[Iron deficiency anaemia: clinical presentation, biological diagnosis and management]. / Anémies ferriprives: signes d'appel, diagnostic et prise en charge.

The iron deficiency is the first cause of anaemia. In healthy young adult, anemia is well tolerated because of its progressive installation. The most common symptoms of anemia are pallor, fatigue and dyspnea. In biological exams, anemia is classically associated with microcytosis and hypochromia. The origins of microcytic anemia are iron deficiency, inflammatory aetiologies, thalassemia and sideroblastic anaemia. The iron-deficiency diagnosis includes two explorations: biological and clinical. The biological exploration is based on interpretation of serum biologics tests as blood iron, ferritin, transferrin with saturation, total iron-binding capacity and its soluble receptors. This interpretation is simple if it is not associated with clinical disorders influencing the internal iron cycle. The clinical exploration must always be followed by a careful assessment of the underlying cause as blood loss. The most common causes in women of reproductive age are gynaecologic. In men and menopausal women, the gastrointestinal tract bleeding is source of anemia. Therapeutic management of anemia is oral iron therapy. Etiological diagnostic of microcytosis is essential before iron therapy. If not, the treatment could be inefficient or it could mask or delay the etiological diagnostic.

[Health care units in the French blood banks, why?]. / Les structures de soins dans les établissements de transfusion sanguine, pourquoi faire ?

In France for several years, many patients have been treated in Blood Transfusion Centers belonging to the EFS. This partnership between public hospitals and EFS is appreciated by the patients who find a competent staff in transfusion and apheresis process, in a more pleasant environment than in hospital. There is a total of 93 Health Care Units in Blood Transfusion Centers. Sixty-three of these Health Care Units perform only transfusions and bleeding. In the remaining 30 Health Care Units apheresis, peripheral blood hematopoietic stem, cell harvesting, plasmatic exchanges and extracorporeal photopheresis are also performed. Despite the perfect fit between hospital needs, comfort and easiness for patients, an economical problem remains. At the present time, the reimbursement rate by national health insurance is below the real cost. If unsolved, this discrepancy could force an end to this beneficial partnership.

[Role of vitamin deficiency in pancytopenia in Djibouti. Findings in a series of 81 consectutive patients]. / Place des étiologies carentielles dans les pancytopénies à Djibouti a propos de 81 patients consécutifs.

The purpose of this study of patients with pancytopenia in Republic of Djibouti was to identify etiologic factors and attempt to define diagnostic and therapeutic strategies adapted to local conditions. Clinical, biological and radiological assessment was performed in 81 patients hospitalized for pancytopenia. There were 56 men and 25 women. Mean hemoglobin, leukocyte and platelet rates were 56,5 +/- 22,7 g/l, 2,1 +/- 0,7.g/l and 56,2 +/- 24,7 g/l respectively. Vitamin deficiency was the most common cause of pancytopenia (49%), followed by hypersplenism (9%), HIV infection (6%) and leishmaniasis (6%). Vitamin-deficient patients had significantly more severe anemia and thrombopenia and significantly higher mean corpuscular volume than patients with pancytopenia related to other causes. Hemoglobin rate lower than 40 g/L and platelet rate lower than 35 G/L showed a positive predictive values of 90% and 100% respectively for a vitamin deficient pancytopenia. Vitamin deficiency is the most frequent etiology of pancytopenia and causes the most severe cytopenia in Djibouti. Rapid vitamin supplementation after minimal etiologic assessment including a myelogram is an effective treatment strategy for this public health problem.

Proliferation of human progenitor cells in a long-term culture system is more efficiently sustained by the addition of Flt-3 ligand or megakaryocyte growth and development factor than by Kit ligand.

INTRODUCTION: We compared the effects of the early-acting growth factors (GF), Flt-3 ligand (FL), c-Kit ligand (KL), and leukemia inhibitory factor (LIF), and the late-acting GF, granulocyte-colony stimulating factor (G-CSF) and megakaryocyte growth and development factor (MGDF), added alone in human long-term marrow culture (LTMC). MATERIALS AND METHODS: The GF were used in primary cultures of mononuclear cells (MNC) and in cocultures of CD34+ cells on murine preestablished MS-5 stromal layers. GF activity was assessed as nonadherent and adherent progenitor cell production and cobblestone area formation at week 5. RESULTS: In this system, only FL, KL, and MGDF significantly stimulated early stages of hematopoiesis, whereas only G-CSF stimulated the proliferation of mature progenitor cells within the granulo-monocyte lineage and no effect was observed with LIF. FL displayed the strongest activity, and MGDF was more efficient than KL, both in primary cultures of MNC and in cocultures of CD34+ cells. However, the stimulatory effects of these GF used alone were dependent on the presence of a stromal layer. CONCLUSION: These LTMC data emphasize the particular roles for FL and MGDF in the stimulation of primitive hematopoiesis.

Persistent decrease in proliferative potential of marrow CD34(+)cells exposed to early-acting growth factors after autologous bone marrow transplantation.

Post-graft hematopoiesis is characterized by long-term quantitative deficiency in marrow progenitor cells in both autologous and allogenic settings. In order to evaluate the function of post-graft progenitor cells, the proliferative capacity of marrow CD34(+) cells was evaluated in 10 patients 6 months after autologous bone marrow transplantation (ABMT) for non-Hodgkin's lymphoma and compared to that of 10 patients before ABMT and 10 normal controls. Immuno-selected CD34(+) cells were cultured for 7 days in liquid serum-free medium with a combination of early-acting GF consisting of stem cell factor, IL-3 and IL-1beta. Clonogenic efficiency of unselected cells for CFU-GM and BFU-E was decreased in post-graft patients compared to pre-graft and control patients. However, clonogenic efficiency of selected CD34(+) cells for CFU-GM was not different in post-graft, pre-graft and control patients but BFU-E values of post-graft patients remained lower than those of control patients. Decreased percentages of CD34(+) CD38(-) cells were observed in both post-graft and pre-graft patients while those of CD34(+) c-kit(+) cells were similar in all three patient groups. After 7-day liquid culture, expansion yields of total progenitor cells were significantly lower in post-graft patients (147 +/- 28%) than in pre-graft (255 +/- 27%) and control patients (246 +/- 23%). Post-graft deficiency in progenitor cell expansion was particularly marked for BFU-E (61 +/- 24%) compared to pre-graft patients (220 +/- 82%) and to controls (349 +/- 82%). These results indicate impaired proliferative potential of marrow CD34(+) cells several months after ABMT involving erythroid progenitor cells and/or commitment towards erythroid lineage from a more immature stage (pre-CFU).

Evaluation of performance of white blood cell reduction filters: an original flow cytometric method for detection and quantification of cell-derived membrane fragments.

BACKGROUND: Contamination of blood products by white blood cells leads to a risk of transmission of infectious agents, particularly abnormal prion protein, the probable causative agent of new-variant Creutzfeldt-Jakob disease. Blood product filtration could reduce this risk, but the filtration systems might generate potentially infectious membrane fragments. We developed an original flow cytometric method that allows the detection and quantification of membrane fragments in filtered products and the evaluation of the quantity of destroyed cells. METHODS: This method has four technical requirements: cytofluorometric acquisition of forward scatter parameters on a log scale, use of a fluorescent aliphatic reporter molecule (PKH26-GL) to identify membrane fragments, quantification with fluorescent beads, and the drawing up of a standard curve on the basis of cells destroyed by freezing/thawing to generate cell debris (i.e., quantity of membrane fragments measured versus quantity of destroyed cells). RESULTS AND CONCLUSIONS: This original method can be used to test new filtration devices and it allows optimization of the filtration process or comparison of different filtration systems. We tested the method with three commercial white cell removal filters. We demonstrated that it is possible to evaluate the filter quality, particularly the likelihood of fragment removal during the filtration process.

Association between the SDF1-3'A allele and high levels of CD34(+) progenitor cells mobilized into peripheral blood in humans.

Some patients unexpectedly fail to mobilize sufficient numbers of haematopoietic progenitor cells (HPCs) into the peripheral blood for autologous transplantation. Considering the important role of the chemokine stromal cell-derived factor 1 (SDF-1) in HPC homing, we investigated a possible relationship between SDF1 gene polymorphism and HPC mobilization capacity in 63 patients with malignancy. Some 67% of the good mobilizers (> or = 50 CD34(+) cells/microl) and only 36% of the intermediate/poor mobilizers were SDF1-3'A allele carriers (P = 0.032). In multivariate analysis, the presence of the SDF1-3'A allele was the only factor predictive of good CD34(+) cell mobilization (P = 0.025). This is the first report showing the involvement of genetic factors for HPC mobilization in humans and suggests a significant role for SDF-1 in this process.

Determination of ochratoxin A in red wine and vinegar by immunoaffinity high-pressure liquid chromatography.

A method is described for the determination of ochratoxin A (OTA) in red wine and vinegar using an acidic chloroform extraction, an immmunoaffinity clean-up step, and a high-performance liquid chromatographic determination with fluorescence detection. The detection limit was estimated at 0.002 microg/liter. The mean recovery factors were found at 91.3 and 96.6% for wine and vinegar, respectively. Thirty-one samples of red wine originating from Mediterranean sea countries and 15 samples of vinegar were examined for the presence of OTA. All red wine samples contained OTA. Seventy-two percent of these samples were found to be contaminated over 0.1 microg/liter. Among them, nine samples contained ochratoxin A in the range of 0.5 to 3.4 microg/liter, 12 samples in the range of 0.10 to 0.50 microg/liter (median: 0.176 microg/liter), and 9 samples in the range of 0.010 to 0.100 microg/liter (median: 0.041 microg/liter). All 15 vinegar samples showed the presence of OTA. The most contaminated ones were three balsamic vinegar samples containing 0.156 microg/liter, 0.102 microg/liter, and 0.252 microg/liter of OTA. In the remaining 12 samples, ochratoxin A levels ranged from 0.008 microg/liter to 0.046 microg/liter (median: 0.012 microg/liter). These data are in good agreement with the hypothesis that wine originating from Southern countries might contain significant OTA concentration and showed the possible occurrence of traces of OTA in vinegar.

Early secondary acute myelogenous leukemia in breast cancer patients after treatment with mitoxantrone, cyclophosphamide, fluorouracil and radiation therapy.

BACKGROUND: The topoisomerase II-targeted drugs, epipodophyllotoxins and anthracyclines, have been shown to induce therapy-related AML (t-AML) characterized by a short latency period after chemotherapy, the absence of prior myelodysplastic syndrome and stereotyped chromosome aberrations. Few reports have been published on patients treated with the anthracenedione mitoxantrone which also targets topoisomerase II. We observed 10 cases of such t-AML over a 7-year-period in breast cancer patients treated with mitoxantrone combined with fluorouracil, cyclophosphamide and regional radiotherapy, and in three cases with vindesine. PATIENTS AND METHODS: We retrospectively analyzed patients referred to our hospital for AML with a past history of polychemotherapy for breast cancer, including mitoxantrone, either as adjuvant (8 patients)/neoadjuvant (1 patient) therapy or for metastatic disease (1 patient). We studied the probability of developing t-AML in a prospective series of 350 patients treated with an adjuvant FNC regimen (mitoxantrone, fluorouracil, cyclophosphamide) and radiation therapy. RESULTS: The median age was 45 years (range 35-67). t-AML developed 13-36 months (median 16) after beginning chemotherapy for breast cancer, and 4-28 months (median 10.5) after ending treatment. As described in t-AML following treatment with epipodophyllotoxins or anthracyclines, we found a majority of FAB M4, M5 and M3 phenotypes (7 of 10), and characteristic karyotype abnormalities that also can be found in de novo AML: breakpoint on chromosome 11q23 (3 patients), inv(16)(p13q22) (2 patients), t(15;17)(q22;q11) (1 patient), t(8;21)(q22;q22) (1 patient) and del(20q)(q11) (1 patient). The prognosis was poor. All patients died of AML shortly after diagnosis. Since two patients had been enrolled in a prospective trial for the treatment of breast cancer which included 350 patients, the probability of developing t-AML was calculated to be 0.7% from 25-40 months, using the Kaplan-Meier method (95%, confidence interval (95% CI): 0.1-4.5). CONCLUSIONS: The combination of mitoxantrone with cyclophosphamide, fluorouracil, and radiation therapy can induce t-AML, as with other topoisomerase II-targeted drugs. Despite a low incidence, the prognosis appears to be poor.

A rapid single-laser flow cytometric method for discrimination of early apoptotic cells in a heterogenous cell population.

A recently reported cytometric method described the possibility of discriminating apoptotic from necrotic cells using FITC-labelled annexin V and propidium iodide (PI). Nevertheless, the brightness of PI-staining and its extensive spectral emission overlap with phycoerythrin (PE) does not permit the study of a subset of a heterogenous cell population with single laser instrumentation. The surface staining of a subset with PE in a heterogenous cell population therefore requires another exclusion dye to detect necrotic cells. We used 7-amino-actinomycin D (7-AAD) that can be excited by the 488 nm argon laser line. 7-AAD emits in the far red range of the spectrum and 7-AAD spectral emission can be separated from the emissions of FITC and PE. The fluorescence parameters allow characterization of necrotic (7-AAD+ annexin V-FITC+ cells), apoptotic (7-AAD-annexin V-FITC+ cells) and viable cells (7-AAD- annexin V-FITC- cells) in a subset of PE+ cells. The value of this method was demonstrated by measuring apoptosis and necrosis in a model of HL-60 cells exposed to different inducers of cell death. The method was validated by fluorescent cell sorting in combination with morphologic examination of the sorted cells. The technique we present is particularly valuable in a clinical setting because it enables rapid multiparameter analysis of necrosis and early apoptosis in combination with cell surface phenotyping with a single laser. We present the effects of haemopoietic growth factor deprivation on myeloid progenitor CD34+ cells as an example of its application.

All-trans-retinoic acid up-regulates CD38 but not c-Kit antigens on human marrow CD34+ cells without recruitment into cell cycle.

Retinoids, especially all-trans-retinoic acid (ATRA), are well known for their differentiating activity on HL-60 cells. Moreover ATRA induces CD38 antigen overexpression on these cells. In this study we examined the effects of ATRA on purified normal CD34+ cells from adult human marrows incubated with ATRA (1 microM) or stem cell factor (SCF) after 7 d liquid cultures in serum-deprived medium. Before and after the incubation, CD34+ cells were studied by flow cytometry to evaluate the cell-surface expression of CD38 and c-Kit antigens and the cycle status of these cells using high-resolution analysis (DNA content v Ki-67 antigen expression) to clarify the functional meaning of antigenic variations. When compared with control cultures, ATRA-treated cells displayed changes in their immunophenotypic profile. Particularly relevant was the up-regulation of CD38 antigen with a mean (+/-SEM) fold increase of 21 +/- 0.1 (P=0.028) for geometric mean fluorescence intensity (GMFI), without modulation of c-Kit expression. SCF only down-regulated expression of c-Kit with a fold decrease of 4.6 +/- 0.9 for GMFI (P=0.043). Unlike SCF, ATRA did not induce CD34+ cells to entry into cell cycle despite increased levels of surface CD38 antigen. Moreover morphological and functional assays did not argue for an ATRA-induced maturation process. Contrary to steady-state cells, CD34+ cells treated with pharmacological doses of ATRA alone displayed CD38 over-expression without change in c-Kit levels and cycle status, suggesting an absence of maturation pressure.

Cytokines released in vitro by stromal cells from autologous bone marrow transplant patients with lymphoid malignancy.

Marrow stromal cells of patients treated by autologous bone marrow transplantation (ABMT) for malignancies have been assessed for their ability to secrete granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), transforming growth factor beta1 (TGFbeta1) and macrophage inflammatory protein-1alpha. (MIP-1alpha). Long-term marrow cultures were established from 10 patients prior to and 3 months after ABMT, from 7 patients 1 yr after ABMT and from 11 controls. Cytokines in culture supernatants of stromal layers (SL) were evaluated by enzyme-linked immunosorbent assay (ELISA). Significant differences between patient groups and controls were apparent in baseline production of GM-CSF, SCF, MIP-1alpha and TGFbeta1. After IL-1beta addition in cultures, G-CSF production was reduced in pretransplant and post-transplant patients compared to controls. The production of TGFbeta1, LIF, IL-6 and more particularly SCF were reduced in post-transplant patients, while elevated levels of GM-CSF and MIP-1alpha were observed in these patients only when the values were corrected for the number of cells growing in the SL. These results indicate a prolonged stromal defect in growth factor production following ABMT for the early-stage acting cytokines IL-6, LIF and SCF as well as for G-CSF, but not for GM-CSF, while the production of the 2 inhibitors shows different pathways.

In vitro effect of stem cell factor on human clonogenic marrow progenitors after myeloablative treatments.

Abnormal hematopoiesis, including a deficiency of marrow progenitors and particularly of erythroid progenitors, has been described after autologous stem cell transplantation (ASCT), persisting for several years. In order to explain this deficiency, a resistance of marrow progenitors to stem cell factor (SCF) after ASCT was investigated. Marrow samples were harvested from pregraft patients at graft collection prior to ASCT, transplanted patients 6-24 months after high-dose therapy and control patients. CD34+ cells were cultured in a serum-free clonogenic assay with increasing doses of SCF. The clonogenic efficiency without SCF was lower for BFU-E in treated groups than in controls, whereas it was not different for CFU-GM. With increasing doses of SCF a dose-dependent effect was found on the numbers of both CFU-GM and BFU-E in all groups, although the maximal number of BFU-E remained lower in treated groups. However, the SCF dose that induced 50% of maximal BFU-E growth (D50) was similar in all groups. Furthermore, a dose-dependent effect on the size of BFU-E was found in all groups, with no difference in the proportion of large colonies. Thus, clonogenic erythroid progenitors from patients who have received myelotoxic treatments remain sensitive to SCF, with no evidence for a chemotherapy-related resistance.

Changes in the functional capacity of marrow stromal cells after autologous bone marrow transplantation.

Marrow stromal cells were evaluated several months after autologous BMT for their capacity to support both normal hemopoiesis and secrete the main growth factors involved in its control, G-CSF, GM-CSF, IL-3 and SCF. Stromal layers (SL) were obtained by long-term marrow cultures (LTMC) established from 15 patients (9 with hematologic malignancies and 6 with solid tumors) 3 months after autologous BMT and were compared to pre-graft patients. After irradiation, both post-graft and pre-graft SL were recharged with the same inoculum of normal marrow cells. As compared to pre-graft values, CFU-GM production on post-graft SL was significantly increased during the first 2 weeks of culture whereas it was decreased from week 3 to week 8. These findings were only observed in patients with hematologic malignancies and not in patients with solid tumors. Growth factor secretion was evaluated by ELISA in the supernatants of unstimulated and IL-1-stimulated SL from 10 post-graft patients, 13 pre-graft patients and 5 normal controls. In any group of patients, IL-3 was undetectable either spontaneously or after IL-1-stimulation. As compared to controls, secretion by IL-1-stimulated SL was not different for GM-CSF in pre- and post-graft patients but tended to be decreased for G-CSF in post-graft patients. SCF secretion, which was not induced by IL-1, appeared dramatically decreased in both pre- and post-graft patients. The capacity of post-graft SL to support CFU-GM growth in LTMC was correlated at week 1 with G-CSF secretion and from week 3 to week 8 with SCF secretion. These results suggest that microenvironment remains qualitatively damaged several months after BMT involving a decreased capacity both to support early hemopoiesis and to secrete SCF, particularly in patients grafted for hemopoietic malignancies.

The mechanisms involved in the impairment of hematopoiesis after autologous bone marrow transplantation.

Hematopoiesis after autologous bone marrow transplantation (BMT) is characterized by a prolonged and severe deficiency of marrow progenitors for several years, especially of erythroid and megakaryocyte progenitors, while the peripheral blood cells and marrow cellularity have reached relatively normal values within a few weeks. These anomalies are comparable to those reported for allogeneic BMT, despite the absence of any allo-immune reaction or post-graft immunosuppressive therapy. Post-graft hematopoietic impairment is the consequence of quantitative and qualitative changes involving both stem cell and stromal compartments which are expressed by an impaired capacity of stem cell self-renewal and commitment towards erythroid and megakaryocytic lineages. Besides the toxicity of conditioning regimens, hematopoietic reconstitution using autologous grafts is particularly dependent on a combination of factors related to the patient, such as underlying disease and pre-graft chemotherapy regimens, and to the graft processing itself, such as in vitro purging with chemotherapeutic agents.

Granulocyte-macrophage colony-stimulating factor accelerates hematopoietic recovery after autologous bone marrow or peripheral blood progenitor cell transplantation and high-dose chemotherapy for lymphoma.

The use of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) as an adjunct to autologous bone marrow transplantation (ABMT) or peripheral blood progenitor cell (PBPC) transplantation was evaluated in 59 lymphoma patients. Patients were divided into three groups. In group I (n = 21) patients received rhGM-CSF (5 micrograms/kg daily) at the time of PBPC collection and during the recovery phase post-infusion. In group II (n = 12) patients received rhGM-CSF as an adjunct to ABMT. In group III (n = 26) they were grafted with bone marrow without rhGM-CSF. Administration of rhGM-CSF (groups I and II) significantly reduced the time to myeloid engraftment, the number of febrile days and the median duration of antibiotics administration and of hospital stay when compared with the group in which patients did not receive rhGM-CSF. The only difference between ABMT and PBPC, given with rhGM-CSF support, was observed in the duration of hospitalization (group I > group II, P < 0.05). These data show that rhGM-CSF is highly effective in reducing the duration of aplasia following BMT and PBPC transfusion, and there appears to be little difference in efficacy between these techniques, provided that patients also receive rhGM-CSF.