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1 SPECIAL ARTICLE Evaluation of the Biosite Ò Quantitative Whole Blood D-dimer Assay and Comparison With the biomérieux VIDAS Ò D-dimer Exclusion Test Validation and Utility For Use in the Central Laboratory and at the Point of Care Elizabeth Lee Lewandrowski, PhD, MPH and Elizabeth M. Van Cott, MD Abstract: The Biosite D-dimer is a new, rapid assay utilizing EDTA whole blood or plasma that can be used at the point-of-care or in a central laboratory. Most coagulation assays, including many D-dimer assays, require citrated plasma, but the D-dimer has not been previously studied using citrated plasma specimens. Seventyfour citrated plasma specimens on which D-dimer testing was requested for clinical testing were assayed on both the VIDAS and the D-dimer systems. A subset of 29 of these specimens also had an EDTA specimen collected simultaneously and were assayed using the D-dimer system. Diagnostic agreement between citrate and VIDAS citrate, EDTA and VIDAS citrate, and citrate and EDTA was $93%. Correlation coefficients (R 2 ) were 0.86 for citrate versus VIDAS, 0.93 for EDTA versus VIDAS, and 0.96 for citrate versus EDTA. Diagnostic agreement was not changed after adjusting for the dilutional effects of citrate. Bland Altman bias plots showed no bias between citrate and EDTA results if citrate results were adjusted for the dilutional effects of citrate. results, whether EDTA, citrate, or adjusted citrate, tended to underestimate the VIDAS result at the low end and overestimate at the high end. In conclusion, the results suggest that citrate is a suitable alternative to EDTA for the system. These data suggest that the D-dimer compares favorably with the VIDAS, albeit the cutoff values are different. Further studies are warranted to confirm the possibility that D- dimer is a suitable alternative to VIDAS. Key Words: D-dimer, point of care, whole blood test (Point of Care 2005;4: ) From the Department of Pathology, Massachusetts General Hospital, Clinical Chemistry and Coagulation, Boston, Massachusetts, USA. Reagents for this study were supplied at no charge by Biosite Diagnostics Inc. The authors received no remuneration for the study or for preparation of the manuscript. Elizabeth Lee Lewandrowski (or members of the author s family) have received occasional speaking honoraria or research support from Biosite Diagnostics. Reprints: Elizabeth Lee Lewandrowski, PhD, Department of Pathology, Massachusetts General Hospital, Clinical Chemistry and Coagulation, Gray 5, Fruit Street, Boston, MA USA ( Elewandrowski@ partners.org). Copyright Ó 2005 by Lippincott Williams & Wilkins Multiple studies are available concerning the use of D-dimer testing in the evaluation of deep venous thrombosis (DVT) and pulmonary embolism (PE). 1,2 In 2004, the United States Food and Drug Administration (FDA) approved the biomérieux VIDAS D-dimer exclusion assay (VIDAS D-dimer) for the exclusion of both DVT and PE. Currently, the VIDAS D-dimer is the only brand available that has been specifically approved for this claim. However, the role of D-dimer testing in the evaluation of DVTand PE continues to be unclear. 1 3 A new rapid whole blood assay for D-dimer (Biosite D-dimer test [ D-dimer]) was recently introduced by Biosite Diagnostics (San Diego, CA, USA) for the quantitative measurement of cross-linked fibrin degradation products containing D-dimer in EDTA whole blood and plasma specimens. The instrument performs a number of other immunodiagnostic tests designed for use on whole blood specimens either in the central laboratory or at the point of care. This study reports an evaluation of the D-dimer test with a comparison with the VIDAS D-dimer assay. In addition, because coagulation assays usually require blood collection using citrate tubes, we report for the first time an evaluation of D-dimer using citrated plasma. METHODS AND MATERIALS Testing for D-dimer on the VIDAS and systems was performed according to the manufacturers specifications. The test characteristics of the two assays based on manufacturers package insert data are shown in Table 1. The D-dimer test is a fluorescence immunoassay for the quantitative determination of cross-linked fibrin degradation products containing D-dimer. This assay uses an EDTA whole blood or plasma sample. Unlike the VIDAS system, the D-dimer test has not been specifically approved for the exclusion of DVT or PE. The is a point-of-care testing device that performs a variety of immunoassay tests, including cardiac markers (myoglobin, CK-MB, troponin I, BNP), drugs of abuse, a stroke panel (recently introduced in the European market), and D-dimer. The platform is also used in central laboratories where rapid whole blood testing is desired. Test results for the D-dimer assay are available in 15 minutes, utilizing a single-use disposable test cartridge. Point of Care Volume 4, Number 3, September
2 Lee Lewandrowski and Van Cott Point of Care Volume 4, Number 3, September 2005 TABLE 1. Comparison of the VIDAS D-dimer and the D-dimer Assays Based on the Manufacturers Package Insert Data Feature VIDAS Assay type Fluorescence immunoassay Enzyme-linked fluorescence immunoassay Antibody 3B6 monoclonal antibody Anti-FbDP monoclonal antibody Sample type EDTA whole blood/plasma d plasma Platform Point of care/central lab Central lab Turnaround time 15 min 35 min not including centrifugation Centrifuge time NA 2 15 min depending on instrumentation Sample volume 250 ml 200 ml Reportable range ng/ml 45 10,000 ng/ml Expected normal values 90%, 400 ng/ml, 95%, 600 ng/ml 96%, 500 ng/ml Manufacturers suggested cutoff,400 ng/ml,500 ng/ml Quality control (QC) frequency Electronic QC each day, liquid QC each lot or every 30 days, two internal controls in every cartridge Liquid QC each kit and each recalibration (60 tests per kit) Calibration frequency Each reagent lot Each reagent lot and every 14 days Precision Total imprecision of 15.4% at 128 ng/ml Total imprecision of 5.7% at 264 ng/ml to 6.1% at 2990 ng/ml to 7.1% at 7283 ng/ml The VIDAS D-dimer test is an automated enzyme-linked fluorescent immunoassay that utilizes a citrated plasma sample. Test results are available in approximately 35 minutes (not including centrifugation time). It is generally believed that the VIDAS D-dimer is a gold standard method against which new D-dimer methods can be compared. Sample Collection and Storage Seventy-four citrated plasma specimens on which D-dimer testing on the VIDAS system was requested for clinical testing were obtained as discarded samples. These samples were centrifuged and tested immediately on the system. Of the 74 samples, 29 also had simultaneously drawn EDTA anticoagulated whole blood samples available as discarded specimens in the hematology laboratory. These 29 samples were subsequently run on the system. All these specimens were stored at room temperature and were tested on the within a maximum of 2 hours from the time of the initial VIDAS D-dimer result. An audit of 290 consecutive D-dimer results from the VIDAS system in routine laboratory use was also performed using test records in the laboratory information system. Diagnostic agreement, linear regression analysis, and Bland Altman plots were used to compare paired and VIDAS D-dimer results. RESULTS The reportable range and accuracy of 4 meters was verified by analysis of 2 control samples and 5 calibration samples representing a D-dimer range from 173 to 4360 ng/ml. In all 4 cases the correlation coefficients (R 2 ) were greater than The precision of the D-dimer test was validated on 4 meters by repetitive analysis (5 10 replicates each) of 2 control samples and 3 calibration verification samples representing a range of approximately 100 too 4300 ng/ml. The coefficients of variation ranged from 2.6 to 13.1%. Of note, the lowest value tested frequently gave results less than 100 ng/ml, and therefore precision at this low range could not be reliably determined. However, the values ranged from less than 100 to 133 ng/ml, indicating clinically acceptable precision at the low end of the reportable range. Overall imprecision of the 4 instruments was greater at the lower end of the reportable range (2.6%, 12.6%, 13.1%, and 12.3% at approximately 369 ng/ml) and better at the upper end of the reportable range (3.5%, 4.1%, 2.6%, and 3.9% at approximately 4120 ng/ml). At the Massachusetts General Hospital, we perform approximately 6000 D-dimer tests per year using the VIDAS system 24 hours per day. An analysis of 290 consecutive test results revealed that 56 cases (19.3%) were requested from the emergency department (ED) whereas the remaining 234 cases (80.7%) came from hospital inpatient units. Of the 56 cases from the ED, 18 (32.1%) were above the cutoff level (,500 ng/ml) for the VIDAS and 38 (67.9%) were below the cutoff. The range of results from the 56 ED cases was 83 to 3498 ng/ml. All these values are within the reportable range of the Biosite on the high end or would have been reported as negative on the low end. ED testing for D-dimer included 39 females (69.6%) and 17 males (30.4%). The total turnaround time for D-dimer testing for the ED using the central laboratory (including pre- and postanalytic phases) was consistently greater than 2 hours. Excluding transportation time, the average turnaround time from the receipt of the specimen by the central laboratory for 176 consecutive specimens received from the ED and inpatient floors was 60 minutes. Figure 1 shows a comparison of the VIDAS D-dimer using citrated samples with the Biosite system using citrate (Fig. 1A) and EDTA (Fig. 1B) anticoagulants. blood collection tubes contain 10% citrate solution when filled with blood, whereas the EDTA blood collection tubes contain dried EDTA. Consequently, the citrate tubes are expected to 134 q 2005 Lippincott Williams & Wilkins
3 Point of Care Volume 4, Number 3, September 2005 VIDAS and D-dimer Systems FIGURE 2. Comparison of the D-dimer assay using EDTA and citrate anticoagulant. FIGURE 1. Comparison of the VIDAS D-dimer with the D-dimer assay using citrate (A) and EDTA (B) anticoagulants. have a slight dilutional effect on the D-dimer result when compared with EDTA. For that reason, comparisons for the citrate results were performed with and without adjusting for the 10% dilution. The comparisons showed the following statistics: A. citrate = 1.15 VIDAS 147 (R 2 = 0.86) citrate adjusted for dilution = 1.27 VIDAS 162 (R 2 = 0.86) B. EDTA = 1.33 VIDAS 204 (R 2 = 0.93) Figure 2 shows a comparison of the D-dimer using both citrate and EDTA specimens. The comparison yielded the following statistics: A. EDTA = 1.07 citrate + 52 (R 2 = 0.96) EDTA = 0.98 adjusted citrate + 52 (R 2 = 0.96) Figure 3 shows the diagnostic agreement between the VIDAS D-dimer and the D-dimer using citrate anticoagulant (Fig. 3A) or EDTA (Fig. 3B), and between the D-dimer using citrate and EDTA (Fig. 3C) using the manufacturers suggested cutoff values (VIDAS D-dimer, 500 ng/ml fibrinogen equivalent units, D-dimer, 400 ng/ml). In all 3 cases, the diagnostic agreement was $93% using the manufacturers suggested cutoff points. In the case of the VIDAS (citrate) versus the (citrate), the diagnostic agreement was 96% on 74 patient samples. Diagnostic agreement was unchanged after adjusting the citrate results for the dilutional effect. Table 2 shows the discrepant cases from each of the 3 comparisons shown in Figure 3. In all but 2 cases (VIDAS 721 vs citrate 236, and citrate 103 vs EDTA 434), the discrepancies resulted from one or more of the comparative values being close to the suggested cutoff points. By examining all the comparative data between the VIDAS and the systems we were not able to find a more optimum cutoff than that suggested by Biosite Diagnostics (,400 ng/ml), indicting that this value is appropriate if it is assumed that the VIDAS system is the gold standard for comparison. Figure 4 shows Bland Altman bias plots comparing citrate (adjusted) with VIDAS (Fig. 4A), EDTA with VIDAS (Fig. 4B), and citrate (adjusted) with EDTA (Fig. 4C). The bias plot comparing unadjusted citrate with VIDAS looks essentially the same as Figure 4A with adjusted citrate (data not shown). The bias plot comparing unadjusted citrate with EDTA does show that unadjusted citrate underestimates the EDTA result across the entire range of results, as expected due to dilution by citrate solution in the blood collection tube (data not shown). Figure 4C shows that after adjusting the citrate results for the dilutional effect, there is no bias seen versus EDTA. The Bland Altman figures suggest that (citrate [not shown], adjusted citrate [Fig. 4A], or EDTA [Fig. 4B]) overestimates VIDAS results in the high range and underestimates VIDAS results in the low range, which may be at least partly why the manufacturer chose a lower cutoff of 400 ng/ml. The lower cutoff to maintain adequate sensitivity may come partly at the expense of reduced specificity, in that more normal individuals will be above the cutoff with than with VIDAS (10% vs 4% per Table 1). Nevertheless, with assessment for venous thromboembolism, sensitivity is the critical requirement, rather than specificity. q 2005 Lippincott Williams & Wilkins 135
4 Lee Lewandrowski and Van Cott Point of Care Volume 4, Number 3, September 2005 DISCUSSION Venous thromboembolism including DVTwith or without PE is common, resulting in significant morbidity and mortality (more than 50,000 deaths per year in the United States). A number of conditions predispose to thromboembolism, including postoperative states, trauma, pregnancy, immobility, oral contraceptives, as well as congenital and acquired hypercoagulable states. Nevertheless, the majority of patients presenting with signs and symptoms of thromboembolism ultimately are found not to have the condition. In addition, many patients with DVT and even small pulmonary emboli are asymptomatic. In patients with untreated DVT approximately 5 to 20% ultimately develop PE. 4 For these reasons, diagnostic testing for thromboembolism is essential. Testing for DVT may include various combinations of laboratory tests (D-dimer) and radiologic procedures, including ultrasound and either magnetic resonance or computed tomographic (CT) venography. A number of diagnostic studies are also available for the diagnosis of PE, including D-dimer, chest CT, lung scanning, and pulmonary angiography. Assays for D-dimer may be used as a rapid initial test for thromboembolism and are relatively inexpensive when compared with radiologic studies. A value for D-dimer of less than 500 ng/ml using the VIDAS D-dimer test will rule out both DVT and PE in the majority of cases. In outpatients and ED patients with a low likelihood of PE, a normal D-dimer value is sufficient to exclude PE from the differential diagnosis. 5 For this reason, the VIDAS D-dimer test was recently approved by the FDA for the exclusion of DVT and PE. The sensitivity and specificity of D- dimer assays from other manufacturers varies depending on the method. 1 3 As a result, the VIDAS D-dimer test is considered the current gold standard against which other manufacturers assays can be compared. A number of studies have shown that a negative VIDAS D-dimer result using a cutoff point of less than 500 ng/ml yields a high negative predictive value for DVT and PE, ranging from % depending on the study. 3 A number of conditions other than DVT and/or PE may result in an elevated D-dimer level, including disseminated intravascular coagulation, myocardial infarction, and a variety of other conditions. According to the manufacturer of the VIDAS D- dimer, although the sensitivity (.99%) and negative predictive value (approximately 100%) of their assay is high, the specificity using a cutoff of less than 500 ng/ml is only 33%. Therefore, testing for D-dimer in patients with suspected DVT/PE is useful as a rule-out test, but the value of a positive test result must be interpreted in conjunction with clinical and radiologic findings. Patients suspected of having either DVT or PE may present to the ED or they may present during a hospitalization for some other condition. Rapid testing for D-dimer may be FIGURE 3. Diagnostic agreement between the VIDAS D-dimer and the D-dimer using citrate (A) and EDTA (B) anticoagulant and between the D-dimer using citrate and EDTA (C). TABLE 2. Values (ng/ml) of Discordant Samples From Three Comparisons Vidas Vidas EDTA EDTA q 2005 Lippincott Williams & Wilkins
5 Point of Care Volume 4, Number 3, September 2005 VIDAS and D-dimer Systems FIGURE 4. Bland-Altman figures showing comparison between the VIDAS D-dimer and the D-dimer using adjusted citrate anticoagulant values (A) or EDTA (B) and adjusted citrate versus EDTA (C). useful in either situation as an aid to guide subsequent studies or to discharge selected patients from the ED. In many institutions it is difficult for the central laboratory to provide rapid turnaround time for testing originating from the ED due to preanalytic and various logistic considerations. In our institution, preanalytic delays from the ED (including specimen transportation time) result in a total average turnaround time for all laboratory tests of 220 minutes 5 and D-dimer testing is no exception. A significant percentage of D-dimer tests in our institution (32%) originate from the ED (approximately 2000 tests/year out of 85,000 ED visits, or 2.4% of all ED patients). Of these about one third are above the VIDAS cutoff and two thirds are below the cutoff value. Point-of-care testing in the ED for a selected menu of tests may decrease ED length of stay, decrease the number of hours the ED is on divert status, and lower overall cost. 4 Because patients presenting with signs and symptoms of DVT and/or PE are relatively common, it is reasonable to conjecture that rapid point-of-care testing for D-dimer, if accurate, may yield economic and medical benefits, albeit this has never been documented in the peer-reviewed literature. Several manufacturers offer rapid tests for D-dimer that are potentially suitable for use at the point of care (eg, Biosite Diagnostics, Dade Stratus CS, Roche Diagnostics). The Biosite D-dimer is a rapid (15-minute) whole blood assay that is easy to perform, with a reportable range of 100 to 5000 ng/ml. Our analysis of 56 D-dimer test results from the ED using the VIDAS system showed a range of results from 83 to 3498 ng/ml, indicating that in most cases the Biosite assay would produce a quantitative result within the linear reportable range of the system among ED patients. Using a cutoff of 400 ng/ml, the Biosite D-dimer test using either citrated or EDTA anticoagulated whole blood showed good agreement with the VIDAS method, which requires citrated plasma (diagnostic agreement $ 93%) and showed good analytic correlation (R 2 ), although some bias was noted. Because the Biosite D-dimer is rapid, easy to use, can be performed on whole blood, and compares favorably with the gold standard VIDAS D-dimer, it is especially well suited for applications at the point of care. In addition, the Biosite can also be used to test for CK-MB, myoglobin, troponin I, and BNP, all of which have been shown to be useful as point-ofcare tests in the ED setting. In conclusion this study demonstrates that the D-dimer assay using a cutoff of less than 400 ng/ml compares favorably with the VIDAS D-dimer assay using a cutoff of less than 500 ng/ml. The VIDAS assay has been approved by the FDA for the exclusion of DVT and PE. The data presented here suggest that citrate is a suitable alternative to EDTA for the D-dimer, and clinical trials are warranted to determine further if the D-dimer is an acceptable alternative to the VIDAS assay. Furthermore, the D-dimer test is faster, uses whole blood, and is suited for use at both the point of care and the central laboratory. REFERENCES 1. Stein P, Hull R, Patel K, et al. D-dimer for the exclusion of acute venous thrombosis and pulmonary embolism. Ann Intern Med. 2004;140: Dempfle C, Zips S, Ergul H, et al. Evaluation of 23 quantitative assays as basis for the development of D-dimer calibrators. Thromb Haemost. 2001; 85: Heim S, Schectman J, Siadaty M, et al. D-dimer for deep venous thrombosis: a metaanalysis. Clin Chem. 2004;50: Goldhaber S. Pulmonary thromboembolism. In: Kasper D, Braunwald E, Fauci A, et al, eds. Harrison s principles and practice of internal medicine. 16th ed. New York: McGraw Hill; 2005: Lewandrowski E, Corboy D, Lewandrowski K, et al. Implementation of a point-of-care satellite laboratory in the emergency department of an academic medical center: impact on test turnaround time and patient length of stay. Arch Pathol Lab Med. 2003;127: q 2005 Lippincott Williams & Wilkins 137
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