Aetiological diagnosis

While testing to establish the aetiological diagnosis of congenital thrombophilia should not be carried out routinely, it is essential to determine the following:

  • in which patients a "thrombosis test" should be performed
  • at what time this test should be performed

Routine screening for congenital thrombophilia is not indicated in the event of:

  • a thromboembolic venous accident following immobilisation
  • surgery
  • malignant disease
  • arterial thrombosis accompanied by risk factors for atherosclerosis

Thrombophilia screening should be performed in patients aged under 60 years presenting an episode of DVT or PE. Screening for cancer should not be overlooked in patients without a previous history and presenting an initial DVT or PE over the age of 50 years.

Given the metabolic modifications occurring during an acute episode of DVT or PE, particularly in inflammatory syndrome, it may be preferable to postpone testing because of the potential effects on the values of the test parameters.

Since pregnancy may also affect the values of certain parameters, testing should be carried out some time before or after pregnancy.

In addition, since anticoagulant treatments can also affect the measured values, antithrombin assays should be performed in the absence of treatment with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH), and assays of protein C and of protein S should be carried out after (one month) administration of vitamin K antagonists.

Aetiological diagnosis is designed to establish a known cause of congenital thrombophilia. First-line screening is performed to identify:

  • antithrombin deficiency
  • protein C deficiency
  • protein S deficiency
  • resistance to activated protein C and screening for associated factor V Leiden mutation
  • factor II gene mutation G20210A

Antithrombin (formerly "antithrombin III") deficiency:

Antithrombin (AT) deficiency, discovered by Egeberg in 1965, though less common, is the form of congenital thrombophilia associated with the highest level of thrombotic risk.

Classification of AT deficiency is complex.
Basically, diagnosis is founded on determination of AT activity. If such activity is reduced, determination of antigen levels may be used to differentiate between quantitative deficiency (parallel decrease in activity and in antigen) and qualitative deficiency (decreased activity alone, with normal antigen levels).

When interpreting results, it is essential to bear in mind all factors likely to reduce AT levels (heparin treatment, oestrogen-progestogen use, pregnancy) and, where necessary, to verify levels some time after such situations.
Screening for molecular abnormalities may only be carried out at specialised centres.

Protein C deficiency

Diagnosis of protein C deficiency is based upon first-line measurement of protein C activity. Antigen measurement, used as a second-line test, in the event of decreased protein C activity, may be used to distinguish between quantitative deficiency (type I deficiency) and qualitative deficiency (type II deficiency). In quantitative deficiency, a parallel decrease occurs in activity and in antigen levels while in qualitative deficiency, activity is reduced but antigen levels remain normal.

In interpreting the results, it is essential to bear in mind certain clinical settings in which protein C levels may be modified. Levels are increased during pregnancy and inflammatory syndromes. Since protein C synthesis is dependent upon vitamin K, protein C levels are reduced in patients on vitamin K antagonists. Assay of protein C may be performed one month after discontinuation of such treatment. It is not affected by heparin therapy.
Screening for molecular abnormalities may only be carried out at specialised centres.

Protein S deficiency

Numerous classifications of protein S deficiency have been proposed.
Protein S circulates in both an active free form and an inactive form bound to C4b-BP, a protein found within the complement system.
First-line diagnosis of the deficiency involves determination of the activity of protein S and of free protein S antigen. Assay of total protein S antigen may be carried out for second-line support of the diagnosis where activity and/or free antigen are reduced.

In interpreting the results, attention must be paid to certain clinical settings in which protein S levels may be modified. Protein S levels are reduced during pregnancy, with the use of oestrogen-progestogen and in inflammatory syndromes. Since protein S synthesis is dependent upon vitamin K, protein S levels are reduced in patients on vitamins K antagonists. Assay of protein S may be performed one month after discontinuation of such treatment. This value is unaffected by heparin therapy.
Screening for molecular abnormalities may only be carried out at specialised centres.

Resistance to activated protein C / factor V Leiden mutation

Resistance to activated protein C is associated with a mutation affecting one of the sites of cleavage of factor V by activated protein C.
Diagnosis is based upon a screening test involving determination of clotting time in the presence of activated protein C. In normal patients, clotting time is prolonged beyond a certain level but is shorter in patients with factor V Leiden mutation.

In the event of resistance to activated protein C, screening for factor V Leiden mutation is necessary. This test provides confirmation and indicates whether the mutation is heterozygous or homozygous.

Prothrombin gene mutation G20210A

This mutation, described in 1996 by Poort, occurs in the untranslated region of the prothrombin gene (factor II). It is associated with prothrombin levels higher than those found in subjects without the mutation. However, there is extensive overlap in prothrombin levels found in the two populations (subjects with and without the mutation) and determination of prothrombin levels is insufficient to confirm diagnosis, which is therefore based on screening for prothrombin gene mutation G20210A, and this test also indicates whether the mutation is heterozygous or homozygous.

Combined prothrombin gene mutation G20210A and factor V Leiden mutation is not uncommon and is associated with increased risk of thromboembolic accident, and it is consequently important to carry out simultaneous screening for both mutations.


Homocystein is a sulphated amino acid. Raised homocystein levels have been shown to be associated with increased risk of venous and arterial thromboembolic accident.

Hyperhomocysteinaemia may be either acquired or congenital. A deficiency in vitamin B12 or in folate may be accompanied by a moderate increase in circulating concentrations.

Two enzymes are involved in homocystein metabolism: methylene tetrahydrofolate reductase (MTHFR) and cystathionine beta-synthase (CBS). A mutation affecting either of these enzymes can cause congenital hyperhomocysteinaemia.

A mutation, mutation C677T, results in a thermolabile variant of MTHFR associated with moderately raised levels of homocystein (< 100 µmol/L, generally < 50 µmol/L). Screening for this mutation is straightforward using molecular biology methods; however, because of the marked prevalence of this mutation in the general population (approximately 30% in the heterozygous state), the value of this screening procedure is considered by some to be limited.

Mutations in CBS, though rare, can result in extremely high levels of homocystein associated with instances of venous thrombosis, as well as arterial thrombosis, such as myocardial infarction in young subjects (as early as 20 years in some cases). Many mutations may be responsible, and screening for these abnormalities must be carried out at specialised laboratories.

Increased levels of factor VIII

There is some debate concerning the value of first-line testing of factor VIII levels.
High factor VIII levels may be acquired or congenital.
Diagnosis is based upon determination of the clotting activity of factor VIII.


Some types of dysfibrinogenaemia have been associated with increased thrombotic risk.
Diagnosis is based upon measurement of functional fibrinogen activity and of fibrinogen antigen levels. Dysfibrinogenaemia is characterised by dissociation between levels of functional fibrinogen and levels of fibrinogen antigen.

In thrombophilia testing, it is no longer necessary to include assay of factor VIII and dysfibrinogenaemia or hyperhomocysteinaemia screening (recommendations of the GEHT [Groupe d'Etude sur l'Hémostase et la Thrombosis - French Haemostasis and Thrombosis Study Group] - France - 2009).