We conducted a multicenter study of 101 patients with congenital dysfibrinogenemia (CD) to characterize the incidence of hemorrhagic and thrombotic events as well as complications of pregnancy and ...surgery. At the time of diagnosis, 10.9% and 13.9% had experienced major bleeding and thrombotic events, respectively. During a mean follow-up of 8.8 years after CD diagnosis, the incidence of major bleeding and thrombotic events was 2.5 and 18.7 per 1000 patient-years, respectively, with estimated cumulative incidences at age 50 years of 19.2% and 30.1%. We identified 111 pregnancies with an overall incidence of spontaneous abortions and postpartum hemorrhage of 19.8% and 21.4%, respectively. The risk of postpartum hemorrhage was associated with a previously identified bleeding phenotype (odds ratio, 5.8; 95% CI, 1.2 to 28.0). Among 137 surgical procedures analyzed, 9 (6.5%) were complicated by abnormal bleeding. Propositi vs relatives, sex, mutation hotspots, fibrinogen levels, and activity:antigen ratios were not associated with the risk of thrombotic or bleeding outcomes. In conclusion, the results of our study, the largest in genotyped CD and the first including long-term history, indicate that propositi with CD and their relatives carry not only a high risk of major bleeding, including postpartum hemorrhage, but also of thrombotic event.
•Major bleeding, thrombosis, and postpartum hemorrhage are frequent in propositi and relatives with congenital dysfibrinogenemia.•Hotspot mutations were not predictive of either phenotype or outcome.
Abstract 1745
The diagnosis of Polycythemia Vera (PV) and Essential Thrombocytemia (ET) relies on a set of criteria gathered in the 2008 WHO classification; however, it may occasionnaly be difficult, ...especially in the absence of molecular markers such as Jak2V617→F or Mpl mutations. Having previously reported a significant increase in circulating platelets microparticles (PMP) in patients bearing Myeloproliferative neoplasms versus healthy donors (V.Tintillier-Colin, ASH Annual Meeting 2009, Abstr. 1906), our purpose has been to assess the usefulness of blood PMP count as an additional criteria in dubious cases of ET and PV.
Patients and methods: We performed PMP counts in 100 newly diagnosed and still untreated patients (55 ET and 45 PV in accordance to WHO criteria), 40 secondary or reactive states (erythrocytosis and thrombocytosis, 20 cases each) and 75 controls matched for age and sex. The PMP were characterized by their size and co-expression of Annexin V and CD41; their concentration was measured on plasma extracts using the FC500 flow-cytometer (Beckman-Coulter™). Pre-analytical and testing procedures complied with the recommendations of the ISTH Standardization Sub-committee with each test performed in duplicate.
The 3 groups were homogeneous regarding age and sex-ratio. Mean platelet counts were 703.109/l in the TE group, 652.109/l in the cohort with secondary thrombocytosis and 247.109/l in the control group. Mean Hb level was 18.0g/dl in the PV group, 18.1g/dl in the secondary erythrocytosis group and 15g/dl in controls. The median value of PMP concentration is much higher (p < 0.05, Krusall-Wallis test) in the TE group (5900/μl) than in the reactive thombocytosis group (1283/μl), this latter being no different from the control group (996/μl). In the ET group, PMP count is similarly increased whether patients beared Jak2V617→F mutation or not. Using the ROC statistical method, we defined a threshold value of 3400 PMP/μl able to discriminate ET from reactive thrombocytosis with 93% specificity and 67% sensitivity.
Similarly, the median value of PMP count was neatly higher in the PV group (3258/μl) than in secondary erythocytosis (671/μl) and healthy controls (996/μl) (p < 10−5, Krusall-Wallis test). The ROC method fixed a 1300/μl threshold separating PV from secondary erythrocytosis with 89% sensibility and 63% specificity.
We report an evaluation of the blood PMP count in a quite large series of newly diagnosed untreated ET and PV patients compared to patients with secondary thrombocytosis or erythrocytosis and healthy volunteers; this study emphasizes the interest of the circulating PMP count in order to differentiate MPN neoplasms from normal subjects as well as patients with reactive conditions. This test, quick, reproducible and cheap, especially shows its value as an additional diagnostic criteria in ET or PV when clonal markers are lacking.
No relevant conflicts of interest to declare.
Abstract 1906
Poster Board I-929
Microparticles (MP) are plasma membrane vesicles bearing potent procoagulant proteins and released into the circulation by various blood and endothelial cells during ...cellular activation and apoptosis. Their concentration has been shown markedly increased in vascular and thromboembolic diseases and in several types of cancer. Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PM) are Bcr-Abl-negative chronic myeloproliferative disorders (MPD) with common biological and clinical features, especially thrombocytosis and more or less effective megakaryocytic proliferation. ET, PV and PMF are also characterized by an increased risk of thrombotic complications. Given the involvement of megacaryocytopoiesis in this group of diseases, we chose to focus on circulating platelet-derived microparticles (PMP) whose count can be established by flow cytometry. Our goals were to look for an increased release of PMP in MPD and if so, try to establish a correlation between thrombotic events recorded retrospectively and PMP level.
Plasma samples were collected from 85 patients with MPD (40 ET, 28 PV, 17 PMF) at any time of the disease course except blastic phase and 31 healthy age-paired controls ; the quantity of PMP (positive for the platelet marker CD41 and the microparticle marker AnnexinV) was measured by flow cytometric analysis using the FC500 cytometer from Beckmann-Coulter™. Pre-analytic and testing procedures complied with the recommendations of the ISTH Standardization Subcommittee and besides, our laboratory participates in a multicenter program to standardize the enumeration of cellular microparticles.
The number of PMP is significantly higher in ET, PV and MFP patients (median: 4862 MPP/mL, 3289 MPP/mL and 6114 MPP/mL respectively) than in controls (median 1310 MPP/mL) p=0.000018, p=0.0013, p=0.0009. Neither in MPD patients nor in control group PMP correlates with platelet count and appears thus as an independent parameter. ROC method allows to fix a cut-off at 3121 MPP/mL to discriminate MPD from controls. This value has a sensibility of 75 % and a specificity of 94%. Interestingly, the total number of PMP is not different between untreated patients (mean=7246 MPP/mL) and those who received myelosuppressive therapy (mean=6320 MPP/mL). Regarding thrombotic events, we failed to demonstrate any significant difference between patients with previous thrombosis and those without; however, the small size of the series and the retrospective assessment are possible bias.
Patients with Phi negative MPDs have a high number of circulating PMP, irrelevant to platelet count or treatment by Hydroxy-urea or aspirin. A role for microparticles in the development of these diseases and in the occurrence of thrombotic complications can be hypothesized but requires further studies -;preferably prospective- on larger cohorts of patients.
Charpentier:Schering-Plough: Research Funding.
Numerous mutations in
,
or
lead to congenital fibrinogen disorders (CFDs), but their epidemiology is not well characterized. The aim of this study was to evaluate the molecular epidemiology of CFD ...and to develop a genotyping strategy.
Genetic data from 266 unrelated CFD patients genotyped at our laboratory and from a CFD open access database (
= 1,142) were evaluated. We developed a step-wise screening strategy for the molecular diagnosis of CFD and prospectively tested this strategy on 32 consecutive CFD probands.
We identified 345 mutated alleles overall, among 187 heterozygous, 63 homozygous and 16 compound heterozygous individuals. Afibrinogenemia was almost always caused by null mutations (98.6%), mainly in
(85%). Hypofibrinogenemia was mainly caused by missense mutations of
or
(54.2%). Dysfibrinogenemia was almost always caused by heterozygous missense mutations (99.3%) in
and
. Hotspot mutations were prevalent among quantitative (33.1%) and qualitative fibrinogen disorders (71.1%). The mutational cluster at our laboratory was similar with that reported in the CFD open access database. The proposed step-wise genetic screening strategy proved efficient in both the development and validation samples for CFD: the screening of
exons 2, 4, 5 and
exon 8 and search for the 11 kb deletion of
led to the identification of approximately 80% of mutated alleles, including 15 new mutations.
The described molecular epidemiology of CFD is complex. The proposed step-wise genetic screening strategy may provide an efficient way to identify causative mutations analysing a minimal number of exons.
Rationale: Essential Thrombocythemia (ET) is a Philadelphia-negative myeloproliferative neoplasm characterized by an increased risk of thrombosis. Previous reports suggest a role for microparticles ...(MP) in the pathogenesis of thrombosis in ET. MPs are small plasma membrane vesicles bearing potent procoagulant proteins and phospholipids. They are released into the circulation by various blood and endothelial cells after cellular activation and/or apoptosis. The recently described somatic calreticulin (CALR) exon 9 mutations in almost 20% of ET defines a lower-risk thrombosis ET subtype. We extensively studied phenotype and procoagulant activity of plasma MPs in order to assess MP contribution to the thrombotic risk in CALR versus JAK2 (V617F) mutated ET patients.
Patients and methods: We analyzed MP count, phenotype and procoagulant activity in 45 JAK2 V617F+ and 15 CALR+ consecutive and newly diagnosed ET patients in accordance to WHO criteria recruited between november 2010 and april 2013. After given informed consent, blood samples were obtained in all patients before initiating any cytoreductive therapy. Pre-analytical and testing procedures complied with the recommendations of the ISTH Standardization Sub-Committee and each test was performed in duplicate . Using flow cytometry (FC500 flow-cytometer, Beckman-Coulter), MPs were characterized and measured in platelet-free plasma samples. MPs were characterized by their size and co-expression of bound Annexin V and the following cell-specific monoclonal antibodies: CD41 (Platelet-MP, PMP), CD235a (Red cell-MP, RMP), CD14 (Monocytes-MP, MoMP), CD11b (Granulocytes-MP, GMP), CD144 (Endothelial-MP, EMP), CD62P and CD41 (P-Selectin+ PMP), CD142 and CD41 (Tissue Factor (TF)+ PMP, TF+PMP). MP-associated procoagulant activity was also measured using a-thrombin generation assay (Zymuphen MP-activity). Statistical analysis was performed with SPSS software. Results are expressed as median interquartile range.
Results: Patients characteristics: The platelet count (109/L) was higher in CALR+ than in JAK2+ patients (866 666 – 918, 659 571 – 807 respectively, p= 0.049), and all other clinical and hematological characteristics were also distributed in agreement with previous reports. Furthermore, CALR+ patients were preferentially distributed in the lower risk categories of the IPSET-thrombosis and IPSET-survival scores than in JAK2+ patients (p<0.00001 and p= 0.04 respectively).
The main results are summarized in table 1. MP count and MP/Platelets ratio were significantly lower in CALR+ than in JAK2+ patients. PMP count, PMP percentage of all MPs and PMP/Platelets ratio were lower in CALR+ than in JAK2+ patients. Furthermore, CALR+ patients had lower PMP surface P-Selectin. MP-associated procoagulant activity/Platelets ratio was significantly lower in CALR+ than in JAK2+ patients. There were no significant differences in TF-carrying PMP or in other cell-derived MP (RMP, MoMP, GMP, EMP).
Conclusion: Our results demonstrate a decreased in circulating procoagulant PMP in CALR+ ET-patients compared to JAK2+ patients and lower platelet activation in CALR+ ET patients as measured by P-Selectin expression on PMP. Thus, as CALR+ patients have lower thrombotic-risk according to the literature and the IPSET-survival and IPSET-thrombosis scores observed in our study, the lower level of procoagulant PMP could account, at least in part, for a lower thrombotic risk of CALR+ ET patients.
Table 1main resultsCALR +JAK2 V617F +p valueMP (/µL)3289 1662-42404961 2697-66870.04MP/Platelets3.6 2.8-5.36.9 4.7-9.60.01PMP (/µL)3100 2068-38875702 3423-102570.004PMP/Platelets3.38 2.97-6.337.55 5.12-12.660.002% PMP90 84-9293 90-960.03MP-activity(nM)/platelet0.015 0.008-0.0280.029 0.019-0.0410.05P-Selectin+ PMP (/µL)195 152-219747 383-9650.001RMP (/µL)4 1.7-8.24 2.5-9.7nsMoMP (/µL)43 27-5274.5 22-135nsGMP (/µL)31.5 24-4830 11-55nsEMP (/µL)99.4 32-11095.6 51-140nsTF+ PMP (/µL)22.3 9.8-11527.1 20.8-94.4ns
No relevant conflicts of interest to declare.
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