Factor XIII Assays

Factor XIII [FXIII] is a heterodimer consisting of two A subunits and two subunits:

  • The A subunit is the active part of the molecule and functions as a transglutamidase cross-linking the lysine of one g-chain to the glutamine residue of another g-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]. The B subunit has no enzymatic activity and functions as a carrier for the A subunit preventing its proteolytic degradation within the plasma and in the binding of FXIII to the fibrin clot. The gene for the FXIII B subunit [F13B] maps to chromosome 1 [1p31-q32.1]


FXIII is activated by thrombin to generate XIIIa - the active enzyme.

 

Principles of FXIII Assays

Screening Tests: Clot solubility Assays

Activated FXIIIa stabilises the fibrin clot and the clot is insoluble in 5M urea, 2% acetic acid or  1% monochloroacetic acid (MCA). If the cross-linking does not occur then the clot is soluble in either 5M urea, 2% acetic acid or 1% MCA. A variety of methods exist for the clot solubility screening test.

 

Methods

Urea Method: The plasma sample [patient and control] are clotted by the addition of an excess of calcium and incubated at 37C 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 24hours at either room temperature or 37C. If the clot has dissolved this suggests FXIII deficiency and a formal FXIII assay should be undertaken.

Acetic Acid/Monochloroacetic acid (MCA) Method: The plasma sample [patient and control] are clotted by the addition of an excess of calcium and incubated at 37C for 30 minutes. An alternative approach involves clotting the plasma with thrombin and saline. The clot is removed and placed in either 2% acetic acid or 1% MCA and incubated for 24hours at either room temperature or 37C. If the clot has dissolved this suggests FXIII deficiency.

 

ELISA Assays

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 and if it is low the B subunit is measured as the B subunit will not with low a normal A subunit but the converse is, of course not true.

Laurell Rocket Immunodiffusion

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.

Functional FXIII Assays

  1. 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 Ca 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 and the amount of substrate incorporate 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% FXIII.

  2. Chromogenic assays are also available for measuring FXIII.

 

Thromboelastograpy [TEG]

The TEG can also detect significant FXIII deficiency – the TEG shows reduced clot formation and increased Fibrinolysis.
[Nugent, D.J. (2006) Prophylaxis in rare coagulation disorders -- factor XIII deficiency. Thromb Res, 118 Suppl 1, S23-28.]

 

Interpretation

Low factor XIII levels may be seen in:

  • Individuals with inherited FXIII deficiency
  • FXIII inhibitors - rare but seen in association with Isoniazid
  • Henoch-Schoenlein purpura (HSP)
  • In patients on cardiopulmonary bypass
  • Chronic inflammatory bowel disease
  • Levels fall in pregnancy - severe FXIII deficiency is associated with recurrent miscarriage
  • Excessive activation, as seen in DIC, exposure to some snake venoms and caterpillar toxins

 

Reference Ranges

The concentration of subunit A in plasma is 15 mg/mL, while that of subunit B is 21 mg/mL. Much of FXIII circulates in blood in association with fibrinogen

 

Comments

  1. The clot solubility assays detects only the most severe forms of FXIII deficiency. The calcium/urea method appears to sensitive to levels of FXIII of 1-5U/dl whilst the thrombin/acetic acid method is sensitive to level of 10 U/dl.
  2. In cases of suspected FXIII deficiency - measurement of FXIII levels should be undertaken. As the A subunit is the active subunit - most labs measure FXIIIa initially and if this is low may also measure FXIIIb.

 

Useful Links & References

  1. Bereczky, Z., Katona, E. & Muszbek, L. (2003) Fibrin stabilization (factor XIII), fibrin structure and thrombosis. Pathophysiol Haemost Thromb, 33, 430-437.
    Ichinose, A. (2001) Physiopathology and regulation of factor XIII. Thromb Haemost, 86, 57-65.
  2. Lorand, L. (2005) Factor XIII and the clotting of fibrinogen: from basic research to medicine. J Thromb Haemost, 3, 1337-1348.
  3. Muszbek, L., Ariens, R.A. & Ichinose, A. (2007) Factor XIII: recommended terms and abbreviations. J Thromb Haemost, 5, 181-183.
  4. Nugent, D.J. (2006) Prophylaxis in rare coagulation disorders -- factor XIII deficiency. Thromb Res, 118 Suppl 1, S23-28.
  5. Peyvandi, F., Duga, S., Akhavan, S. & Mannucci, P.M. (2002) Rare coagulation deficiencies. Haemophilia, 8, 308-321.
  6. Factor XIII: www.emedicine.com/med/TOPIC3491.HTM
  7. Factor XIII deficiency: www.emedicine.com/ped/topic3040.htm
  8. Williams, M.D., Chalmers, E.A. & Gibson, B.E. (2002) The investigation and management of neonatal haemostasis and thrombosis. Br J Haematol, 119, 295-309.
  9. Bolton-Maggs, P.H., Perry, D.J., Chalmers, E.A., Parapia, L.A., Wilde, J.T., Williams, M.D., Collins, P.W., Kitchen, S., Dolan, G. & Mumford, A.D. (2004) The rare coagulation disorders--review with guidelines for management from the United Kingdom Haemophilia Centre Doctors' Organisation. Haemophilia, 10, 593-628.

 

Data Interpretation

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