HEPARIN QUESTIONS
· When exogenous AT is desired, how should it be added in the test?
· Compare hirudin with heparin. How can hirudin level be determined?
· Do you have standards and controls for measuring unfractionated heparin?
HEPARIN ANSWERS
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.
All of the heparin kits can. Obviously, when measuring LMW heparin samples, LMW heparin standard, like Fragmin, must be used. Remember we currently have calibrators available for LMW heparin and UF heparin.
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).
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.
PRODUCT-SPECIFIC QUESTIONS
All of our heparin kits are for the chromogenic determination of UF and LMW heparin in human plasma, and measure the ability of heparin to catalyze the inhibition of FXa by antithrombin. Coamatic Heparin is a one-stage assay optimized for a wide range of instruments that does not require the addition of exogenous antithrombin. It features the use of substrate S-2732, simple reagent preparation, few components, and a simple, straight-forward assay procedure and is usually performed with undiluted plasma. Coatest Heparin is a two-stage assay with automated protocols available on a wide range of instruments. The assay requires the addition of antithrombin (included in the kit) and utilizes S-2222. Coatest Low Molecular Weight Heparin is mainly intended for the non-automated lab and allows rapid and reliable manual determination in a one-stage procedure. Like Coamatic Heparin, it uses S-2732, and although slightly dependent on the patient’s AT concentration, no exogenous AT is added.
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.
It is recommended to add exogenous antithrombin when children below the age of one year are being tested. Although exogenous AT has been shown to be needless for patients with AT levels between 35 – 135%, pre-term newborns can have levels as low as 30%. Sufficient studies of Coamatic Heparin have not been performed on infants, so as a precaution exogenous AT should be added. Also, for measuring heparin activities in serum, AT is needed since endogenous AT activity will be very low.
· When exogenous AT is desired, how should it be added in the test?
Add to the assay an equal volume of 1 IU/ml AT as the plasma volume. AT, 10 IU, can be bought from DiaPharma. Whenever a sample is tested with exogenous AT, it should be measured against a standard curve also run with exogenous AT.
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. For further explanation, see the Chromogenix Training Course binder for the apple analogy.
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.
· Compare hirudin with heparin. How can hirudin level be determined?
Hirudin is the active anticoagulant obtained from the leech, and is now produced by recombinant technology. It is the most potent and specific known inhibitor of thrombin. It inactivates thrombin by blocking the substrate binding groups following the formation of a 1:1 stoichiometric complex. All proteolytic functions of the enzyme are blocked, as is the activation of factors V, VIII, XIII, and the binding of thrombin to platelets. Heparin works indirectly, requires antithrombin as a cofactor, is not effective against thrombin that is already bound to the fibrin clot, and can be inactivated by PF4 or plasma proteins. Heparin therapy also leads to HIT in 5 – 15% of patients. Conversely, hirudin is the prototypical direct inhibitor of thrombin. It is a more potent anticoagulant and affects clot-bound thrombin, does not require any cofactors, and is not inactivated by PF4 or plasma proteins. Research has suggested that hirudin my provide a small advantage over heparin in situations such as acute coronary syndromes, but cost-benefit analyses are still needed. Hirudin levels can be determined chromogenically using substrate S-2366.
· Do you have standards and controls for measuring unfractionated heparin?
Yes.
Originally, the method called for using the CHR Heparin protocol, but it was recently updated to reflect an upgrade in software. You should use AT III protocol 2 and change the settings as listed in the application provided by Chromogenix.
No, running Coatest LMWH on the MLA 900C is not possible due to the measuring principles of the MLA. The instrument measures the reaction rate during a defined period of time, while checking that the reaction runs linear. Coatest LMWH uses another principle, however. At the same time that substrate hydrolysis takes place, the enzyme amount is gradually reduced due to inhibition by Heparin-AT complex. Time curves of the reaction are therefore not linear, and unfortunately will not be accepted by the MLA 900C. If you are interested in automating this test, you may want to consider purchasing Coatest Heparin instead, as it is more “instrument friendly”. A validated method for Coatest Heparin on the MLA 900C is available, and the test can be used for determination of UF as well as LMW heparin.