HEMATOLOGY EBOOK

D-dimer is a fibrin degradation product, a small protein fragment present in the blood after a blood clot is degraded by fibrinolysis. It is so named because it contains two crosslinked D fragments of the fibrinogen protein. D-dimer concentration may be determined by a blood test to help to help diagnose thrombosis. Since its introduction in the 1990s, it has become an important test performed in patients suspected of thrombotic disorders. While a negative result practically rules out thrombosis, particularly in young and healthy patients, a positive result can indicate thrombosis but does not rule out other potential causes. Its main use, therefore, is to exclude thromboembolic disease where the probability is low. In addition, it is used in the diagnosis of the blood disorder disseminated intravascular coagulation

Principles

Principles of D-dimer testing

Coagulation, the formation of ablood clot of thrombin, occurs when the proteins of the “coagulation cascade” are activated, either by contact with damaged blood vessel wall (extrinsic pathway) or by activation of high-molecular-weight kininogen by a number of stimuli. Both pathways lead to the generation of thrombin, an enzyme that turns the soluble blood protein fibrinogen into fibrin, which aggregates into proteofibrils. Another thrombin-generated enxyme, factor XIII, then crosslinks the fibrin ptoteofibrils at the D fragment site, leading to the formation of an insoluble gel which serves as a scaffold for blood clot formation. The circulating enzyme plasmin, the main enzyme of fibrinolysis, cleaves the fibrin gel in a number of places. The resultant fragments, “high molecular weight polymers”, are digested several times more by plasmin to lead to intermediate and then to small polymers (fibrin degradation product or FDPs). The crosslink between two D fragments remains intact, however, and these are exposed on the surface when the fibrin fragments are sufficiently digested. The typical D-dimer containing fragment cotains two D domains and one E domain of the fibrinogen molecule. D-dimer are not normally present in human blood plasma, except when the coagulation system has been activated, for instance because of the presence of thrombosis or disseminated intravascular coagulation. The D-dimer assay depends in the binding of a monoclonal antibody to a particular epitope on the D-dimer fragment. Several detection kits are commerically available; all of them rely on a different monoclonal antibody against D-dimer. Of some of these it is known to which area on the D-dimer the antibody binds. The binding of the antibody is then measured quantitatively by one of various laboratory methods.

Indications

D-dimer testing is of clinical use when there is a suspicion of deep venous thrombosis (DVT) or pulmonary embolism (PE). In patients suspected of disseminated intravascular coagulation (DIC), D-dimers may aid in the diagnosis.

For DVT and PE, there are various scoring systems that are used to determine the a priori clinical probability of these diseases; the best-known were introduced by Wells et al. (2003).

• For a very high score, or pretest probability, a D-dimer will make little difference and anticoagulant therapy will be initiated regardless of test results, and additional testing for DVT or pulmonary embolism may be performed.
• For a moderate or low score, or pretest probability:
  • A negative D-dimer test will virtually rule out thromboembolism: the degree to which the D-dimer reduces the probability of thrombotic disease is dependent on the test properties of the specific test used in your clinical setting: most available D-dimer tests with a negative result will reduce the probability of thromboembolic disease to less than 1% if the pretest probability is less than 15-20%
  • If the D-dimer reads high, then further testing (ultrasound of the leg veins or lung scintigraphy or CT scanning) is required to confirm the presence of thrombus. Anticoagulant therapy may be started at this point or withheld until further tests confirm the diagnosis, depending on the clinical situation.

In some hospitals, they are measured by laboratories after a form is completed showing the probability score and only if the probability score is low or intermediate. This would reduce the need for unnecessary tests in those who are high-probability.

Test properties

Various kits have a 93-95% sensitivity and about 50% specificity in the diagnosis of thrombotic disease.

• False positive readings can be due to various causes: liver disease, high rheumatoid factor, inflammation, malignancy, trauma, pregnancy, recent surgery as well as advanced age
• False negative readings can occur if the sample is taken either too early after thrombus formation or if testing is delayed for several days. Additionally, the presence of anti-coagulation can render the test negative because it prevents thrombus extension.
• Likelihood ratios are derived from sensitivity and specificity to adjust pretest probability.

History

D-dimer was originally described in the 1970s, and found its diagnostic application in the 1990s.

The euglobulin lysis time (ELT) is a test that measures overall fibrinolysis. The test is performed by mixing citrated platelet-poor plasma with acid in a glass test tube. This acidification causes the precipitation of certain clotting factors in a complex called the euglobulin fraction. The euglobulin fraction contains the important fibrinolytic factors fibrinogen, PAI-1, tPA, plasminogen, and to a lesser extent alpha 2-antiplasmin. The euglobulin fraction also contains factor VIII. After precipitation, the euglobulin fraction is resuspended in a borate solution. Clotting is then activated by the addition of calcium shloride at 37 C. Historically, subsequent amount of fibrinolysis was determined by eye, by observing the clot within the test tube at ten minute intervals until complete lysis had occured. Newer automated methods have also been developed. These methods use the same principle as the older technique, but use a spectrophotometer to track clot lysis as a function of optical density.

Fibrinolysis is the process wherein a fibrin clot, the product of coagulation, is broken down. Its main enzyme plasmin cuts the fibrin mesh at various places, leading to the production of circulating fragments that are cleared by other proteases or by the kidney and liver.

Physiology

Plasmin is produced in an inactive form, plasminogen, in the liver. Although plasminogen cannot cleave fibrin, it still has an affinity for it, and is incorporated into the clot when it is formed. Plasminogen contains secondary structure motifs known as kringles, which bind specifically to lysine and arginine residues on fibrin(ogen). When converted from plasminogen into plasmin, it functions as a serine protease, cutting C-terminal to these lysine and arginine residue. Fibrin monomers, when polymerized, form protofibrils. These protofibrils contain two strandm the fibrin monomers are covalently. Within a single strand, the fibrin monomers are covalently linked through the actions of coagulation factor XIII. Thus, plasmin action on a clot initially creates nicks in the fibrin, further digestion leads to solubilization. Tissue plasminogen activator (t-PA) and urokinase are the agents that convert plasminogen to the active plasmin, thus allowing fibrinolysis to occur. t-PA is released into the blood very slowly by the damaged endothelium of the blood vessels, such that, after several days (when the bleeding has stopped), the clot is broken down. This occurs because plasminogen became entrapped within the clot when it formed; as it is slowly activated, it breaks down the fibrin mesh. t-PA and urokinase are themselves inhibited by plasminogen activator inhibitor-1 and plasminogen activator inhibitor-2 (PAI-A and PAI-2). In contrast, plasmin further stimulates plasmin generation by producing more active forms of both tPA and urokinase. Alpha 2-antiplasmin and alpha 2-macroglobulin inactivate plasmin. Plasmin activity is also reduced by thrombin-activatable fibrinolysis inhibitor (TAFI), which modifies fibrin to make a less potent cofactor for the tPA-mediated plasminogen.

Measurement

When plasmin breaks down fibrin, a number of soluble parts are produced. These are called fibrin degradation products (FDPs). FDPs compete with thrombin, and so slow down the conversion of fibrinogen to fibrin (and thus slows down clot formation). This effect can be seen in the similar results are also seen after administration of DDAVP or after severe stress. A more rapid detection of fibrinolytic activity, especially hyperfibrinolysis, is possible with thromboelastometry (TEM) in whole blood, even in patients on heparin. With various assays an enhanced fibrinolysis becomes visible in the curve signature and from the calsulated values e.g. the maximum lysis parameter. A spcial test for the identification of increased fibrinolysis (APTEM) compares the TEM profile in the absence or presence of the fibrinolysis inhibiotr aprotinin. In severe cases of activated fibrinolysis, this assay confirms the syndrome already in less thn 15 min during the early phases of clot formation.

Role in disease

Few congenital disorders of the fibrinolytic system have been documented. Nevertheless, excess levels of PAI and alpha 2-antiplasmin have been implicated in the metabolic syndrome and various other disease states. However, acquired disturbance of fibrinolysis (Hyperfibrinolysis), is not uncommon. Many trauma patients suffer from an overwhelming activation  of tissue factor and thus massive hyperfibrinolysis. Also in other disease states hyperfibrinolysis may occur. It could lead to massive bleeding if not dianosed and treates early enough. The fibrinolytic system is closely linked to control of inflammation, and plays a role in disease states associated with inflammation. Plasmin, in addition to lysing fibrin clots, also cleaves the complement system component C3, and fibrin degradation products have some vascular permeability inducing effects.

Pharmacology

Fibrinolytic drugs are given after a heart attack to dissolve the thrombus blocking the coronary artery, experimentally in stroke to reperfuse the affected part of the brain, and in massive pulmonary embolism. The process is called thrombolysis. Antifibrinolytics, such as aminocaproic acid (ε-aminocaproic acid) and tranexamic acid are used as inhibitors of fibrinolysis. Their application may be benefecial in patients with hyperfibrinolysis because they arrest bleeding rapidly if the other components of the haemostatic system are not severely affected. This may help to avoid the use of blood products such as fresh frozen plasma with its associated risks of infections or anaphylactic reactions. The antifibrinolytic drug aprotinin was abandoned after identification of major side effects, especially on kidney.