COAGULATION TEST HANDBOOK      
 
 

From: Elizabeth M. Van Cott, M.D., and Michael Laposata, M.D., Ph.D., "Coagulation." In: Jacobs DS et al, ed. The Laboratory Test Handbook, 5th Edition. Lexi-Comp, Cleveland, 2001; 327-358.

Index of Tests

Antiphospholipid Antibody (Lupus Anticoagulant and/or Anticardiolipin Antibody) [CO003100]

Related Information

Synonyms Lupus Inhibitor

Applies to Anticardiolipin Antibody; Antiphosphatidylserine Antibodies; Beta-2-Glycoprotein I; Cardiolipin; Circulating Anticoagulant; Lupus Anticoagulant; PTT

Abstract Antiphospholipid antibodies are associated with an increased risk of thrombosis, thrombocytopenia, and recurrent fetal loss.

Specimen Lupus anticoagulant: plasma; anticardiolipin antibody: serum

Container Lupus anticoagulant: blue top (sodium citrate) tube; anticardiolipin antibody: red top tube

Collection Routine venipuncture. If multiple tests are being drawn, draw blue top tubes after any red top tubes but before any lavender top (EDTA), green top (heparin), or gray top (oxalate/fluoride) tubes. Immediately invert gently at least 4 times, mixing thoroughly. Blue top tubes must be appropriately filled. Deliver immediately to the laboratory.

Storage Instructions Separate plasma (or serum) from cells as soon as possible. Plasma (or serum) may be stored on ice for up to 4 hours; otherwise, store frozen. Platelet count must be <10 x 109/L in plasma prior to freezing, or false-negative lupus anticoagulant results may occur.

Causes for Rejection Blue top specimen received more than 4 hours after collection; clotted specimen; blue top not filled; patient on hirudin, danaparoid, or argatroban anticoagulation

Turnaround Time Lupus anticoagulant: less than 1 day if negative, longer if positive because confirmatory assays need to be performed; anticardiolipin: often several days because testing is batched.

Special Instructions Notify laboratory if patient is on heparin, including subcutaneous low-dose heparin or low-molecular weight heparin. In some assays, heparin may cause false-positive lupus anticoagulant results. Therefore, heparin must first be removed from the specimen by the laboratory. Other assays contain a heparin neutralizer that tolerates specimens containing up to 1 unit/mL heparin. Results can be interpreted correctly in patients on Coumadin®. Hirudin, danaparoid, or argatroban anticoagulation may cause false-positive results in some assays.

Reference Interval Negative for lupus anticoagulant; <15 units of anticardiolipin antibody

Use If an antiphospholipid antibody is suspected, assays for both lupus anticoagulant and anticardiolipin antibody should be performed. Used to evaluate hypercoagulable states, recurrent miscarriage, thrombocytopenia, or prolonged PTT. Lupus anticoagulants may or may not prolong the PTT.

Limitations Factor VIII inhibitors can cause false-positive lupus anticoagulant1 tests (a factor VIII assay showing normal factor VIII levels rules out this possibility). The transient presence of antiphospholipid antibodies may accompany infections or drugs; vide infra.

Methodology

Anticardiolipin antibody: Enzyme-linked immunosorbent assay (ELISA) using cardiolipin, a phospholipid, as the antigen. Newer ELISA assays are available that test for anti-beta 2-glycoprotein I antibodies or antiphosphatidylserine antibodies.

Lupus anticoagulant: To improve sensitivity, two screening tests are suggested.2 These tests are clotting-time based assays, such as the Russell viper venom time, PTT-based assays, kaolin clotting time, or dilute prothrombin time (tissue thromboplastin inhibition test). Lupus anticoagulants prolong various clotting times in the laboratory because they bind to phospholipid and thereby interfere with the ability of phospholipid to serve its essential cofactor function in the coagulation cascade. Lupus anticoagulant screening assays usually have a low concentration of phospholipid to enhance sensitivity. Any abnormal (prolonged) screening result is repeated after a 1:1 mixture of patient plasma with normal plasma to demonstrate that the clotting time remains prolonged upon mixing. Confirmatory assays are performed if the screening assay remains abnormal after the 1:1 mixture. Confirmatory assays typically demonstrate that upon addition of excess phospholipid, the clotting time shortens toward normal. The "platelet neutralization procedure" is a confirmatory assay in which the source of the excess phospholipid is freeze-thawed platelets. Note: The routine PTT may or may not be prolonged, depending on the amount of phospholipid in the reagent. In addition, elevated factor VIII can normalize an otherwise prolonged PTT. PTT-based lupus anticoagulant screening assays have a low concentration of phospholipid to enhance sensitivity. When the PTT is prolonged, a PTT mixing study may be a useful first test. When lupus anticoagulants are present, the PTT remains prolonged upon mixing with an equal volume of normal plasma.

Additional Information The two principal types of antiphospholipid antibodies are lupus anticoagulants and anticardiolipin antibodies. They are present in 0% to 5% of the general population and in 12% or more of patients with thrombosis.3,4 Antiphospholipid antibodies are acquired autoantibodies directed against phospholipid-protein complexes. These antibodies are associated with an increased risk for arterial or venous thrombosis,3,5 thrombocytopenia,6 and fetal loss.7 Associations with cardiac valve disease, livedo reticularis, and other features are also recognized.8 The mechanism of thrombosis is not entirely clear, although a number of mechanisms have been proposed. In a recent prospective study involving individuals with antiphospholipid antibodies, the incidence of thrombosis per year was 1% in individuals with no history of thrombosis, 4% in patients with systemic lupus erythematosus, 5.5% in patients with a history of thrombosis, and 6% in individuals with high titer IgG anticardiolipin antibody (>40 units).9

The diagnosis of antiphospholipid antibody syndrome requires a positive test in the antiphospholipid antibody panel (lupus anticoagulant and/or anticardiolipin antibody) on two separate occasions, at least 6 weeks apart, in the setting of thrombosis, thrombocytopenia, or recurrent miscarriage.10

Anticardiolipin antibodies recognize cardiolipin bound to beta 2-glycoprotein I. Most lupus anticoagulants recognize phospholipid bound to prothrombin, but others recognize phospholipid bound to beta 2-glycoprotein I or other proteins. Rarely, prothrombin levels become decreased as a result of a lupus anticoagulant, and an increased risk for bleeding may develop.11 As cardiolipin is the antigen used for syphilis screening tests (VDRL, Venereal Disease Research Laboratories; and RPR, rapid plasma reagin), false-positive syphilis tests may occur in patients with anticardiolipin antibodies. Conversely, true syphilis infections can cause positive anticardiolipin antibody test results.

Despite the prolonged clotting times, bleeding is not a typical feature associated with these antibodies. Thrombocytopenia, if present, is usually mild. Patients may have either a lupus anticoagulant or an anticardiolipin antibody or they may have both antibodies. A high percentage of patients with systemic lupus erythematosus (SLE) or related autoimmune diseases have these antibodies. These antibodies may also develop in patients without an underlying disorder. The antibodies can appear transiently in association with certain medications (eg, hydralazine, phenytoin) or infections. The human immunodeficiency virus (HIV) is commonly associated with positive tests for antiphospholipid antibodies.12 Infection-associated antibodies may not be associated with clinical symptoms of antiphospholipid antibody syndrome, and they tend to recognize phospholipid rather than the phospholipid-protein complexes described above.13

Heparin treatment in patients with lupus anticoagulants can be complicated by the fact that lupus anticoagulants may prolong the baseline PTT and/or accentuate the PTT prolongation when heparin is added. As such, heparin may be monitored with antifactor Xa assays. If the antifactor Xa assay demonstrates that the heparinized PTT is not affected by the lupus anticoagulant, cautious use of the PTT may be considered for that patient.

Footnotes

1. Goudemand J, Caron C, De Prost D, et al, "Evaluation of Sensitivity and Specificity of a Standardized Procedure Using Different Reagents for the Detection of Lupus Anticoagulants,"Thromb Haemost, 1997, 77(2):336-42.

2. Brandt JT, Triplett DA, Alving B, et al, "Criteria for the Diagnosis of Lupus Anticoagulants: An Update,"Thromb Haemost, 1995, 74(4):1185-90.

3. Ginsburg KS, Liang MH, Newcomer L, et al, "Anticardiolipin Antibodies and the Risk for Ischemic Stroke and Venous Thrombosis,"Ann Intern Med, 1992, 117(12):997-1002.

4. Doig RG, O'Malley CJ, Dauer R, et al, "An Evaluation of 200 Consecutive Patients With Spontaneous or Recurrent Thrombosis for Primary Hypercoagulable States,"Am J Clin Pathol, 1994, 102(6):797-801.

5. Vaarala O, Manttari M, Manninen V, et al, "Anticardiolipin Antibodies and Risk of Myocardial Infarction in a Prospective Cohort of Middle-aged Men,"Circulation, 1995, 91(1):23-7.

6. Galli M, Finazzi G and Barbui T, "Thrombocytopenia in the Antiphospholipid Syndrome,"Br J Haematol, 1996, 93(1):1-5.

7. Yasuda M, Takakuwa K, Tokunaga A, et al, "Prospective Studies of the Association Between Anticardiolipin Antibody and Outcome of Pregnancy,"Obstet Gynecol, 1995, 86(4 Pt 1):555-9.

8. Hogan WJ, McBane RD, Santrach PJ, et al, "Antiphospholipid Syndrome and Perioperative Hemostatic Management of Cardiac Valvular Surgery,"Mayo Clin Proc, 2000, 75(9):971-6.

9. Finazzi G, Brancaccio V, Moia M, et al, "Natural History and Risk Factors for Thrombosis in 360 Patients With Antiphospholipid Antibodies: A Four-Year Prospective Study From the Italian Registry,"Am J Med, 1996, 100(5):530-6.

10. Wilson WA, Gharavi AE, Koike T, et al, "International Consensus Statement on Preliminary Classification Criteria for Definite Antiphospholipid Syndrome,"Arthritis Rheum, 1999, 42(7):1309-11.

11. Bajaj SP, Rapaport SI, Fierer DS, et al, "A Mechanism for the Hypoprothrombinemia of the Acquired Hypoprothrombinemia-Lupus Anticoagulant Syndrome,"Blood, 1983, 61(4):684-92.

12. de Larranaga GF, Forastiero RR, Carreras LO, et al, "Different Types of Antiphospholipid Antibodies in AIDS: A Comparison With Syphilis and the Antiphospholipid Syndrome,"Thromb Res, 1999, 96(1):19-25.

13. McNally T, Purdy G, Mackie IJ, et al, "The Use of an Antibeta-2-Glycoprotein-I Assay for Discrimination Between Anticardiolipin Antibodies Associated With Infection and Increased Risk of Thrombosis,"Br J Haematol, 1995, 91(2):471-3.

References

Galli M and Barbui T, "Antiprothrombin Antibodies: Detection and Clinical Significance in the Antiphospholipid Syndrome,"Blood, 1999, 93(7):2149-57.

Galli M, Finazzi G and Barbui T, "Antiphospholipid Antibodies: Predictive Value of Laboratory Tests,"Thromb Haemost, 1997, 78(1):75-8.

Martinez CE, Rivera GB, and Aguilar LD, "Anticardiolipin Antibodies in Serum and Cerebrospinal Fluid From Patients With Systemic Lupus Erythematosus,"J Investig Allergol Clin Immunol, 1997, 7(6):596-601.

Roubey RAS, "Antiphospholipid Antibody Syndrome,"Arthritis and Allied Condition, Koopman WJ, ed, Baltimore, MD: Lippincott Wilkins & Wilkins, 1997, 1393-406.

Triplett DA, "Protean Clinical Presentation of Antiphospholipid-Protein Antibodies (APA),"Thromb Haemost, 1995, 74(1):329-37.

 

     
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