Abstract Number: PB0375
Meeting: ISTH 2021 Congress
Background: The Fibrin Polymerization (FP) and the Coag-Lysis (CL) assays are fully automated, for the turbidimetric measurement of the Fibrin Polymerization profile and of the Coagulation-Fibrinolysis balance, on STA®-R prototype. The FP is used to improve venous thromboembolism (VTE) diagnosis(1) and the CL assay could help discriminate patients on their clot properties, in bleeding/thrombotic tendency(2).
In some clinical indications such as prevention of thrombosis or VTE treatment, patients could receive low-molecular-weight heparin (LMWH). FP and CL are adversely affected by LMWH. Polybrene (Pb) and Heparinase I (Hep) have been shown to be effective for heparin neutralization(3).
Aims: To design automated FP and CL assays, with reagent neutralizing LMWH, allowing to restore Fibrin and/or Lysis parameters in samples from patients treated with LMWH. Select the best neutralizing agent based on tests performances.
Methods: FP and CL were assessed on STA®-R prototype using the PPP Reagent Low® with (CL) or without (FP) tissue Plasminogen Activator. The reagents were reconstituted with different concentrations of Pb or Hep (Table 1).
Normal Pooled Plasma (NPP) were spiked with different levels of tinzaparin, enoxaparin or dalteparin (Table 1) to cover an anti-FXa activity range up to 1.85IU/mL.
| Performances | Sample | LMWH Anti-Xa Concentrations | Neutralizing agent | Analytical Result |
|---|---|---|---|---|
| Dose-response | NPP | ≤ 0.10 IU/mL | CL : Pb : [0-30] µg/mL ; 9 levels Hep : [0.07-0.27] IU/mL ; 4 levels |
Relative deviation to CL and FP |
| FP : Pb : [0-85] µg/mL ; 11 levels Hep : [0.13-0.27] IU/mL ; 3 levels |
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| Neutralization performance on spiked NPP | Spiked plasmas | Tinzaparin : [0-0.89] IU/mL ; 5 levels | CL: Hep : [0.07-0.27] IU/mL ; 4 levels FP: Hep : [0.13-0.27] IU/mL ; 3 levels |
Relative deviation to CL and FP |
| Tinzaparin : [0-1.64] IU/mL ; 6 levels | CL : Pb : [0-10] µg/mL ; 4 levels FP : Pb : [0-20] µg/mL ; 6 levels |
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| Dalteparin : [0-0.94] IU/mL ; 3 levels Enoxaparin : [0-0.94] IU/mL ; 3 levels |
CL and FP : Hep : 0.13 IU/mL | |||
| On board stability of reagent | Spiked plasmas | Tinzaparin : [0-0.63] IU/mL ; 2 levels | CL and FP : Hep : 0.13 IU/mL | Relative deviation of T4 hours to T0 |
Evaluation of neutralizing agents in FP and CL reagent with citrated plasma samples.
Results: Table 2 provides summary of assay performances.
| Performances | Results |
|---|---|
| Dose-response | Pb had no impact on CL and FP assays of NPP at concentrations ≤ 10 µg/mL and ≤ 30 µg/mL respectively. Hep had no impact (< 10%) on CL and FP assays of NPP regardless to the tested concentrations. |
| Neutralization performance on spiked NPP | 20 µg/mL of Pb neutralized the effect of tinzaparin up to 0.78 IU/mL on FP assays but modified lysis parameters of CL above 8.5 µg/mL. 0.13 IU/mL of Hep completely neutralized the effect of tinzaparin at 0.89IU/mL but only up to 0.43 IU/mL of dalteparin and enoxaparin on CL and FP assays. |
| On board stability of intermediate reagent | On board stability with 0.13 IU/mL of Hep was up to 2h on CL and FP assay with and without tinzaparin. |
Summary of performance evaluation.
Conclusions: Our results show that 0.13IU/mL of Hep can neutralize the anticoagulant effect up to 0.89IU/mL for tinzaparin and up to 0.43IU/mL for dalteparin and enoxaparin. At these concentrations, neutralization allowed to restore FP and CL parameters in plasma samples. Performance is reduced with Pb.
These in vitro results indicate that Hep is the neutralizing agent of choice to abolish LMWH interference and assess FP or CL profile of patients under LMWH treatment.
(1) Contant G, et al. Res Pract Thromb Haemost 2017.
(2) Pieters M, et al. J Thromb Haemost. 2018.
(3) Hutt ED, et al. J Lab Clin Med 1972.
To cite this abstract in AMA style:
Bildan M-, Lambert M, Fontaine S, Mathieu O, Contant G. In vitro Neutralization of Low Molecular Weight Heparins for the Measurement of Fibrin Polymerization Profile and Coagulation-fibrinolysis Balance [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/in-vitro-neutralization-of-low-molecular-weight-heparins-for-the-measurement-of-fibrin-polymerization-profile-and-coagulation-fibrinolysis-balance/. Accessed June 25, 2022.« Back to ISTH 2021 Congress
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