Roger S. Gilbert, DO, Justin B. D-dimers are formed by the breakdown of fibrinogen and fibrin during fibrinolysis. D-dimer analysis is critical for the diagnosis of deep vein thrombosis, pulmonary embolism, and disseminated intravascular coagulation. Modern assays for D-dimer are monoclonal antibody based.
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Roger S. Gilbert, DO, Justin B. D-dimers are formed by the breakdown of fibrinogen and fibrin during fibrinolysis. D-dimer analysis is critical for the diagnosis of deep vein thrombosis, pulmonary embolism, and disseminated intravascular coagulation.
Modern assays for D-dimer are monoclonal antibody based. The enzyme-linked immunosorbent assay ELISA is the reference method for D-dimer analysis in the central clinical laboratory, but is time consuming to perform. Recently, a number of rapid, point-of-care D-dimer assays have been developed for acute care settings that utilize a variety of methodologies. In view of the diversity of D-dimer assays used in central laboratory and point-of-care settings, several caveats must be taken to assure the proper interpretation and clinical application of the results.
These include consideration of preanalytical variables and interfering substances, as well as patient drug therapy and underlying disease. D-dimer assays should also be validated in clinical studies, have established cut-off values, and reported according to the reagent manufacturers recommendations. The hemostatic system acts to coordinate the delicate balance between bleeding and clot formation.
Formation of a blood clot, or thrombus, is essential to prevent bleeding in the event of vascular injury; however, inappropriate thrombus formation can cause significant morbidity and mortality.
Arterial and venous systems can develop thrombi, which may cause local obstruction with associated ischemic symptoms but may also break off, or embolize, into the circulation and become lodged in distant vessels. This process is called thromboembolization. For example, thrombi that originate in the heart can embolize and become lodged in the small vessels of the brain, leading to a stroke or transient ischemic attack. Similarly, a thrombus that originates as a deep venous thrombosis DVT in the large veins of the lower extremities may embolize to the lungs, resulting in pulmonary embolism PE or infarction.
Despite the significant morbidity and mortality associated with this disease, the coagulation assays that are currently available for diagnosis and prognosis of thromboembolic events are not yet ideal, although they have improved substantially in recent years. Under normal conditions, blood maintains its fluidity as it is propelled through the circulatory system; however, in the event of an injury, clotting must occur to reduce loss of blood from the intravascular space.
Hemostasis refers to the series of complex physical processes that occur between the blood vessels, platelets, coagulation factors, and other elements that promote clotting and thereby prevent blood loss.
Efforts to prevent hemorrhage after vascular injury begin with constriction of the blood vessels to reduce the flow of blood to the affected area. Platelets aggregate at the affected site by binding the prothrombogenic subendothelial collagen fibers that invariably become exposed after vessel injury.
Also, platelet clumping is bolstered further by the binding of plasma protein fibrinogen, which results in an initial platelet plug that temporarily prevents blood loss from the intravascular space.
Clumped platelets undergo multiple biochemical changes that result in alteration of platelet morphology and release of extracellular chemicals that activate neighboring platelets and plasma coagulation factors. Fibrin formation and degradation. Fibrinogen is transformed to fibrin monomers through the cleavage of 2 small fragments ie, fibrinopeptides A and B from the molecule by thrombin.
During this process, the negative charge of the E domain of fibrinogen red circles is converted to a positive charge, permitting spontaneous polymerization of the fibrin monomers into a polymer stabilized by hydrogen bonds. Thrombin also activates a circulating transglutaminase enzyme, factor XIII, which stabilizes the initial fibrin polymer by catalyzing the formation of crosslinked covalent bonds between adjacent D domains green circles.
Plasmin, a component of the fibrinolytic system, is formed from its circulating inactive precursor, plasminogen, through the activity of a serine protease, tissue plasminogen activator TPA , released by injured endothelial cells. Plasmin cleaves fibrin into a variety of smaller fragments termed fibrin degradation products FDPs.
The D-dimer, formed from 2 adjacent cross-linked fibrin monomers, is one of the major FDPs. Plasmin also proteolyzes fibrinogen and other plasma proteins.
A circulating enzyme, alphaantiplasmin, inactivates plasmin to localize fibrinolysis to the site of injury. These clotting modalities are counteracted by a number of mitigating mechanisms that prevent excessive or inappropriate clotting.
Also, endogenous antithrombotic agents, such as antithrombin and proteins C and S, impede platelet activation and inhibit activated coagulation factors. These mechanisms orchestrate a balance between clotting the blood and maintaining its fluidity, whereas dysfunction of these systems may lead to a propensity for inappropriate thrombus formation or bleeding.
Fibrin thrombi are primarily comprised of fibrin polymers that are normally broken down by the fibrinolytic system almost as soon as they are formed. The fibrinogen degradation products FDPs that result from clot disintegration are currently the most widely used indicators of thrombosis.
Also, laboratory evaluation of thrombosis relies on the assessment of levels of factors, such as fibrinogen, that are consumed during coagulation. The major effector of the fibrinolytic system, namely, plasmin, is generated from an inactivated precursor protein called plasminogen by tissue plasminogen activator TPA.
The breakdown of fibrinogen and fibrin by plasmin is counterbalanced by multiple enzymatic modulators such as thrombin-activatable fibrinolysis inhibitor TAFI that modifies fibrin to make it more resistant to plasmin breakdown. Plasmin-mediated cleavage of fibrinogen and fibrin produces a heterogeneous assortment of various-sized breakdown products. However, only fragments originating from fibrin polymers that had undergone factor XIII mediated cross-linking will have an intact covalent bond between 2 adjacent D domains ie, D-dimers Figure 1.
Therefore, the D-dimer fragment, which contains 2 D domains and an E domain, provides the unique target epitope for a fibrin-specific degradation product that is recognized by most reagent antibodies used in the laboratory assessment of thrombosis. Clinical measurement of FDPs began in the early s, using staphylococcal clumping, latex fixation and agglutination, hemagglutinin inhibition, immunoelectrophoresis, immunodiffusion, and other techniques. Significant improvement of sensitivity and specificity occurred in the s with the development of monoclonal antibody-based assays that rely on monoclonal immunoglobulins that target specific D-dimer epitopes not found on FDPs or on non—cross-linked fibrin fragments.
The early monoclonal assays were primarily qualitative latex slide agglutination assays using latex microparticles coated with monoclonal antibodies specific for D-dimer epitopes. Macroscopically visible agglutination of the particles occurs when they are incubated with plasma containing D-dimers. These unenhanced slide-based latex agglutination assays are rapid and inexpensive, but they lack sufficient sensitivity for detection of D-dimers in critical clinical situations, particularly pulmonary embolism and acute venous thrombosis.
Therefore, monoclonal antibody assays have been replaced by other more sensitive and specific methodologies. Currently, measurement of FDPs largely has been replaced by a variety of more-sensitive commercial D-dimer testing platforms that have been designed for central clinical laboratories and point-of-care applications Table 1 and Table 2.
The central laboratory quantitative D-dimer assays were initially based on enzyme-linked immunosorbent assay ELISA technology but have more recently been adapted to coagulation analyzers and clinical chemistry analyzers, with an endpoint based on immunofluorescence, latex-enhanced immunoturbidimetry, or chemiluminescence. Although these assays are highly sensitive and are economical to perform when analyzing large numbers of specimens, the combination of specimen transportation time and analytical time often results in a prolonged response time of more than 40 minutes.
An additional assay is FDA approved to aid in the diagnosis of thromboembolic disease with the addition of 1 or more radiographic procedures. Several additional central laboratory D-dimer assays are available in Europe and other parts of the world but have not received FDA clearance for application in the United States. Currently, the laboratory testing proficiency program of the College of American Pathologists CAP lists 14 different quantitative assays from 7 manufacturers.
The increasing overcrowding of urgent care facilities and the desire for improved patient satisfaction and decreased patient wait time have led to a great interest among physicians for rapid point-of-care POC D-dimer assays to screen patients for thromboembolic disease. Generally, these assays are homogenous, monoclonal antibody-based sandwich types, with a detection method based on hemagglutination, fluorescence, chemiluminescence, or other technology.
A few semiquantitative methods remain in use for POC situations. Whole blood is of the usual specimen type, and the POC D-dimer assays have a short specimen turnaround time of 5 to 20 minutes. Overall, the global D-dimer testing market had an estimated value of 1. We also will discuss the clinical applications of the D-dimer assay. This method involves loading plasma specimens into microtiter wells coated with antibodies that have high affinity binding for D-dimers.
After incubation, a labeled antibody is then added, and the quantity of bound, labeled substance is measured via colorimetric reaction. The labor-intensive and time-consuming constraints typical of conventional ELISA assays make them impractical for routine clinical laboratory use, which has spurred the development of more rapid, automated, and highly sensitive modified ELISA assays.
Latex-enhanced immunoturbidimetric assay is a cost-effective, rapid test that has analytical sensitivity comparable to conventional ELISA. This automated microparticle assay passes a beam of monochromatic light through a suspension of latex microparticles that are coated by monoclonal antibodies specific for D-dimer epitopes.
Because the nm wavelength of the light beam is greater than the diameter of the latex microparticles, only a minimal amount of light is absorbed by the latex microparticle solution at baseline.
However, when plasma is added to the suspension, any D-dimer present in the specimen causes the latex microparticles to agglutinate, and the congregating aggregates have diameters greater than the wavelength of the light passing through the solution. The resulting increased absorbance of light is measured photometrically and is directly proportional to the amount of D-dimer present in the test specimen.
Citrated plasma and buffer are incubated with anti-D-Dimer antibody-coated magnetic particles that are washed, magnetically separated, and incubated with isoluminol-labeled anti-D-dimer antibody. Chemiluminescence is measured following a second magnetic separation and washing. The relative insensitivity of latex agglutination assays spurred the development of more-sensitive POC testing, including slide-based D-dimer assays that take advantage of red blood cell RBC agglutination principles.
A widely used example often observed in acute care settings includes the SimpliRED D-Dimer assay Agen Biomedical Limited , which incubates a drop of whole blood with a hybrid, bispecific monoclonal antibody that recognizes D-dimer and an RBC membrane antigen.
D-dimers present at concentrations higher than the cut-off level will cause visible RBC agglutination that must be visually interpreted by trained personnel. Despite this disadvantage, the test is rapid, inexpensive, and has a reported high sensitivity. It uses a laminated card that consists of a thin, porous, antibody-coated membrane sandwiched between an underlying absorbent pad and an overlying plastic layer with a central well. Specimen plasma is pipetted into the well and enters the membrane, where D-dimer antigens may bind to the antibodies coating the membrane.
This step is followed by the addition of colloidal 4-nm-in-diameter gold particles complexed with reagent immunoglobulins that target a different D-dimer epitope. After washing the solution, any remaining gold particles will impart a red color that is directly proportional to the amount of D-dimer in the solution and can be interpreted visually to yield a qualitative result or can be evaluated by a reflectometer or card reader to yield a quantitative result.
These commercially available qualitative solid-phase immunochromatographic tests rely on a single-use plastic device that houses an acetate cellulose chromatography membrane with immobilized gold-conjugated monoclonal antibody. Several automated fluorescence immunoassays are widely used for D-dimer analysis in POC settings.
Several automated, rapid, quantitative enzyme immunoassays are commercially available for D-dimer analysis. Thromboembolic disease is caused by dysregulation of complex hemostatic regulatory mechanisms, which leads to the formation of thrombi that may cause local vascular occlusion or may embolize to occlude distal vessels.
Because of its severe clinical consequences, many breakdown products of the coagulation system have been studied as potential diagnostic markers for thromboembolic disease. D-dimers represent specific breakdown products of cross-linked fibrin clot formation; to our knowledge, D-dimers are the only clinically beneficial biomarkers for routine use in patients with DVT, PE, and disseminated intravascular coagulation DIC.
Circulating D-dimers are also elevated in patients with coronary artery disease and other cardiovascular diseases, cancer, trauma, pregnancy, infectious and inflammatory diseases, severe renal disease, recent surgical procedure s , advanced age, and many other conditions, health factors, and diseases.
The diverse array of POC and central laboratory D-dimer assays has generated confusing, often contradictory streams of interassay comparative studies in patients with thromboembolic disease. In these studies, the sensitivity, specificity, negative and positive predictive values, and other parameters of D-dimer assays have been compared with the results of radiographic studies and with other clinical and laboratory data to attempt to determine the most efficacious and cost-effective means of diagnosis and treatment monitoring of thromboembolic disease.
These studies have been particularly relevant in recent years because of the increasing demand for rapid test turnaround time. None of the patients with negative D-dimer assay results had developed subsequent VTE by the 3-month follow-up visit. Interlaboratory variation is a serious problem that further compromises the value of the D-dimer assay. Although small, single-center studies often reveal high sensitivity, specificity, and negative predictive values for commercial D-dimer assays, the results of larger studies have shown that the situation is less ideal.
The Coagulation Resource Committee of CAP has taken an active role in the investigation and resolution of this problem. Depending on the testing method, D-dimer results may be reported using 2 different types of units: fibrinogen equivalent unit and D-dimer unit.
The accuracy of the D-dimer assay is further compromised by individual patient heterogeneity in D-dimers determined by patient age, genetic influences, disease factors, hereditary and acquired coagulation deficiencies, the size of the blood clot, and the timing of specimen collection in relation to the thrombotic event. In this study, the specificity of the D-dimer results in specimens from patients of all ages increased with age-adjusted cutoff values but was most dramatic in patients aged 80 years or older, in which specimens the unadjusted cut-off was In these circumstances, laboratories have many opportunities to advance patient care by assuring the appropriate clinical use, performance, and reporting of the D-dimer assay.
This assay should be requested only in an appropriate clinical context and should be used with caution in patients with recent trauma or surgical procedure s , pregnancy, malignant neoplasms, cirrhosis, or severe infection, as well as those who recently received anticoagulation therapy or fibrinolytic drugs.
Using assays that have been validated in clinical studies and have established cut-off values.
Overview of Innovance® D-DIMER of New D-Dimer Reagent
Sysmex J Int. D-Dimer is a specific marker for cross-linked fibrin degradation products, and its presence in human plasma is an indicator of fibrinolytic activity. Elevated D-Dimer levels are observed in all diseases and conditions associated with increased coagulation activation, e. D-Dimer measurement is therefore widely used in the diagnostic work-up of thromboembolic disease, and its determination is used for various diagnostic purposes. The major application of D-Dimer testing is in the exclusion of thromboembolic events, such as for outpatients suspected of having deep vein thrombosis DVT or pulmonary embolism PE by following a non-invasive diagnostic algorithm[1 ], [2 ].
D-dimer is formed during activation of the coagulation system and is commonly assayed in order to diagnose disseminated intravascular coagulation, deep vein thrombosis, and pulmonary embolism. Enzyme-linked immunosorbent assay has been validated as the reference method, but it is a time-consuming procedure. A total of plasma samples from apparently healthy individuals and samples from patients were collected for linearity, precision, and correlation studies. Testing the precision of low- and high-controls yielded CV values of 2. Thus the Innovance D-dimer method showed acceptable precision and linearity, and the assay results showed acceptable correlation with the STA Liatest D-dimer method. The Innovance method was relatively unaffected by potential interfering substances such as bilirubin and hemoglobin. In conclusion, the Innovance D-dimer assay is suitable for monitoring D-dimer concentrations in various clinical conditions and should be useful in clinical laboratories.