Physiological basis of routine coagulation studies

This chapter is relevant to Section Q3(i) of the 2017 CICM Primary Syllabus, which expects the exam candidates to "outline the methods for assessing coagulation (including TEG, ROTEM)". It is identical to the content of Question 9 from the first paper of 2017 and Question 8 from the second paper of 2014, which asked the candidates to outline how the tests assessed coagulation (and yes, they included TEG and ROTEM). In a panic, some of the candidates apparently produced elaborate diagrams of the coagulation cascade, as if to ward themselves against evil. To prevent this sort of thing in the future, this short summary offers enough detail to satisfy even the pickiest of marking criteria.

In short:

An excellent resource for this information is PracticalHaemostasis.com. You really do not need anything more than that. If for whatever reason one is distrustful of online resources not originating from peer-reviewed journals, one could use Curry & Pierce (2007) or Raber (1990).

PT: Prothrombin Time

Judging from the name, this would have to be something related to prothrombin. Prothrombin, of Factor II, is activated by a number of different mechanisms and forms the final stage of the common pathway of coagulation, where everything converges on thrombin. 

Basically, you add "tissue factor" to a sample of plasma, and measure the time it takes for the sample to clot. The tissue factor is usually a recombinant human tissue factor, but historically all sorts of weird stuff has been used (rabbit brain puree, etc). This activates the extrinsic pathway, which in turn activates the final common pathway; so the PT actually measures both pathways. Typically, tissue factor is added first; as the sample contains citrate there is no calcium available to initiate the clotting cascade. When calcium is added, the timer begins (apparently the manual method of running this test literally involves standing there with a stopwatch, looking at the sample as you manually tilt it through a 90-degree arc). Thankfully it's quite quick: the normal time from calcium to full glug is 10-13 seconds. What you're looking for at the end is the  formation of a fibrin clot (as the test is performed on acellular plasma, there are no platelets here). The assessment of whether or not the clot has formed classically comes from observing that the flow in the tilting test-tube has stopped (or by a number of other mechanised techniques which use various optical methods).

Prothrombin Time

Normal range	10-13 seconds
Therapeutic values	20-30 seconds for therapeutic warfarin anticoagulation (corresponds to INR 2.0 to 3.0)
Purpose	Test for factor deficiency (II, VII, X) or warfarin therapy
Coagulation pathway assessment	Extrinsic pathway (Factor VII) Common pathway (Factors X and II) Thus, PT captures most of the Vitamin K dependent factors (Factor IX is also vitamin-K dependent, but is not tested.)
Collection method	Citrated blood tube
Blood sample processing	Sample is centrifuged; plasma is analysed
Laboratory or point of care method	Laboratory method: recombinant tissue factor is added to the sample, a timer is started and the sample is observed manually or in an automated analyser until flow in the moving sample tube has stopped
Diagnoses or errors associated with abnormalitiers	Vit K-dependent factor problems: warfarin FVII problems: warfarin, haemophilia Factor Xa problems: apixaban, rivaroxaban Thrombin problems: dabigatran,  lepirudin, heparin (yes, heparin)
Single best reference	Hood & Eby, 2008

Testing the PT is a way of looking at the function of the Vitamin K-dependent enzymes; namely II, VII and X. Specifically, the rate of the extrinsic pathway is mainly influenced by the amount of Factor VII you have. It has a short half-life and depends on Vitamin K. However, if there is a dysfunction at any step along the pathway, the test will be abnormal, and this includes scenarios where Factor Xa or thrombin itself has been disabled by a directly acting agent. In those settings (eg. dabigatran or apixaban overdose), the PT will be elevated. 

Similarly, the attentive reader will be ready to point out that heparin works by an antithrombin-mediated interference in the activity of Factor Xa and thrombin, which means that heparin could also affect your PT. However, we never see this in practice, and always associate heparin use with a raised aPTT. The reason for this is that most commercially available PT reagents incorporate some mechanism of disabling heparin, for example heparinase.

aPTT: Activated Partial Thromboplastin Time

This is a test of the intrinsic and common pathways which takes advantage of the fact that those aforementioned pathways require the phospholipid surface of a platelet to work their magic. To provide this surface without having to use actual platelets, you add some "partial thromboplastin" to the blood sample,. Thromboplastin in an anachronism - it 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), and thus there is no extrinsic pathway factor activation in the test.

The phospholipid is a necessary precondition, but on its own this will not be enough for the protease cascade to proceed. In order to kick off the intrinsic pathway, some sort of negatively charged substance must be added, which could active Factor XII.  In the laboratory, instead of natural FXII activators like collagen, we instead use kaolin ( a clay mineral) or silica. When the calcium is added, the timer starts (as without calcium none of this stuff would work, and all the reagents can be safely mixed together first). 

As you can see, Factor VII is really the only factor of note which is not being tested by this pathway. A deficiency of any of the other factors will be revealed by aPTT. 

activated Partial Thromboplastin Time

Normal range	30-40 seconds
Therapeutic values	The aPTT target for systemic anticoagulation (eg. for PE or DVT) is usually 50-90 or 60-100 seconds
Purpose	Test for factor deficiency (XII, XI, X, IX, II) or heparin therapy
Coagulation pathway assessment	Intrinsic pathway (Factor VII) Common pathway (Factors X and II)
Collection method	Citrated blood tube
Blood sample processing	Sample is centrifuged; plasma is analysed
Laboratory or point of care method	Laboratory method: source of phospholipid (eg. cephalin) and negatively charged FXII activator (eg. kaolin) are added. Timer starts when calcium is added to reverse the citrate.
Diagnoses or errors associated with abnormalitiers	Factor deficiencies (except Factor VII), eg. liver disease Factor Xa problems: apixaban, rivaroxaban Thrombin problems: dabigatran,  lepirudin, heparin  Antiphospholipid: an antibody to the phospholipid source, eg. lupus anticoagulant

Mixing studies

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)

ACT is basically the aPTT but without any control over the phospholipid dose. A fresh whole blood sample is used and the patient's own platelets are the source of the phospholipid. 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 or so. 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
• Hypothermia

Activated Clotting Time

Normal range	110-130 seconds
Therapeutic values	For ECMO, 200 seconds For bypass, 400 seconds
Purpose	Rapid test of all clotting function parameters, eg. to make adjustments to critically important therapeutic anticoagulation
Coagulation pathway assessment	Whole clotting cascade: intrinsic, extrinsic, common pathway and platelet function
Collection method	Whole fresh blood
Blood sample processing	Sample does not require processing
Laboratory or point of care method	Point of care automated test. A contact activator (eg. kaolin) is added to the whole blood sample. Endogenous platelets are used as the source of phospholipid.
Diagnoses or errors associated with abnormalitiers	Any coagulopathy could give rise to an elevated ACT, i.e. it is not specific for any source of clotting dysfunction.  Most useful when there is one single predictable source of clotting dysfunction (eg. heparinisation), in which case it can be used to titrate the heparin dose to effect

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. However, the longer term ECMO circuits can tolerate a shorter ACT. Moreover, the ECMO patient may have numerous reasons to become coagulopathic, and so an ACT is probably the wrong test to be using (heparin is not the only thing these people got going on). Ergo, aPTT is usually the recommended method of testing heparinisation in ECMO.

Thrombin time

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 centrifuged plasma Thrombin converts fibrinogen to fibrin, and thus the addition of lots of thrombin should cause lots of fibrin to appear. It is so simple that some might say it does not even merit a diagram:

This reaction is usually quite rapid; a normal thrombin time is around 13-15 seconds. The thrombin is naturally 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 whatsoever, as all it can do is reduce the availability of prothrombin (a vitamin K dependent factor). As exogenous thrombin is being added here, there should be no way for warfarin to affect the outcome. 

Thrombin Time

Normal range	13-15 seconds
Therapeutic values	There are no currently accepted direct thrombin inhibitor therapeutic range recommendations, but theoretically TT could be used to monitor these drugs (eg. dabigatran)
Purpose	Test for factor deficiency (XII, XI, X, IX, II) or warfarin therapy
Coagulation pathway assessment	End of the common pathway (specifically, fibrinogen) Tests for the effect of any soluble mediators which might interfere with thrombin activity (eg. dabigartan, lepirudin)
Collection method	Citrated blood tube
Blood sample processing	Sample is centrifuged; plasma and analysed.
Laboratory or point of care method	Laboratory method: exogenous thrombin is added to the plasma; timer is started immediately (there is no need to add calcium, as it is not a cofactor here).
Diagnoses or errors associated with abnormalitiers	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. Amyloidosis

Thrombin time seems like a logical way to assess how well a direct thrombin inhibitor is performing. However, it appears that this test is too sensitive for this purpose. Drugs like dabigatran hang around in the bloodstream, and continue to inhibit the added thrombin in the sample. TT values in excess of 200 seconds were collected in the course of a study by Chin et al (2014). A diluted TT test is available for this purpose, where donated plasma is added to the sample. 

Reptilase time

Reptilase time and thrombin time are closely related. Reptilase is actually the enzyme secreted vigorously 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
• Amyloidosis

Ecarin clotting time

This is a meizothrombin generation test, specifically 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

Anti-Xa levels are an assay for the activity of low molecular weight heparin, as well as some of the 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. 

This can be tested using various sorts of assays. For example, 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. 

TEG and ROTEM

TEG and ROTEM are discussed in greater detail in the revision notes for the Second Part exam, purely because that's where most of those questions are asked.  The chapters "Viscoelastic tests of clotting function"  and "Intepretation of abnormal ROTEM data" are probably more of interest for people who are expected to interpret these data and act on them. For the First Part exam, however, the following low-fi summary will suffice:

Viscoelastic Tests of Clotting Function

Normal range	Multiple variables are measured by each test, including times and amplitudes
Therapeutic values	At this stage there are no guidelines for the use of TEG or ROTEM to routinely monitor therapeutic anticoagulation
Purpose	Simultaneous assessment of multiple domains of clotting function
Coagulation pathway assessment	Intrinsic, extrinsic, common protease pathways; Fibrinogen (amount and function) Platelet function
Collection method	Whole blood
Blood sample processing	Sample does not require processing
Laboratory or point of care method	Point of care automated test. Sample is added to a viscometer which assesses clot formation indirectly by the changing deformability of the sample in different reagents
Diagnoses or errors associated with abnormalitiers	Factor deficiency, fibrinogen deficiency, poor platelet function (or thrombocytopenia), hyperfibrinolysis