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A Rapid and Quantitative D-Dimer Assay in Whole Blood and Plasma on the Point-Of-Care PATHFAST Analyzer ARTICLE in PRESS MODEL 6 TR-03106; No of Pages 7 ARTICLE IN PRESS Thrombosis Research (2007) xx, xxx–xxx intl.elsevierhealth.com/journals/thre REGULAR ARTICLE A rapid and quantitative D-Dimer assay in whole blood and plasma on the point-of-care PATHFAST analyzer Teruko Fukuda a,⁎, Hidetoshi Kasai b, Takeo Kusano b, Chisato Shimazu b, Kazuo Kawasugi c, Yukihisa Miyazawa b a Department of Clinical Laboratory Medicine, Teikyo University School of Medical Technology, 2-11-1 Kaga, Itabashi, Tokyo 173-8605, Japan b Department of Central Clinical Laboratory, Teikyo University Hospital, Japan c Department of Internal Medicine, Teikyo University School of Medicine, Japan Received 11 July 2006; received in revised form 7 December 2006; accepted 28 December 2006 KEYWORDS Abstract The objective of this study was to evaluate the accuracy indices of the new D-Dimer; rapid and quantitative PATHFAST D-Dimer assay in patients with clinically suspected Deep-vein thrombosis; deep-vein thrombosis (DVT). Eighty two consecutive patients (34% DVT, 66% non-DVT) Point-of-care testing; with suspected DVT of a lower limb were tested with the D-Dimer assay with a Chemiluminescent PATHFAST analyzer. The diagnostic value of the PATHFAST D-Dimer assay (which is immunoassay based on the principle of a chemiluminescent enzyme immunoassay) for DVT was evaluated with pre-test clinical probability, compression ultrasonography (CUS). Furthermore, each patient underwent contrast venography and computed tomogra- phy, if necessary. The sensitivity and specificity of the D-Dimer assay using 0.570 μg/ mL FEU as a clinical cut-off value was found to be 100% and 63.2%, respectively, for the diagnosis of DVT, with a positive predictive value (PPV) and negative predictive value (NPV) of 66.7% and 100%, respectively. The correlation between the results of PATHFAST D-Dimer and VIDAS D-Dimer was acceptable (y=1.134×+0.003, r=0.902). The test reproducibility was good (CV%: from 4.0% to 5.0% for plasma and from 7.1% to 7.5% for whole blood) and the total imprecision was very good (CV%: 3.6–5.7%). Whole blood as well as plasma can be used as samples in this assay (y=1.013×−0.010, r=0.971 for heparinized specimens; y=1.068×+0.003, r=0.989 for citrated speci- mens). Because of its high sensitivity and NPV PATHFAST D-Dimer assay can be useful for the rapid rule out of DVT in patients admitted with suspected thrombosis. © 2007 Elsevier Ltd. All rights reserved. Abbreviations: DVT, deep-vein thrombosis; CUS, compression ultrasonography; PPV, positive predictive value; NPV, negative predictive value; ELISA, enzyme-linked immunosorbent assay; POCT, point-of-care testing; ROC, receiver operating characteristic; AUC, area under the curve; CI, confidence interval; FEU, fibrinogen equivalent unit. ⁎ Corresponding author. Tel.: +81 3 3964 1211; fax: +81 3 5944 3354. E-mail address: [email protected] (T. Fukuda). 0049-3848/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2006.12.021 Please cite this article as: Fukuda T et al. A rapid and quantitative D-Dimer assay in whole blood and plasma on the point-of-care PATHFAST analyzer. Thromb Res (2007), doi:10.1016/j.thromres.2006.12.021 ARTICLE IN PRESS 2 T. Fukuda et al. patients were diagnosed as having a DVT. Patients Introduction with any of the following criteria were excluded: previous episode of DVT, stable symptoms at Suspected deep-vein thrombosis (DVT) is a common presentation or prophylactic anticoagulants already condition, with a lifetime cumulative incidence of 2 applied at presentation. Heparinized or citrated to 5% [1]. Compression ultrasonography (CUS) and blood samples (n=124; 52 women and 72 men; age contrast venography are the most reliable methods range 24–52 years) were obtained from apparently for diagnosis of DVT [2]. Contract venography is the healthy subjects who had given informed consent gold standard for diagnosis of DVT [3] but it is not among the staff of the company Mitsubishi Kagaku ideal because of its invasive nature and the risks Iatron, Inc., Chiba, Japan. Blood samples were associated with contrast media. CUS requires skilled collected into 10 mL vacuum tubes containing investigators and its sensitivity and specificity can 0.129 M trisodium citrate or 9 IU lithium heparin. vary considerably depending on which part on the All whole blood samples were tested within 6 h. In lower extremity the examination is performed. order to obtain plasma samples, blood samples D-Dimer is a mixture of cross-linked fibrin deg- containing anticoagulants were centrifuged at radation products which is a marker of endogenous 2000 ×g for 15 min to obtain platelet poor plasma. fibrinolysis and thus it can be detected in patients The plasma samples were tested either within 6 h or with DVT. Retrospective studies have shown a high stored frozen at −40 °C or −80 °C prior to use. A negative predictive value of a D-Dimer concentra- stability check indicated that plasma samples tion for the exclusion of DVT when taking into stored at −40 °C or −80 °C were stable over a account a defined cut-off value for the used assay period of at least 3 months and longer. [4]. Moreover, various studies have proven the safe- ty of withholding anticoagulation in those patients Diagnosis of deep venous thrombosis with a negative D-Dimer test result and a normal ultrasonography [5,6]. The DVT diagnostic work-up used in our institution is Several D-Dimer assays including enzyme immu- based on pre-test clinical probability, according to noassays, latex assays and immunoturbidimetric the score proposed by Wells et al. [14] and assays are currently available but their clinical compression ultrasonography (CUS), according to efficiency can differ markedly. Among them, en- standard criteria [15]. Furthermore, each patient zyme immunoassays show a high sensitivity and underwent contrast venography, and computed negative predictive value (NPV) in the presence of tomography. DVT in comparison to latex assays [7–13]. Some of these assays can be used as point-of-care tests D-Dimer testing (POCT) for an emergency need and among them the VIDAS D-Dimer assay is one of the well-established methods for diagnosis or exclusion of DVT. The The PATHFAST™ D-Dimer assay (Mitsubishi Kagaku newly developed PATHFAST D-Dimer assay is a Iatron, Inc., Tokyo, Japan) is a fully automated, sensitive and quantitative method based on the rapid and quantitative chemiluminescence enzyme principle of enzyme immunoassay using a chemilu- immunoassay for measurement of degradation minescent substrate. Whole blood as well as plasma products of cross-linked fibrin (D-Dimer) in human can be used as samples and the reaction time takes whole blood and plasma samples. Both heparin and only 5 min. In this study, our aim has been the citrate can be used as anticoagulants to obtain evaluation of the diagnostic value of the PATHFAST plasma. The PATHFAST analyzer can automatically D-Dimer assay when used as a screening test to distinguish whole blood and plasma samples by exclude DVT. means of a sample recognition sensor. Results obtained with whole blood samples are corrected Materials and Methods optionally by input of the individual hematocrit value in percent (%) in the PATHFAST analyzer. A default hematocrit value (40%) is used by the Clinical samples PATHFAST analyzer, when no value adjustment is performed. When whole blood is tested, the This study included 82 consecutive outpatients (40 manufacturer recommends to start the PATHFAST women and 42 men; age range: 23–85 years; D-Dimer assay within 5 min after whole blood women, 56.1±18.0 years; men, 46.8±17.7 years) sample has been dispensed into a sample well of a referred to our hospital for diagnostic work-up for PATHFAST cartridge. The PATHFAST D-Dimer assay is suspected DVT of a lower limb. Twenty eight a one-step sandwich immunoassay method Please cite this article as: Fukuda T et al. A rapid and quantitative D-Dimer assay in whole blood and plasma on the point-of-care PATHFAST analyzer. Thromb Res (2007), doi:10.1016/j.thromres.2006.12.021 ARTICLE IN PRESS A D-Dimer assay in whole blood and plasma on the point-of-care PATHFAST analyzer 3 automated on PATHFAST analyzer which permits to standard methods for proportions. The 95% single tests with ready-to-use reagents including confidence intervals (CI) were calculated according magnetic particles covalently conjugated with a D- to the binomial distribution. The correlation was Dimer monoclonal antibody and alkaline phospha- calculated according to Spearman and a regression tase-conjugated D-Dimer monoclonal antibody. The analysis was performed using the method of Passing D-Dimer antibodies used by Mitsubishi Kagaku and Bablok [16]. Iatron, Inc. were raised against degradation pro- ducts of cross-linked fibrin as an immunogen. The Comparison method antibodies recognize neo-antigens of degradation products of cross-linked fibrin prepared after The Biomerieux VIDAS® D-Dimer assay [17] was used digestion by plasmin. After the immuno-reaction as the comparison method in this study. This assay of D-Dimer antigen contained in the blood sample was performed according to the manufacturer's and the reagents for 5 min during which an immune instructions. The VIDAS D-Dimer assay is an enzyme complex is formed, bound/free separation is per- immunoassay with a detection limit of 45 ng/mL FEU formed by Magtration® procedure. Chemilumines- (0.045 μg/mL FEU) and an upper assay range of cent signal is measured after the addition of a 10,000 ng/mL FEU (10 μg/mL FEU). A cut-off value of chemiluminescent substrate (CDP-Star®) to the 500 ng/mL FEU (0.500 μg/mL FEU) is used for that immune complex.
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