Two novel variants of uncertain significance in GP9 associated with Bernard-Soulier syndrome: Are they true mutations?

Bernard-Soulier syndrome (BSS) is an autosomal recessive major thrombocytopathy, the symptoms of which are mainly marked by mucocutaneous bleeding. This rare disease, initially described in the 1970s, is the result of an abnormal formation of the glycoprotein complex Ib-IX-V (GP Ib-IX-V), a platelet receptor of von Willebrand factor. A large number of mutations, sometimes involving the GP9 gene, have been described as possibly responsible for the disease. We report here the case of a BSS patient who presented with persistent thrombocytopenia (31x109/L) and decreased surface expression of GPIb-IX-V on large platelets with anisocytosis. Thorough molecular analyses disclosed two previously unreported GP9 variants, respectively c.230T>A (p.Leu77Gln) and c.255C>A (p.Asn85Lys). Both are likely to modify the conformation of GP-IX interactions with other glycoproteins of the Ib-IX-V complex and thus proper expression of this complex on the membrane of platelets.

Microfluidics in flow cytometry and related techniques.

Technological advances in laboratory automation are now well understood and applied as they considerably improved the speed and robustness of haematological laboratory data, in the companion fields of blood analyzers and flow cytometry. Still rather confidential is the field of microfluidics, mostly confined so far to academic settings and research laboratories. The literature in the field of microfluidics is growing and applications in hematology range from cell counting to flow cytometry, cell sorting, or ex vivo testing. A literature search allows to identify many innovative solutions developed to master the specific physics of fluid movements in microchips. Miniaturization also dwells on findings that have emerged from different areas such as electronics and nanoengineering. This review proposes an overview of the major principles guiding developments in microfluidics and describes a necessarily limited and nonexhaustive series of specific applications. Readers are strongly encouraged to consult the documents referred to in the references section to learn more about this world knocking at our door and possibly liable to revolutionize our profession of hematology biologists in a not so far future.

Lymphocyte expansion after unrelated cord blood allogeneic stem cell transplantation in adults.

Limited information is available regarding the incidence and features of lymphocyte expansions after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Large granular lymphocytes (LGL) expansions have been reported after bone marrow or peripheral blood, but not after unrelated cord blood (UCB) allo-HSCT, associated with indolent clinical courses and favorable outcomes. Here, we considered 85 recipients of UCB allo-HSCT to more broadly define the impact of lymphocytosis, not limited to LGL. Sustained lymphocytosis was observed in 21 (25%) patients at a median onset of 12.6 months and with a median duration of 12 months. Immunophenotypic analysis showed predominantly CD8+ T and/or polyclonal B-cell expansions. Three patients only had monoclonal T-cell expansion. CMV reactivation was significantly more frequent in the group of patients with lymphocytosis (76% vs 28%, P=0.0001), but was not associated with survival. Conversely, 2-year disease-free survival and overall survival were significantly higher for lymphocytosis patients (85% vs 55%, P=0.01 and 85% vs 63%, P=0.03, respectively). In conclusion, expansion of T or B lymphocytes after UCB allo-HSCT in adults is not a rare event. Although occurring relatively late after transplant, this feature is predictive of a better outcome for the patients.

New parameters on the hematology analyzer XN-10 (SysmexTM) allow to distinguish childhood bacterial and viral infections.

INTRODUCTION: Complete blood counts (CBC) performed for infected children admitted for fever mostly disclose leukocytosis. Yet, the recently developed XN-10® provides novel CBC parameters which could be useful to ascertain infection and discriminate between bacterial and viral etiologies. These were the main objectives of the study presented here. METHODS: Blood samples from 90 children, 1 month to 5 years old, admitted to an emergency unit for fever benefited from a CBC, C-reactive protein, and procalcitonin assays. For 58, a bacterial infection was documented while a viral cause was disclosed for 32. Concomitantly, 30 healthy children of the same age range were selected as a control group. RESULTS: Complete blood counts parameters and leukocyte differentials allowed to statistically significantly disclose infection, compared to reference children, in the age group of 1-5 years old. Among the eight novel discriminant parameters, a particular interest appeared for Neutr-RI and Delta-He. They both were successfully incorporated in a score together with age and immature granulocytes (IG). ROC curves and AUCs were calibrated using a Hosmer-Lemeshow test. Moreover, novel lymphocyte parameters allowed to segregate bacterial and viral infections in the whole group of 90 febrile children. CONCLUSION: Complete blood counts is the most broadly performed rapid laboratory investigation. Here, we show that XN-10® provides complementary information allowing to confirm infection in febrile children, moreover discriminating between bacterial or viral origin.

Acute lymphoblastic leukemia relapsing after first-line pediatric-inspired therapy: a retrospective GRAALL study.

The outcome of adult patients with Philadelphia chromosome-negative acute lymphoblastic leukemia (Ph- ALL) relapsing after pediatric-inspired front-line therapy is ill known. Here 229 relapsing Ph- ALL younger adults (18-63 years) treated within the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003/-2005 trials were considered. Salvage regimens consisted of potentially curative therapies in 194 cases, low-intensity therapies in 21, allogeneic stem cell transplant (allo-SCT) in 6 and best supportive care in 8. Overall, 77 patients received allo-SCT after relapse. The median follow-up was 3.1 years. A second complete remission (CR2) was achieved in 121 patients (53%). In multivariate analysis, only younger age <45 years (P=0.008) and CR1 duration ⩾18 months (P=0.009) predicted CR2. Overall survival (OS) at 2 and 5 years was 19.3% (14-24%) and 13.3% (8-18%), respectively. In CR2 patients, disease-free survival (DFS) at 2 and 5 years was 29.0% (21-38%) and 25% (17-33%). In multivariate analysis, CR1 duration ⩾18 months and allo-SCT after relapse were associated with longer DFS (P<0.009 and P=0.004, respectively) and longer OS (P=0.004 and P<0.0001, respectively). In conclusion, although younger adults relapsing after pediatric-inspired ALL therapies retain a poor outcome, some of them may be cured if CR1 duration ⩾18 months and if allo-SCT can be performed in CR2. New therapies are definitely needed for these patients.

Impact of diet and nutraceutical supplementation on inflammation in elderly people. Results from the RISTOMED study, an open-label randomized control trial.

BACKGROUND & AIMS: Eating habits may influence the life span and the quality of ageing process by modulating inflammation. The RISTOMED project was developed to provide a personalized and balanced diet, enriched with or without nutraceutical compounds, to decrease and prevent inflammageing, oxidative stress and gut microbiota alteration in healthy elderly people. This paper focused on the effect on inflammation and metabolism markers after 56 days of RISTOMED diet alone or supplementation with three nutraceutical compounds. METHODS: A cohort of 125 healthy elderly subjects was recruited and randomized into 4 arms (Arm A, RISTOMED diet; Arm B, RISTOMED diet plus VSL#3 probiotic blend; Arm C, RISTOMED diet plus AISA d-Limonene; Arm D, RISTOMED diet plus Argan oil). Inflammatory and metabolism parameters as well as the ratio between Clostridium cluster IV and Bifidobacteria (CL/B) were collected before and after 56 days of dietary intervention, and their evolution compared among the arms. Moreover, participants were subdivided according to their baseline inflammatory parameters (erythrocytes sedimentation rate (ESR), C-Reactive Protein, fibrinogen, Tumor Necrosis Factor-alfa (TNF-α), and Interleukin 6) in two clusters with low or medium-high level of inflammation. The evolution of the measured parameters was then examined separately in each cluster. RESULTS: Overall, RISTOMED diet alone or with each nutraceutical supplementation significantly decreased ESR. RISTOMED diet supplemented with d-Limonene resulted in a decrease in fibrinogen, glucose, insulin levels and HOMA-IR. The most beneficial effects were observed in subjects with a medium-high inflammatory status who received RISTOMED diet with AISA d-Limonene supplementation. Moreover, RISTOMED diet associated with VSL#3 probiotic blend induced a decrease in the CL/B ratio. CONCLUSIONS: Overall, this study emphasizes the beneficial anti-inflammageing effect of RISTOMED diet supplemented with nutraceuticals to control the inflammatory status of elderly individuals.

Flow cytometry in hematological nonmalignant disorders.

Multiparameter flow cytometry (MFC) has become an integral part of the diagnosis and classification of hematological malignancies. However, several nonmalignant or premalignant disorders may benefit from this technology in hematology laboratories. This review provides information on the normal immunophenotypic characteristics of peripheral blood leukocyte subsets and their modifications in several clinical conditions. The usefulness of MFC and the specific markers that can be investigated in hyperlymphocytosis, infection, hypereosinophilia, paroxysmal nocturnal hemoglobinuria, and large granular lymphocyte disorders is described. Mention is also made of the developments of MFC for analyses of red blood cells or platelets.

Viability and stress protection of chronic lymphoid leukemia cells involves overactivation of mitochondrial phosphoSTAT3Ser727.

Chronic lymphoid leukemia (CLL) is characterized by the accumulation of functionally defective CD5-positive B lymphocytes. The clinical course of CLL is highly variable, ranging from a long-lasting indolent disease to an unpredictable and rapidly progressing leukemia requiring treatment. It is thus important to identify novel factors that reflect disease progression or contribute to its assessment. Here, we report on a novel STAT3-mediated pathway that characterizes CLL B cells-extended viability and oxidative stress control. We observed that leukemic but not normal B cells from CLL patients exhibit constitutive activation of an atypical form of the STAT3 signaling factor, phosphorylated on serine 727 (Ser(727)) in the absence of detectable canonical tyrosine 705 (Tyr705)-dependent activation in vivo. The Ser(727)-phosphorylated STAT3 molecule (pSTAT3Ser(727)) is localized to the mitochondria and associates with complex I of the respiratory chain. This pSer(727) modification is further controlled by glutathione-dependent antioxidant pathway(s) that mediate stromal protection of the leukemic B cells and regulate their viability. Importantly, pSTAT3Ser(727), but neither Tyr705-phosphorylated STAT3 nor total STAT3, levels correlate with prolonged in vivo CLL B cells survival. Furthermore, STAT3 activity contributes to the resistance to apoptosis of CLL, but not normal B cells, in vitro. These data reveal that mitochondrial (Mt) pSTAT3Ser(727) overactivity is part of the antioxidant defense pathway of CLL B cells that regulates their viability. Mt pSTAT3Ser(727) appears to be a newly identified cell-protective signal involved in CLL cells survival. Targeting pSTAT3Ser(727) could be a promising new therapeutic approach.

Methodological aspects of minimal residual disease assessment by flow cytometry in acute lymphoblastic leukemia: A French multicenter study.

Background: Minimal residual disease (MRD) assessment provides a powerful prognostic factor for therapeutic stratification in acute lymphoblastic leukemia (ALL). Multiparameter flow cytometry (MFC) has the potential for a rapid and sensitive identification of high risk patients. Our group has previously published that MRD levels analyzed by clone specific Ig/TcR-QPCR and MFC were concordant at a sensitivity of 10-4 . Here we report the MFC methodological aspects from this multi-center experience. Methods: MRD was assessed by MFC in 1030 follow-up samples from 265 pediatric and adult patients with de novo ALL treated in the FRALLE, EORTC or GRALL clinical trials. MRD assessment as applied by the eight participating MFC laboratories is described in detail regarding cell preparation, leukemia-associated immunophenotype (LAIP) markers and data analysis. Samples were obtained from bone marrow (BM) and peripheral blood (PB). Immunostaining was performed after erythrocyte lysis or Ficoll enrichment. Results: This study confirms the applicability of MFC-based MRD assessment in 97% of patients with ALL at the 10-4 cut-off. MRD values after Ficoll enrichment and erythrocyte lysis were found comparable. Higher MRD values were obtained in BM than in PB, especially for B-lineage ALL. Conclusions: Measurement of MRD by MFC at the 10-4 cut-off is applicable within a few hours for almost all patients and using a comparable analytical strategy allows for multicenter collaborative studies. The method can be introduced in a strategy aimed at defining the risk of failure of patients with childhood or adult ALL. © 2014 Clinical Cytometry Society.

Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes-proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS.

Definite progress has been made in the exploration of myelodysplastic syndromes (MDS) by flow cytometry (FCM) since the publication of the World Health Organization 2008 classification of myeloid neoplasms. An international working party initiated within the European LeukemiaNet and extended to include members from Australia, Canada, Japan, Taiwan and the United States has, through several workshops, developed and subsequently published consensus recommendations. The latter deal with preanalytical precautions, and propose small and large panels, which allow evaluating immunophenotypic anomalies and calculating myelodysplasia scores. The current paper provides guidelines that strongly recommend the integration of FCM data with other diagnostic tools in the diagnostic work-up of MDS.