Antiphospholipid antibody profile (LA & ACA)

Initial screening for antiphospholipid antibodies (APA) comprises analysis for lupus anticoagulant (LA) and anticardiolipin antibodies (ACA).

Lupus anticoagulants (LA) are classified as antiphospholipid antibodies (APA), although they are in fact directed against phospholipid-binding proteins, in particular, β2 glycoprotein I and prothrombin. The presence of persistent LA has a greater association with thrombosis, pregnancy morbidity and recurrence than the criteria antibodies detected in solid-phase assays (aCL & aβ2GPI). LA are a heterogeneous group of autoantibodies that are detected by inference based on their behaviour in phospholipid-dependent coagulation assays after other possible causes of elevated clotting times have been excluded. LA detection involves use of screening, confirmatory and mixing tests.

Screening tests commonly employ dilute phospholipid to accentuate the in vitro anticoagulant effect of LA, which if present, will prolong the clotting time. Screening tests can be prolonged for reasons other than LA, (i.e. factor deficiencies, anticoagulant therapy), so all elevated screening tests receive follow-up analyses to help define the nature of any abnormality. The confirm test generally involves performing the screening test in an identical fashion except that the phospholipid concentration is markedly increased. This has the effect of partially or completely overwhelming the LA and thus leads to a shorter clotting time than the screening test, thereby evidencing phospholipid dependence. Clotting times are converted to ratios to mitigate for issues of analytical variability. Correction of the screen ratio by the confirm ratio by ≥10% is considered consistent with the presence of a LA, providing that other causes of elevated clotting times are excluded. Diagnostic specificity is improved by performing the screen and confirmatory tests on 1:1 mixtures of test and normal plasma to evidence inhibition and reduce interferences, although the inevitable dilution effect can compromise this aspect of analysis.

Antibody heterogeneity and reagent variability necessitate use of at least two assays, of different analytical principle, to achieve acceptable detection rates. First-line assays are dilute Russell's viper venom time (dRVVT) and LA-responsive APTT, a pairing that will detect most clinically significant antibodies. Our laboratories employ a dilute APTT (dAPTT) in this respect, which employs a silica activator and a low concentration of phospholipid comprised of a composition of phospholipid types that is LA-responsive. The confirm test involves addition of concentrated platelet-derived phospholipid. dRVVT analysis employs diluted FX activator from the venom of Russell's viper (Daboia russellii), a low concentration of phospholipid comprised of a composition of phospholipid types that is LA-responsive and calcium ions. The confirm test involves an identical reagent except that the same phospholipid preparation is employed at a higher concentration. All elevated dAPTT & dRVVT screen ratios are reflexed to receive the confirm test, and the screen and confirm mixing tests, and are reported with interpretive comment. Patients with LA may be postitive in one or both of the dAPTT & dRVVT test medleys.

Anticardiolipin antibodies (ACA) are detected and quantified using indirect enzyme-linked immunosorbent assay (ELISA). The ACA produced as part of the autoimmune response in antiphospholipid syndrome are in fact directed to a cryptic epitope in Domain I of β2glycoprotein I (β2GPI) that is exposed during a conformational change induced in β2GPI when it binds to negatively charged phospholipid. ACA in patient sera are captured in microtitre plates coated with cardiolipin and β2GPI. Unbound material is then washed off and a solution of antibody to human immunoglobulin that is conjugated to an enzyme is added to ‘tag’ onto any captured ACA. Unbound conjugate is washed off and a substrate for the enzyme is added, the product of the enzyme-substrate reaction being coloured. Colour intensity is in direct proportion to the degree of conjugate-binding, itself proportional to the amount of ACA capture and thus, ACA concentration.
Clinical details: 
The antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterised clinically by vascular thrombosis and/or pregnancy morbidity. APS is diagnosed in patients who present with these clinical signs and symptoms and demonstrate the persistent presence of antiphospholipid antibodies.

Criteria antibodies for diagnosis of APS are lupus anticoagulant, anticardiolipin antibodies and/or β2 glycoprotein I antibodies. Persistence of one or more of these antibodies in the presence of appropriate clinical manifestations secures diagnosis of APS, although association and recurrence are higher in patients with multiple-positivity.
Reference range: 

Ratio (dRVVT & dAPTT)
GPL U/ml (IgG ACA)
MPL U/ml (IgM ACA)

Ratio (dRVVT & dAPTT) GPL U/ml (IgG ACA) MPL U/ml (IgM ACA)
Sample type and Volume required: 
External requests: serum - 400µL x 1 aliquot
Citrated platelet poor plasma - 1mL x 2 aliquots
Internal requests: please refer to EPR label

Turnaround time: 
7 - 12 days
Special sample instructions: 

The citrate samples should be analysed or manipulated & stored in the laboratory within 4 hours of venepuncture. Please ensure sample tubes are filled exactly to the fill-line as underfilling creates a dilution error and leads to inaccurate results.

Diagnostic Haemostasis and Thrombosis Department
St Thomas': 020 7188 2797; Guy's: 020 7188 7188 ext. 53860
St Thomas' Hospital
North Wing - 4th and 5th Floors
Westminster Bridge Road
London SE1 7EH

Laboratory opening times

Guy's Hospital
Southwark Wing - 4th Floor
Great Maze Pond
London SE1 9RT

Outside core hours, contact Duty Haemostasis Biomedical Scientist
For clinical advice or interpretation of results, please contact the laboratory in the first instance.

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Last updated: 08/03/2017