A randomized study comparing the effect of GM-CSF and G-CSF on immune reconstitution after autologous bone marrow transplantation.

Haemopoietic growth factors (HGFs) have been shown to accelerate recovery from severe neutropenia after autologous bone marrow transplantation (ABMT) but their effect on immune reconstitution is not well defined. The present study compares, through randomized trial, the in vivo effect of GM-CSF and G-CSF administration on the immune recovery of patients who underwent ABMT. For that purpose, we have sequentially analysed 14 different T, B and NK lymphoid cell subsets using appropriate dual staining during the first year following transplant (days +6, +17, +31, +66, +90, +120, +180, +360).

Incidence of chromosome numerical changes in multiple myeloma: fluorescence in situ hybridization analysis using 15 chromosome-specific probes.

The presence of complex karotypes with frequent numerical and structural abnormalities has been reported in 20 to 50% of multiple myeloma (MM) patients. This variability is mainly due to the difficulty of conventional cytogenetics to obtain tumor metaphases representative of all possible neoplastic clones in MM. To gain insight into the real incidence of numerical chromosome changes in MM we have studied by fluorescence in situ hybridization technique 15 different human chromosomes, 1, 3, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, X, and Y, in a series of 52 MM patients.

CD5+ B cells in Graves' disease: correlation with disease activity.

In the present paper the distribution of peripheral blood CD5+/CD19+ (CD5+B) and CD5-/CD19+ (CD5-B) B-lymphocytes in Graves' disease (GD) patients is analyzed in order to correlate them with disease activity, interleukin-2 (IL-2) and IL-6 binding to T and B cells as well as with anti-thyrotropin (TSH) receptor antibodies and the thyroid hormone serum levels. The combination of flow cytometry and 3-color immunofluorescence revealed a remarkable increase in the absolute numbers of CD5+ B cells in hyperthyroid-untreated GD patients (218 +/- 137 x 10(6)/l vs. 66 +/- 69 x 10(6)/l in healthy subjects, p < 0.

Value of colony forming unit-granulocyte macrophage assay in predicting relapse in acute myeloid leukaemia.

Aim: To evaluate the validity of the colony forming unit-granulocyte macrophage (CFU-GM) assay for predicting relapse in patients with acute myeloid leukaemia (AML).

Methods: The study population comprised 32 patients with AML in remission, followed for a median of 18 months. A mean of four studies was carried out per patient.

Analysis of natural killer-associated antigens in peripheral blood and bone marrow of multiple myeloma patients and prognostic implications.

The aim of this study was to analyse the expression of NK-associated antigens in both peripheral blood and bone marrow lymphocytes from a large series of newly diagnosed multiple myeloma patients. 112 patients with untreated multiple myeloma (MM) were included in the study. 36 sex- and age-matched healthy volunteers were used as controls for peripheral blood (PB) studies and 14 for the bone marrow (BM) studies.

Performance evaluation of the FACSCount System: a dedicated system for clinical cellular analysis.

Flow cytometers are instruments that can determine multiparameter data simultaneously and have a great potential in providing unique information about cells. The FACSCount System is designed as the first dedicated flow cytometer for the clinical laboratory. Its current configuration provides CD4, CD8, and CD3 absolute counts from 100 microliters of whole blood.

Antigen phenotype and cytotoxic activity of natural killer cells in hemodialysis patients.

The antigen phenotype and cytotoxic activity of peripheral blood natural killer (NK) cells have been studied in 35 patients on hemodialysis, evaluating the influence exerted by different types of dialysis membranes and diverse clinical and biologic features of end-stage renal disease. Two-color immunofluorescent analyses with different monoclonal antibodies and flow cytometry were performed to identify the NK cells. Functional cytotoxic assays were simultaneously performed in the same patients.

General concepts about cell sorting techniques.

Research involving cell analysis frequently requires isolation of certain cell types or subcellular components either as a final objective or as a preparative tool for further assays. At present, there are a high number of cell sorting methods that are suitable for being used in the clinical laboratory. These methods can be divided into two major groups: (1) bulk sorters and (2) single-cell-based sorters.

Astrocyte differentiation in primary culture followed by flow cytometry.

Astrocyte proliferation and differentiation in primary culture were followed by flow cytometry. The time-courses of the percentages of astrocytes in the different cell-cycle phases suggest that astrocytes proliferate during the first 10 days in culture thereafter reaching confluence. Two types of astrocytes are identified immunocytochemically: one growing in the bed monolayer, identified as type-1 astrocytes, and another growing on the top of the monolayer, identified as type-2 astrocytes.

Phenotypic changes in acute myeloid leukaemia: implications in the detection of minimal residual disease.

Aim: To explore the role of phenotypic changes as possible limiting factors in the immunological detection of minimal residual disease in patients with acute myeloid leukaemia (AML).

Methods: 20 relapses were evaluated, with special attention to changes in the criteria used for the definition of a phenotype as "aberrant". In all cases the same monoclonal antibody and fluorochrome were used at diagnosis and in relapse.

Immunophenotype and DNA cell content in multiple myeloma.

In this paper three different areas of the biology of multiple myeloma (MM) are reviewed: (1) the immunophenotypic characteristics of plasma cells (PC), (2) the changes in the immunoregulatory cells, and (3) the cell DNA content of PC. Myelomatous PC display a heterogeneous phenotype not only between different patients but also within each patient consistent with the fact that the neoplastic clone is able to undergo a certain degree of differentiation. In addition, PC generally lack surface B cell associated antigens and infrequently show reactivity for non-lineage restricted markers.

Expression of lymphoid-associated antigens in mast cells: report of a case of systemic mast cell disease.

In this study the expression of 'classically' considered lymphoid-associated antigens (CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD19, CD20, and CD22) was explored both in peripheral blood (PB) and bone marrow (BM) mast cells (MC) in a case of systemic mast cell disease (SMCD) by means of using multiple stainings and a direct immunofluorescence technique. CD2 and CD22 were expressed in both PB and BM MC, all the remaining lymphoid-associated markers were negative. Our results suggest that the reactivity for both CD2 and CD22 in PB and BM MC would be aberrant.

Adrenalectomy induces a decrease in the light scatter properties and amylase content of isolated zymogen granules from rat pancreas as analyzed by flow cytometry.

The effect of glucocorticoid deprivation induced in male rats by adrenalectomy on the pancreatic zymogen granules was studied. Zymogen granules were purified from control, sham-operated and adrenalectomized animals studied 1, 3 and 7 days after surgery. The zymogen granules were characterized by flow cytometry, and in each granule the size (based on the forward or low angle light scatter (FSC) parameter), membrane complexity (based on side or 90 degrees light scatter (SSC) parameter) and amylase content were evaluated.

Changes in adult rat liver mitochondrial populations at different energy states analyzed by flow cytometry.

The present work studies the changes in green fluorescence intensity after Rh-123 staining of the low (LFP) and the high fluorescence populations (HFP) in isolated mitochondria from rat liver. The results show that the HFP represents a mitochondrial compartment less sensitive to changes in energy states. In addition, it is concluded that the use of Rh-123 to monitor changes in mitochondrial membrane potential should be undertaken with caution because, under certain circumstances, there is no correlation between the Rh-123 intensity of fluorescence due to its uptake by mitochondria and previously reported changes in the mitochondrial membrane potential.

In vitro bromodeoxyuridine-labelling of single cell suspensions: effects of time and temperature of sample storage.

The present study was aimed to explore how the in vitro BrdUrd-labelling of rat thymocytes might be affected by both the time elapsed between obtaining the sample and the beginning of the labelling (0, 15, 30 or 60 min) and the effect of the temperature of storage (4 degrees C versus room temperature). Single cell suspensions obtained after in vivo labelling with BrdUrd were used as controls. The S phase fraction was calculated by flow cytometry both according to BrdUrd-immunolabelling and DNA content.

Phenotypic analysis of CD34 subpopulations in normal human bone marrow and its application for the detection of minimal residual disease.

In the past, studies on CD34+ cells have been based on the use of monoclonal antibodies conjugated with different fluorochromes that show different fluorescence intensity and yield variable results. Moreover, most of these studies have neither specifically focused on adult human BM samples nor have they used combinations to explore specifically the phenotype of myeloid committed CD34+ cells. The aim of the present study has been to characterize the normal human CD34+ precursor cells from adult BM in order to identify missing or extremely rare phenotypes that can be used for detecting minimal residual disease (MRD) in patients with AML.

Analysis of isolated zymogen granules from rat pancreas using flow cytometry.

Rat pancreatic zymogen granules were analyzed using flow cytometry to determine their heterogeneity with respect to different characteristics such as size (FSC), internal complexity (SSC) and membrane permeability to lipophilic and cationic dyes using rhodamine-123 as probe. Differences in the chemical composition of the membrane were determined using FITC-labeled lectins (concanavalin A, Wheat germ agglutinin (WGA) and Tetragonolobus purpureus) displaying specific binding for different carbohydrates (D-mannose, N-acetyl D-glucosamine and L-fucose, respectively). Finally, the amylase content of zymogen granules was also analyzed using an anti-amylase antiserum.

Effect of ethanol consumption on adult rat liver mitochondrial populations analyzed by flow cytometry.

In the present study, the effects of administering ethanol to adult male rats on the distribution of the low fluorescence population (LFP) and high fluorescence population (HFP), and the rhodamine-123 fluorescence intensity of these groups of mitochondria are analyzed by flow cytometry. Our results show that ethanol administration to adult male rats induces a redistribution of the HFP and LFP mitochondrial populations leading to an increase of the less functional HFP mitochondria. In addition, ethanol induced an increase in the mean intensity of green fluorescence of the HFP that is probably related to an increased number of rhodamine-123 binding sites per mitochondria resulting from mitochondria enlargement.

Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL).

Criteria for the immunological classification of acute leukemias are proposed by a recently established European group designated EGIL. The main aims of EGIL are to establish guidelines for the characterization of acute leukemias based on marker expression and provide a uniform basis for the diagnosis of the various types of these hemopoietic malignancies which should be helpful for future multinational clinical and laboratory investigations. Within the two major types (B and T cell lineage) of acute lymphoblastic leukemia (ALL), several groups are delineated according to the degree of cell differentiation.

Hypothyroidism prevents postnatal changes in rat liver mitochondrial populations defined by rhodamine-123 staining.

The effect of hypothyroidism on the percentages of low fluorescence population (LFP) and high fluorescence population (HFP) rhodamine-123-stained mitochondria, respiratory parameters, and ATPase activity were studied in liver mitochondria from early newborn rats. Hypothyroidism prevented the decrease in the percentage of HFP and the subsequent increase in LFP that occurs immediately after birth. This effect coincides with the impairment of mitochondrial respiratory function, as shown by the low respiratory control ratio and the low activity of F0,F1-ATPase found in hypothyroid newborns.

Clinical, biological, and immunophenotypical characteristics of B-cell chronic lymphocytic leukemia with trisomy 12 by fluorescence in situ hybridization.

The clinical, biological, and immunophenotypical characteristics of B-cell chronic lymphocytic leukemia (B-CLL) patients with trisomy 12 detected by fluorescence in situ hybridization (FISH) using a chromosome 12 alpha-centromeric probe (D12Z3) were analyzed in the present study. From a total of 104 consecutive B-CLL patients, 21 (20%) displayed trisomy 12, the percentage of trisomic cells ranging from 13% to 76%. From the clinico-biological point of view, patients with trisomy 12 were associated with atypical CLL morphology (43% vs 10%, P = 0.

Immunological detection of blast cell subpopulations in acute myeloblastic leukemia at diagnosis: implications for minimal residual disease studies.

The aim of the present study was to analyze the incidence of AML cases displaying more than one blast cell subpopulation by immunophenotype at diagnosis, since, any of them, although minimal, can be responsible for the relapse. For this purpose we have prospectively investigated the immunophenotype of blast cells from 40 de novo AML patients at diagnosis with a large panel of monoclonal antibodies in double and triple staining combinations analyzed at flow cytometry. The discrimination between the different cell populations was based on: (1) the existence of aberrant phenotypes; (2) differences in light-scatter characteristics; and (3) the expression of differentiation-associated antigens (CD34, CD117, HLADR, CD33, CD15, CD14, CD11b and CD4).

In vitro autonomous proliferation in ANLL: clinical and biological significance.

In acute non-lymphoblastic leukemia (ANLL) progenitor cells frequently display a certain degree of autonomous growth. The aim of the present work was to analyze the autonomous proliferative capacity of leukemic progenitors in both de novo and secondary to myeloproliferative disorders (MPD) and myelodysplastic syndromes (MDS), acute myeloid leukemias and to correlate with clinical and biological characteristics of the disease. Clonogenic assays with and without leukocyte conditioned medium with PHA (LCM-PHA) were performed and the autonomous proliferation index (API) calculated in a series of 50 patients (34 de novo ANLL, eight secondary to MPD and eight secondary to MDS).