A modified, optimized kinetic photometric assay for the determination of blood coagulation factor XIII activity in plasma.
BACKGROUND
Blood coagulation factor XIII (FXIII) is a zymogen that is transformed into an active transglutaminase by thrombin and Ca(2+). FXIII plays an essential role in fibrin stabilization and in the protection of fibrin from proteolytic degradation. No convenient method has been available for the measurement of FXIII activity in plasma. The aim of the present study was to improve and optimize a kinetic photometric FXIII assay originally developed in our laboratory.


METHODS
In the assay, FXIII was activated by thrombin and Ca(2+). Fibrin polymerization was prevented by an inhibitory tetrapeptide. Glycine-ethyl ester and a glutamine residue of a synthetic dodecapeptide served as acyl acceptor and acyl donor transglutaminase substrates, respectively. The amount of ammonia released during the reaction was monitored using glutamate dehydrogenase and NADPH.


RESULTS
The use of a new glutamine substrate and optimization of activator and substrate concentrations increased sensitivity. Substitution of NADPH for NADH and introduction of an appropriate blank eliminated systemic overestimation of FXIII activity. The recovery of FXIII was 96%, the assay was linear up to 470 U/L, the detection limit was 1 U/L, and the imprecision (CV) was <8% even at very low FXIII activities. A reference interval of 108-224 U/L (69-143%) was established. The results correlated well with results obtained by an immunoassay specific for plasma FXIII.


CONCLUSIONS
The optimized FXIII assay is a simple, rapid method for the diagnosis of inherited or acquired FXIII deficiencies and increased FXIII concentrations. It can be easily adapted to clinical chemistry analyzers.
Kárpáti
L
L
Department of Clinical Biochemistry and Molecular Pathology, and Cell Biophysics Research Group of the Hungarian Academy of Sciences, Medical and Health Science Center, University of Debrecen, Debrecen H-4012, Hungary.
Penke
B
B
Katona
E
E
Balogh
I
I
Vámosi
G
G
Muszbek
L
L
D000328:Adult; D000368:Aged; D000641:Ammonia / Q000032:analysis; D001800:Blood Specimen Collection; D005175:Factor XII Deficiency / Q000097:blood; D005176:Factor XIII / Q000032:analysis*; D005969:Glutamate Dehydrogenase / Q000737:chemistry; D006801:Humans; D007118:Immunoassay; D007202:Indicators and Reagents; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D010783:Photometry; D012016:Reference Values; D012680:Sensitivity and Specificity; D011503:Transglutaminases
D007202:Indicators and Reagents; D000641:Ammonia; D005176:Factor XIII; D005969:Glutamate Dehydrogenase; D011503:Transglutaminases