Immunophenotypic analysis of bone marrow B lymphocyte precursors (hématogones) by flow cytometry.

The aims of this flow cytometry study were to quantify B lymphoid precursors known as hématogones across age and clinical conditions and to study the immunophenotypic profile of these benign immature B cells. A total of 406 consecutive marrow specimens were analyzed for hématogones using 4-color flow cytometry during a 19 month period (60% males and 40% females). The age range was 3 months to 89 years. Hématogones were present in 80% of the specimens. Morphologic analysis of the smears from each patient showed small numbers of hématogones (<13% of total cellularity). The B cell population was defined by CD19 + CD45 bright positivity, coexpression of other B lineage markers: CD20, CD22, CD10, CD29, CD38 and CD58 in addition to HLA-DR and CD34. In our study we found a significant decline in hématogones with increasing age but a broad range was found at all ages. Marrow from some adults contained relatively high numbers. Diagnosis in these patients included cytopenias, infections, and neoplastic diseases. Distinction of hématogones is critical for disease management particularly after therapy of paediatric B acute lymphoblastic leukaemia to monitor for minimal residual disease.

[Study of antigenic profile of blasts in acute lymphoblastic leukemia: flow cytometric analysis of 152 cases]. / Etude du profil antigénique des blastes au cours des leucémies aiguës lymphoblastiques : analyse de 152 cas par cytométrie en flux.

The aim of this study was to characterize the antigenic profile of blasts in acute lymphoblastic leukaemia (ALL) and to determine possible phenotypic aberrancies in a series of 152 patients with acute leukaemia diagnosed non myeloid leukaemia in cytology. Samples were analyzed by EPICS XL flow cytometer (Beckman Coulter) after staining with monoclonal antibodies(Beckman Coulter). Based on criteria of EGIL (European Group of Immunological Leukaemia), cases were classified as: acute lymphoblastic leukaemia (ALL, 52,6%); 75% cases of ALL belong to lymphoid B lineage. 80% of ALL B were CD10 positive, marker of best prognosis. In 10,5% of cases, biphenotypic leukaemia is diagnosed (lymphoid/myeloid). In 25% of cases aberrancies in phenotype were found. Flow cytometry has wide field of applications to characterize blast cells from patients with acute leukaemia: to establish diagnosis of lineage responsible in proliferation, to determine the stage of maturation, to predict prognosis for a better adaptation of adequate treatment, to follow evolution of disease after chemotherapy and to study minimal residual disease.

[Platelet-leukocyte aggregates as a marker for platelet activation in platelet concentrates]. / Complexes leucoplaquettaires en tant que marqueur d'activation dans les concentrés plaquettaires standards.

OBJECTIVES: Several in vitro laboratory tests to assess the quality control of platelet concentrates (PC) are available. Some of them have a good correlation with the platelet recovery index. To assess the quality control of standard PC prepared in our blood bank, we measured the blood gas and the degree of platelet activation. MATERIALS AND METHODS: SPC were prepared by the PRP method. Fifty-five SPC (45 SPC at day one of storage and 20 SPC at day five of storage) were analysed. Blood gas (pH, PO(2), PCO(2) and bicarbonate concentration) in the SPC were measured by blood gas automate. Platelet activation profile were determined by measuring the percentage of platelet expressing the CD62p (% CD62) and the percentage of platelet-leukocyte aggregate (% PLA). RESULTS: The pH values of all studied SPC were comprised between 7.0 and 7.6. SPC at day 1 of storage have a significantly higher pH than those at day 5 of storage (7.5+/-0.05 versus 7.3+/-0.14; p<0.001). The % CD62p were higher in SPC at day five compared to the SCP at day one without reaching a statistical significance (28.4+/-15% versus 24.3+/-9.7%, p=0.052). The percentage of PLA were higher in SPC at day one compared to SCP at day five although this difference is not statistically significant (22.2+/-7.5% versus 17.9+/-8.0%; p=0.23). CONCLUSION: Preparation and storage procedure adopted in our centre did not significantly affect the quality SPC. Our study is the first to assess the PLA in PC. Studies assessing the PLA are warranted to appreciate the clinical impact of this parameter.

[Ovary tumor in chronic myeloid leukaemia. Case report]. / Tumeur ovarienne et diagnostic de leucémie myéloïde chronique. A propos d'un cas.

Granulocytic sarcoma or chloroma is a neoplasia consisting of myeloid precursors in an extramedullary site. Its appearance in chronic myeloid leukaemia is unusual. We report a case of ovary tumor associated with chronic myeloid leukaemia in chronic phase.

Identification of the translocation t(15;17) in acute myeloid leukemia (AML) initially classified as FAB M1: case report and review of the literature.

We report a case of a patient aged about 53 years, who initially presented with hematological disorders (WBC: 44000/mm3, Hb: 11g/dl, Pit: 127000/mm3) without tumoral syndrome. The Wright-Giemsa stained bone marrow and peripheral blood smears showed a population of blast cells characterized by cells with high N/C and strongly basophilic cytoplasm without granules. The nuclei were predominantly round. Nuclear chromatin was fine and contained small nucleoli. Cytochemisty was positive for peroxidase activity. Immunophenotyping showed myeloid typical markers of granulocytic lineage (MP0+, CD13+, CD33+, CD117+, CD34-). The karyotype revealed the expression of t(15;17) chromosomal translocation. The diagnosis of acute myeloid leukaemia (AML) was then evoked initially. The cytological features corresponded closely to the M1 subtype as defined in the FAB classification. The patient was treated with induction therapy according to the 7/3 protocol. One month later, he was discharged from hospital on hematological and cytogenetic remission. He died at home because of a heart attack. From the biological findings the patient was retrospectively diagnosed as having promyelocytic leukemia (hyperbasophilic form).

[Haematological characteristics, FAB and WHO classification of 153 cases of myeloid acute leukaemia in Tunisia]. / Caractéristiques hématologiques et classification FAB et OMS de 153 cas de leucémies aiguës myéloïdes en Tunisie.

A complete blood analysis with a careful morphologic examination of peripheral blood and bone morrow smears completed by cytochemical reaction will help to classify the most acute myeloid leukaemia (AML). Actually, the study of other cytogenetis and immunophenotypic markers are now necessary to confirm diagnosis. The World Health Organisation WHO classification (2001) incorporates theses approaches. The purpose of this study is a bio-clinical review according to the WHO recommendations in 153 cases of LAM diagnosed between January 1998 and December 2003. The patients were aged 2 months to 90 years with sex ratio (M/F) of 1,22. The morphologic conclusion was difficult in 12% cases. Presence of dysplasia is noted in 50% of cases with multilineage dysplasia in 42% of cases. Our results showed cloned chromosomal abnormalities in 57% of cases (t(8;21): 12%, t(15;17) : 10%, Inv16: 1,3%, 11q23: 2,6% et complex karyotype: 14,3%). In 69% of cases with multilineage dysplasia, the karyotype was normal. 3 cases of LAM were noted at patients treated for breast cancer with chirurgic chemotherapy and radiotherapy 3, 4 et 5 years after treatment (LAM3 with t(15;17), LAM4 with genetic abnormalities of chromosomes 3, 5, 7, 8, 9, 14 et 16 et LAM 6 with genetic abnormalities of chromosomes 4, 7, 12, 14, 19 et 21). In WHO classification, cytology is essential in diagnosis of LAM even if the karytype have an important prognostic value. Research of signs of dysplasia lineage after lineage constitutes an important microscopic work and it is difficult to quantify dysplasia when the lineage is poor.

[Thrombotic thrombocytopenic purpura in a newborn]. / Purpura thrombotique thrombocytopénique chez un nouveau-né.

We report the case of a newborn presenting with hemolytic anemia, thrombocytopenia, hyperbilirubinemia, and renal failure in the first hours of life. An early plasmatherapy was undertaken, with good outcome. The specific von Willebrand factor-cleaving protease activity (ADAMTS 13 for a disintegrin and metalloprotease with thrombospondin type 1 repeats) was found to be low. This is the specific biologic diagnostic element of congenital thrombotic thrombocytopenic purpura (TTP). This disease of constitutional thrombotic microangiopathy is rare. The prognosis, usually life-threatening, was completely transformed given the better understanding of the pathogenesis of the disease and therapeutic progress.

[Juvenile myelomonocytic leukemia: A three-case series]. / Leucémie myélomonocytaire juvénile : à propos de trois cas.

Juvenile myelomonocytic leukemia (JMML), previously known as juvenile chronic myeloid leukemia (JCML), is a rare, myelodysplastic-myeloproliferative disease typically presenting in early childhood. This disorder is difficult to distinguish from other myeloproliferative syndromes such as chronic myeloid leukemia (CML) because of the similarities in their clinical and bone marrow findings. However, because of its unique biological characteristics such as absolute monocytosis with dysplasia, absence of Philadelphia chromosome or BCR-ABL fusion protein, hypergammaglobulinemia, and raised fetal hemoglobin level, this disorder does not satisfy the criteria for inclusion in the CML or chronic myelomonocytic leukemia (CMML) group, as seen in adult patients. We describe three cases of JMML, who had very similar clinical and laboratory findings.

[Hematologic abnormalities in infantile visceral leishmaniasis]. / Anomalies hématologiques au cours de la leishmaniose viscérale infantile.

The clinical and biological manifestations of visceral leishmaniasis are often confusing, most particularly because it can mimic and lead to a variety of hematological disorders. The aim of this study was to investigate the hematologic abnormalities observed in infantile visceral leishmaniasis from January 2000 and December 2013. The study included 35 children with a mean age of 3.5 years. Clinical manifestations were dominated by splenomegaly, fever, and paleness, defining the classic triad in 16% of our patients. Anemia was present in all patients. Leukopenia was found in 51% of the cases. Thrombocytopenia was observed in 48% of our patients and 36% had pancytopenia. All cases were confirmed by the presence of Leishman bodies (amastigotes) in the bone marrow smears. Quantitative and qualitative megakaryocyte abnormalities were found. Similarly, dysgranulopoiesis was observed in 31% of the cases, eosinophilia was present in 6%, erythroid hypoplasia in 3%, and erythroid hyperplasia in 34%. Different features of dyserythropoiesis were revealed in 71% of the patients with images of hemophagocytosis in 6% and multiple dysplasias in 9%. The knowledge of these hematological abnormalities associated with infantile visceral leishmaniasis can assist us in searching for Leishman bodies in the bone marrow smears to provide a diagnosis more quickly without necessarily resorting to more sophisticated tests.

Treatment of haemophilia A in Tunisia: efficacy and inhibitor study.

Cryoprecipitate is the principal type of factor VIII (FVIII) concentrate used for treating haemophilia A in Tunisia. Allergic reactions, viral transmission, and inhibitor formation remain the most serious complications of FVIII therapy. The aims of the study presented here were to evaluate the efficacy of FVIII therapy, to investigate the inhibitor prevalence, and the factors which may affect inhibitor formation in our haemophilia A patients. Plasma samples were screened for FVIII inhibitors by the Bethesda method. 30 minutes FVIII recovery was also determined for each patient. In this prospective study, 18 previously treated haemophilia A patients, four with severe (FVIII concentration <2%) and 14 with moderate haemophilia, were closely followed up during administration of 223 FVIII concentrates (cryoprecipitate and/or fresh frozen plasma). The median age of the patients involved in the study was 13.5 years (range 5 to 53). Clinical response to FVIII was consistently good to excellent. In the majority of cases, actual and predicted FVIII recovery correlated' well. Adverse reactions were not observed. Five patients, aged less than 18 years and minimally treated (>36 FVIII exposure days), were found to have low titre FVIII inhibitors (<10 Bethesda units) at the end of the study. Inhibitor activity was detected in one patient with severe and in four patients with moderate haemophilia. In conclusion, FVIII therapy was effective, well tolerated, and low titre inhibitors identified did not preclude continued on demand FVIII therapy. Our study has also demonstrated that patients' age and treatment regimen do not affect inhibitor formation. Further studies are necessary to confirm these findings.

[Hereditary sideroblastic anemia: a rare diagnosis]. / Anémie sidéroblastique héréditaire: une maladie rare.

Hereditary sideroblastic anemia is a very rare disease recessive and X-linked that affect heme biosynthesis by deficit or decreased of delta aminolevulinic acid synthase (ALAS) activity. We report a case of a six-month-old boy, admitted in the hospital for anemic syndrome. The hemogram showed anemia (hemoglobin: 4.5 g/dL), frankly hypochronic microcytic and a regenerated (mean corpuscular hemoglobin concentration: 26 g/dL, mean cell volume: 53 fl, reticulocytes: 10 x 10(9)/L) with red cells morphologic disorders in smears (anisopoikylocytosis) without attack of the other lineages; white blood cells: 11 x 10(9)/L (neutrophils: 64% and lymphocytes: 35%); platelets: 350 x 10(9)/L. Examination of bone marrow showed an important erythroid hyperplasia (about 69%) with dyserythropoiesis. Perls stain revealed intense siderosis with 90% of ringed sideroblasts and a large number of siderocytes. Exploration of ALAS2 and ABC7 genes on the DNA of the infant was not found abnormalities. Treatment with pyridoxine corrects moderately the anemia. By the way, we proposed to remind that iron deficiency, inflammatory syndrome and thalassemia are the common microcytic anemia. However, it's mandatory to explore other causes if diagnosis is not solved.

[Screening of hemoglobinopathies and molecular analysis of beta-thalassemia in Central Tunisia]. / Dépistage des hémoglobinopathies et analyse moléculaire des beta-thalassémies en Tunisie centrale.

BACKGROUND: Previous investigations have permitted to locate 16 beta-thalassemic mutations in different samples of the Tunisian population. One of them (IVS I nt 2: T--G) had been found only in the central region of Tunisia. Our research was carried out in this part of the country to estimate the prevalence of this mutation and to establish a prenatal diagnosis using appropriate probes. POPULATION AND METHODS: One thousand one hundred and five blood samples taken from 1987 to 1990 from healthy blood donors and 346 samples taken from 1985 to 1992 from patients were analysed. Detection of hemoglobinopathies was carried out by means of specific hematological tests and different electrophoretic and chromatographic techniques. Mutations were detected by means of the usual techniques of molecular biology. RESULTS: Sickle cell anemia and beta-thalassemia were the most frequent in the samples studied. The molecular analysis carried out on eight patients native of the Essouassi-El-Djem region point out that all these patients carry the same point mutation (IVS I nt 2: T-G) detected for the first time in 1988 in a patient native of the same region. One of these patients, aged 43, who did not suffer from anemia and did not show the usual symptoms of beta O thalassemia, had one hemolytic attack at the age of 17. CONCLUSIONS: The high number of persons carrying Hb S and beta-thalassemia trait increase the risk of appearance of homozygous forms. The presence of the same mutation IVS I nt 2: (T-G) in all beta O-thalassemic patients from Essouassi-El Djem region may indicate that it may have its origin there. The heterogeneity of clinical phenotype of these patients shows the difficulty of establishing a unique strategy of prenatal diagnosis by DNA analysing which can be applied in all cases.

[Cytological features of acute leukaemia in the central region of Tunisia]. / Aspects cytologiques des leucémies aiguës: à propos de 193 cas colligés dans la région centrale de Tunisie.

In Tunisia, because of an absence of population registry, data on acute leukaemia are scarce. We studied the epidemiological and cytological characteristic of 193 patients with acute leukaemia. Haemograms were carried out and slides for peripheral blood and bone marrow were prepared for each patient. The age range of the patients was 10 months to 83 years with a predominance of males (ratio: 1.27). As regards type of leukaemia, 40.4% had acute lymphoblastic leukaemia, 51.8% had acute myeloblastic leukemia and 7.8% were unclassified. Diagnosis was made at less than 10 years in 31.6% of cases and 72% of these were the lymphoblastic type. Anaemia (Hb < 11 g/dL was found in 85% of cases, thrombocytopenia (platelets < 100 000/mm3) in 80.5% and hyperleukocytosis (WBC > 100 000/mm3) in 14.5% of cases with blasts in peripheral blood in 92% of cases.

[Hepatitis C virus antibodies in 34130 blood donors in Tunisian Sahel]. / Les anti V.H.C chez 34130 donneurs de sang du Sahel Tunisien.

Since January 6th 1994 to december 31 1997. We researched hepatitis C Virus antibodies by second and third generation ELISA in 34,130 bloods donors living in "Sahel Tunisien". 193 were positive (0.56%). Only 171 of them were secondary tested by immunoblot assay (anticore, anti NS5, anti NS3, anti NS4). Which was positive in 53 cases (30.9%); in determined (presence of only one antibody) in 78 cases (45.6%) and negative, in 40 cases (23.3%). There was a significant relation between a ratio over than 2.5 in ELISA and immunoblot positivity. Immune response to different hepatitis virus antigens were heterogeneous with predominant in determined profile. (78/171 cases). Most of donors of the last profile had either anti NS5 (32/78) or anti NS3 (33/78) and we excluded them even through usually negative in P.C.R and associated with a very low risk of contamination.

Biosorptive uptake of methylene blue using Mediterranean green alga Enteromorpha spp.

Batch biosorption experiments were carried out for the removal of methylene blue, a basic dye, from aqueous solution using raw and dried Enteromorpha spp., Mediterranean green alga. A series of assays were undertaken to assess the effect of the system variables, i.e. contact time, solution pH and sorbent amount. The results had showed that sorption capacity was optimal using 6-10 solution pH range (i.e. maximum adsorption capacity of 274 mg/g). The minimum sorbent concentration experimentally found to be sufficient to reach the total removal of the dye molecules from the aqueous solution was 5 g/L. Besides, equilibrium data were fitted using five linearisable isotherm models. The related results showed that the experimental data were very well represented by the Langmuir model for the linear regression analysis and both the Langmuir and Redlich-Peterson isotherm models for the non-linear analysis. In both cases, such modelling behaviour confirms the monolayer coverage of methylene blue molecules onto energetically homogenous Enteromopha surface. In addition, an exhaustive comparative study was done to situate this marine biomass among other proposed sorbents.