By Lisa Fratt

Deep vein thrombosis (DVT) and pulmonary embolism (PE) are two of the more perplexing and troubling conditions that may be present in patients who visit the emergency department. DVT, a blood clot in a leg or pelvic vein, may be more readily diagnosed than PE, as its clinical symptoms are fairly clear. Patients with DVT often present with a cold or swollen limb that may lack a pulse. DVT can lead to the more serious and potentially fatal PE when a blood clot breaks off and travels to the lung. PE is often characterized by more generic symptoms such as shortness of breath, chest pain, and coughing up blood that can be attributed to thousands of other conditions. It is critical to rule out PE in emergency department patients, as patients with untreated PE can die within 1 hour.

The HEMOCHRON Response from ITC performs a variety of coagulation tests at the point of care.

The murky diagnostic picture is complicated by the conditions’ serious clinical outcomes. Together, DVT and PE are the leading causes of preventable in-hospital mortality in the United States, with at least 30,000 PE deaths annually. Accurate diagnosis and treatment of PE with anticoagulation medication is critical, as undiagnosed PE has a 30% mortality rate; mortality falls to 3% with diagnosis and treatment. To further complicate things, recent research indicates that the true prevalence of both conditions is underestimated.

Diagnosing DVT and PE
Diagnosing DVT and PE can hinge on a delicate balance among clinical observations, lab tests, and imaging studies. “The problem with diagnosing DVT and PE is that it is more of a ruling-out process than a positive diagnostic process,” says Susana Vidan, PhD, manager of customer support for American Diagnostica Inc in Stamford, Conn.

The lab enters the diagnostic picture with test kits to measure D-dimer. D-dimer tests present a number of issues for laboratorians and health care professionals. Marcia Zucker, PhD, director of clinical research for International Technidyne Corp in Edison, NJ, explains, “The negative predictive value of D-dimer is excellent. It can easily rule out PE. The big problem with the current state of the art is the high frequency of false positives with D-dimer.”

Zucker continues, “There is an issue with the clinical utility of D-dimer. Health professionals [may not be] trained to understand the limitations of the procedure. It’s common for laboratorians to assume that there is no difference between negative and positive predictive value. But it’s rarely true.” That is, although the negative predictive value of D-dimer is excellent, its positive predictive value is not as good. The true benefits of the D-dimer assay lie in its negative predictive value, which, in turn, translates into cost savings by eliminating the need for expensive imaging exams for negative low-risk patients. These patients are able to avoid invasive testing to rule out DVT or PE.

On the other hand, a positive result leads to additional testing to rule out or diagnose DVT or PE. A variety of imaging tests, such as a leg vein ultrasonography, a pulmonary angiography, a ventilation perfusion scan, a lung scintigraphy, or a computed tomography (CT) scan, may be ordered to rule out or diagnose DVT or PE. Unfortunately, current D-dimer assays produce a fair number of false positives, which means a fair number of patients without DVT or PE must undergo additional testing on the basis of a positive D-dimer result.

The Simplify Rapid D-dimer Test from American Diagnostica Inc is used to rule out DVT and PE.

Hospitals can trim the number of false positives and subsequent imaging tests for DVT or PE by implementing a diagnostic classification algorithm in the emergency department. Indeed, “One trend in D-dimer testing is to use a diagnostic algorithm to determine a patient’s probability of PE or DVT,” says Vidan. The pretest probability (PTP) scoring consists of a series of questions that evaluate the patient’s risk for DVT and PE and divides patients into three risk categories: low, medium, and high. The algorithm can boost the value of the D-dimer test. “If hospitals train ER personnel to use the classification algorithm, the combination of the algorithm and D-dimer can have a 100 percent negative predictive value,” says Vidan. Low-risk patients with a negative D-dimer can be released from the emergency department with a high degree of confidence that DVT or PE is not present. Medium- or high-risk patients require imaging studies regardless of the D-dimer assay’s results to confirm the diagnosis.

The D-dimer Market
D-dimer assays are not new to the market; what is fairly new is the widespread use of the tests. “We are seeing rapid growth in the use of D-dimer tests,” confirms Giovanni Russi, corporate product manager for hemostasis for Instrumentation Laboratory in Lexington, Mass. Russi calculates that D-dimer demand is growing 20%–30% annually.

Laboratories evaluating their D-dimer options will find multiple types of D-dimer assays from several different manufacturers on the market. Some assays are based on lateral flow, some are single-test cartridges, and some are ELISA- or latex-based. All of the kits on the market are moderate complexity, according to Clinical Laboratories Improvement Act (CLIA) classification. “The most powerful tests are quantitative tests,” says Russi.

Single-test-cartridge assays are probably the easiest to use. The test works like a pregnancy test. On the plus side, the 10-min test is easy to perform and requires minimal intervention or oversight on the laboratorian’s part. Moreover, results are clearly objective—either negative or positive—and the test can be used with whole blood or plasma. Some clinicians, however, prefer a quantitative test result with a numerical measure.

Another option is the red blood cell aggregation assay. This requires a bit more lab intervention than the cartridge assay. It is completed manually on a plastic slide, with the laboratorian adding a small drop of blood and a drop of reagent to the slide. Typically, the antibody recognizes red blood cells (RBCs) and D-dimer, so the D-dimers in the blood cause the aggregation (cross-linking) of RBC. The amount of clumping correlates with the concentration of D-dimers in the blood. On the positive side, the whole blood test can be completed in 3 minutes; however, aggregation assays yield subjective results. It is critical that the laboratorian have experience and/or training with the test to confidently call a positive, says Vidan.

Latex-agglutination tests may be the most familiar D-dimer assay and are quite similar to red blood cell aggregation assays. The main difference? Latex-agglutination assays rely on latex beads coated with antibody. D-dimers in the plasma aggregate the latex beads. These assays can be performed manually or they can be automated. The manual assays are a valuable option, but most rely on the subjective interpretation of the results by the operator, thus introducing a variable in the outcome of the results. In addition, most manual assays are not quantitative and do not report a numeric result.

The automated latex-agglutination assays offer the advantage of automation, ease of use, and a quantitative D-dimer result in a few minutes. The automated latex assays are in general more sensitive than the manual options and show better clinical performances once used for the exclusion of DVT and PE.

There are some perceived disadvantages of latex-agglutination tests on the clinical side. Some latex-agglutination assays may not be as sensitive as other tests, says Vidan. Russi counters, “At proper cutoff and the negative predictive value and sensitivity are 100 percent. The primary benefit of using latex technology is the full automation on coagulation instruments, with time to result in a few minutes.”

The final D-dimer option is the ELISA test. Like all ELISAs, the D-dimer ELISA is a quantitative test that provides a concentration of D-dimers. Quantitatively oriented clinicians often prefer the ELISA test, as it produces a “number” for D-dimer concentration. For the lab, however, the ELISA test can be more time-consuming than other alternatives, with some tests taking 35 minutes or longer. An ELISA reader is required to complete the test. Finally, in many labs more experienced technicians carry out ELISAs. More skilled laboratorians may work only the day shift, so the test may not be available 24/7.

In the last few years, a number of scientific journals have included articles in which the relative sensitivity of automated latex D-dimer assays compared to the ELISA technique were debated. The most recent studies have demonstrated that the latest generations of automated latex assays seem to have a clinical performance similar to ELISA methods.

The Meaning Behind the Numbers
Although clinicians and lab professional may prefer quantitative test results, the quantitative results yielded by a D-dimer assay can be somewhat murky. The major hitch comes in the form of cutoff values or standardization for the D-dimer assay. “There is no gold standard. Each test has a different cutoff value, so there is no way to correlate or cross-reference results among different tests,” explains Vidan. Laboratorians, who may have been trained at different institutions using different tests, need to understand the value of the particular test used in the hospital and not rely on a generic D-dimer value, because that number does not apply to other tests.

“The tentative steps taken by the scientific community have not yet succeeded in standardizing D-dimer,” says Russi. The reason? D-dimer is part of a heterogenous mixture of fibrin degradation products known as XL-FbDP. The different antibodies used in the variety of commercially available D-dimer assays have different specificity for fibrinogen, fibrin, and their derivatives. As a result, the different D-dimer assays have different normal ranges and a different definition of abnormal results.

To complicate the picture, the D-dimer concentration is expressed by the different manufacturers either as ng/mL or as FEU (fibrinogen equivalent unit)/mL. The relation between the two units is approximately 1: 2, but this brings more confusion about the cutoff value to be used for the exclusion of DVT and PE.

For these reasons, each assay will have a different cutoff value for DVT and PE. As a result, the hospital must evaluate the clinical performances of a D-dimer assay in relation to the negative predictive value and the sensitivity of the assay to rule out DVT and PE at a certain D-dimer cutoff value. The clinical performances of the D-dimer assays have to be addressed on the basis of the results coming from management studies conducted on that specific test. Information about these results can be either provided by the manufacturers and/or published in scientific journals.

Because the D-dimer assay is a tool for the exclusion of DVT and PE, it is important to be aware of the negative predictive value (NPV). NPV represents the percentage of patients with negative D-dimer (below the cutoff) and no confirmed cases over all D-dimer negative patients. The NPV of what is commonly considered the gold standard, venography, is 97%–98%. It is therefore expected that the D-dimer assays have a NPV of at least 98% at the selected cutoff value. An NPV of 100% would indicate that none of the samples with D-dimer below the cutoff is a false negative, thus supporting the exclusion of DVT and PE.

The specificity of the assay is less important than the NPV, but it gives an indication of the number of false-positive patients. The higher the specificity, the lower the number of false-positive patients, which, in turn, indicates the predictive power of the assay for DVT and PE. Commercially available assays have specificity in the range of 25%–50%. The main reason is that high D-dimer levels can be a consequence of conditions other than the presence of DVT and PE. Consequently, it is important that a patient with a positive D-dimer (above the cutoff) is always followed with a confirmatory investigation that typically is performed with objective techniques such as imaging studies. Specificity may become important when comparing different assays on the same patient population. In this case, a higher specificity would imply less false-positive patients undergoing more expensive and time-consuming investigations.

DVT/PE Treatment and the Lab
Treatment for DVT and PE typically consists of anticoagulation medications such as heparin or warfarin, and inpatients may be monitored to measure activated clotting time (ACT). Once again, as in diagnosis, there are some gray areas for the lab. “The key issue with ACT assays is the lack of standards among instruments. Results are not equivalent among different systems. Every system on the market will give a different result for a given patient,” says Zucker. Consequently, laboratorians need to understand that results are relative, not specific, according to Zucker. Given the complexities of these systems, it is important for the lab to develop and adhere to quality-assurance programs to ensure accurate results and readings.

Moreover, if the hospital implements a new ACT, the test must be evaluated for use in DVT and PE treatment, and the new test should be compared to the current standard. Finally, the lab must complete correlation and bias analysis to determine any necessary changes in target-treatment times.

Although DVT and PE are fairly common, they are difficult to diagnose. By educating laboratorians and clinicians about the utility and limits of D-dimer assays and ACT testing, the lab can play a key role in reducing unnecessary imaging tests and facilitating rapid and accurate diagnosis.

Lisa Fratt is a contributing writer for Clinical Lab Products.