by Robert Janetschek, MS, MT (ASCP) and Robert Greenfield, Ph.D.

American Diagnostica’s DVV Reagents for Lupus Anticoagulants testing

Lupus Anticoagulants (LAs) are among the most baffling and challenging pathological conditions tested for within the coagulation laboratory. LAs are heterogeneous collections of antibodies, which frequently interfere with common in-vitro phospholipid-dependent coagulation tests, without necessarily interfering with in-vivo clotting factor activity. Historically classified as anti-phospholipid antibodies, a more accurate description is that they are antibodies directed against plasma proteins, which also bind to phospholipid surfaces.

     The condition’s name itself, Lupus Anticoagulants, is also somewhat confusing as the majority of patients diagnosed with LAs do not suffer from the autoimmune disease Systemic Lupus Erythematosus (SLE) and rarely develop any systemic autoimmune disorders. However, the presence of LAs can be found in a significant number of patients (approximately 50 percent) suffering from SLE. LAs affect approximately 2 to 4 percent of the general population, with a greater prevalence of expression in women than men. Many times, their presence is only accidentally discovered, usually as a prolonged Activated Partial Thromboplastin Time (APTT) detected during a routine surgical screening procedure. However, in today’s environment of managed health care, where the use of preoperative coagulation testing is often discouraged, the unexpected detection of LAs within the general population is also decreasing.

Clinical pathology
     For the most part, there are no clinical implications with the condition other than the need for the explanation of a longer than normal APTT. However, minorities of patients with LAs do exhibit a hypercoagulable state, which may be demonstrated by recurrent venous and arterial thromboses, multiple spontaneous miscarriages, unexplained migraine headaches, sporadic bleeding episodes, thrombocytopenia and possibly even stroke.

     Unfortunately, the exact etiology of LAs is also unclear. Studies within the past five years have only begun to unravel the secrets of LAs and their associated clinical pathologies. It has been hypothesized that the binding of certain proteins to phospholipids alters the protein conformation, leading to exposure of neoepitopes on the proteins. LAs have been shown to be directed against a number of phospholipid proteins, including -2 glycoprotein, prothrombin and annexin V. They are usually of the IgG and IgM isotypes, but IgA isotypes, or mixtures, have also been reported. A number of drugs, including procainamide, hydralazine, isoniazid, quinidine, phenytoin and penicillins (in children) have also been associated with LAs, or lupus-like syndromes. Most studies have indicated that the risk of complications in patients with drug-induced LAs is minimal, or absent.

Guidelines for diagnosing LAs
     The accurate laboratory diagnosis of LAs can be problematic, as no one single test has been proven to be 100 percent accurate and precise. However, through the efforts of the Scientific and Standardization Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies (SSC Subcommittee), a working panel stemming from the International Society on Thrombosis and Haemostasis (ISTH), some degree of inter-laboratory consistency is finally emerging. Perhaps one of the most significant of the guidelines proposed by the SSC Subcommittee encompasses the employment of an “integrated test system,” which includes the use of screening and confirmatory tests to aid in the clinical diagnosis. Consequently, a series of in-vitro diagnostic tests may now be utilized for the diagnosis of LAs in those patients suspected of, or presenting clinical manifestations of, the condition.

     The primary, and certainly the most useful assays which can be routinely employed within the coagulation laboratory, are the routine phospholipid-antibody based clotting assays, such as the activated partial thromboplatsin time (APTT), the kaolin clotting time (KCT), the dilute prothrombin time (dPT) and dilute Russell’s viper venom time (dRVVT) tests. Typically, a prolongation of a clotting time above the limits of a normal quality control sample is indicative of a positive finding for LAs. The SSC Subcommittee guidelines recommend that at least two different clot-based screening assays be performed, followed by a confirmatory assay employing a reagent containing a high phospholipid concentration. The high phospholipid-containing reagent should shorten the clotting time, thereby confirming the presence of phospholipid-dependent antibodies (i.e., LAs).

DVV reagents and controls
     In accordance with these industry recognized guidelines, American Diagnostica Inc. of Greenwich, Conn. offers clinical laboratories the DVV line of reagents and quality control materials to assist in the screening and confirmation of LAs. DVVtest, based on the dilute Russell’s viper venom time (dRVVT) test, is a screening reagent which incorporates venom, phospholipid and calcium chloride into a single vial, providing a one-step clotting assay to test for the possible existence of LAs within the clinical sample being evaluated. The direct activation of factor X by Russell’s viper venom bypasses the contact and intrinsic coagulation factors, excluding interferences of factors VIII, IX, XI and XII and their respective inhibitors.

     DVVconfirm reagent verifies the presence of LAs in a clinical specimen by combining Russell’s Viper Venom, concentrated phospholipid and calcium chloride into one distinct vial. DVVconfirm is a comparative assay, specifically designed for use in conjunction with DVVtest screening reagent. DVVconfirm reagent will correct the prolonged result of a DVVtest if LAs are present and can distinguish LAs from factor VIII and other intrinsic inhibitors. The test offers a demonstrated 100 percent positive predictive value. DVVtest and DVVconfirm reagents are available in two separate and convenient packaging configurations. They feature negligible interference from heparin, and extended reconstituted stability of five days when stored at 2° C to 8° C. Both tests are suitable for manual and semi-automated procedures, as well as for use on automated coagulation instruments. Automated applications are currently available for a broad line of coagulation instruments, including those from Instrumentation Laboratory, Dade Behring, MLA, Diagnostica Stago and Sysmex.

     DVVtroll normal and abnormal quality control materials are also available to fully support both DVVtest and DVVconfirm. The normal control is a lyophilized preparation from a multi-donor pool of normal plasmas, with added buffers and stabilizers. The abnormal control is a lyophilized preparation derived from known positive LAs donors, in a buffered solution, tested and shown to be positive for LAs in accordance with the revised criteria of the SSC Subcommittee. Source plasmas for both controls are processed by means of a uniform approach consistent with established protocols to ensure platelet-poor plasma and have been tested to be non-reactive for HbsAg, HIV-1 and HCV.

     Recent advancements have started to resolve the LAs testing quandary. The DVV line offers simple and reliable assays for LAs testing that can be incorporated into virtually any clinical testing facility.

Robert Janetschek is marketing services manager for diagnostic products and Robert Greenfield is director of research and development at American Diagnostica Inc. They can be reached at 203-661-0000, or rjanetschek@amdiag.com.