Factor XIII Activity Assays Research Articles

Seizures in a Term Newborn.

Seizures in a Term Newborn.

Clinical Validation of an Automated Fluorogenic Factor XIII Activity Assay Based on Isopeptidase Activity.

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0–135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom® FXIII Activity, a functional ammonia release assay, and the HemosIL™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays.

Molecular diagnosis of factor XIII deficiency, data from comprehensive coagulation laboratory in Iran

: Diagnosis of factor XIII (FXIII) deficiency (FXIIID) as a rare bleeding disorder is a challenge worldwide. Thus, in the present study, we used different methods including two molecular methods for detection of FXIIID. This study was conducted on individuals suspected to FXIIID. All individuals were checked by two routinely used methods of clot solubility test in Iran and two other clot solubility tests as well as FXIII activity and antigen assays. Molecular analysis was performed by PCR-restriction fragment length polymorphism (PCR-RFLP) and tetra-primer amplification refractory mutation system (T-ARMS)-PCR for only FXIIID mutation in southeast Iran (p.Trp187Arg), previously associated with severe FXIIID. Out of 151 individuals, 26 had abnormal clot solubility test with all four methods. PCR-RFLP revealed that 27 patients were homozygotes for p.Trp187Arg, whereas 12 were heterozygotes. Molecular analysis revealed that in routinely used clot solubility combinations, two homozygotes (∼8%) were missed, whereas in two other combinations, one patient (∼4%) was missed. One false positive result was observed in routinely used methods, whereas further combinations don't have false positive. T-ARMS-PCR had three discrepancies with PCR-RFLP and sequencing confirmed that the results of T-ARMS-PCR were false. FXIII antigen assay diagnosed all homozygotes, whereas in FXIII activity assay, two homozygotes had higher than 5% FXIII activity that inconsistent with severe deficiency. It seems that clot solubility test is not enough sensitive and specific and molecular analysis is the most reliable method for detection of FXIIID in areas such Iran with one or few specific mutations.

GOOD CORRELATION BETWEEN TWO DIFFERENT AUTOMATED FACTOR XIII ASSAYS AND PRACTICAL RECOMMENDATIONS

Coagulation factor XIII (FXIII) can be quantitatively measured by using modern automated coagulation analyzers. It was uncertained whether different methods of FXIII quantitative measurement are correlated. Two different analytical methods were used for comparison in this study. This study selected a neurosurgical patient population for FXIII measurement, expecting a wide spectrum of results from haemostatic changes in vivo. A correlation study between FXIII activity (Berichrom F XIII, Dade Behring Marburg GmbH) and FXIII antigen assay (HemosIL™, Instrumentation Laboratory Company, Milano Italy) was performed using two different automated coagulation analyzers and test principles. FXIII assays using these two methods were performed on blood specimen from 84 patients taken pre and post neurosurgical procedures. A total of one hundred and thirty eight blood samples were included in this correlation study. There was a significant mean different of FXIII antigen levels pre and post intracranial surgery (p&lt; 0.05). A significant, positive and good correlation between FXIII antigen and activity assays were documented by both methods, r = 0.86, and p&lt;0.001. FXIII antigens are correlated with FXIII activity assays indicating the reliability and robustness of both methods for quantitation of FXIII in the blood. These two methods are recommended for both diagnostic and research purposes.

Agreement between factor XIII activity and antigen assays in measurement of factor XIII: A French multicenter study of 147 human plasma samples.

Factor XIII (FXIII) deficiency is a rare hemorrhagic disorder whose early diagnosis is crucial for appropriate treatment and prophylactic supplementation in cases of severe deficiency. International guidelines recommend a quantitative FXIII activity assay as first-line screening test. FXIII antigen measurement may be performed to establish the subtype of FXIII deficiency (FXIIID) when activity is decreased. The aim of this multicenter study was to evaluate the analytical and diagnostic levels of performance of a new latex immunoassay, K-Assay® FXIII reagent from Stago, for first-line measurement of FXIII antigen. Results were compared to those obtained with the Berichrom® FXIII chromogenic assay for measurement of FXIII activity. Of the 147 patient plasma samples, 138 were selected for analysis. The accuracy was very good, with intercenter reproducibility close to 7%. Five groups were defined on FXIII activity level (<5% (n = 5), 5%-30% (n = 23), 30%-60% (n = 17), 60%-120% (n = 69), above 120% (n = 24)), without statistical differences between activity and antigen levels (P value >0.05). Correlation of the K-Assay® with the Berichrom® FXIII activity results was excellent (r = 0.919). Good agreement was established by the Bland and Altman method, with a bias of +9.4% on all samples, and of -1.4% for FXIII levels lower than 30%. One patient with afibrinogenemia showed low levels of Berichrom® FXIII activity but normal antigen level and clot solubility as expected. The measurement of FXIII antigen using the K-Assay® is a reliable first-line tool for detection of FXIII deficiency when an activity assay is not available.

Safety and Efficacy of Recombinant Factor XIII (FXIII) in Patients with Congenital FXIII A-Subunit Deficiency, Results from the Mentor™2 Trial

Safety and Efficacy of Recombinant Factor XIII (FXIII) in Patients with Congenital FXIII A-Subunit Deficiency, Results from the Mentor™2 Trial

Diagnosis of factor XIII deficiency

Background: Factor XIII (FXIII) deficiency is an extremely rare bleeding disorder with estimated incidence of one per two million. All routine coagulation tests are normal in FXIII deficiency (FXIIID), which complicates the diagnosis of this disorder. Precise diagnosis of FXIIID requires more specific tests, including qualitative tests as well as quantitative tests such as FXIII activity, antigen assays, and finally molecular studies to confirm FXIIID.Objective: This study was conducted to present different quantitative and qualitative methods as well as molecular approaches for screening and diagnosis of FXIIID with advantages and disadvantages of each method.Methods: All relevant English-language publications were searched in Medline (until 2015).Results and discussion: Clot solubility assay is the most widely used method for detection of FXIIID but it is not standardized. The sensitivity of this method is dependent upon different factors mainly clotting factors and the solubilizing agents; therefore, FXIII activity assay is recommended for screening of FXIIID. Among FXIII activity assays, photometric assay is more common but FXIII activity is overestimated in this assay due to lack of sample blank in commercial assay, which can have fatal consequences in severe FXIIID, for which fluorometric assay is an appropriate alternative preventing the overestimation observed in photometric assay. There are different methods for measurement of FXIII-A2, FXIII-B2, and FXIII-A2B2 as well as detection and quantification of FXIII inhibitor, which are mentioned in detail in this review. There are no mutational hotspots in FXIII-A and FXIII-B genes with a few recurrent mutations in some populations; therefore, full sequencing of FXIII genes has remained a main molecular approach for confirmation of FXIIID.Conclusion: Familiarity with different methods for diagnosis of FXIIID and their advantages and disadvantages can help in appropriate and timely diagnosis of this disorder to prevent misdiagnosis of FXIIID and its fatal consequences.

Laboratory Diagnosis of Factor XIII Deficiency, Routine Coagulation Tests with Quantitative and Qualitative Methods.

Factor XIII (FXIII) deficiency is a severe bleeding disorder with normal routine coagulation tests that makes diagnosis of the disorder complicated. After normal results in routine coagulation tests, clot solubility test, and FXIII activity, antigen assays along with molecular methods can be used for precise diagnosis of disorder. In the present study, we described routine coagulation tests along with clot solubility test and FXIII activity and antigen assays. Data were collected from all relevant publications until 2015. All routine coagulation tests including prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), and platelet count are normal in FXIII deficiency (FXIIID) but different conditions such as blood collection, transport, and storage can result in abnormal results in these first line screening tests. In addition to these tests, clot solubility tests as the most common screening tests of FXIIID can influenced by different factors including clotting and solubilizing agents. Different commercial kits are available for FXIII activity and antigen assays with different sensitivity and specificity which could affect diagnosis of FXIIID. Results of routine coagulation tests as well as clot solubility tests along with specific coagulation tests can affect diagnosis of FXIIID; therefore, all steps of these tests should be under control.

Factor XIII: Congenital Deficiency Factor XIII, Acquired Deficiency, Factor XIII A-Subunit, and Factor XIII B-Subunit

Factor XIII (FXIII) is a transglutaminase consisting of 2 catalytic A subunits and 2 noncatalytic B subunits in plasma. The noncatalytic B subunits protect the catalytic A subunits from clearance. Congenital FXIII deficiency may manifest as a lifelong bleeding tendency, abnormal wound healing, and recurrent miscarriage. Acquired FXIII deficiency, with significant reductions in FXIII levels, has been reported in several medical conditions. The routine screening tests for coagulopathies-prothrombin time, activated partial thromboplastin time, and thrombin time-do not show abnormalities in cases of FXIII deficiency. A quantitative, functional, FXIII activity assay that detects all forms of FXIII deficiency should be used as a first-line screening test. Treatment consists of recombinant FXIII or FXIII concentrate. If these are unavailable, then fresh-frozen plasma and cryoprecipitates may be used. Factor XIII has a long half-life; therefore, the patients can lead near-normal lives with regular replacements. Patients with acquired FXIII deficiency with inhibitors need immunosuppressive therapy in addition to factor replacements.

Rapid Determination of Blood Coagulation Factor XIII Activity Using Protein Arrays for Serodiagnosis of Human Plasma

We developed a novel on-chip assay using protein arrays for quantitative and rapid analysis of blood coagulation factor XIII (FXIII) activity in human plasma. FXIII is activated by concerted action of thrombin and Ca(2+) and plays essential roles in hemostasis, angiogenesis, and wound healing. We fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane layer of well-type arrays and determined FXIII activity by analyzing biotinylated fibrinogen with Cy3-conjugated streptavidin. We determined optimal concentrations of Ca(2+), thrombin, and 5-(biotinamido)pentylamine (BAPA) for the on-chip activity assay, and the detection limit was 0.01 Lowey U/mL (9.9 pM). Using the on-chip activity assay, hepatocellular carcinoma patients (n = 24), but not hepatitis (n = 24) or liver cirrhosis patients (n = 41), had significantly lower FXIII activities (p < 0.001) than normal individuals (n = 41), indicating that FXIII activity is a possible diagnostic marker for hepatocellular carcinoma. In addition, we have successfully used this activity assay to reveal individual variations (37-57%, n = 65) in the inhibition rate of FXIII activity by isoniazid, the first-line antituberculosis agent. Thus, our optimized on-chip FXIII activity assay provides a quantitative and high-throughput approach to investigating the role(s) of FXIII in human diseases. Moreover, it has a strong potential to be applied toward FXIII-related personalized medicines.

Quantification of coagulation factor XIII activity by a thio-NADH based assay using factor XIII immuno-depleted plasma as a diluent for calibration

Accurate determination of factor XIII (FXIII) activity is crucial for replacement therapy. FXIII activity is typically determined using a coupled enzymatic reaction that measures nicotinamide adenine dinucleotide hydride (NADH) consumption at 340 nm. Here, we describe the development of a prototype for a novel FXIII activity assay for detection at 405 nm by replacing NADH with thio-NADH, and the application of FXIII immuno-depleted plasma as a diluent for calibration. Performance data show up to two-fold lower susceptibility of the prototype assay to interferences from hemolyzed, icteric, and lipemic samples when compared to a NADH assay format. In addition, the use of FXIII immuno-depleted plasma as diluent for calibration improved recovery almost two-fold in the lower measurement range. The novel prototype assay correlates well with a conventional assay (r=0.98, y=0.99·x+2.17% FXIII, n=173). The described prototype assay has the potential to (a) increase trueness of measurement of low levels of FXIII, (b) improve robustness due to reduction from interferences, and (c) can be used on a broad range of coagulation instruments due to its detection at 405 nm.

Factor XIII Deficiency

Factor XIII (FXIII) is a tetrameric zymogen (FXIII-A (2)B (2)) that is converted into an active transglutaminase (FXIIIa) by thrombin and Ca (2+) in the terminal phase of the clotting cascade. By cross-linking fibrin chains and alpha (2) plasmin inhibitor to fibrin, FXIIIa mechanically stabilizes fibrin and protects it from fibrinolysis. Severe deficiency of the potentially active A subunit (FXIII-A) is a rare but severe hemorrhagic diathesis. Delayed umbilical stump bleeding is characteristic, and subcutaneous, intramuscular, and intracranial bleeding occurs with a relatively high frequency in nonsupplemented patients. In addition, impaired wound healing and spontaneous abortion in women are also features of FXIII deficiency. The extremely rare B subunit deficiency results in milder bleeding symptoms. FXIII concentrate is now available for on-demand treatment and primary prophylaxis. A quantitative FXIII activity assay is recommended as a screening test for the diagnosis of FXIII deficiency. For classification purposes, FXIII-A (2)B (2) antigen in the plasma is first determined, and if decreased, further measurement of the individual subunits is recommended in the plasma and FXIII-A in platelet lysate. Analytical aspects of FXIII activity and antigen assays are discussed in this article. There are no hot-spot mutations in the F13A1 and F13B genes, and the majority of causative mutations are missense/nonsense point mutations.

Phenotype-genotype characterization of 10 families with severe a subunit factor XIII deficiency.

Factor XIII (FXIII) deficiency is a very rare severe autosomal bleeding disorder with a frequency of 1:2,000,000 in the general population and only a few patients have been genetically characterized so far. We report a phenotype-genotype characterization of 10 unrelated Iranian patients. Two FXIII (transglutaminase) activity assays showed no FXIII activity, except a conserved residual activity in patients receiving prophylactic substitution treatment. FXIII antigen concentrations measured by two immunoassays were comparable. Genotype characterization identified four novel mutations (2 missense and 2 small deletions) and two previously reported missense mutations in the FXIII A subunit gene (F13A). Molecular modeling was carried out to reveal the structural consequences of the missense mutations, that caused the replacement of an arginine residue involved in the formation of structurally important extensive hydrogen-bonded network. The replacements [c.320G>A (p.Arg77His) in the beta-sandwich, c.868C>T (p.Arg260Cys), c.869G>A (p.Arg260His) and c.1236G>T (p.Arg382Ser) in the core domain] resulted in the loss or impairment of such H-bonded network. Energy decomposition analysis demonstrated that this situation leads to the instability and perhaps to the incorrect folding of the A subunit, that would explain the development of severe FXIII deficiency.