[Measurement of d-dimers in non-valvular atrial fibrillation. First prospective Tunisian study]. / Intérêt du dosage des d-dimères dans la fibrillation atriale non valvulaire. Première série prospective tunisienne.

Atrial fibrillation can expose to thrombo-embolic complications. Many biomarkers have been tested to refine the stratification of thrombo-embolic risk. The study aim was to assess the interest of the d-dimer testing in this pathology. We conducted a prospective observational study including 50 cases and 19 controls, enrolled at the cardiology department of the Mongi Slim Hospital, from July to November 2012. The d-dimer assay was performed on mini-VIDAS(®) and analyzed according to clinical, echocardiographic and biological data. The mean age of patients was 61.8±10.6years. The mean CHA2DS2-VASc score was 2.58±1.31. The average of D-dimer levels was 590±506ng/ml in patients and 225.26±112.95ng/ml in controls (P=0.02). No significant difference has been found between the d-dimer level and age, sex, type and etiology of atrial fibrillation, the CHA2DS2-VASc score, the left atrial surface. Among patients on acenocoumarol, d-dimer levels was significantly higher in patients with an INR<2 compared to those with an INR≥2 (P=0.004). We identified a positive d-dimer threshold (300ng/ml) in patients distinguishing them significantly with controls (P<0.001). In conclusion, the measurement of d-dimers could help clinicians to identify patients with atrial fibrillation having an increased coagulability and, therefore, an increased thrombo-embolic risk. It could be complementary to the determination of INR in monitoring anticoagulation therapy: d-dimers level refines the thrombo-embolic risk and INR measurement assesses the level of anticoagulation and the bleeding risk.

[Anti-erythrocyte and anti-HLA immunization in hemoglobinopathies]. / Immunisation anti-érythrocytaire et anti-HLA au cours des hémoglobinopathies.

AIM: Evaluate the anti-erythrocyte and anti-HLA immunization rates in hemoglobinopathies. PATIENTS AND METHODS: Cross-sectional study (October 2009-March 2010) on 83 patients followed for hemoglobinopathies. The irregular antibodies research is realized by two techniques: indirect Coombs and enzymatic technique on gel cards. The search for anti-HLA class I antibodies is done by complement dependent lymphocytotoxicity. RESULTS: The mean age was 30 years (14-64 years), the sex ratio M/F is 0.84. Our series included 42 cases of sickle cell disease (29 homozygous sickle cell anemia and 13 sickle-thalassemia) and 41 cases of thalassemia syndromes (26 major and 15 intermediate). The anti-erythrocyte alloimmunization rate is 10.84% without difference between thalassemia syndromes and sickle cell disease. The autoimmunization rate (22.89%) is higher in thalassemia syndromes (41.46%) than in the sickle cell disease (7.14%) (P<0.001). The anti-HLA immunization rate is 31.6% without difference between thalassemia syndromes and sickle cell disease. The young age, transfusion at a young age and the total number of transfusions are the factors that increase the risk of anti-erythrocyte autoimmunization. No clinicobiological parameter does influence the anti-erythrocyte and anti-HLA alloimmunization. There is no significant association between anti-erythrocyte and anti-HLA immunization. CONCLUSION: The erythrocyte and anti-HLA anti-immunization rates are high in our series. Preventive strategy is needed to ensure optimal blood safety.

[Lineage assignment in acute leukemia: confrontation between cytology and immunophenotyping]. / Diagnostic de lignée dans les leucémies aiguës : confrontation entre cytologie et immunophénotypage.

OBJECTIVE: The determination of the cellular lineage in acute leukemia is a crucial step in the diagnosis and the later therapeutic conduct. In Tunisia, emerging country, some cases of acute leukemias are still treated on the basis of an only cytologic study because of lack of cytometry. Our objective is to realize a confrontation between cytology and flow cytometry in the diagnosis of AL and to analyze discrepancies. PATIENTS AND METHODS: The study concerns 100 cases of AL. A second double-blind examination of the bone marrow smears of acute leukemias is realized by two cytologists and confronted to immunophenotyping. RESULTS: In two cases of AML, flow cytometry reassigned lineage into T ALL and biphenotypic AL. In three cases of ALL the lineage was reassigned into undifferentiated acute leukemia (2 cases) and biphenotypic acute leukemia (1 case). Lineage was not established in four cases, immunophenotyping allowed the diagnosis of B ALL in 3 cases, and of biphenotypic acute leukemia in 1 case. In both cases of discrepant findings, flow cytometry allowed the diagnosis of biphenotypic acute leukemia in a case and of AML in the other one. CONCLUSION: The cytological study remains insufficient in the diagnosis of lineage even with experimented cytologists. Immunophenotyping is essential in lineage assignment and reassignment.

Association between clinical expression and molecular heterogeneity in ß-thalassemia Tunisian patients.

Beta-thalassemia is the most frequent hereditary blood disorder in Tunisia because of its geographic localization and history. This pathology is characterized by a complex multisystem process with genetic and biochemical interactions. The aim of this work was to establish phenotype/genotype association through studying the distribution and the relationship between ß-thalassemia and α-thalassemia mutations and three polymorphic markers: the C → T polymorphism at -158 of the Gγ gene, the RFLP haplotype and the repeated sequence (AT)xTy in the ß globin silencer, in two groups of ß-thalassemia major and ß-thalassemia intermedia (TI) patients. Statistical analysis has shown that moderate expression seen in TI patients was significantly associated to ß(+) -87 (C → G), -30 (T → A) and IVSI-6 (T → C) mutations, haplotypes VIII, IX and Nb and to XmnI polymorphism. The regression analysis of combined genotypes (mutation/XmnI/RFLP haplotype) revealed that they contribute to justify 17.1 % of clinical expression diversity (p < 0.05). Among the studied genotypes the XmnI polymorphism seems to be the most determinant modulating factor, followed by the ß-thalassemia mutation and RFLP haplotype. Our findings highlight the heterogeneity of molecular background of ß-thalassemia that would be responsible of clinical variability.

Frequency of three polymorphisms of the CCL5 gene (rs2107538, rs2280788 and rs2280789) and their implications for the phenotypic expression of sickle cell anemia in Tunisia.

The pro-inflammatory context of sickle cell disease promotes the liberation of cytokines such as CCL5, encoded by a gene located on chromosome 17. Herein, the occurrence of three variations of CCL5 in sickle cell anemia (SCA) and their relations to two major complications - painful crisis and presence of infections - were investigated. 100 SCA Tunisian patients and 100 healthy subjects were included in the case control study. Then the sample of patients was divided into two groups according to the presence or absence of each complication. The polymorphisms, namely g.-403G>A, g.-28C>G and g.In1.+1T>C, were analyzed by PCR/sequencing. Our findings show the presence of eight genotypes, namely GG, GA and AA of g.-403G>A, CC, CG and GG of g.-28C>G, and TT and TC of g.In1.+1T>C. The frequencies of studied single nucleotide polymorphisms (SNPs) and haplotypes in SCA patients do not differ significantly from healthy control group results. There is also no significant association between the analyzed polymorphisms and complications as for painful crisis and presence of infections (p > 0.05). Altogether, our data support the conclusion that the three polymorphisms of CCL5, namely g.-403G>A, g.-28C>G and g.In1.+1T>C, do not seem to be involved in the clinical variability of SCA in Tunisia.

[Medical knowledge in immunological security of red blood cells transfusion in Tunisia: evaluation of a CD-ROM of auto-learning]. / Connaissances médicales en matière de sécurité immunologique en transfusion érythrocytaire en Tunisie : évaluation d'un CD-ROM d'auto-enseignement.

In Tunisia, red blood cells (RBC) transfusion joins in a statutory frame but remains subject to failures because of the misunderstanding of legislation and regulations. Our purpose is to estimate the knowledge of the medical staff in the immunological safety of RBC transfusion before and after reading an auto-education CD-ROM. It is a study of evaluation of an intervention. Eighty physicians participated to the study. The evaluation was done using an anonymous questionnaire, containing seven questions with multiple choices (QMC) relating to several items. The rate of good answers (RGA) calculated by questions and by items took into account the impact of the CD-ROM on the improvement of the answers after reading. The global average mark is 2.9/7. The RGA to questions varies from 22.5 % to 76.3%. All participants answered correctly to more than 50% of all items. Two answered correctly to all items. Among the participants, 31.3% answered to all "important" items, concerning ABO blood groups compatibility and ultimate bedside test. The rate of participation to the final evaluation was 83%. The impact of the CD-ROM was important and statistically significant. In the final evaluation, the global mark raised from 2.9 to 5.8/7, 31.5% (vs 2%) answered correctly all the questions and 95.5% (vs 31.3%) answered correctly all "important" items. This study revealed a misunderstanding of the doctors in immunological safety of RBC transfusions. Auto-teaching by CD-ROM was efficient. An improvement of the knowledge by continuous training is necessary in our country.

Three new mutations account for the prevalence of glucose 6 phosphate deshydrogenase (G6PD) deficiency in Tunisia.

A previous study on G6PD deficiency carried out on Tunisian population, led to the finding of seven different mutations with the prevalence of G6PD A- variant. This present study reports 23 new unrelated deficient subjects studied at the molecular level to determine the mutation that causes G6PD deficiency. Using PCR-SSCP of coding regions followed by direct sequencing of abnormal pattern, three new mutations were detected. Two of them are polymorphic intronic mutations. The first is IVS-V 655C-->C/T, found in four female subjects with mild deficiency of class III variant. The second is IVS-VIII 43 G-->A, found in three male subjects with mild deficiency of class III variant. The third mutation is in the exon region so that it changes the primary structure of the molecule. It is cited for the first time and named G6PD Tunisia. This variant affects the exon 7 of the gene at genomic position 15435 G→T. Its cDNA position is 93 G→G/T, it changes arg 246 to leu. This mutation was found in one heterozygote female with deficiency of class II who have had hemolytic anemia due to ingestion of fava beans. Finally, G6PD Med variant, reported before in three cases, was also found in five other cases (four heterozygote females and one male hemizygote). These findings first enlarge the spectre of mutations to be ten variant mutations, characterizing the Tunisian population and also contribute with hemoglobin gene research in our laboratory to trace the whole genetic map of Tunisian population.

Molecular characterization of erythrocyte glucose-6-phosphate dehydrogenase deficiency in Tunisia.

Screening of G6PD deficiency was carried out on 79 unrelated subjects (32 females and 47 males), all coming from out consultation. DNA from deficient subject (11 females and 30 males) was analyzed for the presence of G6PD mutation. Known mutations were studied by the appropriate restriction enzyme digestion of fragment amplified by PCR. Where the mutation could not be identified in this way, the samples were subjected to SSCP analysis and abnormal fragments were sequenced. Through these methods, seven different mutations have been identified. Among deficient females, eight had the African variant A-(tow of them were homozygous) and three had the Mediterranean variant, one of them was homozygous and have had a haemolytic crisis after ingestion of fava beans showing at birth manifestation of neonatal jaundice. Among deficient males, four were hospitalized and transfused after a haemolytic crisis due to ingestion of fava beans. All of them have had manifestation of neonatal jaundice. Of them, one carried the Mediterranean variant and three others had the African variant A-. Among the remaining deficient males, 15 had A-variant, two had the Aurès mutation. SSCP analysis of nine mild deficient males, revealed the presence of the association of 1311 CT/93 TC in two subjects, a newly described silent mutation in the exon 12 associated with the polymorphism in the intron 11 93 TC in one subject and tow single intronic base deletion. The first is IVS V 17 (-C) found in two subjects and the second is IVS VIII 43 (-G) encountered in four subjects.

[Efficacy of imatinib in FIP1L1-PDGFRA positive hypereosinophilic syndrome]. / Efficacité de l'imatinib dans les syndromes hyperéosinophiliques associés au transcrit FIP1L1-PDGFRA.

Hypereosinophilic syndromes (HES) are a heterogeneous group of disorders characterized by marked peripheral blood and tissue eosinophilia resulting in organ damage. Recent advances in molecular biology have led to the identification of a FIP1L1-PDGFRA fusion gene as a recurrent abnormality in some patients with HES. This fusion gene results from a cryptic 4q12 interstitial deletion involving an 800 kb region. Recent reports indicate that this subtype of HES is imatinib responsive with rapid and complete haematological remissions. Here we report two patients successfully treated with imatinib.

FIP1L1-PDGFRA positive chronic eosinophilic leukemia in Tunisian patients.

Hypereosinophilic syndromes (HES) are a heterogenous group of rare disorders characterized by sustained and otherwise unexplained overproduction of eosinophils with organ involvement and consecutive dysfunction. Detection of the FIP1L1-PDGFRA fusion gene or the corresponding cryptic 4q12 deletion in HES supports the diagnosis of chronic eosinophilic leukemia (CEL) and provides a molecular explanation for the pathogenesis of this disorder. We screened seven Tunisian patients fulfilling the WHO criteria of HES for the presence of the FIP1L1-PDGFRA fusion gene using nested reverse transcription polymerase chain reaction on peripheral blood samples. Four of the seven patients were positive for this fusion gene. Sequence analysis revealed a substantial heterogeneity of the fusion transcripts due to the involvement of several FIP1L1 exons. All patients were male. The median age at diagnosis was 24 years (range, 18-50); one patient had a history of hypereosinophilia of more than 10 years. Two patients had clinically important and symptomatic eosinophilic endomyocardial disease with thrombotic events. Splenomegaly was constant in FIP1L1-PDGFRA positive CEL but not in the other HES patients (only 1/3).

[Cytology and immunophenotyping of acute promyelocytary leukaemia]. / Cytologie et immunophénotypage des leucémies aiguës promyélocytaires.

Acute promyelocytic leukaemia (AML3) is characterized by particular clinical and biological features. We report the cytology and the immunophenotype of 14 AML3 from which 3 were AML3v. A double negativity of HLA-DR and CD34 is found in 12 cases and aberrant expression of CD2 in 2AML3v. Aberrant expression of CD56 and CD22 was shown in, respectively, one case, CD15, CD65 and CD117 expressions were variable. Cytological diagnosis is often evident, although in some cases, it is not typical and immunophenotype will contribute to the diagnosis.

[Blood-transmitted viral infections among haemophiliacs in Tunisia]. / Infections par des virus transmissibles par le sang chez des hémophiles en Tunisie.

In this work, we proposed to evaluate prevalences of hepatitis B and C viruses and Parvovirus B19 among 70 Tunisian haemophiliacs treated with clotting factors imported from Europe and/or locally produced cryoprecipitate; among them 6 (8.6%) are known HIV positive patients. HBs antigen, anti-HBc antibodies and anti-Parvovirus B19 antibodies were detected in 7.1%, 52.9% and 91.8%, respectively. HCV prevalence, defined as positive ELISA with positive Immunoblot and/or PCR was 50.0%. Prevalences of these viral infections in haemophiliacs are higher than prevalences detected among general population and in the control group of the study. HCV infection is less frequent in haemophiliacs born after 1985, the year of introduction of the inactivation procedures in the production of coagulation factors concentrates; it decreases more considerably after 1994, date of introduction of systematic screening of HCV among blood donors. In contrast, despite the inactivation of the factors concentrates and the systematic screening of the blood donations against HBs antigen, since 1973, the risk of HBV infection contamination remains high in the Tunisian haemophiliacs. The introduction in 1995 of hepatitis B vaccination in the national schedule of new-born vaccination may resolve in the future the problem of HBV infection in haemophiliacs and in the other categories of the Tunisian population.

A novel mutation in the SLC19A2 gene in a Tunisian family with thiamine-responsive megaloblastic anaemia, diabetes and deafness syndrome.

Thiamine-responsive megaloblastic anaemia (TRMA) syndrome with diabetes and deafness was found in two patients from a Tunisian kindred. The proband was homozygous for a novel mutation, 287delG, in the high-affinity thiamine transporter gene, SLC19A2. We demonstrated that fibroblasts from this patient exhibited defective thiamine transport. These data confirm that the SLC19A2 gene is the high-affinity thiamine carrier and that this novel mutation is responsible for TRMA syndrome.

[Atypical defibrination syndromes and acute leukemias with a t(9,22) translocation, apropos of 2 cases]. / Syndromes de défibrination atypiques et leucémies aiguës à translocation t(9,22); à propos de deux observations.

We report two cases of atypical defibrination syndromes in patients with respectively acute monoblastic leukemia (chronic myeloid leukemia initially) and acute lymphoblastic leukemia. Hemostasis studies show low fibrinogen level, elevated D-dimers, decreased alpha 2 antiplasmin and factor V, normal antithrombin III values. Plasminogen is below the normal range in one patient. Soluble complexes, which are an important argument for diagnosis of intravascular coagulation disease, are not detected in both patients. Primary or secondary hyperfibrinolysis seems also excluded since euglobulin clot lysis time was normal. Enzymatic proteolysis of fibrinogen (or fibrin) by the blast cells has been reported by some authors; this mechanism could account for the hemostasis abnormalities observed in these two patients.

[Long-term survival and prognostic factors in multiple myeloma treated with conventional chemotherapy. Report of 109 cases]. / Survie a long terme et facteurs pronostiques du myelome multiple traite par chimiotherapie conventionnelle. A propos de 109 cas.

Our study is retrospective. We report the results of conventional chemotherapy ins previosly untreated patients with myeloma. Survival and prognostic factors were analysed in 109 patients diagnosed from 1983 to 1992. The median age was 65 years, 87 patients (80%) were including in the stage III according the Durie Salmon staging system. The median survival time was 27 months and 10 years survival rate is 3.66%. In the univariate analysis, two prognostic variables were retained namely the hemoglobin and creatinine level. The study suggest that conventional therapy is a good treatment for old patients. However, patients younger than 55 years, must benefit from intensive chemotherapy supported by autologous bone marrow, pheripheral blood stem cells, or allogenic bone marrow transplantation. A considerable encrace in duration of remission and survival is possible.

[The 5q(-) syndrome. Report of 2 cases]. / Le syndrome 5q(-). A propos de 2 cas.

A rare and primitive myelodysplastic syndrome 5q(-) is characterised first, by the persistence of the cytogenetic anomaly 5q(-), and second, by its feminine predominance. Among 13 cases of myelodysplastic syndromes, the subject of a substantial and systematic cytogenetic medullar study (1996-1998), this paper is a case study of 2 syndromes 5q(-) diagnosed in two male patients, respectively, aged 41 and 68. The following diagnosis was made on the basis of an aregenerative macrocytic anaemia, a high platelet count, and a megakaryocytic hyperplasia, along with dysmegakaryocytopoiesis. The diagnosis of the 5q(-) syndrome was verified by cytogenetic analysis showing in one of the patients a deletion 5q(-)(q13, q33) and 5q(-)(q14, q34) with trisomy in the second one. Treatment was only limited to a blood transfusion. Subsequently one of the patients developed an advanced case of leukaemia. This paper suggests that a systematic medullar cytogenetic study must be conducted in the case of any refractory anaemia in order to identify the syndrome 5q(-) in individual cases.

[Results of a prospective protocol for the treatment of adult Hodgkin's disease]. / Resultats d'un protocole prospectif de traitement de la maladie de Hodgkin de l'adulte.

To report the results of an adapted protocol of treatment of Hodgkin disease in Tunisian patients. 70 patients (47 males and 23 females, sex-ratio = 2.04) with a mean age of 38.5 years (15 to 75) are enrolled in a therapeutic protocol to the prognostic factors and based on chemotherapy with MOPP/ABV or hybrid associated to radiotherapy. We perform an evaluation of response to chemotherapy after the 4th cycle, after the 6th cycle and then at the end of the protocol. Our population is characterized by the frequency of young patients(34% between 30 and 40 years), histologic types 2 and 3 (45 and 48%) and advanced disease with 60% of stages III and IV. After the 4th cycle, 32 patients(45%) are in complete response and 31(44%) in partial response, while 6 patients(9%) progress under chemotherapy. After 6 cycles, we observe 44 in complete response(72%) including 46% of the bad responders after 4 cycles. At the end of the protocol and on the 58 evaluable patients, 50 remain in complete response(86%). We observe 5 deaths occurred in 3 progressing patients and in 2 patients by infection after chemotherapy. 5-year actuarial and disease-free survival is 60% and 56% and median survival is 83 months. In the univariate analysis, response to chemotherapy represent the unic significant prognostic factor.