This is a brief discussion of the reasoning behind the tests of coagulation function. Additionally, one may wish to review the coagulation cascade, if one (like myself) is unable to reliably recall its various details. An excellent resource for this information is PracticalHaemostasis.com
PT: Prothrombin time
This is a test of the EXTRINSIC PATHWAY as well as the FINAL COMMON PATHWAY
Basically, you add "tissue factor" to a sample of plasma, and measure the time it takes for the sample to clot. The tissue factor activates the extrinsic pathway, which in turn activates the final common pathway; so the PT actually measures BOTH pathways.
The rate of the extrinsic pathway is mainly influenced by the amount of Factor VII you have.
Factor VII has a short half-life and depends on Vitamin K. Testing the PT is a way of looking at the function of the Vitamin K-dependent enzymes; namely II, VII and X.
Factor IX is also vitamin-K dependent, but is not tested.
INR: international normalized ratio
Comparison of a given PT to an average PT. An INR of 2 means blood is clotting twice as slowly as normal.
APTT: Activated Partial Thromboplastin Time
A test of the INTRINSIC PATHWAY, as well as the FINAL COMMON PATHWAY.
Basically, you add some "partial thromboplastin" to the blood sample, together with calcium.
Thromboplastin was a weird surrogate for tissue factor; or rather, it is a tissue factor-like protein, already bound to some phospholipid, and derived from cow placenta. Partial thromboplastin is just the phospholipid part. There isn't any tissue factor there. Thus, there is no extrinsic pathway factor activation in the test.
In order to kick off the INTRINSIC PATHWAY, some sort of negatively charged substance must be added. (in the living tissue, this is collagen exposed by cutting the vessel).
In the laboratory, instead of collagen we use kaolin ( a clay mineral) or silica. The phospholipid and calcium are required for the tenase and prothrombinase complexes of the final common pathway. The calcium also participates in the intrinsic pathway.
APTT WILL NOT PICK UP FACTOR VII DEFICIENCY. Additionally, even if you have 50% less of any given factor, your PT and aPTT should remain roughly normal.
Mixing studies distinguish between factor deficiencies and factor inhibitors.
Lets say your sample of plasma is giving a high PT or aPTT - grab your suspicious plasma sample, and mix it with normal blood, 50:50. Obviously, if some sort of "factor inhibitor" is present, the normal blood will also be affected, and the resulting mixture will give abnormal aPTT and PT results.
If there is a factor deficiency, the mixed sample will result in a normal PT or aPTT.
In short exam-focused point form:
- Patient plasma is mixed 1:1 with normal plasma
- APTT and PT are measured immediately
- Complete (with 10%) correction of PT /APTT suggests a factor deficiency
- Plasma is incubated for 1 hour at 37°C
- The mixing studies are repeated
- A longer APTT after 1 hour incubation suggests a factor VIII inhibitor.
Activated Clotting Time (ACT)
This is a sort of a point-of-care test which is done in situations of extreme anticoaguation, when one must act quickly to make changes to the coagulation profile, rather than waiting around for the lab to report on the tests.
Thus, the ACT is done by the bedside of patients undergoing cardiopulmonary bypass, ECMO, or (less frequently) dialysis. An ACT of 400-600 seconds is required for the bypass circuit not to clot, and this figure is arrived at empirically - most anaesthetists do not report an excess of clot formation with an ACT over 300 seconds. Similarly, most ECMO circuits are happy with an ACT of 200-300. By comparison, the ACT of a totally normal non-anticoagulated patient is around 100-110 seconds.
The ACT is performed fairly crudely, by mixing fresh blood with various random garbage (usually, glass beads or kaolin balls) and then measuring how long it takes for a clot to form. This means the ACT tests the entire spectrum of the coagulation cascade, with the exception of fibrinolysis.
ACT will thus be elevated in a wide range of circumstances; a short list could resemble the following:
- Thrombocytopenia, or platelet dysfunction
- Clotting factor deficiency, or factor inhibitors
- Low fibrinogen
Why can we not just use the APTT in bypass surgery, if we are merely using (vast amounts of) heparin to maintain anticoagulation? Well. With such quantities of heparin, the normal APTT test will never clot. There is simply too much hyperactive antithrombin-III. Thus, we must perform ACT measurements.
The thrombin time (TT) measures the final step of the clotting cascade, the conversion of fibrinogen into fibrin. It involves adding some thrombin (human or bovine) to some of the patient's plasma, ideally platelet-poor plasma. Thrombin converts fibrinogen to fibrin, and thus the addition of lots of thrombin should cause lots of fibrin to apppear. This reaction is usually quite rapid; a normal thrombin time is around 13-15 seconds.
This step is antagonised by antithrombin-III, which means that the presence of heparin should cause a prolonged TT. Warfarin, on the other hand, should have no effect on TT.
Causes of an unusually prolonged thrombin time:
- Heparin therapy
- Low fibrinogen levels
- Dysfunctional fibrinogen (eg. foetal fibrinogen)
- Direct thrombin inhibitors (eg. hirudin, argatroban, dabigatran)
- High levels of abnormal proteins, eg. paraproteins and fibrin degradation byproducts can lead to abnormal TT by interfering with the cleavage of fibrinogen by thrombin.
- Very high fibrinogen levels can paradoxically interfere with TT.
Reptilase time and thrombin time are closely related. Reptilase is actually an enzyme secreted gladly by the South American pit viper (Bothrops atrox) Like thrombin, reptilase catalyses the cleavage of fibrinogen into fibrin, and causes clotting. Unlike thrombin, it is under absolutely no control from normal human homeostatic feedback mechanisms. This makes sense, as it is a reptilian venom, designed to kill mammals.
Thus, reptilase time will not be affected by antithrombin III (and thus, by heparin); it will not be affected by direct thrombin inhibitors such as argatroban or hirudin; it will only react to abnormalities of fibrinogen.
Thus, reptilase time will be abnormally increased in the following circumstances:
- Low fibrinogen levels
- Dysfunctional fibrinogen (eg. foetal fibrinogen)
- High levels of abnormal proteins, eg. paraproteins and fibrin degradation byproducts
- Very high fibrinogen levels
Ecarin clotting time
This is a meizothrombin generation test, used to measure the activity of direct thrombin inhibitors such as dabigatran and hirudin. Ecarin is a metalloprotease acquired from the venom of Echis carinatus. It activates prothrombin, bypassing the intrinsic and extrinsic pathways. ECT is therefore insensitive to the absence of a large portion of the clotting cascade. ECT will be normal in the presence of heparin or warfarin, or in conditions of even very severe factor depletion. However, the ECT will be abnormal in the presence of any direct thrombin inhibitors.
The ECATEM cuvette of the ROTEM analyser contains ecarin, and is equivalent to the ECT.
Anti-Xa levels are an assay for the activity of low molecular weight heparin, as well as some of the new anti-Xa oral anticoagulants (eg. apixaban and rivaroxaban).
Anti-Xa activity measurements will also be upset by unfractionated heparin, because it will still cause the binding of Xa and antithrombin.
In patients receiving LMWH the Xa activity is increased.
This can be tested using various sorts of assays. The patients heparinised blood is added to a known dose of Xa. The heparin in the blood sample binds the Xa and forms complexes with it; the residual Xa is then measured using an artifical substrate for Xa. The amount of residual Xa is thus an indirect measure of the concentration of heparin i.e the more heparin, the less residual Xa) and so it is expressed as a heparin "level", a concentration measured in U/ml.
Because of the routinely subcutaneous dosing of LMWH, the test should be performed at the peak of the activity, which is about 3-4 hours after the injection is given.
As far as oral anticoagulants go, rivaroxaban and apixaban are the main reasons for testing anti-Xa activity.