To convert pmol/L of AMH to ng/ml, divide by 7.14.aCL Antibodies

Anticardiolipin antibodies (aCL) at least 10 GPL unitsaCLs, a subgroup of aPL, are a quantifiable aPL and have been shown to be an independent risk factor for stroke. patients with the highest aCL titers may be at greatest risk for subsequent thrombo-occlusive events.aCL was assayed for the IgG isotope by enzyme-linked immunosorbent assay as previously described9 and standardized via the Second International Standardization Workshop7 and reported as GPL (IgG phospholipid) units. One GPL unit is equivalent to 1 μL/mL of IgG aCL immunoreactivity.We did not find any clinically meaningful or prognostic features based on IgM aCL titers in this cohort. Our data support the importance of aCL isotype specificity, specifically IgG, in predicting clinical complications associated with aCL as others have reported.22 23 In our clinical experience, elevated IgM aCL titers alone are more variable and transient than IgG aCL titers.Antiphosphatidylserine antibodies may also be important5 24 and may have greater specificity for identifying patients with the antiphospholipid syndrome.patients with higher titers of IgG aCL may be at increased risk for subsequent events, even with the best empirical medical therapy (risk factor modification, antiplatelets, anticoagulants, corticosteroids, immunosuppressants, immunomodulation, plasmapheresis). As we did not assay for β2-glycoprotein 1 (the aPL cofactor), further studies will also need to determine whether cofactor dependency is important in refining the prognostic value of aCL isotype and titer.

Antiphospholipid antibody testing is used to help determine the cause of:

Inappropriate blood clot formation (unexplained thrombotic episode, excessive clotting)

Recurrent miscarriage

Low platelet count (thrombocytopenia)

Prolonged PTT test

Depending on a person's signs and symptoms and medical history, a doctor may order one or more types and classes of these

tests to help detect the presence of antiphospholipid antibodies and to help diagnose antiphospholipid syndrome (APS):

Cardiolipin antibodies  (IgG, IgM, and sometimes IgA) are frequently ordered as they are the most common

antiphospholipids. 

Lupus anticoagulant assay  (e.g., RVVT, hexagonal phase lipid neutralization) if a person has a prolonged PTT test

Anti-beta2 glycoprotein I  and anti-phosphatidylserine testing may be ordered along with the other antiphospholipid

antibodies to detect their presence and to provide the doctor with additional information.

If an antiphospholipid antibody is detected, the same test(s) may be ordered 8 to 10 weeks later to determine whether their

presence is persistent or temporary.

Testing may also be performed to help diagnose and/or evaluate a person with an autoimmune disorder as they may be present

with disorders such as systemic lupus erythematosus (SLE or lupus). If a person with an autoimmune disorder tests negative for

antiphospholipid antibodies, testing may be repeated to determine if an antibody has developed in the course of the disease.

^ Back to topWhen is it ordered?

This testing may be ordered when a person has signs and symptoms suggestive of a thrombotic episode, such as pain and

swelling in the extremities, shortness of breath, and headaches. It also may be ordered when a woman has had recurrent

miscarriages and/or as a follow-up to a prolonged PTT test.

When one of the tests is positive, it may be repeated several weeks later to determine whether the antibody is temporary or

persistent. Antiphospholipid testing may be done when clinical signs suggest the presence of antiphospholipid syndrome.

When a person with a diagnosed autoimmune disorder tests negative for antiphospholipid antibodies, one or more of the tests may

be repeated at regular intervals to screen for the development of an antiphospholipid antibody.

^ Back to topWhat does the test result mean?

Care must be taken when interpreting the results of antiphospholipid antibody tests. A negative result means only that the specific

antibody tested was not present at the time of the test. Low to moderate levels of one or more antibodies may occur temporarily

due to an infection or drug or may appear as a person ages. These concentrations are often not considered significant but must be

examined in conjunction with a person's symptoms and other clinical information.

In some cases, a person may have one or more classes of a specific antibody present or absent. For instance, the person may

have significant quantities of IgG and IgM cardiolipin antibodies or may only be positive for the less frequently tested IgA cardiolipin

antibody. Moderate to high levels of one or more antiphospholipid antibodies, which persist when tested again 8 to 10 weeks later,

indicate the likely continued presence of that specific antibody.

If tests indicate the presence of the lupus anticoagulant and it persists when retested, then it is likely that the person is positive for

the lupus anticoagulant. People who have one or more antiphospholipid antibodies and those that are diagnosed with

antiphospholipid syndrome have an increased risk of having recurrent thrombotic episodes, recurrent miscarriages,

and thrombocytopenia.

Test results cannot predict the likelihood of complications, the type, or the severity in a particular person. Some will have a variety

of recurrent problems while others may never experience any difficulties. Examples of this include an asymptomaticindividual who

is diagnosed with antiphospholipid antibodies following a prolonged PTT test that is done for another reason (such as a pre-

surgical screen) and an asymptomatic elderly person who has acquired an antiphospholipid antibody.

Transient antiphospholipid antibodies may be seen in people who have inflammation, autoimmune disorders, infections, or cancer.

^ Back to topIs there anything else I should know?

Occasionally, antiphospholipid antibody testing may be ordered to help determine the cause of a positive VDRL/RPR testfor

syphilis. The reagents used to test for syphilis contain phospholipids and can cause a false-positive result in those with

antiphospholipid antibodies.

False-positive test results may be seen in people who take drugs such as quinidine, procainamide, phenytoin, and penicillin.

Antiphospholipid (APL) antibodies are group of antibodies directed against epitopes on plasma proteins that are uncovered by binding of these proteins to anionic phospholipids on plasma membranes. The most commonly used

tests to detect APL include lupus anticoagulant (LAC), anticardiolipin (ACL) antibodies, and anti-β2 -glycoprotein I antibodies.

Lupus anticoagulantThe International Society of Thrombosis and Haemostasis (ISTH) criteria for lupus anticoagulant detection include 4 mandatory steps, in the following sequence:[1]

1. Prolongation of a phospholipid-dependent clotting assay (activated partial thromboplastin time [aPTT], kaolin clotting time [KCT], diluted Russell’s viper venom test [dRVVT], diluted prothrombin time [dPT]); two tests that have different assay principles should be used, usually the dRVVT and aPTT; this is the screening step

2. Mixing study with 1:1 proportion of patient’s plasma and a normal pooled plasma without preincubation and reassessment of the clotting assay used in step one; if it remains prolonged, an inhibitor is present, either LAC or a specific factor inhibitor; if it corrects, then LAC is excluded and the cause is likely a specific factor deficiency

3. Confirmation that the inhibitory activity is phospholipid-dependent by relative correction of the abnormal clotting time when the concentration of phospholipid is increased in the screening test(s) that yielded abnormal results; this step confirms that the cause of abnormal mixing study is LAC (phospholipid-dependent inhibitor), not a specific factor inhibitor; knowing the clinical history helps in diagnosis—thrombosis in case of LAC or hemorrhage in the case of factor deficiency

4. Exclusion of other coagulopathies that may yield similar results or accompany the LAC presence; specific factor assay might be necessary

The results from the above tests are considered positive when they are above the local cutoff value. For tests in steps 1 and 2, the cutoff value is the 99th percentile of the distribution of the tests performed on plasmas from at least 40 healthy donors younger than 50 years. In the confirmatory tests in step 3, the cutoff value is equal to the mean of the individual percentage of corrections, calculated with the following equation:

[(Screen - confirm)/screen] × 100†

Anticardiolipin antibodiesEnzyme-linked immunoassay (ELISA) is currently the test of choice. The reference range findings are as follows: [2]

Less than 15 immunoglobulin G (IgG) phospholipids units (GPL): Absent or none detected Less than 12 immunoglobulin M (IgM) phospholipids units (MPL): Absent or none detected Less than 12 immunoglobulin A (IgA) phospholipids units (APL): Absent or none detected

If the test is obtained to diagnose antiphospholipid syndrome, only medium to high titers of IgG or IgM antibodies (defined as >40 GPL or MPL, or >99th percentile[3] ) are considered a positive test result.

Anti-β2 -glycoprotein I antibodiesELISA is the test used to detect these antibodies, IgM and IgG isotypes. Per antiphospholipid syndrome diagnostic criteria, these anti-β2 -glycoprotein I antibodies of IgG and/or IgM isotype should be present in the in plasma, in a titer greater than the 99th percentile to be considered a positive test result.

greater than the 99th percentile to be considered a positive test result. [3]

Collection and PanelsLupus anticoagulantA blood serum sample is collected in 0.109 M sodium citrate. Blood should be collected before the start of anticoagulation therapy or after a sufficient period following discontinuation.

Platelet-free (< 10,000/μL) plasma preparation via ultracentrifugation must be done with special care, as it may cause platelet fragmentation and the release of phospholipids, which may significantly affect the results.

Frozen plasma is required if testing is delayed, and frozen plasma must be thawed at 37°C. [1, 4]

Anticardiolipin antibodies and anti - β2 -GPI antibodies

Obtain a 7-mL blood serum sample in a red-topped tube.

Serum is preferred over plasma for testing.

Running samples in duplicate is recommended.

Polystyrene ELISA plates or high-sensitivity plates are preferred.

Cardiolipin should be diluted in ethanol.[5]

Control samples should be collected from at least 40 healthy young (< 50 years) people to determine the local cutoff for the above tests.

BackgroundDescriptionAntiphospholipid (APL) antibodies are group of heterogeneous antibodies directed against epitopes on plasma proteins that are uncovered by binding of these proteins to anionic phospholipids on plasma membranes. These antibodies can be detected with lupus anticoagulant (LAC), anticardiolipin antibodies (ACL), and anti-β2 -glycoprotein I antibodies.

LAC is a functional assay that is used to detect APL antibodies that have anticoagulation activity in vitro. LAC is a misnomer, since it is clinically associated with clotting tendency rather than anticoagulation activity, and only 50% of people with LAC meet the criteria for SLE.

The clinical significance of the above antibodies is their association with thrombosis; once an individual is found to have one of the above antibodies with vascular thrombosis and/or pregnancy morbidity, antiphospholipid antibody syndrome (APS) is diagnosed. APS occurs either as a primary disease or in the setting of another disease such as SLE (so-called secondary APS).

In addition of the above APL antibodies, other APL antibodies exist, such as prothrombin, annexin V, phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine, but their clinical significance remains elusive and experience is limited; thus, none is yet part of the diagnostic criteria of APS.

Indications/ApplicationsTesting for APL antibodies is most appropriate in patients with suspected APS, such as young people with unprovoked venous or arterial thrombosis, early pregnancy loss, or other pregnancy morbidity, especially those with SLE.

Conversely, it is probably reasonable to test for APL antibodies in young people with provoked venous thrombosis and/or recurrent early pregnancy loss. It may also be prudent to check for these antibodies in asymptomatic patients with unexplained prolonged aPTT discovered incidentally during routine testing, although the presence of APL antibodies in this scenario does not confirm the diagnosis of APS, since the patient is asymptomatic. However, the patient may develop symptoms later, and only then can APS be diagnosed. [1]

Persons with APL antibodies are at risk of developing APS, although the presence of these antibodies might be transient, without any clinical significance. Other manifestations, such as thrombocytopenia, livedo reticularis, heart valvular disease, and nephropathy, may be seen in individuals with APS but are still not part of its diagnostic criteria; the presence of these manifestations, especially in people with thrombosis and/or pregnancy morbidity, should alert the physician to suspect APS and to evaluate for the presence of APL antibodies. [6]

ConsiderationsIt is recommended that patients suspected of having APS undergo testing for all 3 antibodies (LAC, ACL, anti-β2 -GPI), although the presence of only one type of these antibodies is sufficient for diagnosis. Nonetheless, patients who are positive for all 3 antibodies have the highest rate of APS-related complications. [7] If only one test is to be chosen, LAC is the assay of choice since it has the strongest correlation with APS manifestation.

To meet the diagnostic criteria for APS, the test results should be positive on two occasions, 12 weeks apart. This is so that persistent pathologic APL antibodies can be differentiated from transient nonpathologic APL antibodies.

Local cutoff values are calculated by performing the test on at least 40 young (< 50 years) healthy donors.

Tests should be performed when the patient is clinically stable, not during an acute thromboembolic event, for 2 reasons: first, an acute event may trigger the production of transient anticardiolipin antibodies; second, acute events increase the acute-phase reactants such as fibrinogen and factor 8, which can alter the results of coagulation tests. [8]

LAC detection tests should be performed before anticoagulation therapy is started or after it has been stopped for a sufficient period. Anticardiolipin or anti-β2 -glycoprotein I antibody testing are generally not affected by anticoagulation therapy.

The concentration of phospholipids affects the LAC detection results. Phospholipids can react with the APL antibodies in the screening tests (step 1; see Lupus anticoagulant) and prevent prolongation of the these tests, and, since platelets are phospholipid-rich, platelet-free plasma is used in these tests to avoid false-negative results, whereas excess phospholipids are added in step 3 (see Lupus anticoagulant) to confirm that the inhibitor is phospholipid-dependent.

These assays differ in their sensitivity and specificity, and their intralaboratory variability remains high. Efforts to standardize these tests are in process.

People with past or current syphilis may have false positive results without being at risk for thrombosis, as is the case with other infection-, medication-, or neoplasm-induced transient antibodies.