Factor XIII Assays
Factor XIII [FXIII] is a heterodimer consisting of two A subunits and two B subunits:
|A subunit||The A subunit is the active part of the molecule and functions as a transglutamidase cross-linking the lysine of one γ-chain to the glutamine residue of another γ-chain to form cross-linked fibrin – a transglutamidase reaction which releases ammonia.
The gene for the FXIII A subunit [F13A] maps to chromosome 6 [6p25-p24].
FXIII is activated by thrombin to generate XIIIa - the active enzyme.
|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]
Principles & Method
1. Clot Solubility Assays
Many laboratories use a screening test for Factor XIII deficiency and only if this is abnormal is a formal FXIII assay performed.
2. ELISA Assays for FXIII
A number of commercial assays exist for the measurement of both the FXIIIA and FXIIIB subunits by ELISA. In general the FXIIIA 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.
3. Laurell Electroimmunodiffusion
The Laurell Immunodiffusion technique can be used to measure FXIII. The agar plate contains an antibody to FXIII and after electrophoresis a rocket shaped precipitin patters 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.
4. Functional FXIII Assays
Functional FXIII assays rely upon the transglutamidase activity of XIIIa. Fibrinogen is immobilised onto the wells of a microtitre plate, the plasma sample is added and the FXIII is activated by the addition of calcium and thrombin. The activation of XIII to XIIIa takes place and the XIIIa is then assayed either by the incorporation of a substrate into the fibrin clot where the amount of substrate incorporated is then measured, or by measuring the amount of ammonia generated when the transglutamidase reaction takes place.
Functional assays have a measurement range from 0 - >250%.
5. Chromogenic FXIII Assays
Chromogenic assays are also available for measuring FXIII [actually FXIIIa] based upon its transglutamidase 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.
The TEG can also detect significant FXIII deficiency – the TEG shows reduced clot formation and increased fibrinolysis.
7. Novel Assays for Factor XIII
A number of novel assays for measuring FXIII have recently been reported and these are listed in the reference section for further reading. At present they are not in common use.
Low Factor XIII Levels are seen in:
- Individuals with inherited FXIII deficiency
- FXIII inhibitors - rare but may be seen in association with Isoniazid
- Henoch-Schoenlein purpura (HSP)
- In patients on and following cardiopulmonary bypass
- Chronic inflammatory bowel disease
- Severe GvHD of the gut
- 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]
- Low factor XIII levels have also been reported in several cases associated with the use of sodium valproate. In these cases the FXIII levels return to normal after the drug is withdrawn. The mechanism is unclear.
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 blood in association with fibrinogen.
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