life-threatening or uncontrolled bleeding. AnXa reverses the anticoagulant effect of FXa inhibitors by binding and sequestering FXa inhibitor. Commercially available anti-FXa assays measure FXa inhibitors using drug specific calibrators and controls. However, there are limitations when these assays are used for measuring AnXa-containing patient samples. One of the limitations is the large sample dilution in the assay setup, which causes dissociation of the inhibitor from the AnXa-inhibitor complex (due to the reversible binding equilibrium of the AnXa-inhibitor), resulting in an erroneous elevation of the anti-FXa activity following AnXa administration. We have developed modified assay parameters for the drug-specific anti-FXa activity assays, apixaban and rivaroxaban, on the ACL TOP Family instruments, which minimizes the effect of sample dilution suitable for analyzing AnXa-containing samples.</jats:p> <jats:p>Methods: Pooled normal human plasma was spiked with different concentrations of apixaban (230, 460, 920 ng/mL, 0.5 - 2.0 μM) or rivaroxaban (218, 436, 872 ng/mL, 0.5 - 2.0 μM) and AnXa at different AnXa:inhibitor molar ratios (0, 0.5,1.0, 2.0). The reversal activity of AnXa for apixaban and rivaroxaban was measured by a modified anti-FXa assay in a 96-well format previously used in the AnXa preclinical and clinical studies. The same set of contrived samples was assessed using the apixaban and rivaroxaban assays on the ACL TOP 700 with HemosIL Anti-Xa with HemosIL Apixaban or HemosIL Rivaroxaban Calibrators and Controls according to the manufacturer's recommendations. The ACL TOP 700 assay parameters were modified with an optimized plasma to reagent volume ratio to reduce the overall sample dilution. The calibration curves for both assays were optimized to report in the 0-100 ng/mL range.</jats:p> <jats:p>Results: When assessed by modified anti-FXa assays in the 96-well format (with an overall 2-fold sample dilution), AnXa dose-dependently reversed the anti-FXa activity of both apixaban and rivaroxaban spiked in plasma. % Reversal increased as a function of the molar ratios (AnXa:inhibitor) with ~50% (1:2), > 95% (1:1), and >99% reversal (2:1) respectively. Conversely, the Anti-FXa setup with the HemosIL Anti-Xa reagents and calibrators showed <75% reversal even with a molar excess of AnXa over the inhibitors (2:1) due to the large sample dilution in the overall reaction mixture. The calibrations of the new modified assays, in the range of 0-100 ng/mL, reported %CV <6% across all levels on the ACL TOP 700. The apixaban and rivaroxaban low controls recovered within the corresponding product insert range. The low controls were diluted to three additional levels and recovered in the modified assays as expected. Preliminary data on the AnXa-containing samples with the modified Anti-Xa assays on the ACL TOP 700 instrument showed similar or slightly lower percent reversal compared to the 96-well format manual method, warranting further verification of the modified test on the ACL TOP Family instruments.</jats:p> <jats:p>Conclusions: The modified parameters for apixaban and rivaroxaban on the ACL TOP Family instruments minimized the dilution effect seen in the commercially released assays for the AnXa-containing samples and the data showed a similar trend in % reversal compared to the 96-well plate manual assays.</jats:p> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>Bui: Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Lu:Portola Pharmaceuticals: Employment, Equity Ownership. Conley:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership.</jats:p> </jats:sec> |
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| spelling | Khan, Anuja Shishko, Krisjela Arcidiacono, Sam Bui, Khanh Lu, Genmin Conley, Pamela B. Winkler, Anne 0006-4971 1528-0020 American Society of Hematology Cell Biology Hematology Immunology Biochemistry http://dx.doi.org/10.1182/blood-2019-126254 <jats:p>Background: Andexanet alfa (AnXa) is a modified human FXa protein approved in the US and EU for reversal of direct factor Xa (FXa) inhibitors (apixaban, rivaroxaban) due to life-threatening or uncontrolled bleeding. AnXa reverses the anticoagulant effect of FXa inhibitors by binding and sequestering FXa inhibitor. Commercially available anti-FXa assays measure FXa inhibitors using drug specific calibrators and controls. However, there are limitations when these assays are used for measuring AnXa-containing patient samples. One of the limitations is the large sample dilution in the assay setup, which causes dissociation of the inhibitor from the AnXa-inhibitor complex (due to the reversible binding equilibrium of the AnXa-inhibitor), resulting in an erroneous elevation of the anti-FXa activity following AnXa administration. We have developed modified assay parameters for the drug-specific anti-FXa activity assays, apixaban and rivaroxaban, on the ACL TOP Family instruments, which minimizes the effect of sample dilution suitable for analyzing AnXa-containing samples.</jats:p> <jats:p>Methods: Pooled normal human plasma was spiked with different concentrations of apixaban (230, 460, 920 ng/mL, 0.5 - 2.0 μM) or rivaroxaban (218, 436, 872 ng/mL, 0.5 - 2.0 μM) and AnXa at different AnXa:inhibitor molar ratios (0, 0.5,1.0, 2.0). The reversal activity of AnXa for apixaban and rivaroxaban was measured by a modified anti-FXa assay in a 96-well format previously used in the AnXa preclinical and clinical studies. The same set of contrived samples was assessed using the apixaban and rivaroxaban assays on the ACL TOP 700 with HemosIL Anti-Xa with HemosIL Apixaban or HemosIL Rivaroxaban Calibrators and Controls according to the manufacturer's recommendations. The ACL TOP 700 assay parameters were modified with an optimized plasma to reagent volume ratio to reduce the overall sample dilution. The calibration curves for both assays were optimized to report in the 0-100 ng/mL range.</jats:p> <jats:p>Results: When assessed by modified anti-FXa assays in the 96-well format (with an overall 2-fold sample dilution), AnXa dose-dependently reversed the anti-FXa activity of both apixaban and rivaroxaban spiked in plasma. % Reversal increased as a function of the molar ratios (AnXa:inhibitor) with ~50% (1:2), > 95% (1:1), and >99% reversal (2:1) respectively. Conversely, the Anti-FXa setup with the HemosIL Anti-Xa reagents and calibrators showed <75% reversal even with a molar excess of AnXa over the inhibitors (2:1) due to the large sample dilution in the overall reaction mixture. The calibrations of the new modified assays, in the range of 0-100 ng/mL, reported %CV <6% across all levels on the ACL TOP 700. The apixaban and rivaroxaban low controls recovered within the corresponding product insert range. The low controls were diluted to three additional levels and recovered in the modified assays as expected. Preliminary data on the AnXa-containing samples with the modified Anti-Xa assays on the ACL TOP 700 instrument showed similar or slightly lower percent reversal compared to the 96-well format manual method, warranting further verification of the modified test on the ACL TOP Family instruments.</jats:p> <jats:p>Conclusions: The modified parameters for apixaban and rivaroxaban on the ACL TOP Family instruments minimized the dilution effect seen in the commercially released assays for the AnXa-containing samples and the data showed a similar trend in % reversal compared to the 96-well plate manual assays.</jats:p> <jats:sec> <jats:title>Disclosures</jats:title> <jats:p>Bui: Portola Pharmaceuticals, Inc.: Employment, Equity Ownership. Lu:Portola Pharmaceuticals: Employment, Equity Ownership. Conley:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership.</jats:p> </jats:sec> Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers Blood |
| spellingShingle | Khan, Anuja, Shishko, Krisjela, Arcidiacono, Sam, Bui, Khanh, Lu, Genmin, Conley, Pamela B., Winkler, Anne, Blood, Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers, Cell Biology, Hematology, Immunology, Biochemistry |
| title | Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers |
| title_full | Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers |
| title_fullStr | Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers |
| title_full_unstemmed | Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers |
| title_short | Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers |
| title_sort | modified anti-fxa assays for measuring the residual activity of apixaban and rivaroxaban in andexanet alfa-containing samples on the acl top family® coagulation analyzers |
| title_unstemmed | Modified Anti-FXa Assays for Measuring the Residual Activity of Apixaban and Rivaroxaban in Andexanet Alfa-Containing Samples on the ACL TOP Family® Coagulation Analyzers |
| topic | Cell Biology, Hematology, Immunology, Biochemistry |
| url | http://dx.doi.org/10.1182/blood-2019-126254 |