[Severe thrombopenia with iron deficiency anemia: about a case and literature review]. / Thrombopénie sévère d'origine ferriprive : à propos d'un cas et revue de la littérature.

Iron deficiency is the leading cause of anemia worldwide, affecting approximately 600 million individuals. Once established, it typically manifests as a hypochromic microcytic anemia, the severity of which varies depending on the degree of deficiency. This anemia is frequently associated with thrombocytosis, but the presence of associated thrombocytopenia is much rarer. Here, we report a case of severe iron deficiency with an atypical presentation of bicytopenia, involving both severe anemia and profound thrombocytopenia, which rapidly resolved following iron supplementation. We then discuss the hypotheses that exist to explain the link between iron deficiency and regulation of thrombopoiesis.

Stability over time of immature platelet fraction and comparison between EDTA and citrated whole blood samples.

BACKGROUND: Immature platelets (IP) are the youngest circulating platelets, released from megakaryocytes, and demonstrating increased dimensions, significant RNA content, and enhanced activity. Immature platelet research focuses on a differential diagnostic help in patients with thrombocytopenia. The objectives of this study were to compare the variability of IP in citrate and EDTA samples, and to determine stability over time. METHODS: Fifty-six patients were included for comparison between EDTA and citrate whole blood sample collection. Among the patients, 28 had thrombocytopenia (platelet count < 150G/L). Platelet measurement impedancemetry and fluorimetry were performed with Sysmex XN-9000. The immature platelet fraction (IPF) and absolute immature platelet count (A-IPC) were determined with a fluorescent method. RESULTS: The mean value of platelet count with fluorescence was, in EDTA sample, 215 ± 171 and, in citrate sample, 153 ± 118 G/L. No significant difference was observed between IPF between EDTA and citrate (7.74 ± 6.68% vs. 8.45 ± 7.37%, p = 0.69), respectively. With the Bland-Altman analysis, the mean difference in the EDTA sample, between 1 and 24 h, was 8.06 ± 6.96% and 8.73 ± 7.12% for IPF, whereas in the citrate sample, between 1 and 6 h, it was 8.60 ± 7.29% and 7.54 ± 6.97%, for IPF. Comparing 1 h EDTA sample with 6 h citrate sample, the variance ratio was 0.974 (95% CI: 0.864-1.084) in IPF. CONCLUSIONS: We confirmed the potential to conduct IP measurements up to 24 h in the EDTA sample and IPF measurements in the citrate sample for up to 6 h. These results may be useful for the use of IPF, which is a promising parameter whose interest in clinical practice and standardization is not yet well defined.

Assessment of Reticulocyte and Erythrocyte Parameters From Automated Blood Counts in Vaso-Occlusive Crisis on Sickle Cell Disease.

Sickle cell disease is a complex genetic disease involving cell adhesion between red blood cells, white blood cells, platelets and endothelial cells, inducing painful vaso-occlusive crisis (VOC). We assessed reticulocyte and erythrocyte parameters in a cohort of confirmed SCD patients, and investigated whether a combination of these routine laboratory biomarkers of haemolysis could be used to predict VOC development. Reticulocyte and erythrocyte parameters were evaluated using the Sysmex XN-9000 analyser. A total of 98 patients with SCD were included, 72 in steady state and 26 in VOC. Among the 72 patients in steady state, 22 developed a VOC in the following year (median: 3 months [2-6]). The following parameters were increased in SCD patients with VOC development compared to SCD patients without VOC development in the following year: reticulocyte count (94.6 109/L [67.8-128] vs. 48.4 109/L [24.9-87.5]), immature reticulocyte count (259 109/L [181-334] vs. 152 109/L [129-208]) reticulocyte/immature reticulocyte fraction (IRF) ratio (6.63 109/(L*%) [4.67-9.56] vs. 4.94 109/(L*%) [3.96-6.61]), and medium fluorescence reticulocytes (MFR) (19.9% [17.4-20.7] vs. 17.1% [15.95-19.75]). The association of a reticulocyte count of >189.4 109/L and an MFR of >19.75% showed a sensitivity of 81.8% and a specificity of 88% to predict VOC development in the following year. Based on our findings, a combination of routine laboratory biomarkers, as reticulocyte count, immature reticulocyte count and fluorescent reticulocyte fraction at steady state, could be used to predict VOC development in SCD.

The Use of Ferritin to Identify Critically Ill Patients With Secondary Hemophagocytic Lymphohistiocytosis.

OBJECTIVES: Thrombocytopenia is a common, multifactorial, finding in ICU. Hemophagocytosis is one of the main explanatory mechanisms, possibly integrated into hemophagocytic lymphohistiocytosis syndrome, of infectious origin in the majority of cases in ICU. The hemophagocytic lymphohistiocytosis is probably underdiagnosed in the ICU, although it is associated with dramatic outcomes. The main objectives of this work were to identify the frequency of secondary hemophagocytic lymphohistiocytosis, and the main prognostic factors for mortality. DESIGN/SETTING: We conducted a retrospective observational study in all adult patients admitted with suspected or diagnosed hemophagocytic lymphohistiocytosis, between January 1, 2000, and August 22, 2012. PATIENTS: A total of 106 patients (42%) had significant hemophagocytosis on bone marrow examination, performed for exploration of thrombocytopenia, bicytopenia, or pancytopenia. MEASUREMENTS AND MAIN RESULTS: The median age was 56 (45-68) and the median Simplified Acute Physiology Score 2 was 55 (38-68). The main reason for ICU admission was hemodynamic instability (58%), predominantly related to sepsis (45% cases). The main precipitating factor found was a bacterial infection in 81 of 106 patients (76%), including 32 (30%) with Escherichia coli infection. Forty six of 106 patients (43%) died in the ICU. They were significantly older, had higher Simplified Acute Physiology Score 2, plasma lactate deshydrogenase bilirubin, and serum ferritin. The fibrinogen and the percentage of megakaryocytes were significantly lower in nonsurvivors when compared with survivors. In multivariate analysis, only serum ferritin significantly predicted death related to hemophagocytosis. A serum ferritin greater than 2,000 µg/L predicted death with a sensitivity of 71% and a specificity of 76%. A decreased percentage of megakaryocytes also predicted patient death in the ICU. CONCLUSIONS: Hemophagocytosis is common in thrombocytopenic patients with sepsis, frequently included in a postinfectious hemophagocytic lymphohistiocytosis setting. Our study reveals that ferritin could be a reliable prognostic marker in these patients, and hold particular interest in discussing a specific treatment for hemophagocytic lymphohistiocytosis.

Spontaneous remission in three cases of AML M5 with NPM1 mutation.

Patients with NPM1-mutated AML M5 who develop spontaneous remission (SR) after antibiotic therapy at diagnosis seem to form a favorable prognosis and chemo sensitive subtype. We report three cases of AML M5 patients with the same genotype that experienced transient SR and are now leukemia free after standard treatment.

Survival of cord blood haematopoietic stem cells in a hyaluronan hydrogel for ex vivo biomimicry.

Haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs) grow in a specified niche in close association with the microenvironment, the so-called 'haematopoietic niche'. Scaffolds have been introduced to overcome the liquid culture limitations, mimicking the presence of the extracellular matrix (ECM). In the present study the hyaluronic acid scaffold, already developed in the laboratory, has been used for the first time to maintain long-term cultures of CD34⁺ haematopoietic cells obtained from human cord blood. One parameter investigated was the impact on ex vivo survival of CD34⁺ cord blood cells (CBCs) on the hyaluronic acid surface, immobilized with peptides containing the RGD motif. This peptide was conjugated by coating the hyaluronan hydrogel and cultured in serum-free liquid phase complemented with stem cell factor (SCF), a commonly indispensable cytokine for haematopoiesis. Our work demonstrated that these hyaluronan hydrogels were superior to traditional liquid cultures by maintaining and expanding the HPCs without the need for additional cytokines, and a colonization of 280-fold increment in the hydrogel compared with liquid culture after 28 days of ex vivo expansion.

Combination of cellular population data and CytoDiff analyses for the diagnosis of lymphocytosis.

BACKGROUND: Differentials with moderate lymphocytosis are common in hematology laboratories and it is important in these cases to discriminate monoclonal from reactive lymphocytosis (RL). Blood smear reflex examination is dependent of the expertise of a cytologist, time-consuming and not always informative. Therefore, rapid and easy orientation parameters are clearly needed to discriminate malignant from RL. METHODS: The differential performed by the Beckman-Coulter analyzers is based on the determination of three parameters (volume, conductivity and scatter of the cell subpopulations) called cellular population data (CPD). This study evaluated CPD in 332 patients with a typical B-chronic lymphocytic leukemia (B-CLL), 90 patients with other B-lymphoproliferative diseases (OLPD) and 55 patients with a proven RL, and established a discriminating protocol to identify these pathologies. Secondly, this approach was evaluated in a prospective study including 102 patients with lymphocyte counts above 3.5 × 10(9)/L and in each case the diagnosis suggested by CPD was compared with conventional flow cytometry (FC) analysis and that obtained using CytoDiff reagent, a combination of six antibodies/five colors which performs a rapid WBC differential by FC. RESULTS: Lymphocyte anisocytosis was observed for malignant and RL. A low lymphocyte volume identifies monoclonal B-cell lymphocytosis and classical B-CLL. CytoDiff analysis is helpful when lymphocyte volume is in the normal range. A ratio B-Ly/total Ly count >0.32 is suggestive of a B-malignancy, whereas a non-cytotoxic T-lymphocyte count above 2.43 × 10(9)/L suggests RL. CONCLUSIONS: The analysis of CPD in combination with CytoDiff analysis shows promise for the rapid and accurate identification of lymphocyte pathologies in routine practice.

S(mu) mutation patterns suggest different progression pathways in follicular lymphoma: early direct or late from FL progenitor cells.

Follicular lymphoma (FL) is a B-cell malignancy characterized by the t(14;18) translocation. Although sensitive to treatment, the disease remains incurable and the reason why tumor cells invariably evade treatment, leading to clinical relapse, is still unknown. Here, we tracked the clonal history of tumor cells by studying mutations introduced by activation-induced cytidine deaminase on the switch mu region of the der(14)t(14;18) during the early phase of the class-switch recombination (CSR) process. We observed frequent intraclonal variations, suggesting that CSR often remains active after the acquisition of the fully transformed phenotype. However, mutations only rarely accumulated over time, but instead showed complex evolutionary scenarios and 2 different progression pathways. The first pathway was a direct and rapid evolution from the dominant clone. The second was indirect, arising from earlier subclones usually after years of remission. A better understanding of these mechanisms might influence the future choice of treatment strategies.

Detection of somatic quantitative genetic alterations by multiplex polymerase chain reaction for the prediction of outcome in diffuse large B-cell lymphomas.

BACKGROUND: Genomic gains and losses play a crucial role in the development of diffuse large B-cell lymphomas. High resolution array comparative genomic hybridization provides a comprehensive view of these genomic imbalances but is not routinely applicable. We developed a polymerase chain reaction assay to provide information regarding gains or losses of relevant genes and prognosis in diffuse large B-cell lymphomas. DESIGN AND METHODS: Two polymerase chain reaction assays (multiplex polymerase chain reaction of short fluorescent fragments, QMPSF) were designed to detect gains or losses of c-REL, BCL6, SIM1, PTPRK, MYC, CDKN2A, MDM2, CDKN1B, TP53 and BCL2. Array comparative genomic hybridization was simultaneously performed to evaluate the sensitivity and predictive value of the QMPSF assay. The biological and clinical relevance of this assay were assessed. RESULTS: The predictive value of the QMPSF assay for detecting abnormal DNA copy numbers ranged between 88-97%, giving an overall concordance rate of 92% with comparative genomic hybridization results. In 77 cases of diffuse large B-cell lymphomas, gains of MYC, CDKN1B, c-REL and BCL2 were detected in 12%, 40%, 27% and 29%, respectively. TP53 and CDKN2A deletions were observed in 22% and 36% respectively. BCL2 and CDKN2A allelic status correlated with protein expression. TP53 mutations were associated with allelic deletions in 45% of cases. The prognostic value of a single QMPSF assay including TP53, MYC, CDKN2A, SIM1 and CDKN1B was predictive of the outcome independently of the germinal center B-cell like/non-germinal center B-cell like subtype or the International Prognostic Index. CONCLUSIONS: QMPSF is a reliable and flexible method for detecting somatic quantitative genetic alterations in diffuse large B-cell lymphomas and could be integrated in future prognostic predictive models.

Recurrent disruption of the Imu splice donor site in t(14;18) positive lymphomas: a potential molecular basis for aberrant downstream class switch recombination.

t(14;18) positive lymphomas are mature germinal center B-cell neoplasms. In agreement with this cellular origin, most have somatically mutated immunoglobulin variable genes and the IGH@ locus has almost always been reorganized by class switch recombination (CSR). However, contrasting with normal B-cells, a majority of cases still express an IgM while the constant genes are normally rearranged only on the non-productive allele. Concurrently, aberrant intra-allelic junctions involving downstream switch regions, with a lack of engagement of the switch mu (Smu), often accumulate on the functional alleles, suggesting some recurrent CSR perturbation during the onset of the disease. To clarify these surprising observations, we addressed the accessibility of the Smu to the CSR machinery in a large series of patients by characterizing the mutations that are expected to accumulate at this place upon CSR activation. Our data indicate that the Smu is mutated in a large majority of cases, often on both alleles, indicating that these cells usually reach a differentiation stage where CSR is activated and where this region remains accessible. Interestingly, we also identified a significant cluster of mutations at the splicing donor site of the first exon of the Smu germline transcripts, on the functional allele. This location suggests a possible relation with CSR perturbations in lymphoma and the clustering points to a probable mechanism of selection. In conclusion, our data suggest that an acquired mutation at the splicing donor site of the Smu transcripts may participate in the selection of lymphoma cells and play a significant role during the onset of the disease.

Intense paraneoplastic neutrophilic leukemoid reaction related to a G-CSF-secreting lung sarcoma.

A white blood cell count more than 50 x 10(9)/l, not related to bone marrow involvement, is termed leukemoid reaction. We report on the first case of an undifferentiated sarcoma of the lung associated with an intense paraneoplastic neutrophilic leukemoid reaction related to the production of granulocyte colony-stimulating factor (G-CSF). A radiography and a computed tomography scan of the chest revealed a well-limited voluminous and heterogeneous low-density mass of the left lung. The patient died of multiorgan failure related to uncontrolled progressive tumor growth after admission and two cycles of chemotherapy. The patient's G-CSF serum concentration was dramatically elevated (6,538 pg/ml) compared to serum levels observed in normal controls and patients with elevated leukocytosis (31 and 387 pg/ml, respectively). The G-CSF concentration dramatically increased after the first cycle of chemotherapy and during the subsequent neutropenia, as a result of the tumor lyses as well as of disruption of the physiological negative feedback mechanism. Adjunction of the patient's serum to CD34+ cell cultures induced a 12.3-fold increase in CD15+ cells, demonstrating the serum's capacity to induce myeloid differentiation.

MALT1 is deregulated by both chromosomal translocation and amplification in B-cell non-Hodgkin lymphoma.

The MALT1 gene was identified through its involvement in t(11;18)(q21;q21), seen in 30% of cases of mucosa-associated lymphoid tissue (MALT) lymphoma. Here, we show that deregulated MALT1 expression may occur in B-cell non-Hodgkin lymphoma (B-NHL) of various histologic subtypes either through translocation to the immunoglobulin heavy chain (IGH) locus or by genomic amplification. First, 2 cases, one case of MALT lymphoma and another of aggressive marginal zone lymphoma (MZL) with t(14;18)(q32;q21), cytogenetically identical to the translocation involving BCL2, were shown by fluorescence in situ hybridization (FISH) to involve MALT1, which lies about 5 Mb centromeric of BCL2. Molecular cloning of both by long-distance inverse polymerase chain reaction showed breakpoints lying 1 to 2 kilobase (kb) centromeric of the first 5' MALT1 exon; both cases showed MALT1 overexpression at either RNA or protein levels. Second, we examined the structure and gene expression profile of genomic amplifications involving 18q21 in a panel of 40 B-NHL cell lines using comparative genomic hybridization to microarrays (array CGH) and gene expression profiling techniques. Using array CGH, 2 peaks of genomic amplification were observed, one centered around BCL2 and the other around MALT1. Ofthe 3 cell lines with MALT1 amplification, 2 showed MALT1 overexpression as assessed by gene profiling, quantitative reverse transcription-polymerase chain reaction (QRT-PCR), and Western blotting. To determine if comparable events occurred in primary MALT and splenic MZL tumors, 40 cases were analyzed by FISH or QRT-PCR; genomic amplification and MALT1 overexpression were seen in 2 cases. Together, these data implicate MALT1 as a dominant oncogene that may play a role in the pathogenesis of B-NHL.

Eosinophilic leukemia associated with t(2;5)(p23;q31).

Chromosomal aberrations have been reported in most malignant hematopoietic disorders such as acute or chronic myeloid leukemia, acute lymphoid leukemia, and myelodysplastic syndromes. Eosinophilic leukemia is a rare hematologic malignancy difficult to distinguish from other forms of idiopathic hypereosinophilic syndrome, so that the diagnosis is often made by exclusion, unless cytogenetic abnormalities can be demonstrated in bone marrow cells. We describe a patient with eosinophilic leukemia whose cytogenetic study shows a t(2;5)(p23;q31). Initial data could suggest a clonal eosinophilia, with an hepatosplenomegaly, severe pancytopenia, and a high level of blood and medullar eosinophilia.