No 3 / 2012 SOLUTIONS THE BULLETIN OF GAMMA-DYNACARE LABORATORIES ANTIPHOSPHOLIPID SYNDROME: DIAGNOSING A COMPLEX CONDITION Antiphospholipid syndrome (APS), sometimes known as Hughes Syndrome, is an autoimmune disease with serious ramifications including venous/arterial thrombosis, and pregnancy complications such as recurrent fetal loss and severe preeclampsia. Sera taken from patients with APS often contain antibodies to cardiolipin, an acidic phospholipid - hence its original name “antiphospholipid syndrome”. To this day, APS remains a complex illness that is inherently difficult to diagnose.1,2 It is believed that APS affects between 1–5% of the population. APS is a major women’s health issue, as 75–90% affected are women. It is estimated APS is the cause of 15–20% of all thrombotic events, 10–25% of recurrent miscarriages, and one-third of the strokes in patients under the age of 50. The syndrome is also thought to be present in 40–50% of patients with systemic lupus erythematosus (SLE).3,4 Pathogenesis of APS Should you wish to receive this bulletin via email, please go to http:// www.gamma-dynacare. com/Newsletters/ Registration.aspx. Thrombosis has a key role in the clinical manifestations of APS. Several mechanisms have been proposed for APS-related thrombosis; however, it is most probably multifactorial in etiology. The thrombotic tendency may be caused by antiphospholid antibodies through the following mechanisms:2 pInhibition of the factors of the anticoagulant system, affecting thrombin formation and antithrombin activity. pImpairment of fibrinolytic activity. pInterference with coagulation factors and complement, particularly the intrinsic and protein C pathway. pDirect effect of antiphospholipid antibodies on cell function, such as platelets, endothelial cells and vascular cells. There is growing evidence that phospholipid antibodies are present in patients months to years before the onset of any clinical symptoms of APS, possibly induced by exogenous stimulus or due to antigen spreading.3,5 Many individuals with APS may never experience a thrombotic event or pregnancy loss.5 Secondary risk factors, co-morbidities, and other factors influence whether a patient actually experiences the clinical manifestations of APS. Thrombosis may be initiated by trigger events, such as surgery, pregnancy, or the use of oral contraceptives.3 Thrombotic events can affect arteries and veins of all sizes, including the microvascular system. The thrombosis can also be recurrent.3 The most frequent thrombotic events are deep-vein thrombosis (DVT) and ischemic stroke (IS). Catastrophic APS is a very rare variant occurring in less than 1% of all APS patients. This is an accelerated form of this syndrome that results in multiple organ failure.2, 3 SOLUTIONS THE BULLETIN OF GAMMA-DYNACARE LABORATORIES Diagnosis The International Consensus Statement for the Classification Criteria for Definite Antiphospholipid Syndrome was updated in 2006 to aid physicians in the diagnosis.2,6 Unfortunately, despite these improved guidelines, diagnosing APS in the clinical setting still remains a challenge. The Sapporo classification criteria were first formulated and published in 1999.2,6 The revised criteria, now known as the Sydney criteria, were proposed at a workshop in Sydney, Australia at the Eleventh International Congress on antiphospholipid antibodies. It was proposed that a patient must have at least one clinical criterion and one positive laboratory criterion to be diagnosed with APS.6 pThe elapsed time of 12 weeks between the initial and confirmatory test is to increase the probability of excluding temporary infection-associated antibodies.2 pIf there are fewer than 12 weeks, or more than 5 years between a positive APS test and the clinical manifestations, the classification criteria should not be used.6 The panel discussed clinical and laboratory features associated with APS that are not included in the revised criteria.3,6 The committee decided that if included, it may decrease diagnostic specificity. These include: pHeart valve abnormalities pLivedo reticularis pThrombocytopenia pNephropathy Clinical criteria6 pVascular thrombosis: PAt least one confirmed clinical episode in an arterial, venous, or small vessel in any tissue or organ that has been confirmed. pPregnancy morbidity: POne or more unexplained deaths of a normal fetus at or beyond the 10th week of gestation. PThree or more unexplained consecutive spontaneous abortions before the 10th week of gestation. POne or more premature births of a normal neonate delivered prematurely before the 34th week of gestation due to severe pre-eclampsia, eclampsia, or severe placental insufficiency (where other causes are excluded). Laboratory criteria6 pPositive results on two or more occasions at least 12 weeks apart for at least one of the following tests: PLupus anticoagulant (LA); PCardiolipin antibodies (aCL) of IgG and/ or IgM isotype in medium or high titre, > 40 units; Pß2 -glycoprotein I (ß2GPI) antibodies of IgG and/or IgM isotype. pNeurological manifestations pIgA Cardiolipin antibodies pIgA ß2 -glycoprotein I antibodies pPhosphatidylserine antibodies pPhosphatidylethanolamine antibodies pProthrombin antibodies pPhosphatidylserine-prothrombin complex antibodies Although the pathophysiology of APS is now relatively well understood, difficulties still persist with making a definitive diagnosis based solely on clinical criteria. The prevalence of the clinical symptoms is high and the differentials for vascular thrombosis and pregnancy morbidity are relatively broad. Laboratory testing can therefore be instrumental in providing definitive diagnosis. APS laboratory testing Laboratory testing for APS includes both functional coagulation assays and immunology testing.2 Lupus anticoagulant (LAC) testing is an in vitro functional assay detecting the effect phospholipid antibodies have on the coagulation cascade. Phospholipid antibody tests are a direct measure of the presence or absence of the antibodies themselves. There are two main classes of antiphospholipid antibodies assays available. Cardiolipin antibodies detect antibodies binding to ß2 -glycoprotein within context of a protein/phospholipid complex whereas ß2 -glycoprotein I antibodies detect antibodies binding to ß2-glycoprotein in absence of phospholipids.2 Predicting future APS-associated events Several studies have been done on the use of these laboratory tests in identifying patients at risk for thrombotic events or severe pregnancy complications: pPositive antiphospholipid lab tests, in the absence of clinical criteria, should only be considered to be at risk factors rather than diagnostic criteria for APS. Patients should be assessed for additional thromboembolic risk factors (e.g., smoking, obesity, hypertension) for risk reduction.5 pClinical studies have shown that testing for the antibody profile (see Sidebar 2) is more useful in identifying thrombotic risk than the result of any individual test.2 pThe presence of both aCL–IgG antibodies and ß2GPI antibodies is believed to identify patients at higher risk for APS.2 pThe presence of LAC has been observed more frequently in patients without clinical events, and may be false positive in the elderly.2 pß2GPI antibodies have been correlated with thrombosis, pre-eclampsia, and eclampsia as well as having a role in young women with ischemic stroke.2 SOLUTIONS THE BULLETIN OF GAMMA-DYNACARE LABORATORIES Lupus anticoagulants (LAC) Cardiolipin (aCL) antibodies The term “lupus anticoagulant” was first coined in 1972 to describe an inhibitor directed against the coagulation cascade phospholipids.2 The name is a misnomer as most people testing positive for “lupus anticoagulant” (LAC) do not have SLE and it has a procoagulant effect in vivo, however the name persists despite attempts to modify. The real limitation of the aCL assay is inter-assay variability. The results for different assays on the market particularly differ in the lower measuring range for the test.1,6,7 The LAC test detects various APA’s on the basis of their interference with the phospholipid dependent steps in the coagulation cascade. Testing for the presence of LAC is performed following strict guidelines set up by International Society of Thrombosis and Haemostasis (ISTH) which include the preparation of platelet-poor plasma, and utilizing screening, mixing and confirmation tests.2,7 The ISTH guidelines state that laboratories are to perform two different screening tests.7 They recommend to perform (a) the dilute Russell’s Viper Venom Time (dRVVT) and (b) the activated partial thromboplastin time (aPTT) using a reagent with low phospholipid content.2 If either test is positive, the results are to be confirmed using a bilayer or hexagonal-phase phospholipid based reagent. Interpreting aCL antibodies test results pOnly medium and high levels, > 40 units of aCL antibodies (IgG or IgM) are included in the diagnostic criteria which improve the specificity.2,6 pLupus anticoagulant paCL IgA is of little diagnostic value; it has more use in classifying patients into diagnostic subgroups for risk since IgA aCL may also be associated with thrombocytopenia, skin ulcers and vasculitis.1,6 pß2GPI glycoprotein I antibodies, IgG and/or IgM ß2 -glycoprotein I (ß2GPI) antibodies ß2GPI antibodies have been incorporated into the 2006 updated criteria and are considered to be the most clinically significant antibodies. The assay shows higher specificity than the aCL assay and can be the only positive test in 3–10% of the APS patients.3,6 Interpreting ß2GPI antibodies test results pIt has been shown that LAC is more consistent with the clinical manifestations of APS than phospholipid antibodies.1,5 pHigh titres of ß2GPI antibodies are associated with a high risk of thrombosis.6 Phospholipid antibodies The challenge with the laboratory tests for phospholipid antibodies (APA) is that there is no diagnostic “gold standard”.2,5 The presence of APA is a necessary inclusion criterion to make the diagnosis, but is not diagnostic of APS. The antibodies may also be found in children with viral infection, other autoimmune disorders, patients with infections, malignancy and even in healthy individuals.2 Patients presenting with clinical symptoms suggestive of APS should have the following tests ordered, particularly those without other common risk factors: pAt lower concentrations, aCL-IgM tends to give false-positive results for APS, particularly in the presence of rheumatoid factor or cryoglobulins.6 Interpreting LAC test results pTesting should not be performed while patients are on anticoagulant therapy. Heparin and vitamin K antagonist treatments may impair the detection of the lupus anticoagulant.7 Tests to Order in Cases of Suspected APS pInterferences by cryoglobulins or rheumatoid factor may cause a false positive in interpretation of IgM ß2GPI antibodies.6 pCardiolipin antibodies, IgG and/or IgM Results of these tests will be adjunctive to the clinical findings and should be not be considered diagnostic of, but rather as risk factors for thrombosis, pregnancy loss and clinical manifestations of APS.2,5 If positive, confirmatory tests should be repeated within 6 to 12 weeks.2,6 SOLUTIONS THE BULLETIN OF GAMMA-DYNACARE LABORATORIES References 1.Tincani A, Andreoli L, Casu C, et al. Antiphospholipid antibody profile: implications for the evaluation and management of patients. Lupus. 2010;19(4):432–435. 2.Devreese K, Hoylaerts M. Challenges in the diagnosis of antiphospholipid syndrome. Clin Chem. 2010:56(6):930– 940. 3.Lockshin MD, Derksen RHWM. New developments in lupus-associated antiphospholipid syndrome. Lupus. 2008;17(5):443–446. 4.APS Foundation of America, Inc. Antiphospholipid antibody syndrome. Available at: http://www.apsfa.org/aps. htm. Accessed June 21, 2010. 5.Roubey RAS. Risky business: the interpretation, use, and abuse of antiphospholipid antibody tests in clinical practice. Lupus. 2010;19(4):440–445. 6.Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006;4(2):295–306. 7.Pengo V, Tripodi A, Reber G, et al. Update for the guidelines for lupus anticoagulant detection. J Thromb Haemost. 2009;7(10):1737–1740. Ç EMAIL [email protected] J PHONE For any questions, please call Gini Bourner, Scientific Director – Hematology at 1.866.790.3515 ext. 5220.