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

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Genetics, diagnosis and clinical features of congenital hypodysfibrinogenemia: a systematic literature review and report of a novel mutation.

Essentials Hypodysfibrinogenemia is rarely reported among the congenital fibrinogen disorders. This first systematic literature review led to identification of 51 hypodysfibrinogenemic cases. Diagnosis based only on functional/antigenic fibrinogen ratio may be insufficient. Family studies show an incomplete segregation of mutation with the clinical phenotypes. SUMMARY: Background Hypodysfibrinogenemia is a rare disease characterized by decreased levels of a dysfunctional fibrinogen. It shares features with both hypo- and dysfibrinogenemia, although with specific molecular patterns and clinical phenotypes. Objectives To better define the genetics, the diagnosis and the clinical features of hypodysfibrinogenemia. Patients/Methods A systematic literature search led to 167 records. After removal of duplicates, abstract screening and full-text reviewing, 56 molecular and/or clinical studies were analyzed, including a novel FGB missense mutation in a woman with a mild bleeding phenotype. Results A total of 32 single causative mutations were reported, mainly in the COOH-terminal region of the γ or Aα chains at heterozygous or homozygous state. Seven additional hypodysfibrinogenemias were due to compound heterozygosity. The hypofibrinogenemic phenotypes were a result of an impaired assembly or secretion or an increased clearance of the fibrinogen variant, whereas the dysfibrinogenemic phenotype was mainly a result of a defective fibrin polymerization and an abnormal calcium or tPA binding. Among 51 identified index cases, a functional/antigenic fibrinogen ratio < 0.7 had a sensitivity of 86% for the diagnosis of hypodysfibrinogenemia. Eleven patients (22%) were asymptomatic at time of diagnosis, 23 (45%) had a mild bleeding phenotype with mainly obstetrical or gynecologic-related hemorrhage and 22 (43%) had experienced at least one thrombotic event, including 23 venous and eight arterial thromboses. Conclusions This first systematic review on hypodysfibrinogenemia shows the heterogeneity of causative mutations and that misdiagnosis could occur in relation to the functional and antigenic fibrinogen levels. Family studies reveal an incomplete segregation of the mutation with the clinical phenotype.

Probing platelet factor 4 alpha-granule targeting.

The storage mechanism of endogenous secretory proteins in megakaryocyte alpha-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet alpha-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in alpha-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify alpha-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in alpha-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze alpha-granule biogenesis and its alterations in the congenital defect gray platelet syndrome.

Common variable immunodeficiency patient classification based on impaired B cell memory differentiation correlates with clinical aspects.

Common variable immunodeficiency (CVID) is a very heterogeneous syndrome defined by impaired immunoglobulin production. The functional classification of CVID patients on the basis of in vitro immunoglobulin production is time consuming and has never shown any predictive value. We propose a classification based on the quantitative repartition of naive/memory B cells according to the dual expression of IgD and CD27. Fifty-seven patients were categorized into three groups: Group MB2 (11 patients, 19%) with normal memory B cells; Group MB1 (19 patients, 33%) with defective switched memory (IgD-CD27+) but normal nonswitched memory B cells (IgD+CD27+); Group MB0 (27 patients, 47%) with almost no memory B cells. In addition, a downexpression of activation markers (CD25, CD21, CD80, CD86) on B cells characterized the group MB1 patients and was associated with an upexpression of activation markers (HLA-DR, CD95, CD57) on T cells. This classification correlates with some clinical aspects showing a higher prevalence of splenomegaly (16/27, 59%), lymphoid proliferation (13/27, 48%) and granulomatous disease (12/27, 44%) in group MB0. Splenomegaly was also frequent in group MB1 (8/19, 42%). In contrast, autoimmunity was observed with similar prevalence in all three groups. Moreover, by analyzing B cell phenotype, immunoglobulin transcript expression, and somatic mutations, we propose different putative mechanisms responsible for impaired B cell activation and memory differentiation in this syndrome.