Introduction
Factor XIII [FXIII] is a heterodimer and circulates in plasma as a pro-transglutaminase consisting of two catalytic A subunits [FXIII-A2] and two non-catalytic B subunits [FXIII-B2] held together by non-covalent bonds. Intracellular Factor XIII including platelet Factor XIII comprises only the FXIII-A subunits and exists as a homodimer of the A subunit [FXIII-A2]. Factor XIII has a number of other functions in addition to the stabilisation of the Fibrin clot and these include the maintenance of pregnancy, bone/cartilage growth and wound healing.
| Subunit | Structure/Gene |
|---|---|
| A subunit | The A subunit is the active part of the molecule and functions as a transglutaminase cross-linking the lysine residue of one γ-chain to the glutamine residue of another γ-chain to form cross-linked fibrin – a transglutaminase reaction which releases ammonia. The gene for the FXIII-A subunit [F13A] maps to chromosome 6 [6p24-p25] and contains 15 exons encoding a 3.9kb mRNA. FXIII is activated by Thrombin in the presence of Calcium ions and Fibrin cleaving the activation peptide A. The B subunit then dissociates from the A subunit in the presence of Calcium and Thrombin and then the cleaved Factor XIIIa undergoes a conformational change exposing the active site which reacts with the fibrin monomers cross-linking the lysine of one γ-chain to the glutamine residue of another γ-chain to form cross-linked fibrin. Factor XIII-A contains both the binding site for Thrombin and Calcium required for catalytic activation. Factor XIIIa also forms covalent bonds with α2-Antiplasmin and TAFI [Thrombin Activatable Fibrinolysis Inhibitor] stabilising the fibrin clot and making it resistant to proteolytic degradation by the fibrinolytic pathway. Factor XIII is also found in platelets and monocytes/macrophages but as a dimer [FXIII-A2] and not complexed to FXIII-B2. |
| B subunit | The B subunit has no enzymatic activity and functions as: 1. A carrier for the A subunit preventing its proteolytic degradation within the plasma 2. In the binding of FXIII to the fibrin clot. The gene for the FXIII B subunit [F13B] maps to chromosome 1 [1p31-q32.1] and consists of 12 exons encoding a 2kb mRNA. |
Factor XIII Nomenclature
| Factor XIII | Nomenclature |
|---|---|
| Plasma FXIII | FXIII-A2B2 |
| Cellular FXIII | FXIII-A2 |
| FXIII Subunit A monomer | FXIII-A |
| FXIII Subunit A dimer | FXIII-A2 |
| FXIII Subunit B monomer | FXIII-B |
| FXIII Subunit B dimer | FXIII-B2 |
| Factor XIII-A gene | F13A1 |
| Factor XIII-B gene | F13B |
Assays - Principles
A. Clot Solubility Assays
Laboratories may use a screening test for Factor XIII deficiency and only if this is abnormal is a formal FXIII assay performed. However the screening test can be misleading and may fail to identify mild-moderate Factor XIII deficient patients [Factor XIII >2-3 IU/dL ]. Clot-solubility tests that use added Thrombin have been reported to have greater sensitivity than other solubility tests. The recommendations are not to use the Clot Solubility Assay for the diagnosis of Factor XIII Deficiency.
Quantitative FXIII activity assays are recommended as first-line screening tests whenever possible due to the insensitivity of the Clot Solubility Assay and the lack of standardisation. The Clot solubility assay cannot be used to monitor FXIII replacement therapy.
| Screening Tests | |
|---|---|
Urea Method |
The plasma samples [patient and control] are clotted by the addition of an excess of calcium and incubated at 37°C for 30 minutes. An alternative approach involves clotting the plasma with Thrombin and saline. The clot is removed and placed in 5M urea and incubated for 24 hours at either room temperature or 37°C. If the clot has dissolved this suggests FXIII deficiency and a formal FXIII assay should be undertaken. Hypofibrinogenaemia and Dysfibrinogenaemia can cause false-positive results and a functional Fibrinogen assay,Thrombin time/Reptilase time should be performed to exclude these disorders before the Urea screening test is performed. |
Acetic Acid |
The plasma samples [patient and control] are clotted by the addition of an excess of calcium and incubated at 37°C for 30 minutes. An alternative approach involves clotting the plasma with Thrombin and saline. The clot is removed and placed in either 2% Trichloroacetic acid or 1% Monochloroacetic acid and incubated for 24hours at either room temperature or 37°C . If the clot has dissolved this suggests FXIII deficiency. |
2. Immunological Assays
A number of assays exist for the measurement of the FXIII-A and FXIII-B subunits and the FXIIIA2B2 complex, by ELISA. In general the FXIII-A subunit is assayed first and if it is low the B subunit is measured. These assays may fail to detect rare qualitative [Type II] variants in which the immunological Factor XIII levels are normal but the protein is functionally defective. In these rare cases a functional FXIII assay is needed.
a. ELISA Assays for FXIII
A number of assays exist for the measurement of both the FXIII-A and FXIII-B subunits by ELISA. In general the FXIII-A subunit is assayed first and if it is low the B subunit is measured as the B subunit will not be low with a normal A subunit but the converse is not true.
b. Laurell Electroimmunodiffusion
The Laurell Immunodiffusion technique can be used to measure FXIII although in practice it rarely is. The agar plate contains an antibody to FXIII and after electrophoresis a rocket shaped precipitin pattern forms along the axis of migration that can be visualised after staining with Coumassie Blue. The height of the peak (the ‘rocket’) is proportional to the concentration of FXIII.
3. Functional FXIII Assays
There are a number of methods for measuring Factor XIII levels and which rely upon the transglutaminase activity of FXIIIa:
a. Ammonia release assay:
Factor XIII is activated by Thrombin and Ca2+. Soluble fibrin generated by the Thrombin accelerates the reaction but an inhibitor of fibrin polymerisation is contained in the assay and which prevents the formation of a fibrin clot. The Factor XIIIa transglutaminase activity between a synthetic amine peptide substrate and a Gln containing oligopeptide leads to the generation of Ammonia [NH4+]. The generation of ammonia is continuously monitored through the action of Glutamate Dehydrogenase [GLDH] that converts NADPH to NADP+. The conversion of NADPH to NADP+ can be monitored by its absorption at 340nm. Ammonia release assays are rapid, readily automated and easily standardised.
b. Amine incorporation assay:
In this assay a high dilution of the plasma sample prevent the formation of fibrin polymers and in addition the use of various peptides inhibits fibrin polymerisation. In these assays the binding of an acyl acceptor substrate amine to an acyl donor glutamine residue of a protein by FXIIIa is measured.
in general the Amine incorporation assays are time consuming and difficult to standardise.
c. Isopeptidase assay:
In specific conditions FXIIIa can exert isopeptidase activity i.e. it can release amines bound to a Gln residue in an oligopeptide. This release can be measured by the use of a N-terminal coupled dye and the florescence is proportional to Factor XIII activity.
5. Chromogenic FXIII Assays
Chromogenic assays are available for measuring FXIII [actually FXIIIa] based upon its transglutaminase activity. The test is based upon the incorporation of a biotinylated amine substrate [5-(biotinamido)pentylamine] into Fibrinogen [immobilised on a microtitre plate] by Thrombin activated FXIII[a]. The incorporated amine substrate is detected with a strepavidin-enzyme conjugate and chromogenic substrate. The colour change is proportional to the concentration of FXIIIa.
6. Thromboelastogram
The TEG can also detect significant FXIII deficiency – the TEG shows reduced clot formation and increased Fibrinolysis.
Interpretation
Low Factor XIII Levels are seen in:
- Individuals with inherited FXIII deficiency
- Factor XIII levels fall in pregnancy. Severe inherited FXIII deficiency is associated with recurrent miscarriage
- Excessive activation, as seen in DIC, exposure to some snake venoms and caterpillar toxins [Lonomia Achelous caterpillar Venom]
- Henoch-Schoenlein purpura (HSP)
- In patients on and following cardiopulmonary bypass
- Chronic inflammatory bowel disease
- Severe GvHD of the gut
Acquired Factor XIII deficiency secondary to an auto-antibody is rare and approximately 50% of cases are idiopathic. Other causes include:
- Autoimmune disease
- Malignancy
- Myelo and Lympho-proliferative disorders
- Various drugs including Isoniazid, Penicillin and Procainamide. In these cases the FXIII levels often return to normal after the drug is withdrawn.
For more information on Acquired Factor XIII deficiency see References: Ichinose A et al 2016 and Yan et al 2018.
Classification of Factor XIII Deficiency
The Factor XIII and Fibrinogen SSC Subcommittee of the ISTH has proposed a Classification for Factor XIII deficiency - see References: Kohler et al 2011.
Reference Ranges
The concentration of subunit A in plasma is 60-130 U/dL and that of subunit B is also 60-130 U/dL. Much of FXIII circulates in plasma in association with fibrinogen.
What Test Next?
Factor XIII deficiency is rare and in individuals in whom a low level is found mutational analysis of both the F13A1 and F13B genes should be undertaken. Screening of family members is essential so that appropriate counselling can be given.
Consideration should also be given to the possibility that the low Factor XIII levels are acquired and the appropriate tests based upon the causes outlined under 'Interpretation' above, performed.