Clinical and Laboratory Validation of the 2nd International Standard of Low Molecular Weight Heparin (IS 2003-01/608) in the Anti-Xa, Anti-LIA Heptest and Prothrombinase Induced Clotting (PiCT) Tests.

Background: Because of the relative insensitivity of the global clot based assays such as the activated partial thromboplastin time (APTT), the low molecular weight heparins (LMWHs) are potency evaluated/optimized in the anti-Xa (AXa) and heptest. A new clot based assay namely, prothrombin induced clotting time (PiCT) is sensitive to the anticoagulant effects of LMWHs and related drugs. As the LMWHs are standardized using the anti-Xa methods, using the International Standards, this study was designed to cross validate the 2nd International Standard for LMWHs(NIBSC 01/608) in various assay methods.

Methods: Commercially available LMWHs, Dalteparin (D), Enoxaparin (E), Tinzaparin (T) and the 1st International Standard (85/600) were crossed referenced against the 2nd International Standard (NIBSC 01/608) using an amidolytic AXa method. Each of these LMWHs were compared in the AXa, adjusted concentration range of 0–1.0 U/ml using the Heptest, AXa, AIIa and PiCT. In addition plasma samples from patients receiving a LMWH, E for therapeutic and interventional purposes were measured using various tests.

Results: The AXa potency adjusted LMWHs (D, E, and T) and 1st International Standard provided superimposable concentration curves in the amidolytic AXa assays. However marked differences in the heptest and PiCT were noted. Major differences were noted in the AIIA levels, even between the two International standards. When patients samples (n=75) from a therapeutic trial (1.0 mg/kg BID/SC) were evaluated, assay based differences were further amplified. The amidolytic AXa assay consistently measured higher AXa levels. When the two standards were cross-referenced with one another in different assays, major differences were noted in the clot-based assays. Even in the AXa assay at equivalent AXa levels, differences were obvious.

Conclusions: These results suggest that both of the International Standards of LMWH are valid for only the cross standardization of the AXa activities of LMWHs. If any of the other methods were used, significantly different results were obtained with each of the individual LMWHs. Thus, the 2nd International Standard should only be used for amidolytic AXa assay for potency referencing purposes. Moreover, the stated potency of the 2nd Internation Standard may need to be readjusted against the 1st Standard to obtain valid results. These standards are of limited value in the clinical monitoring of LMWHs. It is therefore proposed that each of the LMWHs should be cross referenced by its own standard and the clinical monitoring of these drugs should only be carried out utilizing the specific drug used in a given patient. The PiCT test offers a global test which is capable of monitoring the effects of all components of heparins regardless of their affinity to serpines. Moreover, the effect of TFPI released on clotting processes is also measured. Thus, the PiCT test provides a physiologically relevant anticoagulant index.