The Lupus Anticoagulant: Truth or Consequence

Douglas Tripplet, MD, former chair of the Scientific Standardization Committee/Subcommittee of Lupus Anticoagulant/ Phospholipid-dependent Antibodies, described the Lupus Anticoagulant (LA) as a riddle wrapped in a mystery inside an enigma. The riddle is that the blood doesn't clot in the tube. The mystery is that patients appear to have a predisposition toward thrombosis. The enigma results from the dilemma of whether the LA causes the thrombosis or is a result of a previous event.

Conley and Hartmann first described a circulating anticoagulant in a patient with Systemic Lupus Erythematosus (SLE) in 1952.¹ Twenty years later, Feinstein and Rapaport coined the term Lupus Anticoagulant.¹ This can be a difficult diagnosis but one that is very important in determining whether a patient will bleed or thrombosis.

The LA Principle
To understand these findings, it's important to understand the principle of the LA.

The LA is a misnomer and part of a syndrome called anti-phospholipid syndrome (APS). Five percent of patients make antibodies to phospholipids. These phospholipids are anti-cardiolipin antibodies (ACA) and the LA. Phospholipids are polar, ionic compounds composed of 1,2-diacylglycerol and the phosphodiester-linked nitrogenous bases choline, ethanolamine and serine. They occur in organelle and cellular membranes such as mitochondria, endothelial cells, aggregated or activated platelets.²

Antibodies are made in three different classes: IgG, IgM and IgA. IgG is the most common one. ACAs are five times more common than the LA.³ They bind the anionic phospholipid cardiolipin, including antibodies to B2Glycoprotein 1 (B2GP1) and phospholipid-binding proteins of the coagulation cascade such as prothrombin. ACAs are solid phase antibodies.

LAs, on the other hand, bind protein anionic phospholipid complexes and are fluid phase antibodies; that is, they prolong phospholipid-dependent tests. These antibodies have a specific activity against the phospholipid and cannot serve as a surface upon which coagulation enzymes can sequentially interact to form fibrin in the test system. These are two distinct and separate antibodies.⁴ Sixty percent of LA-positive patients have ACA.

Alloimmune vs. Autoimmune Antibodies

APS syndrome can be alloimmune or autoimmune. Alloimmune antibodies are classified as being detected in ACA ELISA testing but not in LA testing. These are not associated with thrombosis and are self-limiting. They can be caused by acute bacterial or viral infections and disappear after two weeks. Others are caused by HIV, malignancies, therapy from quinine, quinidine, calcium channel blockers, procainamide and certain antibiotics. These antibodies will disappear four to six weeks after treatment.

Autoimmune antibodies will appear in connective tissue diseases such as Sjögren's syndrome, rheumatoid arthritis and SLE. They may also arise spontaneously. These antibodies are persistent over time and detected in LA testing.⁵

The LAs were given that name because they were originally found in people that had lupus. The anticoagulant name comes from the fact that the activated partial thromboplastin time (APTT) is prolonged. LAs are antibodies to phospholipids; reagents used for testing are phospholipid based.

PT, APTT Reagents

Prothrombin time (PT) reagents contain a high concentration of phospholipids that will mask the antigen/antibody reaction, whereas APTT reagents contain a low concentration of phospholipids that results in the antibody reaction and prolongs results. This leads to the assumption that the patient would be prone to bleeding; however, LA patients are predisposed to thrombosis. The actual method in which the LA causes thrombosis is unclear.

Seventy percent of thrombotic events are venous and occur in the veins of the lower extremities. Or, they may be hepatic, mesenteric or intracranial. Thirty percent are arterial and include myocardial infarction, strokes, coronary artery disease, deep vein thrombosis (DVT), pulmonary embolism (PE) and transischemic attacks. In addition, there's a large association between the LA and intrauterine death.⁶

Theories of Thrombosis

The most recent theories that predispose patients to thrombosis are complex and involve a sophisticated understanding of complex coagulation pathways. They are as follows:

1. A possible mechanism is an acquired form of resistance to activated Protein C (APC). Studies show impaired factor Va inhibition by APC in the presence of B2GP1.

2. Indirect concentration of prothrombin on cellular or phospholipid surfaces with subsequent excessive local generation of thrombin at sites of injury.

3. Decreased concentration of Protein S, which may be due to anti-phospholipid antibodies/B2GP1 binding of free S to C4B binding protein that occurs as an acute phase reactant. This complex may be removed from the system, causing clotting.

4. Displacement of Annexin V ( a thrombo-protectant protein) from cellular surfaces exposing charges phospholipid and accelerating phospholipid coagulation reactions.⁴

These are the current theories that may cause thrombosis in the presence of antibodies.

Patients with this syndrome who are prone to thrombosis are categorized into several clinical groups:

• Type I: DVT and PT
• Type II: Coronary or peripheral artery thrombosis
• Type III: Cerebrovascular/retinal vessel thrombosis
• Type IV: Mixture of the above
• Type V: Recurrent fetal losses
• Type VI: Normal individuals without manifestations of antiphospholipid syndrome

This grouping is important for treatment. Type I and II should be treated with heparin or LMWH; Type III with long-term heparin plus antiplatelet therapy; and Type V with low-dose aspirin pre-conception and heparin post-conception until delivery.⁷

There are several reasons, however, that may cause a person with an LA to bleed, further complicating the diagnosis. They are:

1. the presence of thrombocytopenia,

2. a qualitative platelet defect that is a functional abnormality,

3. consumption of prothrombin (Factor II) possibly due to accelerated clearance of prothrombin-antithrombin antibody complex from the blood and

4. recurrent fetal abortions.⁸

Laboratory Testing
In the laboratory, we work with little or no clinical picture, so we need to rely on good test ordering algorithms (Fig. 1). It's equally important to work with a good sample.

Look at some pre-analytical situations before encountering an expensive coagulation work-up. The ratio of blood to anticoagulant must be 9:1; if the tube is underdrawn, there will be excess anticoagulant prolonging the tests.

Next, look at the hematocrit; a decreased level will affect the ratio of blood to anticoagulant. One of the common reasons for a prolonged APTT can be due to heparin contamination. The best test to determine residual heparin is the thrombin time, used to determine fibrinogen abnormalities. To distinguish between heparin contamination and fibrinogen abnormalities, a reptilase time should be performed. This will be normal due to heparin and abnormal reflecting a fibrinogen problem.

Another less expensive method is to perform the thrombin time and repeat the test substituting a 0.1 percent solution of Toulidene Blue for the buffer. Toulidene Blue will neutralize any heparin that's present and return the thrombin time to normal.

The presence of platelets in the sample also can greatly affect the result. Platelets are a source of phospholipids; if the sample being tested is not platelet poor plasma, the presence of platelets will increase the amount of phospholipids and shorten the test result.

The plasma should have a platelet count of <10,000. This can be exacerbated if the specimen is frozen/thawed, resulting in burst platelets.

The LA is very complex and difficult to diagnosis. To ensure proper diagnosis, it's best to use a series of tests instead of one singular test (Fig. 2). The Scientific Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies came up with specific recommendations that should be followed before one can diagnosis an LA. They are:

1. The sample must have a prolonged phospholipid-dependent clotting time.

2. There must be evidence of an inhibitor.

3. The inhibitor must be phospholipid dependent.

4. The diagnosis of LA must be confirmed before given to a patient.⁸

In the laboratory, the first evidence of an LA may be a prolonged APTT. This will occur if the testing laboratory uses a reagent that is sensitive to the LA. That particular reagent would have a lower concentration of phospholipids. If the reagent contains a higher concentration of phospholipids, it may mask the reaction, resulting in a normal APTT. These reagents are insensitive to an LA and are, therefore, not a good screen for the LA.

The next test that will confirm the presence of an inhibitor is a mixing study. The principle of the test relies on a 1:1 mix of a pooled normal plasma (PNP) with the patient's test plasma. The APTT is repeated, and if the results are normal, it suggests a factor deficiency. Factors that are deficient in the patient's plasma are replaced by the PNP. When there's no correction, it suggests the presence of an inhibitor.

A mixing study may also be performed at a 4:1 mix to enhance sensitivity. It is suggested to perform an incubated mixing study; this will enhance the presence of a weak inhibitor and make the diagnosis easier.

There are several theories about what's considered a correction of a mixing study. A correction of a mixing study is difficult to describe and must be looked at with regard to the entire coagulation picture. In most cases, if the mix corrects into the normal range, the result is due to a factor deficiency. Other situations include a correction within two seconds of the normal control. There may be a partial correction, so an inhibitor must still be investigated. It's now the task to determine if the inhibitor is to a specific factor or to an LA. When there's an inhibitor to a specific factor, bleeding will occur. If there's an LA, the patient will be predisposed to thrombosis.

Testing in the laboratory must cover two types of pathways: both a clot-based assay and phospholipid testing (Fig. 3). The best test for phospholipid testing is a dilute russel viper venom test (DRVV) (American Diagnostica, Greenwich, CT). The test principle is based on a viper venom activating the cascade at Factor X. This eliminates any problems, including inhibitors or factor deficiencies, that occur prior to Factor X activation.

The test is based on a screen that uses a low concentration of phospholipid resulting in a prolonged result. The screen may also be prolonged due to heparin, coumadin or a decreased level of V or X. The confirmatory test uses a high concentration of phospholipid, which should shorten the result. A normal range ratio of the DRVV screen/DRVV confirm should be established for your instrument/patient population. If the ratio is greater than the normal range, the patient is positive for an LA and testing is complete. If the ratio is normal, testing must proceed to a clot-based assay.

The clot-based assay used is the Staclot LA (Diagnostica Stago Inc., Parsippany, NJ). It is an antigen/antibody test for the LA. The theory of the test is based on the fact that antibodies to LA configure in a hexagonal configuration, while ACA configure in a bilayer configuration. This discriminates between the two types of antibodies. The principle utilizes a clot-based assay that compares a straight APTT with an APTT including a hexagonal phase phosphaidyleanloamine (HPN). If the antibody is present, it will neutralize the effect of the HPN and greatly shorten the time. If the difference between the two APTTs are > 8 seconds, the result is positive for the LA.

To completely cover testing, if both tests are negative and/or if you need to confirm the presence of an inhibitor, a final test that could confirm the presence of an inhibitor is the Tissue Thromboplastin Inhibitor (TTI). It uses different dilutions of thromboplastin reagents in comparison to a normal control. A lupus-like anticoagulant will show inhibitory activity against tissue thromboplastin, resulting in a significant prolongation of the clotting times compared to the normal control. The test confirms the presence of a non-specific inhibitor that may be present due to a chronic, viral or other unexplained condition. It becomes important in cases of an unexplained pre-op prolonged APTT that requires clearance for surgery. The principle of this test uses dilute thromboplastin and is reported in a ratio compared to normal plasma. If the ratio is positive, it confirms the presence of a non-specific inhibitor reflected in a prolonged APTT. This will eliminate the possibility of a surgical bleed due to a prolonged APTT.

Occasionally, an inhibitor is manifested in the PT most frequently seen in children with chronic infections and ingestion of antibiotics. Patients are not likely to bleed or thrombosis. The TTI may be useful in determining these types of inhibitors.

Donna D. Castellone is a technical specialist, Department of Clinical Hematology, New York (NY) Presbyterian Hospital.

References

1. Jensen R. Antiphospholipid antibodies syndrome diagnosis. Clinical Hemostasis Review 1996;10:3.

2. American Diagnostica, "Anti-Phospholipid Antibody Syndrome" informational bulletin, 1999.

3. Petri M. Antiphospholipid antibodies, antiphospholipid antibody syndrome, and lupus. SLE in Clinical Practice Volume 1; Issue 2: 2.

4. ASH Education Program Book, "Diagnosis and Management of Patients with Spontaneously Acquired Inhibitors of Coagulation," New Orleans, LA. Dec. 3-7 1999: 200-201.

5. Fritsma GA, Duncan A. "Thrombosis Risk Testing," Colorado Coagulation Consultants, 1997 pg. 15.

6. Triplett DA and Brandt JT. "Lupus Anticoagulant: Clinical Implication and Laboratory Diagnosis." Bio/Data Corp., Horsham, PA. 1988: 3.

7. Bick and Baker. Seminars in Thrombosis and Hemostasis Review, 1999;25(3):333-350.

8. Brandt JT, Karna LK and Triplett DA. Laboratory identification of lupus anticoagulants: Results of the second international workshop for identification of lupus anticoagulants. Thrombosis and Haemostasis 1995;74(6):1597-1603.