By Robert Janetschek, MSc, MT (ASCP) & RS Greenfield, PhD

Formation of blood clots (thrombi) that block arteries or veins is a life-threatening event that can result in severe medical consequences including myocardial infarction, stroke, and thromboembolism. Therefore, the rapid and accurate clinical diagnosis of blood clots is critically important for patient survival and represents a major challenge for both clinicians and medical laboratory professionals. Formation of undesired clots occurs when one of the arms of the coagulation cascade is spontaneously triggered within an artery or vein. A key step in the development of a thrombus is proteolytic cleavage of fibrinogen that initiates the formation of a fibrin clot. During thrombus formation, hemostatic mechanisms also trigger activation of fibrinolytic (clot lysing) mechanisms, the body’s own countermeasure to clot formation. Plasmin, the main fibrinolytic protease, cleaves fibrin in the clot resulting in formation of a variety of fibrin degradation products (FDPs). One particular form of FDPs is called cross-linked FDPs (XL-FDPs) and the finding of elevated levels of XL-FDPs circulating in the blood is a strong indicator of the occurrence of thrombosis. Thus, rapid testing procedures for the presence of XL-FDPs are critical for the diagnosis and effective management of patients presenting thrombotic symptoms.

Biochemistry & Clinical Significance of XL-FDPs
Injury to an artery or vein due to, for example, disruption of arteriosclerotic plaque can initiate the coagulation cascade. This ultimately leads to the activation of prothrombin to thrombin, the key protease responsible for fibrin clot formation. Thrombin converts fibrinogen to fibrin monomers, which polymerize to form a soluble fibrin-gel complex. This soluble fibrin-gel complex is further transformed by covalent cross-linking of fibrin monomers by activated Factor XIII, which is a transglutaminase-like enzyme. The cross-linking of fibrin monmers by FXIIIa leads to the formation of a stable, more durable fibrin clot.

As the fibrin clot is formed, hemostatic mechanisms also activate the fibrinolytic system, which is responsible for clot lysis. Tissue plasminogen activator (tPA) cleaves plasminogen to form plasmin, the major clot-lysing protease that rapidly cleaves the cross-linked fibrin causing dissolution of the clot. Small peptidyl by-products, referred to as FDPs, are then produced by the proteolytic cleavage of the cross-linked fibrin clot by plasmin. There are two types of FDPs, non-cross-linked FDPs and cross-linked FDPs (XL-FDPs), where XL-FDPs contain fibrin fragments cross-linked together by the action of FXIIIa. Therefore, the presence of XL-FDPs in the blood is a reliable indicator of recent fibrin clot dissolution activity.

XL-FDPs are increased in disseminated intravascular coagulation (DIC), in surgical and trauma patients, sickle-cell disease, liver disease, severe infection, sepsis, inflammation, malignancy, and pre-eclampsia coagulopathy. Elevated levels of XL-FDPs are found during myocardial infarction, and in some cases used as a predictive indicator. D-dimer, one of the more distinct and specific monomers of XL-FDPs, is a widely used selective marker for venous thromboembolisms (VTE). Levels of the XL-FDPs proteins may also increase during normal pregnancy, but very high levels are often associated with complications. Finally, quantitation of XL-FDPs has been used to monitor thrombolytic therapy with tissue-type plasminogen activator (tPA) and streptokinase (SK). However, the concentration of XL-FDPs detected in a specimen will depend on several factors, such as the severity of the thrombotic episode, the rate of cross-linked fibrin formation and degradation, and the time elapsed between the thrombotic event and drawing of the blood from the patient.

Measurement of XL-FDPs
Although XL-FDPs can be measured using a variety of methodologies, many of these techniques are not easily adaptable to, or compatible with, routine use in the clinical laboratory.

However, advances in assay technologies have lead the way toward improvements in measuring XL-FDPs for use in the clinical laboratory. Sensitive, rapid, and convenient laboratory tests for XL-FDPs are now available. These tests greatly improve the diagnosis and patient management during a thrombotic crisis. For example, the development of highly specific monoclonal antibodies has lead to the development of qualitative, semiquantitative, and quantitative clinical diagnostic assays for these specific degradation products. As a result of these advances, American Diagnostica Inc of Stamford, Conn, recently introduced the new ACTISCREEN™ XL-FDP assay. ACTISCREEN XL-FDP is intended for use as a rapid qualitative, or semiquantitative, procedure to determine the presence of XL-FDPs in human plasma. The assay is a manual immunoagglutination test procedure utilizing latex beads coupled with a murine monoclonal antibody against XL-FDPs. The monoclonal antibody utilized in the assay is highly specific for XL-FDPs and does not cross-react with fibrinogen, factor XIIIa cross-linked fibrin, or non-cross-linked FDPs.

In the simple, 3-minute test procedure, plasma is mixed with the antibody-coated latex beads on a conveniently designed solid support. The XL-FDPs present in the plasma sample will bind to the coated latex beads, resulting in the aggregation of the beads that is unmistakably visible to the human eye. Positive test results can be seen at concentrations of XL-FDPs in plasma above 200 ng/mL (0.20 mg/L), the lower limit of detection for the assay. This results in a highly sensitive test procedure. In addition, a semi-quantitative test result, if desired, can be obtained by testing serially diluted patient samples.

Performance Characteristics
ACTISCREEN XL-FDP was used to test 145 plasma samples from patients suffering from or having a high probability for thrombotic disorders. There was a very high correlation of positives (r = 0.94) and a resulting regression equation of y = 1.19x when compared to another commercially available test procedure. In another study, the effect of anticoagulants used during plasma collection was also determined. Fifty plasma samples were collected using sodium citrate, EDTA, and heparin as the anticoagulant. The correlations between the titers obtained with

ACTISCREEN XL-FDP and the titers based on ELISA obtained XL-FDP values were r = 0.91 for citrated samples, r = 0.73 for EDTA samples and r = 0.78 for heparin samples, respectively. Citrate is the obvious anticoagulant of choice.

In specificity studies, plasma from 162 out of 170 apparently healthy, volunteer blood donors tested negative (95.3% specificity) using ACTISCREEN XL-FDP. With regard to precision, the intra-assay (within-run) reproducibility was determined for 10 replicates of three plasma samples that contained different levels of XL-FDPs. The results were found to be essentially equivalent for all replicates tested at each concentration of XL-FDP. Inter-assay (run-to-run) reproducibility was determined using ten plasma samples with XL-FDP titers ranging from 1 to 16. In 10 runs, the replicates of these specimens did not vary by more than one titer.

Interference studies showed that substances such as bilirubin to 0.2 mg/mL, hemoglobin to 5.0 mg/mL, lipemia (triglycerides) to 30 mg/mL and protein (gamma globulin) to 0.06 g/mL do not affect the results with ACTISCREEN XL-FDP assay. Additional studies have suggested that ACTISCREEN XL-FDP is also insensitive to rheumatoid factor (RF) disturbances.

Key Features & Benefits
The inherent benefits associated with the ACTISCREEN XL-FDP assay are numerous. To begin, the assay is a convenient, economic and easy-to-use 3-miOPnute procedure, requiring no expensive automated instrumentation. Each test kit contains all necessary reagents, quality-control materials and associated testing components in a liquid-stable format, with demonstrated lot-to-lot consistencies and extended shelf-life claims. The compact, operator-friendly kit design contains 60 tests and allows for easy and convenient storage at refrigerated temperatures. Most important, the ACTISCREEN XL-FDP assay features exceptional sensitivity (200 ng/mL cut-off), along with exceptional performance characteristics.

Additionally, the ACTISCREEN XL-FDP assay is sufficiently versatile to be used in a variety of clinical testing facilities and settings. For example, in POLs, large group practices, and freestanding clinics, ACTISCREEN XL-FDP could be employed as a testing device where the diagnostic profile calls for immediate on-site testing. Second, ACTISCREEN XL-FDP is also ideally suited for use in small hospitals, and STAT and satellite laboratories that do not have fully automated testing capabilities or that have limited access to sophisticated diagnostic equipment. The same holds true for regional core laboratories responsible for coordinating a group of smaller laboratories with limited instrumentation. Lastly, in commercial reference and large hospital laboratories, ACTISCREEN XL-FDP can be utilized as a backup to automated procedures, or as an after-hours test.

Robert Janetschek, MSc, MT (ASCP) and RS Greenfield, PhD, can be reached c/o American Diagnostica Inc, PO Box 110215, Stamford, Conn 06911-0215; (203) 602-7777, Fax: (203) 602-2221.