SEARCH

SEARCH BY CITATION

Keywords:

  • d-dimers;
  • diagnosis;
  • deep vein thrombosis;
  • pulmonary embolism

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We compared three rapid d-dimer methods for the diagnosis of venous thromboembolism. Patients presenting to four teaching hospitals with the possible diagnosis of deep vein thrombosis or pulmonary embolism were investigated with a combination of clinical likelihood, d-dimer (SimpliRED) and initial non-invasive testing. Patients were assigned as being positive or negative for deep vein thrombosis or pulmonary embolism based on their three-month outcome and initial test results. The three d-dimer methods compared were: (a) Accuclot d-dimer (b) IL-Test d-dimer (c) SimpliRED d-dimer. Of 993 patients, 141 had objectively confirmed deep vein thrombosis or pulmonary embolism. The sensitivity of SimpliRED, Accuclot and IL-Test were 79, 90 and 87% respectively. All three d-dimer tests gave similar negative predictive values. The SimpliRED d-dimer was found to be less sensitive than the Accuclot or IL-Test. When combined with pre-test probability all three methods are probably acceptable for use in the diagnosis of venous thromboembolism.

The diagnosis of deep vein thrombosis or pulmonary embolism (venous thromboembolism) is problematic because clinical diagnosis alone is unreliable and the gold standard tests (venography or pulmonary angiography) are invasive and expensive (Kearon et al, 1998a). Consequently, non-invasive imaging such as lung scanning or compression ultrasonography is usually performed first. However, a normal compression ultrasound of the proximal veins or a non-diagnostic ventilation perfusion (V/Q) scan does not rule out a diagnosis of clinically significant deep vein thrombosis or pulmonary embolism respectively (Birdwell et al, 1998; Kearon et al, 1998a,b; Wells et al, 1998a; Anderson & Wells, 1999; Perrier et al, 1999). Management studies have demonstrated that further testing is required, but this results in many normal tests, high expense, and inconvenience for the patient because of the low frequency of pulmonary embolism and deep vein thrombosis (Turkstra et al, 1997; Kearon et al, 1998b). A normal d-dimer test can help to exclude venous thromboembolism in many such patients (Wells et al, 1995; Freyburger et al, 1998; Ginsberg et al, 1998; Kahn, 1998; Wells et al, 1998a; Anderson et al, 1999; Perrier et al, 1999).

The d-dimer tests used in most diagnostic laboratories to screen for disseminated intravascular coagulation are latex agglutination assays which are not sufficiently sensitive to exclude venous thromboembolism (Carter et al, 1993). Initial studies using enzyme-linked immunoabsorption assay (ELISA) d-dimer testing showed sensitivities and negative predictive values approaching 100% (Bounameaux et al, 1994; vanBeek et al, 1996; Janssen et al, 1997). However, ELISA testing is not practical for use on a routine basis, particularly in an Emergency Room setting because batch testing is recommended and, consequently, results are not available in a timely fashion. Moreover, ELISA testing is expensive and has a low specificity.

Consequently, more sensitive and rapid latex agglutination tests and point of care tests for d-dimer have been evaluated (Dale et al, 1994; Ginsberg et al, 1995, 1998; Elias et al, 1996; Turkstra et al, 1996; Bernardi et al, 1998; Wells et al, 1998b). Depending on the population studied and the test used, the sensitivities vary from 80% to 100% with negative predictive values generally greater than 90% (Dale et al, 1994; Ginsberg et al, 1995, 1998; Elias et al, 1996; Turkstra et al, 1996; Bernardi et al, 1998; Wells et al, 1998b). Problems with several of these methods, however, include a greater expense compared with standard latex agglutination d-dimer tests, a need for dedicated instrumentation, a lack of suitability of the test for other d-dimer testing indications (e.g. as a screen for disseminated intravascular coagulation) and the lack of automation leading to subjective interpretation of test results and poorer quality control.

In this study, we compare the accuracy of three rapid d-dimer tests for the diagnosis of acute venous thromboembolism. One of these assays, SimpliRED, is a point of care manual method that has only been used for the diagnosis of venous thromboembolism. The other two, Accuclot and IL-Test d-Dimer, are both suitable to other indications for d-dimer testing and, until now, have not been well studied in deep vein thrombosis and pulmonary embolism. Accuclot is a semiquantitative latex-agglutination manual method, whereas the IL-Test d-dimer is a fully quantitative and automated method.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients Patients with suspected deep vein thrombosis or pulmonary embolism from the London Health Sciences Centre (London), Ottawa Civic Hospital (Ottawa), Queen Elizabeth II Health Sciences Centre (Halifax), and the Hamilton Health Sciences Corporation, McMaster and General Divisions (Hamilton), were potentially eligible for the study. Patients were excluded if they had any of the following: a comorbid condition making life expectancy < 3 months; symptoms having resolved for more than 3 d before presentation; contraindication to contrast media; pregnancy; age < 18 years; or refusal of informed consent. The protocol was approved by the Institutional Review Boards at each site, and all patients gave written consent.

Intervention Consenting patients were assigned a clinical probability (low, intermediate or high pre-test probability) for deep vein thrombosis or pulmonary embolism using explicit clinical models as previously described (Wells et al, 1995, 1998a). SimpliRED d-dimer was performed on citrated whole blood and subsequent diagnostic investigations were based on the results of clinical likelihood and SimpliRED d-dimer testing according to previously described algorithms (Anderson & Wells, 1999). (For example, if the pre-test probability was low and d-dimer was negative, a diagnosis of deep vein thrombosis or pulmonary embolism was considered excluded without the need for objective tests.)

All blood samples were collected at the initial visit. Samples were collected into 0·109 mol/l sodium citrate Vacutainer tubes (Becton Dickinson, Rutherford, NJ). An aliquot of this whole blood was tested immediately using the SimpliRED method (according to manufacturer's specifications) and the remainder of the blood was immediately centrifuged. Samples were spun at 1700 g for 20 min, the plasma removed to a plastic tube and centrifuged again to attain platelet poor plasma. This plasma was transferred to plastic tubes for storage at −70°C. Batch tests were then performed at a later date.

Diagnosis of venous thromboembolism All patients were followed for 3 months. A diagnosis of deep vein thrombosis was confirmed by positive compression ultrasound of the proximal veins or ascending venography. A diagnosis of pulmonary embolism was made by high probability V/Q scan or pulmonary angiogram or a non-diagnostic V/Q scan together with objectively confirmed deep vein thrombosis. Patients were also assigned as being positive for deep vein thrombosis or pulmonary embolism if the initial series of investigations was normal but they subsequently developed objectively documented symptomatic deep vein thrombosis or pulmonary embolism in the 3 month follow-up period.

D-Dimer tests Three d-dimer tests were performed. These methods were chosen because they were the most readily available at the time of the study. SimpliRED (Agen Biomedical Limited, Brisbane, Australia), Accuclot D-dimer (Sigma Diagnostics, St Louis, MO) and IL-Test D-dimer (Beckman Coulter, Lexington, MA). SimpliRED testing was performed immediately by registered laboratory technologists in core laboratories at each centre. Accuclot d-dimer and IL-Test d-dimer were performed in the Special Coagulation laboratory at London Health Sciences Centre. For consistency, these two tests were performed in batches by the same technologist. To eliminate bias all d-dimer tests were performed without knowledge of the other test results or the patients' clinical outcomes or pre-test clinical likelihood.

SimpliRED is a qualitative red cell agglutination assay. A chemical conjugate of a monoclonal antibody specific for d-dimer is linked to a monoclonal antibody that binds to the red blood cell surface. The conjugate will coat the RBC's but will not cause agglutination in samples with d-dimer levels < 200 µg/l. d-dimer levels of > 200 µg/l will bind to the conjugate on the RBC's causing cross linking between conjugate groups of adjacent cells resulting in visible RBC agglutination. Any visible agglutination, including trace agglutination, is considered a positive result.

Accuclot d-dimer is latex agglutination assay for the determination of plasma d-Dimer. Plasma is mixed with a latex reagent that contains particles coated with a mouse monoclonal antibody specific for the d-dimer domain. Agglutination will occur in samples in which the concentration of d-dimer is > 250 µg/l. In this study, samples were tested neat without any further titration. Results are again considered positive or negative based on visible agglutination.

IL-Test d-dimer is an automated method for measuring D-dimer quantitatively. All automated testing was performed on the AMAX CS 190 (Sigma Diagnostics). The kit contains a latex reagent, reaction buffer and a calibrator. The latex reagent is a suspension of polystyrene particles of uniform size coated with a monoclonal antibody specific for the d-dimer domain of soluble fibrin derivatives. When plasma containing d-dimer is mixed with the latex reagent plus the reaction buffer, the coated particles will agglutinate. The degree of agglutination is directly proportional to the concentration of d-dimer in the sample. Agglutination is determined by measuring the decrease in light transmitted at 405 nm. The amount of light transmitted is then compared with a calibration curve to produce a result in ng/ml. A group of 24 normal volunteers was run to establish a reference range. Values < 200 µg/l were considered normal. One lot number of reagents was used in all testing to minimize any variations.

Analysis Each patient was classified as positive or negative for deep vein thrombosis or pulmonary embolism as described above. For each patient the d-dimer result was determined with each of the three d-dimer tests. Sensitivity, specificity, negative and positive predictive values were then calculated. The 95% confidence intervals(C.I.) were determined for each of these values using binomial distribution. Because the three tests varied in measured sensitivity and specificity, differences based on receiver–operator characteristic (ROC) curve analysis were considered inappropriate. The methods were compared by testing for differences in paired proportions (McNemar's chi-square test) using splus software.

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

There were a total of 993 consecutive patients (468 for possible deep vein thrombosis and 525 for possible pulmonary embolism) included in the study and 141 (14%) had objectively documented deep vein thrombosis or pulmonary embolism.

Sensitivity for venous thromboembolism was statistically significantly lower for the SimpliRED d-dimer test 79% (95% C.I. 77–82) than for the Accuclot 90% (88–92, P = 0·0035) and the IL-Test 87% (84–89, P = 0·0339) assays (Tables I and II). However, the specificity of SimpliRED d-dimer test 76% (73–79), was higher than for the other two assays 72% (69–75, P = 0·0088) and 71% (68–73, P = 0·0003) (Tables I and II). Although there was a trend towards a higher negative predictive value with the Accuclot assay compared with the SimpliRED test [98% (97–99) versus 96% (94–97)], this difference was not statistically significant (Tables I and II). Of note, SimpliRED had 29 false negative results compared with 14 for Accuclot and 19 for the IL-Test. Only seven patients were diagnosed with venous thromboembolism in the 3 month follow-up period after the initial investigations were negative. Finally, Table III gives a subanalysis of deep vein thrombosis and pulmonary embolism for sensitivity, specificity and negative predictive value.

Table I.   Comparison of D-dimer tests (n = 993).
 SimpliREDAccuclotIL-Test
True positive (n)112127122
False positive (n)205237251
True negative (n)647615601
False negative (n)291419
Sensitivity percentage (95% C.I.)79 (77–82)90 (88–92)87 (84–89)
Specificity percentage (95% C.I.)76 (73–79)72 (69–75)71 (68–73)
Negative predictive value percentage (95% C.I.)96 (94–97)98 (97–99)97 (96–98)
Positive predictive value percentage (95% C.I.)35 (32–38)35 (32–38)33 (30–36)
Table II.   Comparative accuracy of D-dimer tests.
Method comparisonP-value for sensitivityP-value for specificity P-value for negative predictive value
SimpliRED vs. Accuclot0·00350·00880·0834
SimpliRED vs. IL0·03390·00030·4269
Accuclot vs. IL0·22780·06370·0614
Table III.   Subanalysis of D-dimer results for DVT (n = 468) and PE (n = 525).
 SimpliREDAccuclotIL-Test
  1. DVT, deep vein thrombosis; PE, pulmonary embolism.

Sensitivity (%) for DVT789185
Sensitivity (%) for PE809191
Specificity (%) for DVT676361
Specificity (%) for PE797674
Negative predictive value (%) for DVT919693
Negative predictive value (%) for PE979999

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The diagnosis of deep vein thrombosis and pulmonary embolism remains problematic (Kearon et al, 1998a). Negative compression ultrasound testing and non-diagnostic V/Q scans do not necessarily rule out clinically significant deep vein thrombosis or pulmonary embolism (Kearon et al, 1998a). Recently the use of non-invasive testing, d-dimer testing and clinical pre-test probability has simplified the diagnosis of venous thromboembolism and dramatically reduced the need for more invasive testing.

This study, which is the largest study of its kind to date, shows that both the Accuclot and the IL-Test d-dimer methods have a significantly better sensitivity than SimpliRED for venous thromboembolism with sensitivities of 90%, 87% and 79% respectively. However, consistent with other results is a trade-off with lower specificity.

Several previous studies have illustrated the usefulness of sensitive D-dimer testing with the most widely studied of these methods being SimpliRED (Ginsberg et al, 1995; Turkstra et al, 1996; Kearon et al, 1998b; Wells et al, 1998b). The main advantages of SimpliRED are that to date it is one of the most widely studied d-dimer methods and that it can be used as a point-of-care test. When used as a point-of-care test the main potential disadvantage is that it is operator-dependent, which could decrease the accuracy of the test if performed by untrained personnel. This test is also non-quantitative and so it cannot be used as a screening test for disseminated intravascular coagulation or other conditions that can cause elevation of d-dimer levels.

Initial studies using SimpliRED reported sensitivities for the presence of deep vein thrombosis or pulmonary embolism > 90% (Ginsberg et al, 1995, 1998; Turkstra et al, 1996; Wells et al, 1998b). More recent studies, which have used larger patient populations (Janssen et al, 1997; Freyburger et al, 1998; Ginsberg et al, 1998), suggest that the true sensitivity of SimpliRED for venous thromboembolism is < 90% (Janssen et al, 1997; Freyburger et al, 1998; Ginsberg, 1998). This is probably because the gold standard tests were not used in all the earlier studies and so some smaller venous clots went undetected. Other sensitive d-dimer tests have since been shown to have higher sensitivities than the SimpliRED (Janssen et al, 1997; Freyburger et al, 1998; Ginsberg, 1998). Recent work has illustrated that despite reasonable negative predictive values for all patients, a negative SimpliRED result is only able to safely exclude deep vein thrombosis or pulmonary embolism in patients with low pre-test clinical likelihood. This means that d-dimer testing is not an appropriate stand-alone test (Ginsberg et al, 1998; Wells et al, 1998b; Farrel et al, 2000).

Accuclot is a manual semiquantitative latex agglutination method that is potentially susceptible to observer bias. It does have the advantage compared with SimpliRED that it could be used as a screening test for disseminated intravascular coagulation if performed in dilutions to create a higher cut-off for positive results. Hence, it would probably be appropriate as a sole d-dimer method for coagulation laboratories that are investigating for both venous thromboembolism and disseminated intravascular coagulation.

IL-Test is also appropriate for the investigation of both venous thromboembolism and disseminated intravascular coagulation because a quantitative result is obtained. It has the distinct advantage that it is fully automated and may be more appropriate for busy coagulation laboratories that are investigating both of these conditions.

Limitations of our results are the fact we batch tested Accuclot and IL-Test methods by the same technologist, whereas SimpliRED was performed by several different technologists at the time of initial patient presentation. All testing was, however, carried out without knowledge of either the patient's pre-test clinical likelihood or subsequent results of objective tests to avoid bias. Our study may have an incorporation bias potentially favouring SimpliRED because the investigation for venous thromboembolism was influenced by the SimpliRED test result and not the other two.

On the basis of this study, we conclude that the SimpliRED d-dimer is less sensitive than Accuclot or IL-Test methods when used as a screening test for venous thromboembolism. None is sufficiently sensitive to use a negative result in isolation to rule out deep vein thrombosis; that is, it must be used together with pre-test clinical likelihood. However, as has been previously demonstrated for SimpliRED and others, when combined with clinical assessment a negative result will exclude deep vein thrombosis or pulmonary embolism if pre-test clinical probability is low (Ginsberg et al, 1998; Anderson & Wells, 1999). Negative results in patients with moderate or high pre-test clinical probability has the possible potential to exclude deep vein thrombosis if the initial ultrasound is negative or V/Q scan non-diagnostic (Ginsberg et al, 1998; Wells et al, 1999b; Farrel et al, 2000). Further assessment by large management studies is appropriate.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The authors would like to thank Mrs B. Stevenson and Mrs J. Psiuk-Rodgers for their administrative assistance in the preparation of this manuscript. The authors also wish to acknowledge M. Johnson, Mc Master University, M. Freedman, Ottawa Civic Hospital as well as the technologists from Queen Elizabeth 2 Health Sciences Centre, and London Health Sciences Centre, for their help in providing patient samples.

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  • Anderson, D.R. & Wells, P.S. (1999) Improvements in the diagnostic approach for patients with suspected deep vein thrombosis for pulmonary embolism. Thrombosis and Haemostasis, 82, 878886.
  • Anderson, D.R., Wells, P.S., Stiell, I., MacLeod, B., Simss, M., Gray, L., Robinson, K.S., Bormanis, J., Mitchell, M., Lewandowski, B., Flowerdew, G. (1999) Thrombosis in the emergency department. Archives of Internal Medicine, 159, 477482.
  • Bernardi, E., Prandoni, P., Lensing, A.W.A., Agnelli, G., Guazzaloca, G., Scannapieco, G., Piovella, F., Verlato, F., Tomasi, C., Moia, M., Scarano, L., Girolami, A. (1998) d-dimer testing as an adjunct to ultrasonography in patients with clinically suspected deep vein thrombosis: prospective cohort study. British Medical Journal, 317, 10371040.
  • Birdwell, B.G., Raskob, G.E., Whitsett, T.L., Durica, S.S., Comp, P.C., George, J.N., Tytle, T.L., McKee, P.A. (1998) The clinical validity of normal compression ultrasonography in outpatients suspected of having deep venous thrombosis. Annals of Internal Medicine, 128, 17.
  • Bounameaux, H., De Moerloose, P., Perrier, A., Reber, G. (1994) Plasma measurement of d-dimer as diagnostic aid in suspected venous thromboembolism: an overview. Thrombosis and Haemostasis, 71, 16.
  • Carter, C.J., Doyle, D.L., Dawson, N., Fowler, S., Devine, D.V. (1993) Investigations into the clinical utility of latex d-dimer in the diagnosis of deep venous thrombosis. Thrombosis and Haemostasis, 69, 811.
  • Dale, S., Gogstad, G.O., Brosstad, F., Godal, H.C., Holtlund, J., Mork, E., Brandsnes, O., Borch, S.M. (1994) Comparison of three d-dimer assays for the diagnosis of DVT: ELISA, latex and immunofiltration assay (NycoCard d-dimer). Thrombosis and Haemostasis, 71, 270274.
  • Elias, A., Aptel, I., Huc, B., Chalé, J.J., Nguyen, F., Cambus, J.P., Boccalon, H., Boneu, B. (1996) d-dimer test and diagnosis of deep vein thrombosis: a comparative study of 7 assays. Thrombosis and Haemostasis, 76, 518522.
  • Farell, S., Hayes, T., Shaw, M. (2000) A negative SimpliRED d-dimer assay result does not exclude the diagnosis of deep vein thrombosis or pulmonary embolus in emergency department patients. Annals of Emergency Medicine, 35, 121125.
  • Freyburger, G., Trillaud, H., Labrouche, S., Gauthier, P., Javorshci, S., Bernard, P., Grenier, N. (1998) d-dimer strategy in thrombosis exclusion. Thrombosis and Haemostasis, 79, 3237.
  • Ginsberg, J.S., Wells, P.S., Kearon, C., Anderson, D., Crowther, M., Weitz, J.I., Bormanis, J., Brill-Edwards, P., Turpie, A.G., Mac Kinnon, B., Gent, M., Hirsh, J. (1998) Sensitivity and specificity of a rapid whole-blood assay for d-dimer in the diagnosis of pulmonary embolism. Annals of Internal Medicine, 129, 10061011.
  • Ginsberg, J.S., Wells, P.S., Brill-Edwards, P., Donovan, D., Panju, A., VanBeek, E.J.R., Patel, A. (1995) Application of a novel and rapid whole blood assay for d-dimer in patients with clinically suspected pulmonary embolism. Thrombosis and Haemostasis, 73, 3538.
  • Janssen, M.C.H., Heebels, A.E., DeMetz, M., Verbruggen, H., Wollershelm, H., Janssen, S., Schuurmans, M.M.J., Nováková, I.R.O. (1997) Reliability of five rapid d-dimer assays compared to ELISA in the exclusion of deep venous thrombosis. Thrombosis and Haemostasis, 77, 262266.
  • Kahn, S.R. (1998) The clinical diagnosis of deep venous thrombosis. Archives of Internal Medicine, 158, 23152323.
  • Kearon, C., Julian, M.M., Newman, T.E., Ginsberg, J.S. (1998a) Noninvasive diagnosis of deep vein thrombosis. Annals of Internal Medicine, 128, 663667.
  • Kearon, C., Ginsberg, J.S., Hirsh, J. (1998b) The role of venous ultrasonography in the diagnosis of suspected deep vein thrombosis and pulmonary embolism. Annals of Internal Medicine, 129, 10441049.
  • Perrier, A., Desmarais, S., Miron, M.J., De Moerloose, P., Lepage, R., Slosman, D., Didier, D., Unger, P.F., Patenaude, J.V., Bounameaux, H. (1999) Non-invasive diagnosis of venous thromboembolism in outpatients. Lancet, 353, 190195.DOI: 10.1016/S0140-6736(98)05248-9
  • Turkstra, F., VanBeek, E.J.R., TenCate, J.W., Büller, H.R. (1996) Reliable rapid blood test for the exclusion of venous thromboembolism in symptomatic outpatients. Thrombosis and Haemostasis, 76, 911.
  • Turkstra, F., Kuijer, P.M.M., VanBeek, E.J.R., Brandjes, D.P.M., TenCate, J.W., Buller, H.R. (1997) Diagnostic utility of ultrasonography of leg veins in patients suspected of having pulmonary embolism. Annals of Internal Medicine, 126, 775781.
  • VanBeek, E.J.R., Schenk, B.E., Michel, B.C., Vanden Ende, B., Brandjes, D.P.M., Vander Heide, Y.T., Bossuyt, P.M.M., Büller, H.R. (1996) The role of plasma d-dimer concentration in the exclusion of pulmonary embolism. British Journal of Haematology, 92, 725732.
  • Wells, P.S., Hirsh, J., Anderson, D.R., Lensing, A.W.A., Foster, G., Kearon, C., Weitz, J., D'Ovidio, R., Cogo, A., Prandoni, P., Girolami, A., Ginsberg, J.S. (1995) Accuracy of clinical assessment of deep-vein thrombosis. Lancet, 345, 13261330.
  • Wells, P.S., Ginsberg, J.S., Anderson, D.R., Kearon, C., Gent, M., Turpie, A.G., Bormanis, J., Weitz, J., Chamberlain, M., Bowie, D., Barnes, D., Hirsch, J. (1998a) Use of a clinical model for safe management of patients with suspected pulmonary embolism. Annals of Internal Medicine, 129, 9971005.
  • Wells, P.S., Anderson, D.R., Bormanis, J., Guy, F., Mitchell, M., Lewandowski, B. (1998b) SimpliRED d-dimer can reduce the diagnostic tests in suspected deep vein thrombosis. Lancet, 351, 14051406.
Footnotes
  1. Dr Kovacs is an Internal Scholar, Department of Medicine, University of Western Ontario. Dr Wells is a Canada Research Chair.