American Association for Clinical Chemistry
Better health through laboratory medicine
April 2009 Clinical Laboratory News: D-Dimer

 

April 2009: Volume 35, Number 4


D-Dimer
A Non-invasive Triage Test for Patients with Suspected DVT
By Shiu-Ki Rocky Hui, MD, and Alan E. Mast, MD, PhD

 

 

Deep venous thrombosis (DVT) affects more than 250,000 people in the U.S. each year. In particular, pregnant women, the elderly, and patients with malignancy are at increased risk for developing DVT; however, the condition can occur at any age. It mainly affects the large veins in the lower leg and thigh. Undiagnosed and untreated DVT is particularly dangerous because of the risk of a pulmonary embolism. Not only can a clot block blood flow in the lower extremities, but it also can break off and move through the bloodstream, lodging in the lungs, leading to severe damage and even death. Patients can also develop postphlebitic syndrome if DVT causes structural damage to the venous valves.

While timely antithrombotic therapy significantly reduces both DVT morbidity and mortality, the treatment poses the risk of bleeding, making accurate diagnosis of patients with suspected DVT critical. However, diagnosis presents a number of challenges for physicians. The clinical symptoms of lower extremity DVT, such as leg swelling and pain, are non-specific and can be confused with other conditions, including cellulitis, Baker’s cyst, musculoskeletal injury, and lymphedema.

Currently, the gold standard test for diagnosis of DVT is contrast venography, a procedure that requires injection of a contrast material. Because it is time-consuming, expensive, and, perhaps most importantly, risky for patients with poor renal function, contrast venography is only used rarely as a screening test, especially in ambulatory care or emergency settings. Compression ultrasound has become the diagnostic method of choice for suspected DVT, despite the fact that this procedure is also time-consuming and expensive. True DVT is identified in only 10% to 25% of patients receiving a diagnostic evaluation for DVT, although many patients present with the typical symptoms.

A rapid, low-cost, and simple peripheral blood test with good accuracy and reproducibility would provide a valuable tool for physicians to rule out the presence of DVT, as well as reduce the need for more time-consuming and expensive testing. Testing for plasma D-dimer in the ambulatory care or emergency setting has emerged as an excellent non-invasive triage test for patients with suspected DVT. The test has a high negative-predictive value (NPV) for DVT when used properly.

Here we describe how laboratorians can help ensure accurate diagnosis of this common and potentially life-threatening condition.

Biochemistry of D-dimer

Fibrinogen consists of a six-peptide molecule with two terminal D domains and a central E domain (Figure 1). Thrombin cleaves fibrinopeptides A and B from E domain region, producing the soluble fibrin monomer. These monomers are then assembled with end-to-end and side-to-side association to form non-covalent fibrin polymers. Factor XIIIa crosslinks these fibrin polymers by catalyzing isopeptide bond formation between the γ-chains in the D domain of the adjacent monomers, which result in an insoluble cross-linked fibrin clot. When plasmin, the end product of the fibrinolytic system, is formed, it degrades insoluble, cross-linked fibrin via cleavage of the α-, β-, and γ-chains and results in the liberation of a variety of fibrin degradation products (FDPs) with a large range of molecular weights. Among these FDPs is the D-dimer, a fragment with a molecular weight of ~180kDa (Figure 1).

Figure 1
Biochemistry of D-dimer Formation

Fibrinogen is converted to cross-linked fibrin polymers through the activity of thrombin and factor XIII. Cross-linked fibrin is degraded by plasmin to generate degradation products containing D-dimer.

Given this biochemical scheme, the presence of D-dimer in blood corresponds to the in vivo formation and degradation of a fibrin clot. Since 2% to 3% of plasma fibrinogen is physiologically converted to cross-linked fibrin and then degraded, small amounts of D-dimer are normally present in the plasma of healthy individuals. But when the coagulation and fibrinolytic cascades are activated in vivo, as is the case with DVT, the plasma concentration of D-dimer increases dramatically. The converse is also true: plasma D-dimer concentration decreases in response to anticoagulant therapy and the resolution of symptoms. The protein has a plasma half-life of approximately 8 hours and clearance occurs mainly via the kidneys and reticulo-endothelial system.

Measurement of D-dimer

D-dimer contains a neo-epitope that is formed following the cross-linking of adjacent D domains by factor XIIIa. It is this epitope that is recognized by specific antisera used in clinical assays. Koopman and coworkers introduced one of the first clinical assays for D-dimer in 1987, a solid-phase EIA containing a mouse monoclonal antibody that specifically recognized cross-linked fibrin degradation products and not degradation products of fibrinogen or uncross-linked fibrin.

Currently, more than 30 different D-dimer assays are commercially available. While these assays represent a wide range of techniques (Table 1), the fundamental principle of the D-dimer assay has remained largely unchanged: recognition of the unique neo-epitope in D-dimer by specific antisera.

Table 1
Commercially Available D-dimer Assays*

Technique

Examples

Sensitivity

Specificity

Comments

Microplate ELISA

Asserachrom Ddi (Stago)

Enzygnost (Dade-Behring)

High

Low

Gold standard; batch analysis not used in real-time single testing

ELISA and Fluorescence (ELFA)

Vidas DD (bioMerieux)

Stratus D-dimer (Dade-Behring)

High

Low

Similar sensitivity to classic microplate ELISA; suitable for real-time use

ELISA and Chemiluminescence

Pathfast (Mitsubishi)

Immulite (Siemens)

High

Low

Similar sensitivity to classic microplate ELISA; suitable for real-time use

Immunofiltration and Sandwich-type

NycoCard (Nycomed)

Cardiac D-dimer (Roche)

High

High

Less sensitive than classic microplate ELISA; suitable for real-time use

Semi-Quantitative Latex Agglutination

Dimertest latex (IL)

Fibrinosticon (bioMerieux)

Intermediate

Intermediate

Rapid; but insufficiently sensitive to be clinically useful

Manual, Whole- Blood Agglutination

SimpliRED (Agen)

Clearview Simplify D-dimer (Agen)

High-Intermediate

High-Intermediate

Rapid; can be performed on whole blood; observer dependent

Second-Generation Latex Agglutination (immuno-turbidimetric)

TinaQuant (Roche)

Liatest (Stago)

MDA D-dimer (bioMerieux)

High

Intermediate

Rapid; Similar sensitivity to classic microplate ELISA

*Adapted from Righini M, Perrier A, De Moerloose P, Bounameaux H. D-dimer for venous thromboembolism diagnosis: 20 years later. J Thromb Haemost 2008;6:1059–1071.

The classic microplate ELISA was considered the gold standard for measuring D-dimer. However, due to the time-consuming and labor-intensive nature of the ELISA technique, it has limited utility in the rapid diagnosis of DVT, especially in outpatient and emergency room settings. Modified ELISA methods have now replaced the microplate ELISA method.

These newer assays use more robust final detection methods such as fluorescence, chemiluminescence, or time-resolved fluorescence, allowing the assays to be automated and reducing the turn-around time to approximately 30 minutes. Quantitative latex agglutination with photometric or turbidimetric detection provides a fully automated and rapid method for D-dimer assays that can be performed on routine coagulation or clinical chemistry analyzers and eliminates the need for dedicated instruments.

Point-of-care (POC) methods, such as the semi-quantitative latex agglutination-based system, provide very rapid bedside testing that does not require complicated instrumentation. However, since most of these POC systems require subjective visual reading of agglutination, inter-observer variability makes them less sensitive than automated methods and greatly limits their clinical utility. Recently, manufacturers have developed a POC D-dimer assay that eliminates visual reading of agglutination and provides fully automated bedside testing. These POC devices can quantitatively measure D-dimer in plasma and whole blood and have minimal turn-around times. Such POC devices may be valuable for rapid rule-out of DVT in the outpatient clinic without the need of more costly and time-consuming imaging studies.

Standardization of D-dimer Assays

Labs typically determine the diagnostic cutoff values for D-dimer from assay-specific reference curves established using the manufacturer’s calibrators. The numeric results of D-dimer assays are reported either as D-dimer concentration (assays that use purified fibrin fragment D-dimer as the calibrator) or fibrinogen-equivalent units (FEUs)—assays that use purified fibrinogen for preparation of a fibrin clot and degradation by plasmin as the calibrator. Laboratorians can transform D-dimer concentration to FEU based on the concept that one unit of FEU is approximately twice the mass of one unit of D-dimer (2FEU=1 D-dimer). Therefore, multiplying the D-dimer concentration by 2 converts the mass to the approximate FEU concentration.

Walker and Nesheim have demonstrated that there is a wide spectrum of D-dimer-containing FDPs released from a fibrin clot via the fibrinolytic action of plasmin. All of these D-dimer-containing FDPs react with the specific D-dimer antibodies. Therefore, the reported D-dimer concentration or FEU actually represents the measurement of a heterogeneous mixture of D-dimer-containing FDPs, while the “true” D-dimer concentration is the amount of cross-linked D-domains present in a sample, irrespective of the size of the FDPs. Since each assay has its own calibrator that is made from either purified D-dimer or plasmin proteolysis products of fibrin clots, these calibrators do not necessarily mirror the heterogeneous nature of D-dimer-containing FDPs in a clinical sample. As a result, the value of D-dimer measured in a sample can vary greatly depending on which assay was used and how it was calibrated. Currently, there is no established reference method, primary standard, or a standard universal calibrator for the D-dimer assay.

This lack of standardization is well illustrated in the 2007 College of American Pathologists (CAP) survey results. The 196 labs participating in the survey used 12 different methods to measure D-dimer. The reported mean D-dimer value for these assays varied from 269.69 ng/mL to 2,449.92 ng/mL. On the other hand, the intra-assay variability between labs was much lower, with the majority of assays having CV of <10%, indicating that intra-assay precision is generally good.

In addition, manufacturers typically recommended that each lab establish its own reference range. Without a universal standardization of D-dimer, it can be difficult for individual labs to determine a proper cut-off value to rule out DVT. There have been multiple attempts to standardize the various quantitative D-dimer methods; however, none is currently in place. Because of the inherent differences in the various D-dimer assays, laboratorians need to pay close attention to the specific D-dimer methods used in published clinical studies. The same cutoff values may not apply unless the lab uses the same manufacturer’s assay.

D-Dimer for Diagnosis of DVT

False-negative results from a D-dimer assay can lead to serious and potentially fatal clinical consequences. When a lab picks a D-dimer assay for diagnosing DVT, it is important to select an assay with high sensitivity and a low CV at the cutoff to minimize false-negative results. The specificity of the assay should also not be overlooked, because a high rate of false-positive results can lead to unnecessary imaging studies and anticoagulation treatments.

While there are some trade-offs between sensitivity and specificity among the various methods, all the commercially available assays have comparable clinical utility. In general, in the setting where the D-dimer will serve as a first-step diagnostic tool to rule out DVT, labs should select an assay with high sensitivity. However, if the D-dimer test is used in conjunction with other tests, a less sensitive and more specific assay should be used to avoid unnecessary additional testing.

Multiple clinical studies have produced efficient algorithms that integrate D-dimer testing into the diagnosis of DVT in ambulatory care and emergency settings (Figure 2). Wells and co-workers demonstrated that the use of a manual, whole-blood agglutination D-dimer test in combination with a scoring system for clinical pretest probability, known as the Wells score, resulted in a significant reduction in the number of patients having compression ultrasound testing performed. In another study, Bates and co-workers demonstrated that a negative immuno-turbidimetric D-dimer test, along with a low-to-moderate pretest probability effectively rules out the presence of DVT.

Figure 2
Sample Algorithm for Incorporating D-dimer Testing in the Diagnosis of DVT in Ambulatory Care Setting

 

Adapted from Hargett CW, Tapson VF. Clinical probability and D-dimer testing: How should we use them in clinical practice? Semin Respir Crit Care Med 2008;29:15–24.

While the use of D-dimer testing to rule out DVT in the ambulatory care setting is well established, its use in the elderly, pregnant women, and patients with recurrent DVT is less clear. Researchers hypothesized that these patients may have increased D-dimer in the absence of DVT compared to general patients in the outpatient setting. However, several recent studies aimed at resolving these issues have not proven this to be the case.

Carrier and co-workers found that among patients ages 60 to 80, a low-to-unlikely Wells score in combination with a negative D-dimer test (manual, whole-blood agglutination, or immuno-turbidimetric) result had a 99% NPV. However, in patients greater than 80 years, the NPV of a low-to-unlikely Wells score in combination with a negative D-dimer result has an NPV of only 21 to 31%. Chan and co-workers performed a similar study of pregnant patients and found that a low pretest probability and a negative D-dimer (manual, whole-blood agglutination) had an NPV of 100%. Rathbun and co-workers also studied the effectiveness of D-dimer (immuno-turbidimetric assay) in excluding recurrent DVT. They demonstrated that of the patients with a negative D-dimer test at presentation only 0.75% had a confirmed diagnosis of symptomatic venous thromboembolism at 3 months follow-up. Therefore, it appears that measurement of D-dimer is useful for ruling out DVT in these more complicated patient populations.

Making the Right Choice

Despite the lack of assay standardization, D-dimer testing is an effective, inexpensive, and rapid diagnostic tool for ruling out the presence of DVT in ambulatory care and emergency patients. In combination with a low-to-moderate pretest probability, a negative D-dimer test can efficiently eliminate the need for more expensive and time-consuming imaging studies.

In selecting a D-dimer assay, it is important to understand what clinical role D-dimer testing will play. If the intention is to rule out the diagnosis of DVT without the need for further studies, then labs should select an assay with high sensitivity and NPV to minimize false-negative results.

But no matter which assay a lab chooses to use, laboratorians need to emphasize to their physician customers that a proper clinical evaluation and evidence-based pretest probability is vital for the proper interpretation of D-dimer assay results.

SUGGESTED READINGS

  • Bates SM, Kearon C, Crowther M, Linkins L, et al. A diagnostic strategy involving a quantitative latex D-dimer assay reliably excludes deep venous thrombosis. Ann Intern Med 2003;138:787–795.
  • Carrier M, Le Gal G, Bates SM, Anderson DR, et al. D-dimer testing is useful to exclude deep vein thrombosis in elderly outpatients. J Thromb Haemost 2008;6: 1072–1076.
  • Chan WS, Chunilal S, Lee A, Crowther M, et al. A red blood cell agglutination D-dimer test to exclude deep venous thrombosis in pregnancy. Ann Intern Med 2007;147:165–170.
  • Dempfle CE. D-dimer assays: The current status and new assay technologies. Thrombosis Research 2006;118: 569–571.
  • Hargett CW, Tapson VF. Clinical probability and D-dimer testing: How should we use them in clinical practice? Semin Respir Crit Care Med 2008;29:15–24.
  • Koopman J, Haverkate F, Koppert P, Neiuwennhuizen W, et al. New enzyme immunoassay of fibrin-fibrinogen degradation products in plasma using a monoclonal antibody. J Lab Clin Med 1978;109:75–84.
  • Meijer P, Haverkate F, Kluft C, de Moreloose P, et al. A model for the harmonization of test results of different quantitative D-dimer methods. Thromb Haemost 2006;95:567–572.
  • Rathbun SW, Whitsett TL, Raskob GE. Negative D-dimer result to exclude recurrent deep venous thrombosis: A management trial. Ann Intern Med 2004;141:839–845.
  • Righini M, Perrier A, De Moerloose P, Bounameaux H. D-dimer for venous thromboembolism diagnosis: 20 years later. J Thromb Haemost 2008;6:1059–1071.
  • Wells PS, Anderson DR, Rodger M, Forgie MF, et al. Evaluation of D-dimer in the diagnosis of suspected deep-vein thrombosis. New Engl J Med 2003;349: 1227–1235.
  • Walker JB, Nesheim ME. The molecular weights, mass distribution, chain composition, and structure of soluble fibrin degradation products released from a fibrin clot perfused with plasmin. J Biol Chem 1999;274:5201–5212.

Shui-Ki Rocky Hui, MD, is a transfusion medicine fellow at the Blood Center of Wisconsin in Milwaukee, Wis.

 

 

 

Alan Mast, MD, PhD, is an investigator and associate medical director at the Blood Center of Wisconsin and an associate professor of pathology and cell biology, neurobiology and anatomy at the Medical College of Wisconsin in Milwaukee.