Chromogenix Coatest® Heparin

Factor X or anti-Xa Assay. Which Do I Use?

David L. McGlasson, MS, MLS(ASCP)^cm, GA Fritsma, MS, MLS(ASCP)^cm
59th Clinical Research Division
JBSA Lackland, TX

HIT, defined by the presence of heparin-dependent IgG antibodies, is characterized by a decrease in platelet count shortly after starting heparin, which resolves after stopping heparin and is not due to any other apparent cause. Mild HIT occurring within 2-3 days is due to a direct effect of heparin on platelets and is not immune-mediated nor associated with thrombosis. Severe thrombocytopenia, usually occurring a few days later, is associated with both arterial and venous thrombosis and is immune-mediated. In most cases, it results from antibody formation to heparin-platelet factor 4 (PF4) complexes, but in about 10% of cases heparin appears to bind to pre-existing antibodies. Although LMW heparin seems to cause fewer incidences of HIT, it should not replace UF heparin therapy if HIT is already suspected. It may exhibit in vitro and in vivo cross-reactivity with UF heparin-dependent antibodies. Therefore, when HIT is suspected, other anticoagulant options must be explored.

Keep in mind the final concentration of heparin is 0.1 IU/ml plasma. Following the package insert, there is a known concentration of 0.1 IU/ml heparin in the standard, regardless of the dilution, provided the samples are all treated the same way. 100 ml of the 0.1 IU/ml heparin dilution is equivalent to a heparin concentration of 0.1 IU/ml plasma in the assay. This concentration should be compared to the patient-sample. If the analyst takes 100 ml of the heparin dilution (0.1 IU/ml) and dilutes it 1:10, it is comparable to 100 ml of the patient plasma also diluted in the same way. If the analyst get the same absorbance for the sample as for the standard, it contains the same amount of heparin. The important thing is the analyst must always treat patient samples in the same manner as the standards.

In some clinical situations such as sepsis, DIC, and cancer, the plasma itself could contain enzymes that might be able to hydrolyze S-2222™. If that happened, background activity could be seen and would therefore lead to an underestimation of the heparin level in the sample. To rule out background activity in the sample, the assay can be run without FXa and instead, and equal volume of buffer is substituted. The absorbance obtained is then subtracted from the absorbance from the normal run.

The USP states that the activity of heparin sodium and heparin calcium should be determined by both a clotting assay and a chromogenic assay. The chromogenic assay consists essentially in the measurement of the anti-FXa activity of the test preparation against the USP Heparin Sodium Reference Standard. All of the chromogenix heparin kits meet this specification. Antithrombin, FXa, and the chromogenic substrates from Chromogenix are suitable for the USP guidelines. The anti-FXa assays are more specific since they measure the ability of heparin-accelerated antithrombin to inhibit a single enzyme. Either plasma or purified AT can be used. More precise determination of unfractionated heparin and low molecular weight heparin are possible.

When injected subcutaneously, the bioavailability of UF ranges from 10-90%, whereas the bioavailability of LMW heparin is greater than 90% and is independent of dose. LMW heparins exhibit much less binding to plasma proteins than UF heparin, and do not accumulate in the liver or spleen, giving them a longer plasma half-life. The dose-response curve of LMW heparin also tends to be linear.

The therapeutic range for UF heparin is 0.3-0.7 IU/ml, while the range for LMW heparin is less clearly defined. Some clinicians maintain that is 0.4-1.1 IU/ml, or more conservatively, 0.5-1.0 IU/ml (anti-FXa method).

Slow protease-antithrombin interactions are enhanced dramatically in the presence of certain sulfated polysaccharides like heparan sulfate. Heparin is a commercial preparation of heparan sulfate, and binds antithrombin, the major inhibitor of coagulation in plasma and thrombin, thereby catalyzing the thrombin-AT reaction. Binding to antithrombin induces a conformational change in AT that facilitates its reaction with thrombin. Thrombin binds to heparin in a non-specific manner and slides along the chain until it encounters the bound AT. The affinity of heparin to the thrombin-AT (TAT) complex is much lower than to free AT. Heparin will therefore dissociate from the TAT complex, which is rapidly removed from the blood circulation by the liver and the result is a stable protease inhibitor complex, which is rapidly removed and catabolized. The anti-FXa assays are more specific since they measure the ability of heparin-accelerated antithrombin to inhibit a single enzyme. Either plasma or purified AT can be used. More precise determination of unfractionated heparin and low molecular weight heparin are possible.

Antithrombin is the most important natural inhibitor of the coagulation cascade, accounting for approximately 80% of the thrombin inhibitory activity in plasma. By inhibiting the coagulation proteases, especially thrombin, FXa, and FIXa, AT prevents uncontrolled coagulation and thrombosis. Inhibition of antithrombin involves the formation of a stable 1:1 complex between the active domain of the serine protease such as thrombin, and the reactive site of antithrombin, which proteases initially recognize as a substrate. During the cleavage of the reactive site bond in antithrombin, a conformational change occurs in the inhibitor that traps the protease.

Slow protease-antithrombin interactions are enhanced dramatically in the presence of certain sulfated polysaccharides like heparan sulfate. Heparin is a commercial preparation of heparan sulfate, and binds antithrombin and thrombin, thereby catalyzing the thrombin-AT reaction. Binding to antithrombin induces a conformational change in AT that facilitates its reaction with thrombin. Thrombin binds to heparin in a non-specific manner and slides along the chain until it encounters the bound AT. The affinity of heparin to the thrombin-AT (TAT) complex is much lower than to free AT. Heparin will therefore dissociate from the TAT complex, which is rapidly removed from the blood circulation by the liver.