Specific Clotting Factor Assays

Specific clotting factor assays measure the activity of individual coagulation factors. They define the cause of a prolonged PT or aPTT and diagnose inherited deficiencies. They also detect acquired deficiency and inhibitors, and monitor replacement therapy.

Inherited deficiencies assayed include haemophilia A and B, and factor VII, X, V, II, XI and XIII deficiency.

The one stage clotting assay is the most widely used. Patient plasma is mixed with plasma deficient in the factor under test and clotted with an aPTT or PT reagent. The clotting time is read against a calibration curve prepared from reference plasma.

Chromogenic assays measure activity from cleavage of a synthetic substrate. They are used for factor VIII and factor X when the one stage method is unreliable.

Haemophilia A affects about 1 in 5000 male births and haemophilia B about 1 in 25 000 to 30 000. Both are X-linked. Von Willebrand disease is more common, and should be considered when factor VIII is low. Factor XI deficiency is common in people of Ashkenazi Jewish ancestry. Deficiencies of factors II, V, VII, X and XIII are autosomal recessive and rare.

Indications.

• Prolonged aPTT or PT that does not explain itself.
• Bleeding with a normal screen (factor XIII, alpha-2 antiplasmin).
• A family history of haemophilia.
• Suspected acquired haemophilia (sudden bleeding in an older adult or postpartum woman).
• Suspected carrier status.
• Monitoring of replacement therapy.
• A mixing study that has failed to correct.

Specimen. Citrated platelet poor plasma from a clean venepuncture, with the tube filled to at least 90% of nominal volume. Centrifuge twice, test within 4 hours at room temperature, or freeze at minus 70 °C. Avoid heparin contamination from lines.

Reference range. About 50 to 150 IU/dL (0.50 to 1.50 IU/mL) for factors II, V, VII, VIII, IX, X, XI and XII in healthy adults. Factor XIII range is about 70 to 140 IU/dL. Local ranges take precedence.

Severity of haemophilia (factor VIII or IX activity).

• Severe: under 1 IU/dL. Spontaneous joint and muscle bleeding.
• Moderate: 1 to 5 IU/dL. Bleeding after minor trauma.
• Mild: over 5 and under 40 IU/dL. Bleeding after surgery or major trauma.

Factors that raise or lower the result.

• Factor VIII rises with acute inflammation, pregnancy, exercise, stress, oestrogen, liver disease, diabetes and non-O blood group, and is lower in blood group O. A raised level may hide mild haemophilia A or the carrier state.
• Factor IX is lower in neonates (typically 15 to 50 IU/dL at birth) and rises over months.
• Vitamin K-dependent factors (II, VII, IX, X) fall with vitamin K deficiency and warfarin.
• Factor VII has the shortest half life, about 6 hours.

Chromogenic versus one stage assay. About a third of mild haemophilia A mutations give discordant results, with the one stage assay reading lower or higher than the chromogenic assay. Both methods are needed when the clinical picture and result disagree, and genotyping settles the question.

Factor XIII. The clot solubility test detects only severe deficiency (under about 1 to 5 IU/dL). Quantitative ammonia release or amine incorporation assays are needed, and a normal PT, aPTT and fibrinogen is the typical pattern.

Inhibitor assay (Bethesda assay). Serial dilutions of patient plasma are incubated with normal plasma for 2 hours at 37 °C, and residual factor activity is measured. One Bethesda unit is the amount that neutralises 50% of the factor in normal plasma. The Nijmegen modification (buffered normal plasma and factor deficient plasma as diluent) improves specificity at low titres. A titre of 5 Bethesda units or less is low titre, and over 5 is high titre.

Isolated low factor VIII. Haemophilia A, von Willebrand disease (including type 2N), carrier state, acquired haemophilia A, and combined factor V and VIII deficiency (LMAN1, MCFD2).

Isolated low factor IX. Haemophilia B, vitamin K deficiency, warfarin, liver disease (late), nephrotic syndrome, and neonatal status.

Low factor XI. Factor XI deficiency, which correlates poorly with bleeding. Bleeding is typically after trauma or surgery in tissues with high fibrinolytic activity (oral cavity, urinary tract, nose), and level does not predict risk well.

Low factor XII, prekallikrein or kininogen. No bleeding. A prolonged aPTT can be isolated, and factor XII deficiency may be linked with thrombosis in registry data, without proof.

Low factor VII. Early vitamin K deficiency, early liver disease, warfarin, and inherited deficiency. Bleeding risk is imperfectly related to level, and levels under about 10 IU/dL carry greater risk.

Low factor X. Inherited deficiency, vitamin K deficiency, liver disease, and acquired deficiency with amyloidosis (AL type), which can cause severe bleeding.

Low factor V. Liver disease (factor V falls and is not vitamin K dependent), DIC, fibrinolytic therapy, inherited deficiency, and rarely autoantibodies, including after exposure to bovine thrombin.

Distinguishing causes of a combined deficiency. In vitamin K deficiency, factors II, VII, IX and X are low with normal factors V and VIII. In liver disease, factors II, V, VII, IX and X are low, and factor VIII is normal or raised. In DIC, factors V and VIII are both consumed.

Acquired factor inhibitors.

• Acquired haemophilia A is an autoantibody to factor VIII, occurring in older adults, postpartum women, and with autoimmune disease, malignancy and some drugs. It causes severe subcutaneous, muscle and mucosal haemorrhage with a prolonged aPTT that fails to correct on mixing, particularly after incubation.
• Inhibitors in inherited haemophilia develop in about 25 to 30% of patients with severe haemophilia A and 3 to 5% of those with haemophilia B, mainly in the first 50 exposure days.
• Lupus anticoagulant gives a prolonged aPTT with variable, mildly reduced factor activity (the result improves at higher dilution, a sign of a non-specific inhibitor).

Emicizumab. It mimics factor VIII, so one stage aPTT-based assays give falsely normal or very high factor VIII and prolonged-aPTT inhibitor tests are unreliable. A chromogenic assay with bovine reagents is needed to measure factor VIII, and human reagents are affected.

Extended half life products. One stage and chromogenic results can differ by product, so the laboratory method must match the product.

Critical findings needing immediate action.

• Factor VIII or IX under 1 IU/dL with active bleeding, head injury or suspected intracranial haemorrhage needs immediate replacement and specialist contact.
• A new inhibitor, or a prolonged aPTT that fails to correct with active bleeding, needs urgent haematology review. Acquired haemophilia carries a mortality of about 10 to 20% without prompt treatment.
• Factor XIII deficiency with umbilical stump bleeding or intracranial haemorrhage in a newborn.
• Haemophilia carrier with a factor level under 40 IU/dL before delivery needs a delivery plan.

Interferences and pitfalls.

• Heparin, direct oral anticoagulants and lupus anticoagulant can lower aPTT-based factor assays and mimic deficiency, and the result must be checked at several dilutions.
• Underfilled tubes, delay, cooling and haemolysis lower factor VIII and V.
• Factor VIII is raised acutely, so a normal level during inflammation, pregnancy or stress does not exclude mild haemophilia.
• Test results from a carrier at one time point can fall within the normal range, and genotyping is more reliable for carrier status.
• A normal factor VIII with a low von Willebrand antigen and activity ratio indicates von Willebrand disease, and a low factor VIII with a normal von Willebrand antigen needs von Willebrand factor binding assessment for type 2N.

Special groups.

• Neonates: vitamin K-dependent factors and contact factors are low, with factor VIII, V and fibrinogen near normal. Mild factor IX deficiency cannot be diagnosed reliably in the first 6 months, and repeat testing is needed.
• Female carriers: levels under 40 IU/dL cause bleeding symptoms, and levels vary over time because of X inactivation.
• Pregnancy: factor VIII, VII, X and fibrinogen rise, so carrier levels need checking in the third trimester before delivery.
• Older adults: a new unexplained prolonged aPTT with bleeding suggests acquired haemophilia.

Follow up tests.

• Mixing studies with incubation.
• Von Willebrand screen.
• Bethesda inhibitor assay.
• F8 and F9 gene analysis (including intron 22 inversion for severe haemophilia A).
• Carrier testing of female relatives.
• Lupus anticoagulant, clotting screen, fibrinogen and liver function tests.
• Vitamin K-dependent factor assays and quantitative factor XIII assay.
• Serum protein electrophoresis when amyloidosis is suspected.
• Family studies.