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Laboratory Evaluation of Factor XIII Deficiency
FXIII is a hemostatic protein that plays a critical role in stabilizing fibrin networks at the site of injury and thus preventing premature fibrinolysis. In the plasma, FXIII circulates as a tetrameric zymogen composed of two catalytic alpha chains (FXIII-A) and two carrier/inhibitory beta chains (FXIII-B) that serve to stabilize FXIII-A in the plasma pool. In addition to the plasma pool, platelet FXIII pool composed only of FXIII-A homodimers plays an important role in increasing local FXIII concentration at sites of injury. FXIII-A and FXIII-B are produced in different tissues. FXIII-A is produced by the cells in the bone marrow, including monocyte/macrophage lineage cells and megakaryocytes, whereas FXIII-B is produced in hepatocytes. The tetrameric A 2 B 2 complex is subsequently assembled in the plasma. Under the usual circumstances, circulating FXIII-B chains are in 50% excess of the tetrameric complex. FXIII activation requires thrombin-induced proteolysis and is greatly accelerated in the presence of fibrin. Thrombin cleaves inhibitory peptides on the alpha chains causing dissociation of catalytically active FXIII-A 2 from FXIII-B 2 . Once activated, FXIII acts as a transglutaminase, catalyzing the formation of covalent cross-links between specific peptide lysine and glutamine residues on fibrin and alpha-2 plasmin inhibitor. Ammonia is released as a byproduct of these enzymatic reactions. The enzyme has three principle clot stabilizing activities; first it cross-links fibrin γ chains, leading to formation of γ chain dimers thus preventing dissociation of fibrin monomers. Second it forms multiple cross-links between fibrin alpha chains, thus increasing the rigidity of the clot. Finally, it cross-links alpha-2 plasmin inhibitor to fibrin, leading to a decrease in fibrin digestion by plasmin. These tasks are critically important for proper hemostatic function, as evidenced by the severe bleeding seen in cases of FXIII deficiency.
Both activity-based and antigenic FXIII assays play a significant role in evaluating FXIII status in bleeding patients. Initial screening relies on determination of FXIII activity. If the activity is significantly decreased, further classification into quantitative FXIII-A deficiency, qualitative FXIII-A deficiency, and FXIII-B deficiency can be made by performing antigenic tests for FXIII A 2 B 2 , FXIII-A, FXIII-B antigens, and occasionally testing FXIII status in the patient’s platelet lysate. Quantitative FXIII testing has also enabled FXIII replacement monitoring.
Factor XIII Activity Assays
Clot Solubility
Historically clot solubility assays were utilized to screen for FXIII deficiency. The test uses either calcium or thrombin to induce fibrin clot. The clot is incubated for 30 minutes at 37°C to allow FXIII-dependent cross-linking and then placed in either 5 or 8 M urea, 1% acetic, or 1%–5% monochloroacetic acid. Clot dissolution within a 24 hour incubation period is suggestive of an absence of FXIII activity.
Quantitative Factor XIII Activity Assays
Until recently, quantitative determination of FXIII activity was laborious and difficult to automate. The assays relied on FXIII-mediated labeled amine (used in place of lysine residue) incorporation into glutamine residue as a readout of FXIII activity. Rare laboratories could perform the laborious assays in daily practice. Recent introduction of automated, quantitative measurements of the ammonia released as a byproduct of FXIII activity has dramatically improved the prospect for introduction of reliable FXIII tests into the clinical laboratory. Recent introduction of a WHO (World Health Organization) FXIII activity standard for use in calibration of the assay standard material has enabled further harmonization of the assay performance across different platforms and laboratories.
Amine Incorporation Assay
The older methodology measures the incorporation of radiolabeled, fluorescently labeled, or biotinylated low-molecular-weight amines into peptide glutamine residue. In the end point assay, the amount of incorporated label is proportional to the FXIII activity present in the test plasma. The assay demonstrates good analytical sensitivity on the low end of the measurement range and is able to measure FXIII activity down to ∼1%. There are two major difficulties that arise during performance of the assay. First is the need to separate free from incorporated amines on completion of the amine incorporation reaction. This problem is solved by either acid precipitation of acceptor peptides and repeated washings to remove free amines or by attaching the acceptor peptides to solid support to remove the need for acid precipitation. Neither of these solutions lend themselves to easy automation. The second problem is interference of fibrin clot with the performance of the assay. If allowed to form, fibrin clot makes it very difficult to wash out unincorporated amines and interferes with photooptical detection of the fluorescent label through inner filter effect. Newer iterations of the assay prevent fibrin formation through either high plasma dilution or using peptides that interfere with fibrin clot assembly.
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