CD66c expression in B-cell acute lymphoblastic leukemia: strength and weakness.

INTRODUCTION: In B-cell acute lymphoblastic leukemia (B-ALL), testing at diagnosis for BCR/ABL1 gene rearrangements is mandatory for prognostic stratification and treatment decisions. Several diagnostic methods have been proposed using flow cytometry to identify BCR/ABL1(+) B-ALL. METHODS: We evaluated expression of the myeloid antigen CD66c by flow cytometry in B-ALL. We studied 94 patients with B-ALL. The t(9;22)(q34;q11) or BCR/ABL1 rearrangement was detected by cytogenetic analysis or RT/PCR. Myeloid antigens CD66c, CD13, CD33, CD117, Myeloperoxidase, CD15 and CD65 were determined by flow cytometry. RESULTS: Of these 94 cases, 17 (18%) cases displayed BCR/ABL1 gene rearrangements and 38 (40%) cases were CD66c positive. CD66c was the most common myeloid antigen expressed on malignant lymphoblasts. Its expression was correlated with BCR/ABL1 rearrangements (P = 0.0001): sensitivity 82%, specificity 69%, positive predictive value 37% and negative predictive value 95%. Co-expression of CD66c(+) CD13(+) was more frequent in BCR/ABL1(+) B-ALL (29%) than BCR/ABL1(-) cases (4%) (P = 0.0044). Some BCR/ABL1(-) B-ALL cases (including hyperdiploid or cases with normal karyotype) were CD66c positive (31%). CONCLUSION: CD66c expression is correlated, but not specifically, with BCR/ABL1 rearrangement. It would seem better to interpret the absence of CD66c expression with a lack of BCR/ABL1 rearrangement. This myeloid antigen could be interesting in the detection of minimal residual disease.

Characterization and comparison of bone marrow and peripheral blood mononuclear cells used for cellular therapy in critical leg ischaemia: towards a new cellular product.

BACKGROUND AND OBJECTIVES: Autologous transplantation of either bone marrow (BM) or peripheral blood (PB) mononuclear cells (MNC) induces therapeutic angiogenesis in patients with peripheral arterial occlusive disease. Yet, the precise nature of the cellular product obtained from BM or PB and used in these therapeutic strategies remains unclear. MATERIALS AND METHODS: We have analysed the characteristics of BM-MNC and PB-MNC collected without mobilization and implanted in patients with critical limb ischaemia in a clinical trial of cellular therapy including 16 individuals treated by BM-MNC and eight by PB-MNC. These MNCs were characterized by cell counts, viability assessment and enumeration of leucocyte subsets, CD34 stem and endothelial progenitor cells (EPCs) (CD34+/CD133+/VEGF-R2+) by flow cytometry. Mean fluorescence intensity ratios were determined for CD34, CD133 and VEGF-R2 markers. All analyses were simultaneously performed in two laboratories. RESULTS: Accuracy and reliability between both laboratories were achieved. BM-MNCs and PB-MNCs were quantitatively and qualitatively heterogeneous and quite different from each other. Stem cells and EPCs were significantly more present in BM- compared to PB-cell products, but with similar mean fluorescence intensity ratios. A weakly positive correlation was observed between CD34+ cell counts and EPCs levels, confirming the specificity of cell identification. CONCLUSION: A great variability was observed in cell product characteristics according to their origin and also between individuals. These data stress the necessity of optimal characterization of cell products especially in multicentric clinical trials.

[Congenital transient leukemia: a case report]. / Un cas de leucémie congénitale transitoire.

UNLABELLED: Neonates with Down's syndrome have an increased risk for congenital leukaemia, particularly acute megakaryoblastic leukaemia (FAB, M7) which most often resolves spontaneously and is called transient leukaemia. It can be observed in non-constitutional trisomy 21 infants then presenting trisomy 21 on blasts cells. OBSERVATION: We report a transient leukaemia with an isolated pericardial effusion in a phenotypically normal neonate. Trisomy 21 was found on blasts cells. Complete remission remains after 32 months. DISCUSSION: Congenital leukaemias, with trisomy 21 on blasts cells have a good prognosis that justifies observation before using chemotherapy.

[Relevance of cytological and immunophenotypical analysis for the diagnosis of B-cell chronic lymphocytic leukaemia]. / Pertinence des analyses cytologiques et immunophénotypiques dans le diagnostic de la leucémie lymphoïde chronique B.

The objective of this study was to describe the cytological and immunophenotypical parameters evocative of B-cell Chronic Lymphocytic Leukaemia (B-CLL) and their ability to participate to the differential diagnosis of other B-chronic lymphoproliferatives disorders with blood dissemination (B-CLD). Two groups of pathology included 92 patients, 79 patients had a B-CLL and the 13 other had a B-CLD (1 Prolymphocytic Leukaemia, 12 non- Hodgkin's Lymphoma in which 4 Splenic Lymphoma with Villous Lymphocytes or SLVL). The lymphoid morphology was studied on blood smear stained with May Gr nwald Giemsa and the immunophenotypical analysis was performed by flow cytometry. The 72 patients with B-CLL were characterized by a predominance of small mature lymphocytes with a Matutes's CLL score 3 (generally CD5+, CD23+, SmIg poor expression). 4 out of B-CLL with cleaved lymphocytes 5 % showed the same immunological characteristics than the typical B-CLL cases. 3 cases of B-CLL with prolymphocytes between 5 and 55 % showed in 2 cases an immunophenotyping compatible with the diagnosis of B-CLD. The presence of shadow cells of Gumprecht was highly evocative of B-CLL. In conclusion, the cytological analysis remains at the root of any diagnosis and can be sufficient in most cases of typical CLL with the presence of shadow cells of Gumprecht on the blood smear. In case of presence of cleaved lymphocytes, the immunophenotyping becomes essential to confirme the diagnosis of B-CLL. In prolymphocytic cases, the differential diagnosis between mixed CLL and B-CLD (especially Mantle Cell Lymphoma and Marginal Zone B-Cell Lymphoma without villous lymphocytes) needs a multidisciplinary approach (clinical, cytogenetical and histological).

Activated Stat related transcription factors in acute leukemia.

Cell proliferation and differentiation are under the control of cytokines and growth factors. Different signaling pathways are involved in the transmission of a specific signal through successive phosphorylation and dephosphorylation of proteins leading to gene transcription necessary for growth and differentiation. The cytokines and growth factors activate the Stat family of transcription factors. The Jak-Stat pathway is essential for cytokine signal transduction. Dysregulation of this cascade might lead to uncontrolled hematopoiesis. Studies have been carried out to examine the functionality of this pathway in cells from patients with acute leukemia. Members of the Stat protein family (Stat1, Stat3 and Stat5) are constitutively activated in cells collected from some acute leukemias suggesting dysregulation of the Jak-Stat pathway. Evidence of the existence of constitutively activated spliced variants of Stat3 and Stat5 proteins are described. The mechanisms of such activation remain to be clarified.

Study of human cord blood lymphocytes by immobilized metal ion affinity partitioning.

The potential of immobilized metal ion affinity partitioning (IMAP) using dextran-PEG+PEG-IDA-M(II) systems to separate mononuclear cells from cord blood has been evaluated. The distribution of B cells, T cells, monocytes and hematopoietic stem cells between PEG and dextran phases was determined by flow cytometry with fluorochrome-labelled specific antibodies. Comparing these values with the post-Ficoll repartition resulted in the determination of enrichment factors, for each subpopulation, in the different phases. We were able to distinguish the partition pattern of B cells, T cells, monocytes and stem cells in different IMAP systems. Their partition was affected by the nature and the concentration of the metal used, but no specificity in distribution for the subpopulations was found.

STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients.

A signal transduction pathway activated by many cytokines has recently been elaborated. The JAK kinases and the signal transducers and activators of transcription (STAT) factors have been found to be essential components. In this report, we describe the presence of constitutively activated STAT factors in peripheral blood cells from patients with acute leukemia. We used oligonucleotide probes from the beta-casein and IRF-1 gene promoters and the ISRE probe to detect STAT proteins in nuclear extracts from acute leukemia cells in bandshift assays. Specific DNA protein complex formation was observed with the probes from the beta-casein and IRF-1 gene promoters, but not with the ISRE oligonucleotide probe, when cell extracts from acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were investigated. We used nonradioactive oligonucleotides as competitors to show the specificity of the complex formation. Specific antibodies directed against the individual STAT proteins were used in supershift experiments. STAT5- and STAT1-related factors were detected in ALL and STAT1-, STAT3-, and STAT5-related proteins were present in nuclear cell extracts from AML. Since the cells were not treated with cytokines before the nuclear proteins were extracted, we conclude that these factors are constitutively activated in vivo. It is likely that the constitutive activation of STAT proteins is a part of the events of leukemogenesis.

Four cases of spuriously low WBC count due to in vitro leukocyte agglutination: contribution of the hematology analyzer Coulter STKS in detecting this clinically misleading artefact.

Four spuriously lowered WBC counts due to in vitro leukoagglutination were reported from an automated cell counter (Coulter STKS). Leukocyte aggregates (3 to 50 cells), detected in the peripheral blood smears, included different cell types, normal (neutrophils, eosinophils, monocytes, lymphocytes) or abnormal (lymphoma cells). The phenomenon was associated with either a spurious leukoneutropenia or an underestimation of hyperleucocytosis. Leukoagglutination was extensively investigated in 3 cases : as shown in several reports, leukoagglutination may occur with various features, especially due to temperature and anticoagulant dependence. Our four cases reflected this variability. Furthermore, one case was found both temperature-dependent and anticoagulant-independent, a pattern not yet described in the literature. A common STKS graphic pattern was found in our 4 cases, suggesting that hematology analyzers such as Coulter STKS may be useful to detect leukoagglutination. In conclusion, each leukoneutropenia and/or each suggestive graphic pattern must be controlled by means of a blood smear examination in order to rule out the possibility of in vitro leukoagglutination.

Evaluation of the erythrocyte and platelet morphological analysis given by the hematology analyzer Coulter STKS.

The morphological profiles of red blood cells (RBC) and platelets (Plt) derived from the distribution analysis given by the Coulter STKS were evaluated in two groups of patients suffering from non hematological (n = 293) and hematological (n = 257) conditions. The RBC and Plt flags were studied in terms of sensitivity of the morphological analysis and specificity and significance of each flag. When all RBC and Plt flags were considered, the percentages of false negatives and false positives in our subjects were found to be 4.7% and 13.4% respectively, with a global efficiency of 81.8%. The sensitivity of the alarm system was higher than 90% for all types of abnormality, except microcytosis (81%) and Howell-Jolly bodies (57% over a limited number of 7 cases). The specificity of the STKS response was found to be low except for anisocytosis (88.5%) and macrocytosis (86.1%). It was shown that the flags microcytosis and/or hypochromia and macrocytosis were poorly significant when they appeared in isolation (false positive rates of 86.6% and 84.2% respectively). Thus, these alarms could be eliminated from the review criteria. When considering only the flags anisocytosis, NRBCs, micro RBCs/RBC fragments, dimorphic RBC pop, PLT clumps and giant Plt, the percentage of false positives was 8.1%. However, it must be kept in mind that suspect leucocyte flags remained review criteria, resulting in a final false negative rate of 2.5% for RBC and Plt morphological abnormalities.