A Practical Guide to Laboratory Haemostasis


Antiphospholipid Antibodies: Introduction


Antiphospholipid antibodies (APL) disrupt the coagulation process and are important for two reasons:
1. They can prolong phospholipid-dependent coagulation tests e.g. the APTT, giving the impression of a potential bleeding disorder BUT
2. In vivo they may promote thrombosis (Antiphospholipid syndrome [APS]). In regard to b) the presence of the antibody may be entirely asymptomatic (they may occur after, for example, infection).

It is important to identify APLs because they may explain thrombotic episodes and recurrent first trimester miscarriage. In addition if not recognised, APLs can prolong coagulation tests and subsequent factor assays may be misinterpreted as indicating coagulation factor deficiency. Fortunately, in the latter some clue usually exists in the form of non-parallel curves in the factor assay.

The diagnosis of APS requires a combination of at least one specific laboratory test and one or more clinical symptoms:

Clinical Criteria
Vascular thrombosis One or more clinical episodes of arterial, venous, or small vessel thrombosis, in any tissue or organ.
Thrombosis must be confirmed by objective validated criteria (i.e. unequivocal findings of appropriate imaging studies or histopathology).
Pregnancy morbidity a. One or more unexplained deaths of a morphologically normal fetus at or beyond the 10th week of gestation
b. One or more premature births of a morphologically normal neonate before the 34th week of gestation because of:
  (i) eclampsia or severe pre-eclampsia or
  (ii) recognized features of placental insufficiency
c. Three or more unexplained consecutive spontaneous abortions before the 10th week of gestation, with maternal anatomic or hormonal abnormalities and paternal and maternal chromosomal causes excluded.

Laboratory Criteria
At least 2 positive tests for antiphospholipid antibody 12 weeks apart.
Directly ELISA (enzyme-linked immunosorbent assay)

The three characteristics of relevance for direct (i.e. ELISA) detected antiphospholipid antibodies are target, isotype and titre.
To be significant the antiphospholipid antibodies must be:
  a) Directed against either cardiolipin (aCL) or β2-glycoprotein-I (anti-β2GPI)
  b) IgG and/or IgM
  c) Present in medium or high titre (i.e. >40 GPLU or MPLU for aCL or >the 99th percentile for either aCL or anti-β2GPI.

Cardiolipin is found almost exclusively in the inner mitochondrial membrane where it performs an essential role in regulating enzymes involved in mitochondrial energy metabolism. The term 'cardiolipin' originates from the fact that it was first isolated from bovine heart in the early 1940s and formed the basis of the Wasserman test for syphilis.

β2-glycoprotein I (also known as Apolipoprotein H) is a multifunction protein which, in addition to binding to cardiolipin and inducing functionally relevant conformational change, also interferes with platelet aggregation by inhibiting serotonin release and interferes with various steps in the coagulation pathway.
The subset of antibodies that bind β2GPI correlate strongly with the thrombotic complications of the APS.
Indirectly Through their effect on phospholipid-dependent coagulation assays

Antiphospholipid antibodies can affect the phospholipid used in some laboratory tests of coagulation (e.g. the APTT) and produce a prolonged clotting time. This effect may be overcome by adding sufficient excess of phospholipid that the antibody is overwhelmed and the in vitro clotting time shortens.

Demonstration of the presence of an antiphospholipid antibody by coagulation tests requires:
a) Prolongation of a phospholipid dependent coagulation test AND
b) Correction of that prolongation by adding an excess of phospholipid or otherwise eliminating the effect of any APL OR
c) comparison to a phospholipid independent confirmatory test.

The tests used for this purpose include:
a) Dilute Russell Viper venom test (DRVVT)
b) Silica clotting time (SCT)

c) Kaolin clotting time (KCT)
d) The Textarin/Ecarin time
e) Taipan venom time (TVT)
f) Factor V ratio

No one test will detect all lupus anticoagulants and for any given test detection may vary between manufacturers. At least two different tests should therefore be used.

The diagram below shows a flow chart for the investigation of a suspected LA:

When screening for a lupus anticoagulant, two tests based on different principals should be used. Currently the ISTH SSC working party on antiphospholipid antibodies recommends the Silica Clotting Time [SCT] and the dilute Russell Viper Venom Time [dRVVT].

What Test Next

To complete testing for antiphospholipid antibody, patient samples should be tested for antiphospholipid antibodies twice and at an interval of at least 12 weeks. A full blood count should also be checked since antiphospholipid antibodies may cause an immune thrombocytopenia. Antiphospholipid antibodies can also cause acquired prothrombin deficiency and in patients with APL with a history of bleeding it is of value to measure prothrombin [Factor II] levels.

Data Interpretation

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