Factor X or anti-Xa Assay. Which Do I Use?
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
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™ or S-2238™.
What is heparin-induced thrombocytopenia (HIT)? When HIT is suspected in a patient treated with UF heparin, should LMW heparin therapy replace UF therapy?
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.
The dilution of the 0.1 IU/ml heparin solution in Chromogenix Coatest® Heparin can cause some confusion. Please explain the Coatest® Heparin standardization.
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 the Chromogenix Coatest® Heparin package insert, the section Limitations of the procedure states that in some pathological states, plasma alone my hydrolyze S-2222™, and that to determine the interference one should substitute FXa with an equal volume of buffer…Why is this necessary?
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.
I need an assay that complies with the USP monograph for the determination of heparin activity. What test kits are suitable?
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.
What is the difference between Chromogenix Coamatic® Heparin, Coatest® Heparin and the discontinued Coatest® Low Molecular Weight Heparin / Heparin?
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 ofexogenous antithrombin. It features the use of substrate S2732™, 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 was mainly intended for the non-automated lab and allows rapid and reliable manual determination in a one-stage procedure. Like Coamatic® Heparin, it used S-2732™, and although slightly dependent on the sample’s AT concentration, no exogenous AT is added.
Which of DiaPharma’s kits can measure LMW heparin?
All of the heparin kits from DiaPharma can. Obviously, when measuring LMW heparin samples, LMW heparin standard, like Fragmin, must be used unless a specific universal calibrator is used. DiaPharma currently has calibrators available for LMW heparin and UF heparin.
Do you have calibrators and controls for measuring unfractionated heparin?
Yes, though they are currently labeled for research use only. See the UFH calibrators and controls product pages for details.
When exogenous AT is desired, how should it be added in the Chromogenix Coamatic® Heparin 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.
Chromogenix Coamatic® Heparin is optimized for use with and without exogenous antithrombin. When is it recommended to add exogenous AT, and why?
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.
How do the pharmacokinetics of LMW heparins differ from UF heparin, and what are the therapeutic ranges for each?
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).
Describe heparin’s interaction with antithrombin. Why is the anti-FXa method a better way to measure heparin activity?
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.
How does heparin interact with antithrombin? Why is the anti-FXa method a better way to measure heparin activity?
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.
What is the function of antithrombin and what is its interaction with thrombin and heparin?
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.
Protein concentrations in plasma
Component |
Molecular
|
Plasma
|
Plasma
|
| Fibrinogen | 330 | 3000 | 9 |
| Prothrombin | 72 | 150 | 2 |
| Factor V | 330 | 20 | 0.05 |
| Factor VII | 50 | 0.5 | 0.01 |
| Factor VIII | 330 | 0.1 | 0.0003 |
| Factor IX | 56 | 5 | 0.09 |
| Factor X | 59 | 8 | 0.13 |
| Factor XI | 160 | 5 | 0.03 |
| Factor XII | 80 | 30 | 0.4 |
| Factor XIII | 320 | 10 | 0.03 |
| Protein C | 62 | 4 | 0.06 |
| Protein S | 70 | 10 (free) | 0.14 |
| Protein Z | 62 | 2 | 0.03 |
| Prekallikrein | 86 | 50 | 0.6 |
| HMW kininogen | 120 | 70 | 0.6 |
| Fibronectin | 450 | 300 | 0.7 |
| Plasminogen | 92 | 200 | 2 |
| t-PA | 60 | 0.005 | 0.0001 |
| Urokinase | 53 | 0.004 | 0.0001 |
| Antithrombin | 58 | 145 | 2.5 |
| Heparin Cofactor II | 66 | 80 | 1.2 |
| Plasmin Inhibitor | 63 | 60 | 1 |
| Protein C Inhibitor | 57 | 4 | 0.07 |
| α2-Macroglobulin | 725 | 2000 | 3 |