Hemostasis and the most common tests for its monitoring (Part Two)

Coagulation Phase Tests

1. Clotting Time (CT). Assesses the time needed for the hemostatic thrombus to form. In practice, Kowert's clotting time is most commonly used, performed on a slide where a drop of the patient's blood is placed and every 30” the formation of the fibrin thread is checked with the tip of a needle. Normal range 4-6’. It should be emphasized that CT will only be prolonged in severe deficits of one or several coagulation factors (< 1% of its level in plasma) which is not common in medical clinic. CT is not affected by the level of Factor VII (external pathway of coagulation).
2. Internal pathway of coagulation, is most often assessed in our medical practice by aPTT (Activated Partial Thromboplastin Time). The norm is 30-37”. aPTT assesses Coagulation Factors XII, XI, IX, VIII, X, V, and II.
3. Used for assessing the severity of hemophilia: 40-60” mild hemophilia, 60-80” is moderate hemophilia and over 80” severe hemophilia.
4. External pathway, is assessed by PT (Quick Time or Prothrombin Level), 13”-14’’ or 70-100%. It is used to evaluate treatment with Sintrom (oral anticoagulants) and is maintained at 25-35% of normal values (should not fall < 20% as there will be hemorrhagic diathesis). In medical practice, INR (International Normalized Ratio) is widely used, which is the ratio between the current PT value and the PT value of a normal blood sample. Normal INR values are 0.8–1.2. PT assesses factors II, V, VII, X.
5. Common pathway (second stage of coagulation) is assessed with: Thrombin Time (TT) which is the time of conversion of fibrinogen into fibrin, 18-22” and Plasma Fibrinogen, 200-400 mg/dl.
6. Howell Time (ACT): Norm 2’ ± 45” (120’’ ± 45”). Used when treating patients with sodium heparin and maintained at 1.5-2-3 times the norm.

Due to the lack of standardization of techniques and reagents, the normal range of each test depends on the technician and the doctor needs to be aware that this normal range of values is different in different laboratories.

Blood samples taken through traumatic venipunctures or from I/V catheters are a cause for inadequate responses to coagulation tests. A poorly collected blood sample is a far more common cause of inaccurate results than is technical error (Wintrobe 1310).

The lack of stable reference values is another problem of coagulation techniques.

Regulation of hemostasis, mechanisms of coagulation control

Careful control of the coagulation system is extremely important. It suffices to say that the "coagulation potential" of just one cm3 of blood, is enough to cause thrombosis of all the body's fibrinogen in just 10-15 seconds.

(The accurate control of the coagulation system is extremely important. It is enough to say, that the "coagulation potential" of only a cubic centimeter of blood is sufficient to cause thrombosis of the whole body fibrinogen within 10-15 seconds).

Immediately after coagulation, a strict regulatory mechanism is activated that interrupts further coagulation, i.e., its spread. If coagulation were to proceed uncontrollably, it would lead to an autoamplification of the coagulation mechanisms leading to general thrombosis and subsequently to the consumption of coagulation factors (as occurs in pathological conditions in DIC).

What are these factors?

Endothelial factors of coagulation control:

1. Production of prostaglandin PgI₂ by endothelial cells, leading to the release of cyclic AMP and its release will lead to inhibition of platelets.
2. From damaged endothelium, ADP-ase (enzyme) is produced, which destroys ADP (which as mentioned above is a strong inducer of platelet aggregation)
3. From endothelial cells, a relaxing factor is produced, which prevents adhesion and aggregation of platelets.

Other regulators, are factors found in plasma (serum factors). These include: Serine protease inhibitors with the main representative Antithrombin III and Protein C (natural inhibitors).

1. Antithrombin III. It is the most important inhibitory factor of coagulation and has heparin as a cofactor. The antithrombin III-heparin complex will bind with thrombin (factor IIa) forming an inactive complex that allows thrombin to act locally, but not generalize the coagulation process (DIC). It also binds and inhibits factors: Xa, IXa, XIa, XIIa,Va.
2. Protein C which is synthesized in the liver and is Vitamin K dependent.

Other inhibitory proteases include: Heparin cofactor II and α₂-macroglobulin.

Fibrinolysis is the third stage of hemostasis that ensures the dissolution of the hemostatic thrombus after the reconstruction of the damaged blood vessel wall and the restoration of circulation. It is a phenomenon that develops progressively slowly. It begins 2-3 hours after the formation of fibrin and ends after more than 72 hours.

The basis is plasminogen which is synthesized in the liver. It is in inactive form and is stimulated by plasminogen activators (internal, external, and therapeutic) to be converted into plasmin, which is a serum protease (enzyme) with specific fibrinolytic activity (dissolution of fibrin). Plasmin acts on fibrinogen and fibrin (proteolysis) giving fragments X, D, E, Y (fibrin degradation products).

It initially acts on fibrin then on fibrinogen, but the action on fibrinogen is more important. The fibrin degradation products are dimers or trimers e.g., YD, DD (dimers), DDE (trimer) etc. Plasmin activity is inhibited by α₂-antiplasmin.

Fibrinolysis tests: The main test is the Fibrinogen Degradation Products (FDP) Test, which assesses the degradation products of fibrinogen and fibrin from the proteolytic action of plasmin on fibrinogen and fibrin as well as D-dimer. They are especially elevated in DIC and fibrinogenolysis, where these tests have diagnostic significance. Also used are the measurement of fibrinogen, prothrombin level, and the clot lysis time (the whole blood clot lysis). The latter requires only incubation and observation of the blood clot. Normal is > 24 hours, if it occurs sooner it has significant diagnostic value.

Hemostasis can be presented in a simplified manner for better understanding in the following comparison: If a water canal were to have a wall damage (crack), water would leak out. Unlike a blood vessel where initially vasoconstriction occurs and then the entire hemostasis is triggered unconsciously (we do not command the development of hemostasis), the canal walls do not have such mechanisms, but people come and first throw sandbags to close the defect (platelet thrombus) and so that the water does not take these bags away, a net is thrown over them (fibrin). Then, workers come and repair the canal walls (fibroblasts) and when the wall is repaired (vessel damage is closed), the net and bags are removed (fibrinolysis) to establish regular circulation in this canal (vessel).

• There is no single test that can assess all of hemostasis.
• Tests are influenced by the anticoagulant used (e.g., fibrinogen measurement is affected by heparin), by the reagents used and technique (significant variations).
• Minor variations depend on the exact application of the technique by the laboratory technician.
• Specialized laboratories perform a series of much more specific tests for hemostasis.

Quantitative or qualitative damages, congenital or acquired, to any element of hemostasis, have significant consequences for life, such as hemorrhage or thrombosis. Similarly, the formation of autoantibodies against any element of hemostasis is another significant complication with the same consequences.

There are a number of medications that affect hemostasis and are widely used in medical practice.

Antiplatelet agents (inhibitors of platelet function and mistakenly called “blood thinners”), which affect primary hemostasis. These include Aspirin, Plavix etc. Medications that affect secondary hemostasis, anticoagulants, where the most common are heparin and oral anticoagulants (Sintrom, Warfarin). Medications that affect the third phase (fibrinolysis) such as its promoters (urokinase, streptokinase etc) or inhibitors of fibrinolysis or antifibrinolytics (EACA, Tranexamic Acid) etc.