The use of rapid D-Dimer testing as an aid in the diagnosis of venous thromboembolism

By Robert Janetschek MS, MT (ASCP)

pt01.JPG (10165 bytes)      Historically, the rapid diagnosis of venous thromboembolism (VTE) has been problematic for both physicians and medical laboratory professionals for numerous reasons. To begin, non-invasive scan methods (e.g. helicoidal CT-scan, lung scintigraphy, ventilation/perfusion, venous compression ultrasonography) are not always available in all testing facilities and can be somewhat difficult to decipher. Second, invasive procedures such as angiography and phlebography can involve a certain amount of risk, including possible mortality, to the patient. Lastly, in a population of clinically suspect patients, only 25 percent actually have venous thrombosis.

     Healthcare providers have been receiving an improved picture of the assessment of patients with suspected VTE over the past few years with the introduction of D-dimer assays. VTE frequently produces a large clot load within the circulation, and as the clot breaks down it forms fibrin degradation products, including the D-dimer protein. As a result, patients with deep venous thrombosis (DVT) and its sequel, pulmonary embolism (PE), collectively referred to as VTE, are expected to have elevated levels of D-dimer circulating in their blood. Elevated levels of D-dimer are indicative of reactive fibrinolysis and can be found in other clinical conditions as well, including disseminated intravascular coagulation (DIC) and pre-eclampsia. Plus, elevated levels of D-dimer as an indication of fibrinolysis have also been reported in surgery, trauma, sickle cell, coronary and liver disease, severe infection, sepsis, pregnancy, inflammation and malignancy.

     The pathology and clinical significance of D-dimer formation is rather straightforward. During blood coagulation, fibrinogen is converted to fibrin by the activation of thrombin. The resulting fibrin monomers polymerize to form a soluble complex of noncross-linked fibrin. This soluble fibrin complex is further transformed to cross-linked fibrin by the thrombin activated Factor XIII to form an insoluble fibrin clot. As the fibrin clot is formed, production of plasmin, the major clot-lysing enzyme, is also activated. Although fibrin and fibrinogen are both cleaved by plasmin to yield degradation products, only those degradation products from cross-linked fibrin contain D-dimer and are referred to as cross-linked fibrin degradation products (XL-FDPs). As a result, fibrin derivatives found in human plasma or whole blood containing XL-FDPs, including D-dimer, are a specific indicator of the endogenous fibrin removal process.

     In the past, there was a recognized shortage of accurate and rapid D-dimer assays that could be easily adapted for use in the acute care setting. Qualitative and semi-quantitative latex agglutination procedures used to evaluate DIC were not sufficiently reliable or sensitive for use on patients with suspected VTE. As a result, the majority consensus recommended that their role be limited in any diagnostic algorithm for DVT and PE. The classic enzyme-linked immunosorbent assays (ELISA) available displayed excellent sensitivity, but were time and labor intensive, with a lengthy test turnaround-time, making them impractical for use as a STAT, or quick screening test. Consequently, a healthcare professional could, or would, possibly wait several days to obtain a test result and in the meantime, miss the opportunity to institute a definitive therapy for a potentially life threatening VTE.

     Today, innovative new assays for the detection of D-dimer are available that are sensitive and fast, yielding test results in minutes to a few hours. For example, there are novel quantitative ELISA and colorimetric/turbidimetric immunoassays with vastly improved test turnaround times. In addition, highly sensitive, rapid, qualitative immunochromoatographic and agglutination assays, incorporating monoclonal antibodies to D-dimer, are also available. In addition to being quick and sensitive, these qualitative assays have the added advantage of being used as point of care testing (POCT) devices.

     In response to these recent innovations, American Diagnostica, Inc. of Greenwich, Conn. has introduced the new Simplify D-dimer assay. Simplify is a rapid, membrane filtration based immunochromatographic test procedure, intended for use as a clinical aid in the diagnosis of VTE disorders. This innovative assay utilizes the patented D-dimer specific murine monoclonal antibody DD3B6/22, conjugated to colloidal gold particles, to detect D-dimer. The antibody-gold conjugate binds specifically to D-dimer containing molecules in the patient sample to form a complex. The antibody-gold D-dimer complex migrates through the filter membrane in an aqueous phase until it is captured and concentrated at a zone to which a second, D-dimer specific murine monoclonal antibody has been bound. The concentration of the complexes at this zone causes a pink/purple line to appear on the membrane. If D-dimer concentrations are below the assay’s clinically established cut-off of 80 ng/mL, no visible line appears. Uncaptured gold conjugate continues to flow to the end of the membrane, where it is bound at the procedural control zone by an anti-murine antibody. Formation of a pink/purple line in the procedural control zone indicates that the device is working as intended.

     The Simplify assay is a convenient and economic, 2-step, 10-minute qualitative walk-away procedure, which requires no expensive, automated instrumentation. Unlike other qualitative procedures, Simplify is free from the subjectivity of visual interpretation by testing personnel. Simplify assays also allow for added flexibility in patient testing as either whole blood, or plasma from sodium citrate, heparin and EDTA anticoagulants may be used, with no interference from hemolysis, lipemia, bilirubin or rheumatoid factor. In addition, Simplify test strips (10 per kit) offer a 12-month stability, which may be stocked at either refrigerated or ambient temperature, for added convenience in kit location and storage. Most importantly, the Simplify D-dimer assay offers a high negative predictive value along with outstanding sensitivity (80 ng/mL cut-off), specificity and precision when compared to traditional latex agglutination procedures.

     The Simplify D-dimer assay is sufficiently versatile to be used in a variety of clinical laboratories, as well as in hospital emergency rooms, or any patient testing location. For example, in POLs or large group practices, Simplify can be employed as a POCT device where the VTE strategy includes on-site testing. Simplify is also ideally suited for use in small hospitals without automated D-dimer testing capabilities and limited access to additional diagnostic equipment, as well as in regional laboratories responsible for coordinating a group of smaller laboratories with limited equipment. Lastly, in commercial reference laboratories and/or large hospital STAT/Satellite labs, Simplify can be utilized as a backup to automated procedures, or as an after hours test.

     In conclusion, Simplify is a fast, non-invasive exclusion test, accessible to numerous diagnostic-testing sites, which serves as a beneficial tool in the detection and diagnosis of VTE. As discussed, the assay can be performed without the need of elaborate instrumentation, is sensitive, easy-to-use and can reduce invasive and expensive examinations (avoiding dangerous and sometimes unnecessary treatment) thereby saving time, costs and patient inconvenience.

Robert Janetschek is marketing services manager for diagnostic products at American Diagnostica Inc. and can be reached c/o American Diagnostica Inc., P.O. Box 1165, Greenwich CT, 06836-1165. 
Phone • 203-661-0000
Fax • 203-661-7784