D-Dimer and High Molecular Weight Fibrin Degradation Products
Fibrinogen is a blood protein from which fibrin clots are formed upon blood coagulation or thrombotic process. Fibrinogen consists of two identical subunits that contain three polypeptide chains: α, β, and γ. During blood coagulation ﬁbrinogen is first converted into ﬁbrin by thrombin, and these fibrin monomers then polymerize to form fibrin clots. In fibrinolysis, the fibrin clots are digested by plasmin, and ﬁbrin degradation products (FDP) of different molecular weights are released into the bloodstream (Fig. 1).
D-dimer (MW 180 kDa) is the final product of ﬁbrin degradation. It consists of the remnants of all three chains (α, β and γ chains) of fibrinogen cross linked by disulﬁde bonds. The dimeric structure of D-dimer is held by two covalent, intermolecular isopeptide bonds between the γ-chains.
D-dimer in diagnostics. D-dimer levels in healthy individuals are less than 0.5 μg/ml. Elevated levels of D-dimer have been found in the blood of patients with pulmonary embolism (PE), deep vein thrombosis (DVT) and atherosclerosis. The elevated level of D-dimer in blood is believed to be a reliable marker of pathological coagulation that underlies the pathogenesis of most cardiovascular diseases (1, 2). It is widely used to exclude the diagnosis of deep vein thrombosis (3).
Despite the long history of using the D-dimer test in clinical practice, there are a lot of problems concerning the quantitative determination of D-dimer in plasma samples. A patient’s plasma contains a wide spectrum of FDP of different sizes along with D-dimer itself. All of these products possess the “D-dimer antigen epitope”. Therefore, antibodies specific to D-dimer also recognize FDP. However, there is a great variance between the results obtained by different assays. This can be explained by differences in antibody specificities; some antibodies and antibody pairs recognize D-dimer better than FDP and vice versa. So far all standardization and harmonization attempts have not resulted in satisfying results and this is a continuous cause of problems in daily practice (4).
For an accurate determination of all FDP and D-dimer, and for using D-dimer as a standard, MAbs should detect FDP and D-dimer with equal specificity. In addition, assays for D-dimer must not detect fibrinogen whose concentration in plasma is 1000 times higher than that of D-dimer.
Assay development and pair recommendations. For development of D-dimer assays, we offer several monoclonal antibodies specific for D-dimer and FDP. The recommended capture-detection pairs for sandwich immunoassays are shown in Table 1. In addition to antibodies, we offer D-dimer that is produced from clotted ﬁbrinogen by means of plasmin digestion.
Monoclonal antibodies specific to D-dimer and FDP. FDP and D-dimer, the most degraded form of FDP, appear in human blood as a result of proteolytic degradation of fibrin clots. The ratio of these products is not constant but varies from patient to patient (see Fig. 4). To decrease bias in quantitation of these degradation products, we have developed an assay which recognizes both FDP and D-dimer with equal specificity. This concept could potentially be one step forward in the attempt to achieve D-dimer assay standardization.
A quantitative sandwich immunoassay that is equally specific for D-dimer and FDP. Advanced ImmunoChemical offers new MAbs (DD189 and DD255) that recognize D-dimer and high molecular weight fibrin degradation products with equal specificity in a sandwich EIA up to 1 μg/ml antigen concentration (Fig. 3). To be analyzed in a sandwich immunoassay, plasma can be diluted ten-fold with 20 mM Tris-HCl buffer, pH 7.5, containing 0.15 M NaCl.
Both MAbs stained D-dimer in Western blotting under reducing and non-reducing conditions (Fig. 6 A and B).
The ratio of D-dimer and FDP varies between patients. Patient plasma from two different disorders was analyzed using gel filtration. The results show that the ratio of D-dimer and FDP is not constant (Fig. 4). This finding further supports the idea that an immunoassay should equally recognize D-dimer and FDP to allow for a more accurate determination of all products resulting from fibrin degradation.
Antibody recommendations for quantitative sandwich immunoassays The recommended pairs are listed in Table 1. They are specific to cross-linked material (D-dimer and high molecular weight fibrin degradation products) in samples and do not detect fibrinogen (Fig. 5).
Table 1. Recommended pairs to be used in a sandwich immunoassay for D-dimer detecgtion in human plasma (DELFIA immunoassay platform).
* Due to the cross-reactivity of DD4 with fibrinogen, we strongly recommend to use it as the detection antibody. In a sandwich immunoassay, plasma must be diluted at least two-fold with 10 mM Tris-HCl, pH 7.5, 1 M NaCl, 0.1 % Tween 20 in order to avoid nonspecific binding. Each step in the assay should be followed by an incubation and wash: coating with the capture MAb, addition of the sample and addition of the (conjugated) detection MAb.
The results demonstrate that the D-dimer assays do not detect fibrinogen, however, some HMW fibrin degradation products are present in the preparation.
Anti-D-dimer MAbs can be used in Western blotting Anti-D-dimer antibodies can be used in Western blotting to detect D-dimer. All MAbs stained nonreduced D-dimer and some of them stained reduced D-dimer as well (Fig. 6 A and B, respectively).
Bounameaux H, de Moerloose P, Perrier A, Reber G. Plasma measurement of D-dimer as diagnostic aid in suspected venous thromboembolism: an overview. Thromb Haemost. 1994; 71: 1-6.
Rowbotham BJ, Carroll P, Whitaker AN, Bunce IH, Cobcroft RG, Elms MJ, Masci PP, Bundesen PG, Rylatt DB, Webber AJ. Measurement of crosslinked fibrin derivatives–use in the diagnosis of venous thrombosis. Thromb Haemost. 1987; 57: 59-61.