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Etat de la question Le syndrome de Richter (SR) correspond a la transformation d’une leucemie lymphoide chronique en un lymphome agressif, principalement lymphome B diffus a grandes cellules (LBDGC). Ses facteurs pronostiques sont partiellement connus. Notre etude devait confirmer/identifier des caracteristiques cliniques, biologiques et genomiques pronostiques de la survie du SR sous-type LBDGC. Materiel et methodes Parmi 103 patients de 10 centres francais presentant un SR sous-type LBDGC, 58 etaient identifies avec un materiel au diagnostic de qualite suffisante pour etudier la valeur pronostique d’un panel de 13 genes. Nous avons realise un modele de regression a risques proportionnels de Cox puis un modele flexible en appliquant une fonction spline sur la variable performance status selon l’« Eastern Cooperative Oncology Group » (PS ECOG) qui n’etait graphiquement pas proportionnelle dans le temps Resultats La mediane de survie globale (SG) etait de 8 mois. En analyse bivariee, les patients avec PS ECOG > 1, ceux avec des plaquettes 1, profil non mute du gene des chaines lourdes des immunoglobulines (IGVH) et anomalie de TP53 ( Tableau 1 ). En appliquant une fonction spline sur le PS ECOG, son effet sur la survie se limitait aux 12 mois suivant le diagnostic : le hazard ratio estime de facon lineaire avant et apres 12 mois pour faciliter son interpretation etait respectivement 3,98 et 1,04 (intervalle de confiance 95 % 1,79–8,82 et 0,26–4,16 ; p = 0,001 et 0,96). Conclusion Le profil IGVH non mute est identifie comme nouveau facteur pronostique, en plus du PS ECOG, du taux de plaquettes et du statut TP53 sur la biopsie de SR. L’effet du PS ECOG est limite aux 12 mois suivant le diagnostic.

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.

Maintenance therapy with alternating azacitidine and lenalidomide in elderly fit patients with poor prognosis acute myeloid leukemia: a phase II multicentre FILO trial

Resume La granulomatose septique chronique (GSC) est une maladie genetique rare qui conduit a un deficit immunitaire par defaut du metabolisme oxydatif des cellules phagocytaires. Il en resulte des infections bacteriennes et fongiques severes a repetition chez les sujets atteints des le plus jeune âge. On retrouve egalement des atteintes inflammatoires avec la formation de granulomes. Le diagnostic de la maladie repose sur la capacite a mettre en evidence un deficit du pouvoir oxydatif des phagocytes. L’infection pulmonaire est la manifestation la plus frequente de la maladie ; cependant tous les organes peuvent etre touches comme l’œil qui peut etre le siege aussi bien d’une atteinte infectieuse que d’une atteinte inflammatoire, notamment chorioretinienne. Le traitement de la GSC repose, d’une part, sur la prophylaxie des episodes infectieux et, d’autre part, sur la prise en charge adaptee des episodes infectieux et inflammatoires. Actuellement, le seul traitement disponible est l’allogreffe de moelle osseuse, ce qui impose la disponibilite d’un donneur HLA compatible et ne se concoit que dans des situations cliniques precises.

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 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 (PP=0.004, respectively) and longer OS (P=0.004 and P

Harmonemia: a universal strategy for flow cytometry immunophenotyping—A European LeukemiaNet WP10 study

A simplified prognostic score is presented based on the multivariate analysis of 138 refractory/relapsed acute myeloid leukaemia (AML) patients (median age 55 years, range: 19-70) receiving a combination of intensive chemotherapy+Gemtuzumab as salvage regimen. Overall, 2-year event-free survival (EFS) and overall survival (OS) were 29±4% and 36±4%, respectively. Disease status (relapse

The European LeukemiaNet (ELN), workpackage 10 (WP10) was designed to deal with diagnosis matters using morphology and immunophenotyping. This group aimed at establishing a consensus on the required reagents for proper immunophenotyping of acute leukemia and lymphoproliferative disorders. Animated discussions within WP10, together with the application of the Delphi method of proposals circulation, quickly led to post-consensual immunophenotyping panels for disorders on the ELN website. In this report, we established a comprehensive description of these panels, both mandatory and complementary, for both types of clinical conditions. The reason for using each marker, sustained by relevant literature information, is provided in detail. With the constant development of immunophenotyping techniques in flow cytometry and related software, this work aims at providing useful guidelines to perform the most pertinent exploration at diagnosis and for follow-up, with the best cost benefit in diseases, the treatment of which has a strong impact on health systems.

Objectives: Patients with near-tetraploid (karyotype: 81 - 103 chromosomes) acute lymphoblastic leukemia (NT-ALL) constitute about 1% of childhood ALL and data reported on them are limited and controversial. The aim of the study was to enlarge the knowledge on these rarely occurring ALL. Methods: The members of the European Group for Immunophenotyping of Leukemias (EGIL) searched retrospectively their databases for NT-ALL patients. Results: We collected data of 36 European children from seven European countries with NT-ALL diagnosed since 1992. All patients reached complete remission (CR) after induction chemotherapy. Their blasts were negative for peroxidase and BCR-ABL1. Ten children were diagnosed as T-cell ALL (T-ALL) EGIL categories (T-I n = 2, T-II n = 2, T-III n = 3, T-IV n = 3) and four displayed various structural chromosomal abnormalities. Eight of 10 T-ALL remained in 1st CR; one died in CR from sepsis and one is alive in 2nd CR. Median survival was 88 (7-213) months. B-cell precursor (BCP) ALL was diagnosed in 26 children. Thirteen were positive for ETV6-RUNX1 and are alive in 1st CR for 32-147 months. Ten children were ETV6-RUNX1 negative and remained in 1st CR for 16-163 months. One girl with hypodiploid and NT metaphases and ETV6-RUNX1-negative BCP-ALL and one of two boys with NT-BCP-ALL not examined for ETV6-RUNX1 died of infection after stem cell transplantation in 2nd/3rd CR. Secondary myelodysplastic syndrome developed in two patients with NT-BCP-ALL. Conclusions: Our data demonstrate immunophenotypic, cytogenetic, and molecular heterogeneity of NT-ALL and favorable prognosis of most NT-ALL across different immunophenotypic and/or genetic ALL subtypes.

Tandem versus single autologous peripheral blood stem cell transplantation as post-remission therapy in adult acute myeloid leukemia patients under 60 in first complete remission: results of the multicenter prospective phase III GOELAMS LAM-2001 trial

Acute lymphoblastic leukemia ( ALL) in the elderly is characterized by its ominous prognosis. On the other hand, imatinib has demonstrated remarkable, although transient, activity in relapsed and refractory Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL), which prompted us to assess the use of imatinib in previously untreated elderly patients. ALL patients aged 55 years or older were given steroids during 1 week. Ph+ve cases were then offered a chemotherapy-based induction followed by a consolidation phase with imatinib and steroids during 2 months. Patients in complete response (CR) after consolidation were given 10 maintenance blocks of alternating chemotherapy, including two additional 2-month blocks of imatinib. Thirty patients were included in this study and are compared with 21 historical controls. Out of 29 assessable patients, 21 (72%, confidence interval (CI): 53 - 87%) were in CR after induction chemotherapy vs 6/21 (29%, CI: 11 - 52%) in controls (P = 0.003). Five additional CRs were obtained after salvage with imatinib and four after salvage with additional chemotherapy in the control group. Overall survival ( OS) is 66% at 1 year vs 43% in the control group ( P = 0.005). The 1-year relapse-free survival is 58 vs 11% ( P = 0.0003). The use of imatinib in elderly patients with Ph+ ALL is very likely to improve outcome, including OS.

Resume L’interpretation des resultats d’une recherche d’anticorps anti-ovariens (AAO) est soumise a controverse. Il est tentant d’etablir une relation de cause a effet lorsqu’une defaillance du fonctionnement ovarien est perceptible cliniquement. Il est plus difficile d’evaluer la signification de tels auto-anticorps chez des patientes sans signe clinique de defaillance de l’ovaire, pour lesquelles la recherche des anticorps anti-ovariens est effectuee dans le cadre d’une autre maladie auto-immune, specifique d’organe ou non, ou encore dans le contexte d’echecs de tentatives de reproduction. L’interpretation est delicate pour plusieurs raisons : i) les cibles antigeniques potentielles sont multiples dans l’ovaire, ii) le facteur temps peut moduler la reponse immunitaire et les stigmates de l’auto-immunite peuvent etre transitoires, iii) la constatation de la concomitance d’auto-anticorps et d’un signe clinique ne signifie pas obligatoirement une liaison de cause a effet. Le but de cette mise au point est de passer en revue la pertinence clinique des AAO a la lumiere des cibles antigeniques identifiees a ce jour.

The involvement of autoimmune mechanisms in premature ovarian failure has been put forward by numerous investigators. In various other ovarian pathologies, such as idiopathic infertility, polycystic ovary syndrome, or endometriosis, similar mechanisms have been suggested. However, the exact role of autoimmunity in the pathophysiology of these diseases still remains controversial. The diagnosis of autoimmune ovarian disease relies on several clinical, biological and histological findings, but special interest has been focused on antiovarian autoantibodies. The search for these antibodies has been undertaken by several authors and yielded somewhat conflicting results which might be conditioned by methodological differences and by the multiplicity of potential immune targets. These targets, which comprise various steroidogenic enzymes, gonadotrophins and their receptors, the corpus luteum, zona pellucida and oocyte, are reviewed. Further investigation of these targets is required to improve the diagnostic tools that will lead to a precocious and reliable diagnosis of autoimmune ovarian disease, an appropriate clinical surveillance as well as the selection of patients who may benefit from immune-modulating therapy and possibly recover ovarian function and fertility.

Leukocytes are recruited from peripheral blood into milk as part of the inflammatory response to mastitis. However, excessive accumulation of inflammatory cells alters the quality of milk and the proteases produced by polymorphonuclear neutrophils (PMNs) and macrophages may lead to mammary tissue damage. To investigate PMN recruitment and the kinetics of their intracytoplasmic enzymes in inflammation, we generated mastitis in six cows by intramammary infusion of lipopolysaccharide (LPS). Clinical signs of acute mastitis were observed in all of the cows, and normal status was resumed by 316 h. Intracytoplasmic elastase, collagenase, and cathepsin activities were measured within live cells by flow cytometry in peripheral blood leukocytes and milk PMNs before and during the inflammatory process (at 10 time points between 4 and 316 h). The proportion of immature PMNs was appreciated by CD33 surface labeling measured in flow cytometry. Leukopenia was observed in the peripheral blood 4 h postinfusion, concomitant to an increase in somatic cell counts in milk. CD33+PMNs were preferentially recruited from the peripheral blood to milk. Enzymatic activities were detected in PMNs, lymphocytes, and monocytes at levels depending on the cell type, sample nature, and time of collection. Milk PMNs had lower enzymatic activities than peripheral blood PMNs. This study showed that milk PMNs recruited during LPS-induced experimental mastitis have an immature phenotype and significantly lower enzymatic activities than peripheral blood PMNs. This suggests that CD33, an adhesion molecule, may be involved in the egress from blood to milk and that the enzymatic contents of PMNs are partly used during this process.

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

IMDSFlow19: et al., 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.

We document findings on c-kit (CD117) expression in 1,937 pediatric and adult de novo acute leukemia cases, diagnosed in five single European centers. All cases were well characterized as to the morphologic, cytochemical, and immunologic features, according to the European Group for the Immunological Classification of Leukemias (EGIL). The cases included 1,103 acute myeloid leukemia (AML), 819 acute lymphoblastic leukemia (ALL), 11 biphenotypic acute leukemia (BAL), and 4 undifferentiated (AUL). c-kit was expressed in 741 (67%) AML cases, regardless of the French-American-British (FAB) subtype, one third of BAL, all four AUL, but only in 34 (4%) of ALL cases. The minority of c-kit+ ALL cases were classified as: T-cell lineage (two thirds), mainly pro-T–ALL or T-I, and B lineage (one third); cells from 62% of these ALL cases coexpressed other myeloid markers (CD13, CD33, or both). There were no differences in the frequency of c-kit+ AML or ALL cases according to age being similar in the adult and pediatric groups. Our findings demonstrate that c-kit is a reliable and specific marker to detect leukemia cells committed to the myeloid lineage, and therefore should be included in a routine basis for the diagnosis of acute leukemias to demonstrate myeloid commitment of the blasts. c-kit expression should score higher, at least one point, in the system currently applied to the diagnosis of BAL, as its myeloid specificity is greater than CD13 and CD33. Findings in ALL and AUL suggest that c-kit identifies a subgroup of cases, which may correspond to leukemias either arising from early prothymocytes and/or early hematopoietic cells, both able to differentiate to the lymphoid and myeloid pathways.

Skin testing with the suspected compound has been reported to be helpful in determining the cause of cutaneous adverse drug reactions (ADRs), but the value and specificity of these tests need to be determined. In this study, 72 patients with presumed drug eruptions (27 maculopapular, 18 urticarial, seven erythrodermic, nine eczematous, four photosensitivity, three fixed drug eruptions, three with pruritus and one with acute generalized exanthematous pustulosis) were assessed. All had drug patch tests; 46 also had prick tests and 30 had intradermal tests (performed on hospitalized patients using a sterile solution of the suspected drug, diluted sequentially) with immediate and delayed readings. Among these patients, 52 (72%) had a positive skin test reaction, 43%, 24% and 67% in patch, prick and intradermal skin tests, respectively. The results of skin tests varied with the drug tested and with the clinical type of cutaneous ADR, as a significantly higher number of positive patch tests was observed in maculopapular rashes than in urticarial reactions (P = 0.001). This study supports the value of careful sequential drug skin testing in establishing the cause of cutaneous ADR. Guidelines are proposed for performing these tests, and these include the use of appropriate negative control patients to avoid false-positive results.

The less differentiated stage (CD10-) of B-lineage acute lymphoblastic leukaemia (ALL) described as preB1-ALL in the GEIL nomenclature, accounts for less than 10% of ALL. It is classically considered to be associated with translocation (4;11)(q21;q23), and to have a poor prognosis. We report an extensive immunophenotypic, genomic and clinical study of a series of 64 preB-1 ALL patients, representing 6.3% of a cohort of consecutive ALLs. The engagement of preB1-ALL cells in the B-lineage was confirmed by their B-lineage score, equal to or higher than 2. In addition, more than 90% of the cases tested showed rearranged IGH genes. Translocation (4;11) was the most frequent karyotypic anomaly seen, but only accounted for 24% of the preB1-ALL cases tested. Expression of the myeloid associated antigen CD15 was also found with high incidence in this subset. Clinical and biological features at presentation showed more significant differences between preB1- and T-ALL than between preB1- and preB2-ALL (CD10+). However, outcome characteristics of the 22 children with preB1-ALL confirmed the worse prognosis of this entity.

Ribosomal immunotherapy has been successfully used since the 1960s to boost the immune system and provide protection against microbial infections. We have investigated both whether and how these immunostimulants behave as natural immunogens in the mucosa-associated immune system. According to current understanding of the physiology of the mucosal immune response, intestinal Peyer's patches and the related solitary nodules are the primary inductive sites involved in the immune protection of all mucosal surfaces. Sensitised lymphocytes generated at these sites reach the general circulation through lymphatic drainage and relocate in mucosal areas by means of specialised 'high endothelial venules'. We hypothesised that orally administered ribosomal preparations would yield sensitised B cells specific for bacterial antigens from the parent strains. These cells should then be detectable in the peripheral blood after ribosomal intake, and identifiable as plasma cells in mucosae-associated tissues after completing their terminal differentiation. Ultimately, specific IgA should appear in secretions. To this end, we studied the immune responses generated in children and adults after 'Ribomunyl' administration, according to various consecutive protocols. The initial hypothesis was confirmed by the identification of specific B cells in the peripheral blood, plasma cells in the tonsillar tissue and specific IgA in the saliva. An animal model involving the use of twin sheep enabled detection of the specific cells in mesenteric and cervical lymph nodes. Analysis of these data indicates that ribosomal preparations trigger the production of lymphocytes specific for both ribosomes themselves and whole bacterial antigens. This supports the fact that small antigenic motifs are carried as partly synthesised peptides on the ribosomal particles. Therefore, ribosomes boost an array of B cells that are specific for many antigenic determinants of the bacteria from which they are extracted. We were also able to show that the stimulation provided was specific, since no response to other bacteria could be detected. Finally, analysis of the kinetics of this stimulation confirmed that oral immunisation generates rapid and transient secretory responses, building increased numbers of memory cells that are readily available to respond to further challenges by either more ribosomal preparations or potential pathogens.

Lymphocyte multiplication can be inducedin vitroby mitogens or specific antigens, and is usually measured using isotopic methods involving tritiated thymidine. Cellular proliferation can also be analyzed by flow cytometry techniques based on cell cycle analysis through the measurement of DNA content. We applied this method to lymphocytes from 113 individuals, to evaluate lymphocyte proliferation after stimulationin vitroby a mitogen (phytohaemagglutinin, PHA) or a recall antigen (tetanus toxoid), using a kinetic approach with four points sequential measurements of the S and G2 phases over six days of culture. The proportion of cells in S phase after PHA stimulation was significantly higher than in controls overall and as early as on day three of the culture. Activation with a recall antigen significantly induced increasing S phase cell proportions up to day six. These data suggest that flow cytometric assessment of the S phase could be a useful alternative to isotopic methods measuring lymphocyte reactivityin vitro.

International audience; The mTORC1 signaling pathway is constitutively activated in almost all acute myelogenous leukemia (AML) patients. We conducted a phase Ib trial combining RAD001 (everolimus), an allosteric inhibitor of mTORC1, and conventional chemotherapy, in AML patients under 65 years of age at first relapse (clinical trial NCT 01074086). Increasing doses of RAD001 from 10-70 mg were administrated orally on days 1 and 7 (d1 and d7) of a 3+7 daunorubicin+cytarabine conventional induction chemotherapy regimen. Twenty-eight patients were enrolled in this trial. The treatment was well tolerated with

Several groups have published flow cytometry scores useful for the diagnosis or prognosis of myelodysplastic syndromes (MDS), mainly based on the detection of immunophenotypic abnormalities in the maturation of granulocytic/monocytic and lymphoid lineages. As anemia is the most frequent symptom of early MDS, the aim of this study was to identify markers of dyserythropoiesis relevant for the diagnosis of MDS analyzed by selecting erythroblasts in a whole no-lysis bone marrow strategy by using a nuclear dye. This prospective study included 163 patients, including 126 with cytopenias leading to MDS suspicion and 46 controls without MDS. In a learning cohort of 53 unequivocal MDS with specific markers, there was a significant difference between the coefficients of variation of mean fluorescence intensities of CD71 and CD36 in MDS patients compared with controls. These two parameters and the hemoglobin level were used to build a RED-score strongly suggestive of MDS if ≥ 3. Using the RED-score in the whole cohort, 80% of MDS or non-MDS patients were correctly classified. When combined with the flow score described by Ogata et al., this strategy allowed to reach a very high sensitivity of 88% of patients correctly classified.

SUMMARYThe origin of mesangial IgA deposits in IgA nephropathy (IgAN) remains obscure. A significant proportion of deposited immunoglobulin is dimeric (J chain-positive). Previous studies of J chain expression within lymphoid tissue in IgAN have utilized antibodies which other investigators have found to be non-specific. To address this problem, we have developed an in situ hybridization (ISH) method for the detection of J chain mRNA within IgA plasma cells. Tonsils from 12 patients with IgAN and 12 controls were studied using (i) non-isotopic ISH for J chain mRNA, and (ii) combined immunofluorescence (IF) and fluorescent ISH. J chain mRNA-positive cells were identified in germinal centres, and within the subepithelial and interfollicular zones. A greater number of J chain mRNA-positive cells were found in the germinal centres of patients (mean 57.7±4.6 cells/105μm2) compared with controls (mean 36.9±3.5 cells/105μm2) (P < 0.001). Combined IF and fluorescent ISH showed a greater proportion of J chain mRNA-positive interfollicular IgA cells in patient tonsils (3.2±3.4%) compared with controls (21±2.3%; P < 0.02). These results indicate a shift towards dimeric IgA production in the tonsils in IgAN. In addition, the finding of excess numbers of J chain-positive IgA-negative cells within germinal centres suggests that an abnormality may be present at the B cell differentiation stage before IgA switching. These results further highlight immune abnormalities within the tonsil as a central feature of abnormal polymeric IgA biology in this common form of glomerulonephritis.

Darbepoetin (DAR), with or without granulocyte colony-stimulating factor (G-CSF), has proved effective in treating anemia in patients with lower-risk myelodysplastic syndrome (MDS), but its effects on quality of life (QoL) and exercise functioning are less well established. In this phase II study (no. NCT00443339), lower-risk MDS patients with anemia and endogenous erythropoietin (EPO) level

Chronic granulomatous disease (CGD) is a rare genetic immune deficiency due to defective oxygen metabolism in phagocytic cells. It results in recurrent severe bacterial and fungal infections in patients from an early age on. Inflammatory lesions are also observed, with the formation of granulomas. Diagnosis relies on the demonstration of a deficiency in the oxidative properties of phagocytes. Pulmonary infections are the most frequent clinical manifestations of the disease, yet all organs can be involved, such as the eye, with either infections or inflammatory chorioretinal lesions. The treatment of CGD relies on prophylaxis to avoid infections, and on the rapid management of infectious and inflammatory episodes. The only cure to date is allogenetic bone marrow transplant, which requires a compatible donor and can only be considered in certain clinical situations.

Item does not contain fulltext Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as 'normal', 'suggestive of', or 'diagnostic of' MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique. 01 juli 2012

CD49d blockade by natalizumab therapy in patients with multiple sclerosis increases immature B-lymphocytes

Adult and child B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) differ in terms of incidence and prognosis. These disparities are mainly due to the molecular abnormalities associated with these two clinical entities. A genome-wide analysis using oligo SNP arrays recently demonstrated that PAX5 (paired-box domain 5) is the main target of somatic mutations in childhood BCP-ALL being altered in 38.9% of the cases. We report here the most extensive analysis of alterations of PAX5 coding sequence in 117 adult BCP-ALL patients in the unique clinical protocol GRAALL-2003/GRAAPH-2003. Our study demonstrates that PAX5 is mutated in 34% of adult BCP-ALL, mutations being partial or complete deletion, partial or complete amplification, point mutation or fusion gene. PAX5 alterations are heterogeneous consisting in complete loss in 17%, focal deletions in 10%, point mutations in 7% and translocations in 1% of the cases. PAX5 complete loss and PAX5 point mutations differ. PAX5 complete loss seems to be a secondary event and is significantly associated with BCR-ABL1 or TCF3-PBX1 fusion genes and a lower white blood cell count.

Near-tetraploid acute myeloid leukemias (NT-AML) constitute an unusual presentation defined by blasts containing 80–104 chromosomes without aberrant near-diploid metaphases. Only about 60 NT-AML cases have been described so far in the literature.1 Reported cases where detailed information was available can be classified as M2 AML with duplication of t(8;21)(q22;q22) (N=13),1, 2 acute promyelocytic leukemias (M3 AML) with duplication of t(15;17)(q22;q21) (N=4)1, 3 – both categories reported mostly from Asian patients – or acute erythroleukemias M6 AML (N=7)1 and M0-M5 AML putatively originating from pluripotent myeloid progenitors characterized by erythroblastic and/or megakaryocytic (EM) dysplasia (N=17).4 Near-tetraploid blast cells are characterized by their large size on bone marrow smears,2, 3, 4 but individual chromosomal abnormalities are often difficult to analyze because of their 'fuzzy appearance'.1, 4

The involvement of serum anti-ovarian autoantibodies (AOA) in ovarian pathology still remains controversial. In some cases of clinically patent ovarian failure, there seems to be a causal relationship between AOA and the ovarian disease. In patients with various organ-specific or systemic autoimmune diseases, or with unexplained, repeated reproductive failure, but otherwise normal ovarian function, it is even more difficult to determine the significance of AOA for several reasons: i) AOA recognize many different antigenic targets in the ovary ii) the antiovarian response may be transient or variable with time iii) the presence of AOA does not imply their aetiopathogenic role in the disease. The present paper reviews the clinical significance of AOA based on their ovarian targets as far as they have been identified until now.

Pro-T acute lymphoblastic leukemias (ALL), also called T-I ALL, are extremely rare diseases where cells at the earliest defined stage of T-lineage maturation proliferate in the bone marrow. They are characterized by the absence of all T-lineage differentiation markers except intracytoplasmic CD3 and surface CD7. They may express markers of immaturity and a number of myeloid lineage markers, without reaching the score defining biphenotypic acute leukemia (BAL). A literature search was performed on Medline and the few publications devoted to this disorder are reviewed in this manuscript. Attempts at understanding the mechanisms leading to the development of T-I ALL, based mostly on animal experiments are further presented.

The analysis of CD87 (urokinase-type plasminogen activator receptor – uPAR) expression has a potential role in the diagnostic or prognostic work-up of several hematological malignancies, particularly acute leukemia and multiple myeloma. The distribution of CD87 in acute myeloid leukemia (AML) varies according to the FAB subtype (highest expression in M5 and lowest in M0). Functionally, it is conceivable that the expression of CD87 could contribute to the invasive properties of the leukemic cells towards the skin and mucosal tissues as reflected by the clinical behavior of CD87 high cases. The lack of or weaker expression of CD87 on blast cells from ALL patients supports the concept that CD87 investigation might help in the distinction of AMLs from lymphoid malignancies. Among lymphoproliferative disorders, the expression of CD87 is exclusively found in pathological plasma cells. Since plasma cells also coexpress some adhesion molecules such as CD138 and CD56, this observation is consistent with the capacity of these cells to home in the bone compartment. High levels of soluble uPAR appear to represent an independent factor predicting worse prognosis and extramedullary involvement in multiple myeloma.

To assess anti-ovarian antibodies (AOA) in serum samples at various times of in vitro fertilization (IVF) attempts to determine whether ovarian stimulation could result in the production of such autoantibodies in women.Prospective study on 134 patients and 138 IVF cycles using a classical long protocol. For each attempt, four serum samples were obtained, respectively, at the onset of downrelation (S1), end of downregulation (S13), after 7 days of follicular stimulation (S21) and the day of follicular puncture (SP). Five hundred and fifty two samples were tested with an enzyme-linked immunosorbent assay for three isotypes (IgG, IgA, IgM) of AOA.In the whole group, mean concentrations of AOA for each isotype were compared group by group: S1-S13, S1-S21, S1-SP, S13-S21, S13-SP, S21-SP. Not any significant difference was observed whatever the isotype considered.This study shows the absence of influence of endogenous or exogenous ovarian stimulation by gonadotropins on anti-ovarian autoimmunity.

Recurrent unexplained abortions are defined as at least two successive abortions during the first trimester of pregnancy. Implication of autoimmunity processes is now widely recognized. The list of biological assays proposed in recurrent abortions is inspired from immunological exploration of systemic lupus erythematosus in which pregnancy usually induces high risk for the conception product. Other assays derive from immunological hypotheses explaining the tolerance paradox of pregnancy, but many have not yet been established and will still require clinical research protocols.

A project group working under the European Confederation of Laboratory Medicine (ECLM) presents recommendations for standardized procedures for the Feulgen-Rossenbeck-Schiff and the periodic acid-Schiff (PAS) reactions on cytological and histological material. The advantages and disadvantages of such standardized procedures are presented here in a preamble. Both users and manufacturers are encouraged to give their opinions with a view to achieving consensus on these procedures and on how further work on these lines may proceed.

Changes in the lactoferrin and lysozyme concentration of human milk during lactation were determined by microparticle-enhanced nephelometric immunoassays of 360 milk samples collected from 64 lactating volunteers. These 360 samples were colostrum from days 1 to 5 postpartum (142 samples), transitional milk from days 6 to 14 (106 samples), and 112 mature milk samples obtained from days 15 to 28 (34 samples), from days 29 to 56 (50 samples) and from days 57 to 84 postpartum (28 samples). The concentration and percentage of lactoferrin vs. total protein were found to be significantly higher in colostrum (5.8 g/L, 27%) than in transitional milk (3.1 g/L, 22%) or day 15 to 28 mature milk (2.0 g/L, 19%), then increased in day 29 to 56 mature milk (2.2 g/L, 22%) and day 57 to 84 mature milk (3.3 g/L, 30%). The concentration of lysozyme decreased from colostrum (0.37 g/L) to transitional milk (0.27g/L) and day 15 to 28 mature milk (0.24 g/L), then increased in day 29 to 56 mature milk (0.33 g/L) and was highest in day 57 to 84 mature milk (0.89 g/L). The percentage of lysozyme vs. total protein was found to be always rising during lactation: colostrum, 2%; transitional milk, 2%; days 15 to 28, 2%; days 29 to 56, 3%; and days 57 to 84 mature milk, 8%.