Hypoprothrombinemia

Objective.-To review the role of lupus anticoagulants in the pathogenesis of both venous and arterial thromboembolic events, as well as in recurrent spontaneous abortions. The pathophysiology of lupus anticoagulants and associated antiphospholipid antibodies (eg, anticardiolipin antibodies) is also discussed.

Data Sources.-Review of the recent medical literature. Data Extraction and Synthesis.-Key articles in the recent medical literature dealing with lupus anticoagulants and their role in pathogenesis of thromboembolic events were reviewed. Plasma proteins that have an affinity for

binding to "perturbed cellular membranes" have been identified as the antigenic targets for anti phospholipid antibodies. Thus, the concept of antiphospholipid antibodies needs to be reevaluated. Perhaps a better term is antiprotein-phospholipid antibodies. The principal antigenic protein targets are beta^sub 2^-glycoprotein 1, prothrombin, and a wide range of additional proteins that interact with activated cellular membranes, including protein C, protein S, annexin V, etc. Most research reported in the literature has focused on beta^sub 2^-glycoprotein I and human prothrombin.

(Arch Pathol Lab Med. 2002;126:1424-1429)

Autoantibodies have been implicated in a variety of diseases. The diversity of these antibodies has stimulated multidisciplinary interest in linking these antibodies to potential pathophysiologic mechanisms. Antiphospholipid antibodies (APAs) were first described by Wassermann et al in 1906.1 The Wassermann test was a complement fixation procedure using saline extracts from the liver of fetuses with congenital syphilis. Following the introduction of the Wassermann test, a variety of procedures using antigens derived from alcoholic extracts of normal tissue were described. These tests used either complement fixation (Kahn) or flocculation techniques (Venereal Disease Research Laboratory [VDRL], rapid plasma reagin test, and automated reagin test). Pangborn2 was the first to identify an acidic phospholipid as the antigen present in alcohol extracts of bovine heart. This extract was subsequently named "cardiolipin." The antigen used in the VDRL test is a mixture of cardiolipin-lecithin-cholesterol. False-positive tests using the VDRL were classified as either acute or chronic biologic false positives (BFPs).3 The chronic BFP was defined as a positive nontreponemal-- based screening test that persisted for 6 months or longer. In many cases, patients with chronic BFP-serologic tests for syphilis (BFP-STS) were found to have underlying autoimmune disease, including systemic lupus erythematosus (SLE), rheumatoid arthritis, and Sjogren syndrome.4 Chronic BFP-STS may be the first laboratory finding of an autoimmune disease and often precedes the onset of clinical symptoms.

An unusual circulating anticoagulant was first described by Conley and Hartmann in 1952. 5 The title of the article suggested the patients had a hemorrhagic disorder; however, no documentation of significant clinical bleeding was described. Subsequently, Lauren and Nilsson6 reported an association between BFP-Wassermann test and a circulating anticoagulant. These investigators also noted the anticoagulant could be adsorbed by the Kahn reagent, which was used in syphilis testing. Laurell and Nilsson were the first to link the anticoagulant to an antibody that reacted with a lipid antigen. Subsequently, in 1972, Feinstein and Rapaport7 proposed the term lupus anticoagulant (LA) for these antibodies. Lupus anticoagulant is a misnomer, since the vast majority of patients with laboratory findings of LA do not have SLE.8

Harris and colleagues9 at St Thomas Hospital in London, England, were the first to report a sensitive test system to identify anticardiolipin antibodies (ACAs). In contrast to earlier studies, they used a solid-phase radioimmunoassay, which was 200 to 400 times more sensitive than the VDRL. They found 62% positivity among 65 patients with SLE or other autoimmune diseases. There was also a strong correlation of positive ACA results and LA (29 [91%] of 32 patients) and thromboembolic events.

ANTI PHOSPHOLIPID ANTIBODIES

Immunology

The term antiphospholipid antibodies is a misnomer in the majority of patients presenting with clinical and laboratory findings consistent with antiphospholipid antibody syndrome (APS).10 There are 2 distinct groups of antibodies, some of which react only with phospholipids (true antibodies to phospholipids, in most cases infection-induced). These antibodies are usually transient and disappear following convalescence.11

In the case of patients who have APAs in the setting of autoimmune diseases (eg, SLE, primary APS, drug-induced APAs), a variety of plasma protein antigenic targets have been identified (Table 1). These autoantibodies recognize plasma proteins that bind to activated cellular membranes (eg, monocytes, endothelial cells, platelets).12,13 Among the proteins binding to activated cellular surfaces are beta^sub 2^-glycoprotein I (beta^sub 2^-GPI), prothrombin, annexin V, protein C, protein S, and proteins from the kininogen system (high- and low-molecular-weight kininogens).13 An important aspect of the pathophysiology is the density of proteins binding to injured cellular surfaces. Increased density of plasma proteins allows for bivalent bonding of the antibodies, which in turn enhance the prothrombotic nature of the injured cells.14

Antigenic Targets

beta^sub 2^-Glycoprotein I (Apolipoprotein H).-beta^sub 2^-Glycoprotein I is a member of the short-consensus-repeat protein family. It is a 50-kd glycoprotein found in plasma with a concentration of approximately 200 (mu)g/mL.15 beta^sub 2^-Glycoprotein I is characterized by 5 "sushi domains." The fifth sushi domain contains the binding site, which attaches to activated cellular surfaces.16 Lysine-rich segments are found in the fifth sushi domain and are responsible for binding to activated cellular surfaces. The physiologic role of beta^sub 2^-GPI remains somewhat enigmatic. In vitro studies have found beta^sub 2^-GPI inhibits prothrombinase activity, the contact system of blood coagulation, and adenosine 5'diphosphate-induced platelet aggregation. 17 beta^sub 2^-Glycoprotein I may also play a role in clearance of apoptotic cells.18 Patients who have been described with an absence of beta^sub 2^-- GPI have not experienced an increased risk of thrombosis.

Antiphospholipid antibodies bind to beta^sub 2^-GPI primarily in the third and fourth sushi domains.18 However, there is some controversy. A research group recently has proposed sushi domain I as the principal target of autoantibodies with pathogenic consequences.19 Lupus anticoagulants have been shown to enhance binding of beta^sub 2^-GPI to activated phospholipid cellular surfaces.

Prothrombin.-Prothrombin is also a common antigenic target for APAs.20 These antibodies bind to prothrombin, prethrombin I, and fragment I but not to thrombin. Antibodies to prothrombin lead to prothrombin clustering (antigenic density) on surfaces of activated cells. Antibodies to prothrombin may account for approximately two thirds of LAs identified in patients with APS. Although these antibodies tend to have low affinity, there are occasions when high-affinity antibodies to prothrombin lead to hypoprothrombinemia.21,22 Often these cases are seen in children with associated hemorrhagic complications.23 These antibodies have species specificity for human prothrombin. Antibodies to prothrombin have also been described in adults. In many cases, these antibodies are detected in young women with evidence of autoimmune disease (eg, SLE, rheumatoid arthritis).

Recently, commercial enzyme-linked immunosorbent assays (ELISAs) for detection of antibodies to prothrombin have been approved for clinical testing. Levels of calcium in the test system are important because they affect the confirmation of prothrombin.

Antibodies to Oxidized Low-Density Lipoprotein.The association of APAs and oxidized low-density lipoprotein has recently been reported. Typically, the antibodies are b^sub 2^-GPI dependent. In a study of patients with SLE and APA positivity, increased quantities of isoprostanes were found in urine specimens.24 The isoprostanes reflect in vivo lipid peroxidation. Treating these patients with vitamin E resulted in significant decreases in urinary isoprostanes.

Animal models using mice or other animals have found that immunization with beta^sub 2^-GPI or other antigens induces in vivo thrombosis.25 In addition, human antibodies injected into animals also promote thrombosis.

Clinical Complications

The range of clinical complications reported with LA/ ACA involves virtually every organ system.26 Antiphospholipid antibody is one of the most common causes of acquired thrombophilia. Arterial thrombotic events are reported most frequently in the cerebral circulation.27 Approximately one third of strokes in patients younger than 50 years have been attributed to APAs.28

On the venous side of the circulation, an estimated 15% of deep vein thromboses (DVTs) are due to the presence of APAs.29 In addition, a number of other venous sites have been associated with thromboembolic events, including mesenteric, renal, and adrenal veins. Clinically, catastrophic APS (CAPS) may resemble thrombotic thrombocytopenic purpura and hemolytic uremic syndrome.30 Recurrent thromboembolic events tend to have consistency with respect to site (ie, arterial-arterial, venous-venous).

A number of obstetrical complications have been linked to APAs, including maternal DVT, chorea, and eclampsia/ preeclampsia. Recurrent spontaneous abortions have also been linked to APAs.31 Between 5% and 15% of all recurrent spontaneous abortions have been attributed to APAs.

Virtually every clinician will encounter patients with APAs. Neurologic complications include chorea, stroke, migraine, and multi-infarct dementia.32 Dermatologists not infrequently are presented with patients with livedo reticularis and, more uncommonly, ulceration or digital gangrene.33

In patients with stroke, it is important to suspect an underlying presence of APAs.27,28 Patients with APAs may have a Libman-Sacks endocarditis. In many cases of young adults with stroke linked to APA, mitral or aortic valves are the origin of the emboli.34 In APA-positive patients with a history of cerebral events, transesophageal echocardiography should be a standard part of the evaluation.

In the early 1990s, an accelerated form of thrombosis affecting the microvasculature of many organs was identified and linked to APAs.35 Clinically, these patients had multiorgan thrombosis. Precipitating factors initiating CAPS included medications, minor and major surgical procedures, and in some cases, infections. The underlying pathophysiology remains a matter of conjecture. Thrombotic involvement of the microvasculature/capillaries is a consistent finding in patients with CAPS. Endothelial cell activation appears to be a part of the pathophysiologic process.30

The differential diagnosis of CAPS includes thrombotic thrombocytopenic purpura, disseminated intravascular coagulation, and hereditary thrombophilia (eg, factor V Leiden, prothrombin G20210A, protein C deficiency, protein S deficiency, antithrombin deficiency). Table 2 summarizes the antibody-mediated thrombotic conditions.

ANTI PHOSPHOLIPID ANTIBODY SYNDROME

History and Definition

Hughes and colleagues (St Thomas Hospital/London) initially suggested the term anticardiolipin syndrome to define the association of positive ACA results and clinical findings.36 Table 3 summarizes the criteria necessary to establish the diagnosis of APS. To establish the diagnosis of APS, the patient must have at least 1 of the major clinical findings (venous thrombosis, arterial thrombosis, recurrent fetal loss, and thrombocytopenia). In addition, there should be a positive laboratory test result indicating the presence of either ACAs or positive LA testing (eg, prolonged dilute Russell viper venom test [dRVVT], prolonged activated partial thromboplastin time [aPTT]). An important caveat with respect to application of the guidelines to identify APS is the need to retest the patient approximately 8 to 10 weeks following the initial positive laboratory results. This step is important in identifying transient APAs, which often are the result of intercurrent infections. Infection-related APAs are not associated with thromboembolic events or other complications (recurrent spontaneous abortion, livedo reticularis, neurologic findings, etc).

LABORATORY IDENTIFICATION OF LUPUS ANTICOAGULANTS

The appropriate collection and preparation of platelet-- poor plasma (PPP) is an often-overlooked initial step in coagulation testing. A traumatic venipuncture may significantly compromise the blood sample. Separation of PPP from the cellular elements of blood should be done expeditiously. Optimally, the number of residual platelets in the PPP should be less than 5000/ (mu)L. In many situations, the PPP may be frozen (-80 deg C) and tested at a later date. If there are significant residual platelets in PPP, one may see a "platelet neutralization effect" on thawing PPP for LA testing.37

According to the guidelines established by the Scientific Subcommittee for Lupus Anticoagulants/PhospholipidDependent Antibodies, screening coagulation testing must use more than 1 test system (eg, aPTT, dilute prothrombin time, dRVVT).38 Currently, at least 2 of the tests listed are recommended; however, many laboratories choose to do all 3 of these tests. Other tests that have been used include Textarin Time and Taipan Venom Test (Table 4). The Kaolin Clotting Time has also been used as a screening procedure. This test system is exquisitely sensitive to residual platelets that may compromise the test results.

The dRVVT has become a very popular screening test in both the United States and Europe. Thiagarajan et al39 were the first authors to describe the use of a modified RVVT for the diagnosis of LA. Their studies found the dRVVT to be more sensitive than aPTTs and the tissue thromboplastin inhibition test (synonym: dilute prothrombin time). There are a number of commercially available dRVVT test systems. However, there is variability among manufacturers.40 When choosing a dRVVT test system, laboratories must carefully identify the reagent with the best sensitivity and specificity for LA detection. Table 5 summarizes the recommendations of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis: Subcommittee for Lupus Anticoagulants/Antiphospholipid Antibodies.38

In response to the recommendations cited, commercial reagent manufacturers have provided confirmatory reagents using a dRVVT test with excess phospholipid. These reagents fulfill the criterion to shorten or correct the prolonged baseline dRVVT. The same principle of an initial screening reagent with a low phospholipid concentration and a confirmatory reagent with higher phospholipid concentration applies to the other tests used as initial screens to detect a circulating anticoagulant (aPTT, dilute prothrombin time, etc).

In addition to the "integrated dRVVT test systems," Diagnostica Stago developed the Staclot LA test.41 This system uses a 2-step approach to establishing LA diagnosis. The Figure illustrates the sequential steps involved in the Staclot LA procedure. The test uses hexagonal-- phase phospholipid to neutralize an LA, which results in shortening of the clotting time in tube 2. The assay system also contains a heparin neutralizer, which enhances the specificity of Staclot LA.

Another approach to confirm the presence of LA is the Platelet Neutralization Procedure.37 This is a simple test using washed frozen platelets. An aliquot of the freeze-- thawed platelets is added to the patient plasma, and a second aPTT is determined. Addition of the freeze-thawed platelets to LA-positive PPP results in significant shortening of the prolonged baseline value. The test is simple to perform and has relatively high specificity. To correct for the dilutional effect of the freeze-thawed platelets, a second mixture of patient plasma together with buffered saline is used as a control. According to the guidelines established by the Scientific and Standardization Committee Subcommittee on Lupus Anticoagulants/Phospholipid-Dependent Antibodies, 2 or more LA screening tests are required (aPTT, dRVVT, Kaolin clotting time, etc).38

LABORATORY DIAGNOSIS OF ANTICARDIOLIPIN ANTIBODIES

Harris and colleagues9 were responsible for the development of an ELISA assay for the identification of ACA. A variety of commercially manufactured ELISA-based assays for ACA are available. Two types of microtiter plates are used: high-sensitivity plates (irradiated) and low-sensitivity plates. The high-sensitivity plates allow for greater antigenic density on the microtiter plate. Bivalent bonding of the antibodies in the patient plasma is critical to establish appropriate antigen-antibody density on the microtiter plate. Numerous commercial ACA ELISA systems are available. However, there is significant variability. The "high-sensitivity" microtiter plates that have been irradiated allow for greater antigenic density and facilitate bivalent bonding of antibodies present in patient plasma. In most cases, the antigenic target is beta^sub 2^-GPI. However, there are significant antibodies to prothrombin in many patient plasmas. The most important variables in identifying a sensitive ACA system are concentration of target antigen (prothrombin, beta^sub 2^-GPI, annexin V, etc), use of a high-sensitivity ELISA microtiter plate (irradiated), and an appropriate buffer system. Either serum or plasma may be used in testing for ACA.

The term anticardiolipin antibody is a misnomer. As noted, the antibodies recognize proteins bound to the microtiter plate. In infectious disease states, one can see true ACAs (ie, bound to cardiolipin).

Other approaches have been taken to identify ACAs, including flow cytometry.42 Beads are coated with cardiolipin or specific proteins such as prothrombin or b^sub 2^-GPI. Using flow cytometry, one can identify all 3 isotypes (immunoglobulin [Ig] G, IgA, and IgM) in a single procedure. The drawbacks to the use of flow cytometry to identify ACA are requirements for significant laboratory volume of ACA testing, purchase cost of a flow cytometry system, and the current relative lack of commercially available flow cytometry kits to support ACA testing.

Laboratories should carefully evaluate the various ELISA assays available for ACA testing. Based on interlaboratory external proficiency testing, the current state of the art in ACA testing is poor. There are a number of underlying variables that may explain the lack of concordance between laboratories. Among these variables are choice of microtiter plates for ELISA assays (high sensitivity vs lower sensitivity), technical issues within the laboratory, buffering reagents, and variable plate readers.

CONCLUSIONS

1. Preanalytic variables, including preparation of PPP and freeze-thawing effects if plasma is frozen and tested at a later date, can influence the outcome of testing.

2. Establishing the diagnosis of LAs requires use of the guidelines as developed by the Scientific Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies (Table 5).

3. "Integrated test systems" are currently preferable when testing for LA. Examples include commercially available dRVVT (screen)/dRVVT (confirm; eg, increased phospholipid content). Other systems include Staclot LA, dilute prothrombin time/screen, and confirmatory reagent (dilute prothrombin time/high phospholipid). Venom-based assays include Textarin Time and Taipan Venom Time.

4. When performing ELISA assays, systems that use high-sensitivity microtiter plates are preferred. Alternatively, flow cytometry may offer a more sensitive and specific test system.

RECOMMENDATIONS

Lupus Anticoagulants/Antiphospholipid Antibodies

Anticardiolipin antibody and LA assays are appropriate for patients with venous thromboembolism, particularly if the venous thromboembolism is idiopathic or associated with autoimmune disease, or if there is no family history of venous thrombosis.29 Level 1

Anticardiolipin antibody and LA testing may be considered for patients with arterial thrombosis, particularly in a young person or a person with no documented atherosclerosis.26,43 Level 1

Anticardiolipin antibody and LA assays should be considered for patients with unexplained stroke, particularly in a young person or a patient with autoimmune disease.32,34 Level 1

Testing for LA and ACA should be considered for patients with cerebral venous thrombosis.10 Level 3

Women with pregnancy loss that is either recurrent or late in the pregnancy (second and third trimester) should be evaluated for APAs (LA and APA).31,44-46 Level 1

To demonstrate persistence, any positive test (APA or LA) must be confirmed by repeat testing after 6 weeks.-16 Level 2

Lupus Anticoagulants

* Platelet-poor plasma used for LA testing should have a platelet count less than 10000/(mu)L.38 Level 1

The use of commercially available, integrated test systems for measuring LA is recommended, for example, the Staclot LA or dRVVT.41 Level 1

Patients being treated with anticoagulants and specimens containing anticoagulants should not be tested for LA; however, if patients on oral anticoagulants or heparin must be tested, the results must be interpreted with caution.38 Level 2

Antiphospholipid Antibodies

Immunoglobulin G ACA testing is recommended for the evaluation of thrombophilia. Although frequently measured, the risk of incident or recurrent thrombosis associated with IgM ACA and IgA ACA is uncertain. Elevated titers (>40 GIU) are most closely associated with thrombophilia. 14 Level 2

Enzyme-linked immunosorbent assays for anti-prothrombin and anti-P,GPI antibodies may be performed in addition to ACA testing in the evaluation of thrombophilia; however, prospective studies involving the use of assays for anti-prothrombin and anti-P2GPI antibodies for the evaluation for thrombophilia are limited.20 Level 3

For an explanation of the levels cited in this article, refer to Olson.47

Accepted for publication April 8, 2002.

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of commercial reagents for the detection of lupus anticoagulants show marked differences in performance between photo-optical and mechanical coagulometers. Thromb Haemost. 1999;81:758-762.

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47. Olson JD. College of American Pathologists Consensus Conference XXXVI: Diagnostic Issues in Thrombophilia: introduction and general considerations. Arch Pathol Lab Med. 2002;126:1277-1280.

Douglas A. Triplett, MD

From the Department of Pathology, Indiana University School of Medicine; Midwest Hemostasis and Thrombosis Laboratories, and Department of Pathology, Ball Memorial Hospital, Muncie, Ind.

Presented at the College of American Pathologists Consensus Conference XXXVI: Diagnostic Issues in Thrombophilia, Atlanta, Ga, November 9-11, 2001.

Reprints: Douglas A. Triplett, MD, Department of Pathology, Ball Memorial Hospital, 2401 West University Ave, Muncie, IN 47303-3499 (e-mail: dtriplett@chs.cami3.com).

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