Fluorescence Immunoassay of Human D-dimer in Whole Blood ; Journal of Clinical Laboratory Analysis

 

Foto : boditech.co.kr

D-dimer is a mixture of cross-linked fibrin degradation products, which is formed after fibrin clots are degraded by a fibrinolytic enzyme called plasmin. D-dimer reflects global activation of platelet coagulation pathway and the degree of thrombin turnover (1, 2). Thus, D-dimer levels have been the most popularly used parameter for the frontline assessment of suspected deep vein thrombosis (DVT) or pulmonary embolism (PE) for almost 30 years (3, 4). In general, commercial D-dimer assays for blood show a high sensitivity up to 95% but have poor specificity, making it difficult to prove the presence of DVT or PE instead of another clotting complication. 

Additionally, elevated levels of D-dimer have been reported in pregnancy, cancer, inflammation, surgery, the elderly, and remain a confounding factor limiting its use (5–9). However, the negative predictive values are nearly 100% accurate, with negative D-dimer test results of indication of a lack of DVT or PE. Hence, the main clinical application of D-dimer assay is to safely rule out DVT, PE, or disseminated intravascular coagulation (10, 11).

Among the commercially available assay systems for D-dimer, enzyme-linked fluorescence (FL) immunoassays and microlatex immunoturbidimetric assays are popularly used (12, 13). Although their performances satisfy the requirements for D-dimer assays in terms of high diagnostic sensitivity and negative predictive values, these assays have some limitations in application during emergency situations or in analyzing a single sample (14, 15).

Thus, we aimed to develop a new D-dimer assay that was out DVT, PE, or disseminated intravascular coagulation (10, 11).

Among the commercially available assay systems for D-dimer, enzyme-linked fluorescence (FL) immunoassays and microlatex immunoturbidimetric assays are popularly used (12, 13). Although their performances satisfy the requirements for D-dimer assays in terms of high diagnostic sensitivity and negative predictive values, these assays have some limitations in application during emergency situations or in analyzing a single sample (14, 15).

Thus, we aimed to develop a new D-dimer assay that was cost-effective and reliable, and could work with whole blood on a point-of-care test (POCT) platform. Here, we introduce a new D-dimer assay system that provides results in 12 min, has a POCT platform, and a reliable analytical performance compared to automated reference analyzers. This newly developed D-dimer assay system takes advantage of a simple lateral flow immunochromatographic assay (ICA) basis, adds a sandwich-type antibody scheme, and employs FL dye as a tracer to detect the level of D-dimer in samples. The FL-ICA D-dimer assay system consists of a monoclonal antibody coated (mAb) strip in a disposable cartridge, a detection buffer containing FL-labeled mAb, and biotin-bovine serum albumin (BSA), a chip for the calibration curve, and a laser FL scanner.

MATERIALS AND METHODS 

Materials 

Human D-dimer and mAb were purchased from Hytest Ltd. (Turku, Finland), and both streptavidin and biotin– BSA were purchased from Sigma-Aldrich (St. Louis, MO). Sephadex G25 and activated Alexa Fluor 647 were obtained from Amersham Pharmacia Biotech (Piscataway, NJ) and Molecular Probes (Eugene, OR), respectively. Nitrocellulose membrane (NC) was purchased from Millipore (Watertown, MA), sample pad and the absorption pads were obtained from Schleicher and Schuell (Keene, NH). 

Labeling of Antibodies With Fluorescent Dye 

For conjugation of the anti-D-dimer-mAb with a FLdye, 10 μl of a 1 mol/l sodium bicarbonate buffer (pH 8.3) was mixed with 100 μl of anti-D-dimer-mAb (1 mg/l) in phosphate-buffered saline (PBS) and followed by the addition of 1 μl of activated Alexa Fluor 647 (10 g/l) to the mixture. After overnight incubation at 4◦C, the mixture was applied onto a Sephadex G25 column to remove the free dye, and FL-labeled mAb conjugates were collected as elutes after centrifugation of the column at 2,500 rpm for 2 min. The biotin–BSA complex was similarly conjugated with Alexa Fluor 647, and FL-labeled biotin–BSA conjugates were purified with same processes as noted above, and used as an internal control for the assay system. The FL-labeled mAb and the FL-labeled biotin–BSA were mixed together with the assay buffer to form the detection buffer and were kept at 4◦C until use.

Immunoassay Strip, Cartridge, and Scanner 

The FL-ICA test strip was fabricated in-house to fit into a disposable cartridge and a laser FL scanner. The sample pad and the absorption pad were cut to a size of 4 × 20 mm and assembled with mAb- and streptavidin-coated NC onto a polystyrene-backing card. The capture mAb and streptavidin were dispensed as 1-mm-wide lines at the test line and the control line, respectively, using a BioJet dispenser (BioDot, Irvine, CA). The assembled strip was kept in a dry vacuum chamber overnight before being placed into a cartridge (15 × 90 mm), which was designed to fit into the holder of the laser FL scanner. The cartridge was then sealed in a foil pouch containing a desiccant and stored at room temperature. Because the appearance of the test cartridge was unique, a laser FL scanner called i-CHROMATM (BoditechMed, South Korea) was used to measure the distribution of FL intensity along the strip of cartridge. The principle of the i-CHROMATM FL scanner was previously described in detail (16).

Assay Procedure 

The detection buffer was a mixture of FL-labeled mAb (detector Abs) and FL-labeled biotin–BSA (internal control) in PBS. A quantity of 10μl from whole blood (plasma and serum) was added to 150 μl of detection buffer, and 75 μl of the mixture was then loaded onto the sample well of cartridge. After 12 min of incubation for immune reactions, the cartridge was inserted into the laser FL scanner for detection of FL intensity. The scanner converts FL intensity to numeric data, calculates the relative amount, and displays the level of D-dimer in the sample as ng/ml on the screen.

Blood Sample 

Blood samples were obtained from individuals who visited the Kangwon National University Medical Center in Chuncheon, South Korea. Informed consent was obtained from volunteers before their participation in the study. The ages of all adult female and male participants were between 32 and 80. Venous blood was collected in 5 ml vacuum tubes (Becton-Dickinson, Franklin Lakes) containing sodium citrate, K2-EDTA, or sodium/lithium heparin, and was centrifuged at 3,600 × g for 10 min at 4◦C to obtain plasma samples. The blood samples were generally tested within 30 min of collection; otherwise, plasma or serum samples were aliquoted in small volumes and frozen at −70◦C before analysis