Peripheral blood stem cell mobilization in multiple myeloma comparison of two consecutive regimens in a limited resources country.

This study compared retrospectively the effectiveness, toxicity and hematopoietic recovery after autologous peripheral blood stem cell transplantation (ASCT) of two consecutive peripheral blood stem cell mobilization regimens in newly diagnosed MM patients. Patients in group 1 (n=178) were treated with 4 g/m2 of cyclophosphamide (CY) plus G-CSF (5 µg/kg/day). Patients in group 2 (n=117) with 750 mg/m2 of VP16 plus G-CSF (10 µg/kg/day). Optimal mobilization, defined by a target number of 8 × 106 CD34+ cells/kg collected, was achieved in 62.4% and 89.7% of patients in groups 1 and 2, respectively (P<10-4). The median number of aphaeresis sessions was reduced from two in group 1 to one in group 2 (P<10-4). Grade4 neutropenia, febrile neutropenia and IV antibiotic use were significantly more frequent in group 1 than in group 2 (P<10-4). Red blood cell transfusion requirements were significantly greater in group 1 (P=0.007). The switch to VP16-G-CSF10 resulted in a significant reduction of the number of hospitalization days (P<10-4). Neutrophil and platelet recovery after ASCT occurred on days 11 and 12, respectively, in the two groups with no significant differences. VP16+G-CSF10 allowed liberation of resources in the clinical and aphaeresis departments and demonstrated a better effectiveness-safety profile than CY+G-CSF5.

Association of stromal cell-derived factor-1-3'A polymorphism to higher mobilization of hematopoietic stem cells CD34+ in Tunisian population.

We explored the influence of polymorphisms in genes encoding the chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 in a cohort of Tunisian patients with malignant hematologic diseases multiple myeloma [MM], non-Hodgkin's lymphoma [NHL], Hodgkin's disease, and acute myeloid leukemia [AML], who underwent stem cell mobilization for autologous transplantation versus a group of healthy donors for allogeneic transplantation. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLp) analysis was used for rapid identification of genotypes. Significant associations for SDF1-3'A polymorphism were observed exclusively in patients with MM and NHL. While there was a lack of all association of SDF-1 polymorphism with AML patients. However, considering that the ability of mobilization varies among subjects, we have observed that the SDF1-3'A allele was associated with good mobilization capacity. Interestingly, the association was mainly observed among healthy allogeneic transplant donors where the analysis was not biased by background disease or chemotherapy (P=.010; odds ratio=2.603; confidence interval [95%]=1.239-5.466).

Human cytokine expression profile in various conditioned media for in vitro expansion bone marrow and umbilical cord blood immunophenotyped mesenchymal stem cells.

INTRODUCTION: Bone marrow (BM) represents the major source of mesenchymal stem cells (MSCs); however, umbilical cord blood (UCB) MSCs have some advantages over BM, such as a higher differentiation capability and noninvasive collection methods. OBJECTIVES: We compared antigen expression and cytokine-secretion by MSC from BM and UCB expanded with media supplemented with fetal bovine serum (FBS) or human platelet lysate (HPL). MATERIALS AND METHODS: We compared protocols for the expansion of hMSC starting from samples of BM or UCB by morphological analysis, calculation of population doubling numbers, and cytometry techniques using monoclonal antibodies (BD Biosciences). Using the last technique, cytokines were detected in brain homogenate supernatant fluids of MSC cultured in various media, using the Bio-Plex cytokine assay system (BD Biosciences). RESULTS: Calculating the number population doubling (PD) and colony-forming unit-(1)fibroblast (CFU-F) assays showed significantly better expansion with HPL compared with a selected batch of FBS and within fewer days: PD about 5 for 10%HPL versus 25 for fibroblast growth factor2 (FGF2) medium. By flow cytometry, we observed a greater number of BM MSCs compared with UCB MSCs, as well as differences in the expression of some MSC antigens, particularly CD105, CD90, and CD31. Analysis of cytokines: FGFb, RANTES, VEGF, IL-6, IL-8, G-CSF, and GM-CSF showed only some of them to be expressed: namely, IL-6, IL-8, and VEGF. MSCs derived from UCB showed low concentrations of these cytokines compared with MSCs derived from BM.

[Immune recovery after allogeneic stem cell transplantation: study of 19 patients]. / La reconstitution immuno hematologique en post allogreffe de moelle osseuse: etude prospective de 19 cas.

The aim of this study was to access average delays for novogeneration of myeloid and lymphoid cells after allogeneic bone marrow transplantation (BMT) outcome and factors affecting this organization. A prospective analysis over 2 years (01/01/07 to 31/12/08) enrolling 19 children treated with allogeneic intrafamilial bone marrow transplantation. Indications for bone marrow transplantation were: aplastic anemia (3 cases), bemoglobinopathies (9 cases), myelodysplastic syndrome (1 case) and primary immunodeficiency (6 cases). Different conditioning regiments were used according to the indication. The study of immune reconstitution was based on the quantitative determination of immunoglobulin and lymphocyte subpopulation. These tests were routinely requested to 1 month, 2 months, 3 months, 6 months, 9 months and 12 months. The average time of engraftment was 18 days (12-24). A rate of CD4+T lymphocytes>200/mm3 was provided within an average of 2,5 months (1-7). The average time to obtain CD8+T lymphocytes>200/mm3 was 2 months (1-5). The humoral immune reconstitution was made within an average of 2 months (1-4). A report of CD4+/CD8+T lymphocytes>I was obtained within 10 months and a half (1-24). Univaried analysis showed a correlation between the bone marrow sex matched and the faster reorganization of CD8+T cells (p=0.042). A quantity of CD34+>6 10(6)/kg was significantly associated with the recapture of a formula lymphocyte CD4+/CD8+T>1 (p=0.03) Immune recovery post bone marrow transplantation in children begins with myeloid lineage then lymphoid B then lymphoid T The inversion of the report CD4+/CD8+T lymphocytes, seems to be influenced by the high contain of CD34+cells in the graft as well as the type of conditioning.

[A contribution to a study of apoptosis of hematopoietic stem cells CD34+ by flow cytometry before and after cryopreservation]. / Contribution à l'étude de l'apoptose par la cytométrie en flux des cellules souches hématopoïétiques CD34+ avant et après le processus de congélation.

Apoptosis represents a particular form of programmed cell death which appears in all the damaged cells and potentially hazardous. It plays a crucial role in the development of multicellular organisms by assuring and maintaining the cellular homeostasis. Thus, apoptosis intervenes not only in the normal process of organisms' development but also in immune defence and in cancerous cells detection. Indeed, any blockage in the program of the apoptotic machinery would be responsible of some neurodegenerative and auto-immune diseases and could play a crucial role in different steps of carcinogenesis. Some researchers were very interested in studying apoptosis in hematopoietic stem cells CD34+ which could be intended to be reinfused to patients suffering from malignant diseases. They have noted that kinetic study of apoptosis of the hematopoietic stem cells CD34+ after the process of cryoconservation is also necessary. Such study permits to quantify the real and exact number of the viable hematopoietic stem cells CD34+ and therefore to eliminate such risk which would be associated with the reinfusion of apoptotic cells to patients. In this paper, we describe our contribution to hematopoietic stem cells CD34+ study by flow cytometry before and after cryopreservation by using annexin V as a specific probe allowing detection of phosphatidyl serine, one of the major features of apoptosis. But, we have noted a pronounced induction of apoptosis in peripheral mobilized blood compared to cytapheresis (after cryopreservation: 29.79% of apoptotic HSC CD34+ in peripheral mobilized blood but only 11.67% apoptotic HSC CD34+ in cytapheresis). Besides, we have noticed that hematopoietic stem cells CD34+ have had a statute of viability better than other mononuclear cells. These results put in value the reliability, the simplicity and the efficiency of flow cytometry for the analysis of apoptosis in hematopoietic stem cells CD34+ by following the intensity of fluorescence of annexin V.

[Study of the ploidy and cellular cycle of hidradenomas and hidradenocarcinomas: a series of 13 cases]. / Etude de la ploïdie et du cycle cellulaire des hidradenomes et des hidradenocarcinomes: a propos d'une série de 13 cas.

AIMS: To study by flow cytometry (FCM) the ploidy and the cellular cycle of nodular hidradenoma (NH) and hidradenocarcinoma (HC) and to assess the prognostic utility of this technique in such tumors. METHODS: We studied retrospectively 2 HC and 11 NH one of which was considered as an atypical NH. Monoparametric study by FCM was realized on paraffin-embedded material. The extracted cells were marked by Propidium's lodure and cellular cycle was analyzed by the software Mod-Fit LT. RESULTS: Our study showed eleven 100% diploid profiles, 10 of which had low S-phase varying between 2 and 12%. All of these 11 tumors were NH. S-phase was high (23.79%) in a single case that corresponded to the atypical NH. Two tumors showed aneuploid profiles; these corresponded to the 2 HC. CONCLUSION: The results of the cytometric study suit perfectly to those of the histopathologic examination. FCM could so help to establish the prognosis of these tumors. But further studies are necessary to determine the value of this technique.

[Contribution of cell cycle and DNA content study by flow cytometry in the prognosis of superficial bladder cancer: Tunisian experience]. / Apport de l'étude du cycle cellulaire et du contenu en ADN par cytométrie en flux dans le pronostic des tumeurs superficielles de la vessie : expérience tunisienne.

Clinico-pathological study of superficial bladder cancer (pTa/pT1) informs about prognostic factors such as the size of the tumor, its uni or multifocal character, its grade and stage. Presently, these factors constitute the basis of therapeutic decision but do not allow to foresee the prognosis with certainty. Many technics have contributed to a better knowledge of such tumors; however, they have not allowed to fully master prognostic uncertainties. During the last decades, cell cycle and DNA content study by flow cytometry has been developping, bringing an additional prognostic element to various types of tumors. We have decided to study the impact of this technique to the assessment of the prognosis of superficial bladder tumors. The study concerned 65 patients presenting superficial bladder tumors (pTa/ pT1), with a follow-up of at least two years in case of not recurrence and in case of recurrence, having had a second resection with analysis of sections. Flow cytometry was applied to formol-fixed and paraffin-embedded endoscopic resection material of initial tumors by simple-labelling of DNA with propidium iodide. Following cytometric study, 35 (54%) of tumors were aneuploid and 30 (46 %) were diploid. For the diploid ones, S-phase mean value was 14.94% (from 2.72% to 33.43%); and G2M mean value was 8.3 (from 1% to 18%). The presence of an DNA- aneuploid peak had a predictive value of recurrence and progression in stage, with relative risks of 12 and 6.85 respectively. It was also correlated with the histological grade and stage. On the other hand, S-phase and G2M values had no prognostic significance.

Prospective randomised comparison of the COBE spectra version 6 and haemonetics MCS(+) cell separators for hematopoietic progenitor cells leucapheresis in patients with multiple myeloma.

A randomised crossover trial of two separators was undertaken to compare the mononuclear cell, CD34(+) cell and CFU-GM yield, in patients (<61 years) with previously untreated symptomatic multiple myeloma. After first-line therapy, all patients received mobilising chemotherapy (cyclophosphamide 4 g/m(2)) and daily G-CSF. The first leucapheresis was performed on the first day the peripheral blood absolute CD34(+) cell count was > 20 cells/microl. All patients underwent 2 leucaphereses on consecutive days. The patients were randomised to undergo either the first or second leucapheresis using the COBE Spectra. The target duration of the procedure on the COBE Spectra was 2 total blood volumes, and for the Haemonetics MCS(+) it was 20 cycles with four recirculations. Between September 2003 and March 2005, 60 patients were entered in the study. COBE Spectra version 6 processed significantly larger volumes of blood than the Haemonetics MCS(+) (8,845 and 5,680 ml, respectively, P < 0.01). The absolute yield of mononuclear cells (2.1 vs. 1.5 x 10(8)/kg, P = 0.04), CFU-GM (11 vs. 3 x 10(4)/kg, P = 0.01) and CD34(+) cells (3 vs. 1.7 x 10(6)/kg, P = 0.02) were all significantly higher with the COBE Spectra version 6, as were the yields per unit volume of blood processed. In conclusion, our study shows that COBE Spectra Version 6 is faster and has a better yield than the Haemonetics MCS(+), in patients with multiple myeloma.

NA antigen frequencies in the Tunisian population.

The biallelic NA antigen system is of special interest as the NA antigens are frequent targets of neutrophil antibodies, causing alloimmune neonatal neutropenia, blood transfusion reactions, and chronic benign autoimmune neutropenia in infancy. Neutrophils isolated from the peripheral blood of 119 unrelated individuals at the National Blood Center were phenotyped for NA1 and NA2 using a granulocyte immunofluorescence assay. A subsequent analysis of the phenotyping study showed that the NA1 and NA2 antigen frequencies were 0.529 and 0.865 respectively, and that the estimated NA1 and NA2 gene frequencies were 0.313 and 0.632 respectively. In conclusion, it was determined that the Tunisian population is of Caucasian origin. However, to validate this finding, further investigations are necessary.

Contribution of flow cytometry to acute leukemia classification in Tunisia.

The precision of immunological characterization of leukemias was improved by a certain number of technical innovations, particularly hybridoma production and standardization, resulting in monoclonal antibodies and definition of recognised cellular antigens (designated by CD: Cluster of Differentiation). The aim of this work was to determine the immunophenotyping profile of patients with leukemia, by means of a flow cytometric method: 66 blood samples coming from leukemic persons in the Sahel region were studied by flow cytometry, using about thirty monoclonal antibodies all marked with a fluorochrome, in one or two colour systems to assess their distribution according to type (lymphoid B or T / myeloid) and age, and to search for possible co-expressions of markers of different lineages. The marked preponderance of childhood B-ALL in our series is, at least partly, attributable to the age distribution of the Tunisian population. In agreement with studies from other countries, the majority of AML cases occurred among adults. A high proportion of AML cases in our series co-expressed markers of other lineages. Overall, accurate classification of acute leukemias was possible from a simple peripheral blood sample in 62 of 66 cases (93.9%).

[NA, a specific system for polymorphonuclear neutrophils: localization, biochemistry, genetics, frequency role in pathology]. / Na, le système spécifique des polymorphonucléaires neutrophiles: localisation, biochimie, génétique, fréquence et rôle en pathologie.

Among the neutrophil polynuclear specific antigens (NA, NB, ND, NE, ...), NA antigen is the most common. It is a glycoprotein situated on the neutrophils FcRIIIb-receptor and presents 2 forms: NA1 and NA2. The epitope responsible of that polymorphism has got an amino acids composition that is unknown. The first techniques used for their analysis were the microagglutination and the granulocytotoxicity-later, the immunofluorescence, the chemiluminescence and the MAIGA (Monoclonal Antibody Immobilized granulocyte Antigen) were introduced. These last years, more efficient techniques appeared like Flow Cytometry and Polymerase Chain Reaction (PCR) that allowed phenotyping and genotyping of neutrophil polymorphonuclear specific antigens. The studies indicated that NA antigen frequency varies according to the populations and the ethnics. NA2 allelic form is more frequent than NA1 in the caucasian population (88% VS 46%). In human pathology, NA antigen is implicated in the physiopathological mechanisms of the alloimmune and probably auto-immune neutropenies.

[Screening and confirmation of anti-HCV antibodies in Tunisian blood donors]. / Dépistage et confirmation des anticorps anti-VHC chez les donneurs de sang tunisiens.

Antibodies to Hepatitis C virus (HCV) were tested in 43000 Tunisian blood donors by using enzyme immuno-assay. Our results show that 0.7% (304/43000) were anti-HVC positive. Of these 304.78 were confirmed anti-HCV positive (0.18%) by immuno-blot, and 99 displayed an indeterminate profile. Different immune responses were observed: In donors with positive serologic pattern (78/304), 25.6% response towards whole antigens (C + NS3 + NS4 + NS5) was frequently observed (44/78) 56.4%. Reactivity to 2 antigens was observed in 28.2% (22/78) and with 3 antigens in only 15.4% (12/78), with systematic reactivity to core. In donors with indeterminate serologic pattern (99/304) 32.5%, reactivity to non-structural antigen NS5 was the most frequently observed (54/99) 54.5%, reactivity to non-structural NS3 antigen was noted in 27.3% (27/99) and to core antigen in 18.2% (18/99). No donors with isolated reactivity to NS4 were observed in our series.

[A human recombinant erythropoietin produced in human lymphoblastoid cells]. / L'érythropoïétine humaine recombinante produite dans des cellules lymphoblastoïdes humaines.

A human recombinant erythropoietin is produced by transfection of a human EBV immortalised lymphoblastoid cell line by human erythropoietin gene. This cell line was selected because it is eucaryotic, human, able to grow in suspension and neither clonogenic nor tumorigenic. Purification of erythropoietin from culture supernatants of lymphoblastoid cells was described and structural, biochemical and functional characteristics were investigated in several studies. Results obtained for this kind of recombinant erythropoietin were similar to those obtained regarding the protein expressed in CHO cells suggesting that both molecules have the same efficiency in clinical applications.

C4 polymorphism in multiplex families with insulin dependent diabetes in the Tunisian population: standard C4 typing methods and RFLP analysis.

The polymorphism of C4A and C4B genes was investigated in Tunisian patients with insulin dependent diabetes (IDDM) and compared to family members (sibs) and to healthy controls. Multiplex families were analysed. A significant increase in C4AQO (26.86% vs 6.90%) and C4BQO (40.29% vs 8.28%) phenotypes was noted in IDDM patients compared with controls. Using RFLP analysis, we confirmed the high frequency of C4 null alleles. We also observed that most of these alleles were genes deleted in IDDM patients (72.23% vs 20% for CA4QO and 74.07% vs 16.70% for C4BQO). A significant decrease in the C4B long (14.92% vs 67.12%) form of the gene was also demonstrated by RFLP analysis compared with controls. Two haplotypes were frequently associated with IDDM patients in whom the C4A and C4B were deleted genes.

Immunogenetic heterogeneity in type I (insulin-dependent) diabetes among the Tunisian population. HLA-DR antigens and organ-specific autoantibodies (family study).

The frequency of HLA-DR antigens, as well as the prevalence of islet cell insulin autoantibodies and other autoimmunity disorders, were investigated in Tunisian patients with insulin-dependent diabetes mellitus (IDDM) and were compared with family members (sibs) and healthy control subjects. Cytoplasmic islet cell autoantibodies (ICA) were found in 79 of 175 (45.1%) patients with IDDM, in 23 of 126 (18.25%) unaffected first degree relatives of type I diabetes patients and in only two of 146 (1.3%) control subjects. In 79 ICA positive patients with IDDM, 46.8% presented other evidence of autoimmunity by testing for specific autoantibodies. Insulin autoantibodies were found in 86.9% of healthy ICA-positive sibs. A good correlation between HLA-DR3/DR4 heterozygous phenotypes and the presence of ICA in patients with IDDM and their unaffected sibs was observed in the Tunisian population. In fact, this heterozygous phenotype is found in 63.3% of ICA-positive diabetic patients and in 44.4% of ICA-positive unaffected sibs, whereas, HLA-DR3/DR4 antigens were noted in only 22.9% of ICA-negative diabetic patients and in no ICA-negative unaffected sibs. In these studies, we also summarized the distribution of HLA-DR antigens in patients with IDDM who presented autoimmune disorders other than ICA.

Extended HLA haplotypes in multiplex families with insulin dependent diabetes mellitus in Tunisian population: HLA serological typing and RFLP analysis.

Haplotypes including HLA A, B, C, DR, and DQ were compared in a study population comprising 18 Tunisian multiplex families with diabetic children. Eighty haplotypes found in IDDM patients were compared with 148 haplotypes present in healthy family members. RFLP analysis showed that two DR subtypes were significantly more common in the diabetic haplotypes (DR4-DQw8: 82 per cent in IDDM members compared to 0 per cent in healthy members, p less than 0.001 and DR-Dw25: 56 per cent in IDDM patients compared to 16.7 per cent in healthy members, p less than 0.001) and these were in most cases found in haplotype combinations with HLA A2 B44 DR4 DRw53 and HLA A 24 B18 DR3 genes, respectively.

Delta infection in chronic HBs Ag carriers in Tunisia: high prevalence in chronic asymptomatic HBs Ag carriers and in HBs Ag positive cirrhosis.

The presence of hepatitis B virus DNA, delta antigen and anti-delta antibodies was examined in 159 Tunisian chronic HBs Ag carriers: 45 were asymptomatic and 114 suffered from cirrhosis. Serum hepatitis B virus DNA was detected in two (4.5%) asymptomatic HBs Ag carriers and in 11 (10%) HBs Ag positive cirrhosis patients. The prevalence of HDV infection determined by the presence of anti-delta was relatively high in asymptomatic HBs Ag carriers (33%) and in HBs Ag positive cirrhosis patients (21%). Active ongoing HDV infection, detected by serum HD Ag and anti-delta IgM, was shown in five patients with cirrhosis and active hepatitis B virus replication. We conclude that hepatitis delta virus may be endemic in Tunisia and does not always inhibit hepatitis B virus replication.

[Comparison of three methods to evaluate microalbuminuria in insulin-dependent diabetics: radioimmunoassay, laser-nephelometry and Laurell electroimmunodiffusion]. / Comparaison de trois techniques de dosage de la microalbuminurie chez les diabétiques insulino-dépendants: radioimmunologie, néphélémétrie-laser et électro-immuno-diffusion de Laurell.

The early detection of microalbuminuria in insulin dependent diabetes is considered as a sign of initial stage of nephropathy possibly reversible if glycemic balance is well maintained. This detection requires very accurate methods as radio-immuno-assays. Yet, they are so slow that they represent an obstacle to systemic detection. We report in this work an apparaisement of an immuno-nephelemetric and electro-immuno-diffusion of Laurell methods. Results reveal that immuno-nephelemetry and electro-immuno-diffusion are a sensitive and accurate methods (threshold of sensitivity of Laurell method is : 2.5 mg/l and nephelemetry is 1.5 mg/l). Moreover, RIA, immunonephelemetry and Laurell methods are significantly well correlated (r = 0.903: Laurell/Radio-immuno-assay, r = 0.907: Nephelemetry/Laurell). In conclusion immuno-nephelemetry and electro-immuno-diffusion of Laurell are a choice methods to test great lines of samples. Radio-immuno-assay can be used as a reference method of detection of microalbuminuria.

[The value of using HEP/2 cells as compared to sections of rat liver in the detection of autoantibodies using indirect immunofluorescence in human pathology]. / Intérêt de l'utilisation des cellules HEP/2 comparativement aux coupes de foie de rat pour la détection des auto-anticorps par immunofluorescence indirecte en pathologie humaine.

1,400 sera taken from patients suspected of having autoimmune diseases and sent to the laboratory for determination of antinuclear antibodies, are tested by comparative indirect immunofluorescence on 2 subtrata; rat liver section and HEP/2 cells. The 143 positive sera on rat liver sections are also positive on HEP-2. In the 1,010 sera which are negative on rat liver sections, 165 are positive on HEP-2, 113 give a nuclear fluorescence, 26 give a cytoplasmic fluorescence and 26 give a nuclear and cytoplasmic fluorescence. Three positive sera were also used in immunofluorescence on another cells: VERO and MRC 5, as well as dual immunodiffusion versus thymic and splenic cell extracts and 76 p. cent of these sera were found positive with these techniques. This confirms the advantage of the use of HEP/2 cells in demonstrating autoantibodies, especially when they are not detected on rat liver sections, like the anticentromer antibodies. This substratum offers the advantage of detecting not only antibodies directed against nuclear antigens, but also those directed against cytoplasmic antigens.