Coagulation tests

Ok great, so now we know about different conditions and abnormalities, how do we test for these in the clinical lab?

The different coagulation tests run in the clinical lab can get confusing because the names are very similar and the names don’t seem to make much sense. We’ll clear up the nomenclature but the main thing to remember is we’re trying to logically solve a puzzle and figure out what specifically is causing a patient to have a hemostasis disorder.

The light blue top, sodium citrate tube is used for most coagulation studies, why is this? Citrate binds calcium, and with calcium tied up, clotting can’t start.

PT (Prothrombin Time):
PT tests the extrinsic and common pathways of the coagulation cascade. Basically, the test wants to determine how quickly a clot will be formed via the extrinsic pathway.

The test is run by adding commercial tissue factor (TF) and phospholipid (the two combined are sometimes referred to as thromboplastin) along with calcium chloride. Usually in the lab the TF, phospholipid, and calcium will be one reagent. This mixture is added to the patient’s plasma sample and the time is measured until a clot is formed. The normal reference range is 11-14 seconds.

As you recall from our earlier discussion of the extrinsic pathway, by adding TF and phospholipid in the presence of calcium, factor 7 will be activated and the extrinsic cascade will be in motion. Factor 7 has been tricked into being activated by the commercial reagents we added! Why is factor 7 tricked? All of the things it needs to be activated are present. TF is normally present when a vessel breaks, phospholipid is normally part of the platelet structure (in plasma samples the platelets have been removed), and calcium is added because the original calcium was tied up by citrate that coats the light blue top tube.

Ok but why is it called prothrombin time? The classical theory that goes back to the 1930’s postulated that in the presence of sufficient tissue factor, calcium, and fibrinogen, the only factor limiting clotting time was prothrombin. So basically they said, “how long is it going to take prothrombin to bring the race home? What is the prothrombin time?” This was a huge advancement in thought regarding coagulation studies; however, it’s not true but this is how the name came about.

International normalized ratio (INR):
The INR is used to standardize PT results. Since different commercial tissue factor reagents are used to kickstart the PT test, each manufacturer assigns an International Sensitivity Index value (ISI) to its tissue factor reagent. INR can be calculated by this formula: INR = (PT test time/PT control time)^ISI. The INR normal range is 0.8-1.2 seconds.

APTT (Activated Partial Thromboplastin Time) :
APTT tests the intrinsic and common pathways of the coagulation cascade. The test is run by adding phospholipid, an activator like silica or kaolin, and calcium chloride to the patient’s plasma sample and measuring the time until a clot forms. The activator will convert factor 12 to 12a. If you reference above, activating factor 12 will jumpstart the entire intrinsic pathway. In this case, factor 12 has been tricked. Normal range is 25-35 seconds.

Why is it called activated partial thromboplastin time? The term partial thromboplastin refers to a time when the components of thromboplastin were not understood. We now know that thromboplastin is tissue factor and phospholipid, and we also now know that partial thromboplastin is just phospholipid. Back then they called it partial thromboplastin and unlucky for us the name stuck.

PTT (Partial Thromboplastin Time):
PTT is the same test as the APTT just without the activator. This does not stand for prothrombin time test! Don’t get confused! Normal range is same as APTT.

TT (Thrombin Time) :
TT tests the amount of time it takes for thrombin to convert fibrinogen to fibrin. This test bypasses the extrinsic and intrinsic pathways as well as factors 10 and 2, and tests the end of the cascade. Normal range is 12-14 seconds.

Memory trick
Think of this test as “It’s time to add some thrombin, it’s thrombin time!”

Bleeding Time:
This test is no longer used but is seen a lot in references and tests. It was used to measure the amount of time it took for a platelet plug to form and bleeding to stop.

Activated clotting time (ACT):
This is a stat test used in most hospitals to monitor high dose heparin administration during intense operative procedures. It can be run on whole blood so is much quicker than running the APTT.

Russell Viper Venom Time:
Now it’s starting to get kind of weird right? This test actually uses viper venom! Who figured that out? The viper venom directly activates factors 5 and 10. Why is this important? Factor 7 deficient plasma will have normal results but plasma lacking factors 2, 5, or 10 will still have a prolonged clotting time. Why is this? Well, factor 7a normally activates factor 10, so if factor 7 is missing, viper venom can take its place to get the cascade going in a test tube. Ok and what about factors 2, 5 and 10? Since viper venom acts directly on factors 5 and 10, if they’re not present or if factor 2 (prothrombin) is not present, you will not get a clot in the test tube.

The dilute Russell Viper Venom test can also be used as part of a workup to detect Lupus anticoagulant, how does that work? As we just learned, Russel Viper Venom works by activating factors 5 and 10. If you remember from earlier, factor 10a activates prothrombin with the help of factor 5 and phospholipid. The key here is the phospholipid. Lupus anticoagulant will bind to the phospholipid and prolong the clotting time of this test if present. Also since Russel Viper Venom acts further down in the cascade, it can provide better information than the APTT, since it doesn’t involve the intrinsic pathway.

Nerdy Note
The Russell Viper is one of the deadliest snakes in India accounting for many deaths every year. The venom can obviously cause your blood to clot but also has many other menacing properties even if the person survives the bite. The venom can cause kidney failure and can cause major problems in the pituitary gland leading to hormone synthesis failure. The venom has been widely studied worldwide which has led to a byproduct coagulation test.

Reptilase Time:
This test is useful when you have a patient with an increased thrombin time which you suspect may be due to heparin neutralizing thrombin. Reptilase is an enzyme from the venom of Bothrops atrox (snake venom is popular around the coag department!), and it will convert fibrinogen to fibrin. How it acts on fibrinogen is slightly different than thrombin but the point is reptilase will convert fibrinogen to fibrin in the presence of heparin. Why is this important? If a patient has excess bleeding and it’s not clear why, having a prolonged thrombin time and a normal reptilase time will tell you there is too much heparin in the patients system. What if reptilase time is prolonged? This will point you toward a fibrinogen disorder.

Mixing studies:
In mixing studies, normal plasma is added to the patient’s plasma in question. This technique can help distinguish if there is a factor deficiency or if an antibody or antagonist is causing the prolonged clotting time. If the mixing study fixes the clotting problem, it could be a factor deficiency. If the mixing study does not fix the clotting problem, it could be an antibody or some interfering substance that will continue to eat up that factor even when you add more in part of a mixing study.

Adsorbed plasma:
Contains factors 1, 5, 8, 11, and 12

Adsorbed plasma is actually short for barium sulfate adsorbed plasma. The barium sulfate adsorbs factors 2, 7, 9 and 10 leaving you with the ones you need to remember above.

Aged Serum:
Contains factors 7, 9, 10, 11, 12
This to me always sounds like someone has barrels of it in some wine cellar but it is actually serum incubated at 37C for 24 hours. The aging leaves you with the factors above.

Both adsorbed plasma and aged serum were phased out of the clinical lab in the late 80’s and early 90’s.

Ristocetin agglutination test:
Ristocetin is an old antibiotic that is no longer used as an antibiotic because it can cause clotting. It’s important because it can help to diagnose vWF deficiency or Bernard Soulier Syndrome. How does it do this? In vitro (in a test tube) when Ristocetin is added to a blood sample it will cause vWF and GP1b to agglutinate.

D-Dimer and fibrin degradation product (FDP) tests:
The D-Dimer test indicates the amount of fibrin degradation products from stabilized fibrin (crosslinked by factor 13). The fibrin degradation product test detects fibrin products from primary fibrinogenolysis. In DIC both the D-dimer and FDP test will be positive. In primary fibrinogenolysis only the FDP will be positive.

Platelet aggregation studies:
Basic concept – The point of these studies is to see if platelets aggregate in primary and secondary phases of platelet aggregation in the presence or absence of certain isolated compounds. In the studies, measuring is done using light transmission. When platelets aggregate, the solution becomes clearer and transmits more light. When platelets don’t aggregate less light is transmitted.

So in these tests platelets aggregate in two phases. The first phase is in response to the compound or agonist added to the mix, and the second phase is mostly in response to the release of platelet granules which leads to upregulation of a number of processes.

Remember an agonist is defined as a substance that initiates a physiological response when combined with a receptor.

There’s a common platelet aggregation study that floats around and it’s got 25 different curves on it. In the right scenario they can all be important but I’m going to focus on a few that I find to be most important, and to basically inform you of what the tests mean because they can be confusing!

1. Ristocetin’s effect on Bernard Soulier Syndrome

a. In a test tube, Ristocetin will agglutinate vWF and GP1b. Since in Bernard Soulier Syndrome there is no GP1b, there will be no platelet aggregation in this study. Ristocetin is not a platelet agonist!

b. All of the other aggregation studies on Bernard Soulier Syndrome using the agonists ADP, collagen, epinephrine, and arachidonic acid are normal because these agonists directly bind to the platelet causing a shape change that leads to primary aggregation. The need for GP1b has been replaced by the artificial agonists. The platelets then release their granule contents which upregulates processes and causes the second wave of aggregation.

2. Ristocetin’s effect on Glanzmann’s Thrombasthenia

a. In a test tube, Ristocetin will agglutinate vWF and GP1b. A patient with Glanzmann’s will have a half strength aggregation curve with Ristocetin because the patient has vWF and GP1b. What’s happening is the Ristocetin is agglutinating the platelets but in order for aggregation to truly occur the fibrinogen receptor GP2b/3a is needed and patients with Glanzmann’s are lacking GP2b/3a.

b. All of the other aggregation studies on Glanzmann’s Thrombasthenia using the agonists ADP, collagen, epinephrine and arachidonic acid are abnormal (no aggregation) because these agonists can bind the platelet and cause a shape change but without the fibrinogen receptor GP2b/3a no aggregation can occur.

3. ADP, arachidonic acid, epinephrine, and collagen’s effect on storage pool disease

a. In storage pool disease platelet granules don’t release their contents or the contents are defective (the hematology condition Chediak Higashi syndrome can cause storage pool disease). In the first wave of this test you will see primary aggregation due to the addition of the agonists ADP, arachidonic acid, epinephrine, and collagen. There is no secondary aggregation because the storage granules are defective. Normally granules release ADP and other compounds which upregulate thromboxane A2 (TXA2) formation which is a potent inducer of platelet aggregation.

4. Arachidonic acid’s effect on a patient taking aspirin

a. Arachidonic acid is a platelet agonist and thromboxane A2 precursor. The binding of arachidonic acid to the platelet will cause induced platelet aggregation due to increased levels of thromboxane A2. Since aspirin inhibits cyclooxygenase, a precursor of thromboxane A2 downstream from arachidonic acid, arachidonic acid has no effect.

Other Notes of Interest:

Aspirin:
Aspirin inhibits the action of the enzyme cyclooxygenase. Cyclooxygenase is a precursor to thromboxane A2 in platelets and prostacyclin (PGI2) in endothelial cells. Thromboxane A2 is a strong platelet aggregator and PGI2 is a platelet inhibitor. This inhibition of cyclooxygenase is irreversible for the life of the platelet because platelets do not have a nucleus to create more cyclooxygenase. Aspirin’s effect on prostacyclin (PGI2) is less substantial because endothelial cells are nucleated and can regenerate cyclooxygenase. This is what makes aspirin effective. Other platelet aggregating pathways don’t use thromboxane A2 so aspirin will not totally shut down platelet activity.

Specimen Handling

Factors 5 and 8 can deteriorate quickly at room temp. Coagulation tests should be run within 2-4 hours of draw.