Chronic hepatitis C virus infection causes B cell lymphoproliferative disorders that include type II mixed cryoglobulinemia and lymphoma. This virus drives the monoclonal expansion and, occasionally, the malignant transformation of B cells producing a polyreactive natural Ab commonly encoded by the VH1–69 variable gene. Owing to their property of producing natural Ab, these cells are reminiscent of murine B-1 and marginal zone B cells. We used anti-Id Abs to track the stages of differentiation and clonal expansion of VH1–69+ cells in patients with type II mixed cryoglobulinemia. By immunophenotyping and cell size analysis, we could define three discrete stages of differentiation of VH1–69+ B cells: naive (small, IgMhighIgDhighCD38+CD27−CD21highCD95−CD5−), “early memory” (medium-sized, IgMhighIgDlowCD38−CD27+CD21lowCD95+CD5+), and “late memory” (large-sized, IgMlowIgDlow-negCD38−CD27lowCD21low-negCD5−CD95−). The B cells expanded in cryoglobulinemia patients have a “memory” phenotype; this fact, together with the evidence for intraclonal variation, suggests that antigenic stimulation by hepatitis C virus causes the unconstrained expansion of activated VH1–69+ B cells. In some cases, these cells replace the entire pool of circulating B cells, although the absolute B cell number remains within normal limits. Absolute monoclonal VH1–69+ B lymphocytosis was seen in three patients with cryoglobulinemia and splenic lymphoma; in two of these patients, expanded cells carried trisomy 3q. The data presented here indicate that the hepatitis C virus-driven clonal expansion of memory B cells producing a VH1–69+ natural Ab escapes control mechanisms and subverts B cell homeostasis. Genetic alterations may provide a further growth advantage leading to an overt lymphoproliferative disorder.

SUMMARYThe mechanisms leading to a relative dominance of T cells producing type 2 cytokines in certain human immune disorders are still unclear. We investigated the relative susceptibility to apoptosis induced by primary in vitro activation of human type 1 (producing interferon-gamma (IFN-g)) or type 2 (producing IL-4) T cells. Peripheral blood lymphocytes were isolated from patients with immune disorders characterized by expansion of type 2 cells (four with AIDS and hyper-IgE/hypereosinophilia, one with Churg±Strauss syndrome, and one with idiopathic hypereosinophilic syndrome) or from individuals with normal cytokine balances. Cells were stimulated for 16 h with ionomycin and phorbol ester, and apoptosis of cytokine-producing cells was assessed by flow cytometry. T cells with a type-2 cytokine profile, i.e. producing IL-4 alone, were significantly more resistant to activation-induced apoptosis than those producing IFN-g alone. This was observed in AIDS patients, whose type 2 cells were mostly CD8

Background Current methods for multiparameter DNA flow cytometry suffer from several limitations. These include significant modifications of cell morphological parameters, the impossibility to counterstain cells with certain fluorochromes, and laborious tuning of the instrument that, for some procedures, must be equipped with an ultraviolet (UV) laser. To overcome these problems, we developed a novel method for the simultaneous analysis of morphological parameters, four‐color immunophenotyping, and stoichiometric DNA labeling using a bench‐top flow cytometer. Methods The method consists of a mild permeabilization/fixation treatment at room temperature, followed by labeling with fluorochrome‐conjugated monoclonal antibodies (mAbs) and with the DNA dye 7‐aminoactinomycin D (7‐AAD) at 56°C. Results Using this method, we analyzed resting peripheral blood mononucleated cells (PBMC), proliferating T cells cultured in the presence of interleukin‐2 (IL‐2), and lymphoblastoid B cells. Lymphocytes, monocytes, and lymphoblasts treated by this procedure retained differential light scattering (DLS) characteristics virtually identical to those of untreated cells. This allowed regions to be drawn on forward scatter (FSC) and side scatter (SSC) cytograms resolving different cell populations. DLS were preserved well enough to distinguish large lymphoblasts in the S or G2/M phases from small G0/G1 cells. Also, stainability with fluorescein‐isothiocyanate (FITC), R‐phycoerythrin (PE), allophycocyanin (APC)‐conjugated mAbs was generally preserved. DNA labeling with 7‐AAD was of quality good enough to permit accurate cell cycle analysis. Conclusions The method described here, which we called integral hot staining (IHS), represents a very simple, reproducible, and conservative assay for multiparameter DNA analysis using a bench‐top flow cytometer. Last but not least, the cytometer tuning for multiparameter acquisition is straightforward. Cytometry 44:120–125, 2001. © 2001 Wiley‐Liss, Inc.

The accurate determination of cell cycle, immunophenotypes and morphology at single-cell level is not fully achieved by current flow cytometry protocols. Acetone, a coagulant fixative/permealizing agent, is widely used in static cytometry, but is impractical in flow cytometry because of its shrinking effect. We sought for conditions of acetone treatment that could permit the simultaneous analysis of physical parameters, surface and intracellular immunostaining, and DNA content. We evaluated different experimental conditions (concentration, duration of fixation, temperature, presence of proteins) to test the capacity of acetone fixation/permeabilization to preserve cell physical parameters (forward and side scatters, FSC, and SSC) and immunophenotyping while allowing stoichiometric DNA staining. The commonly used ethanol fixation technique was used as reference method. To detect phenotypes and DNA content simultaneously, we employed 7-aminoactinomycin D (7-AAD) as ''intercalating'' dye for DNA in spite of, or just for, its controversial ability in stoichiometric DNA staining. Cells were resting peripheral blood monucleated cells (PBMCs), T-and B-cell blasts obtained by PBMCs stimulation, and the human cell lines Ramos and Shep. Acetone fixation, preserving both the recovery and the physical parameters of cells, is drastically influenced by temperature of treatment and is practicable only when the protocol is realized at 88C. Under this condition, acetone maintains the immunophenotypic fluorescences (realized before or after the fixation) better than ethanol. Stoichiometric DNA staining of acetone processed cells, the variation coefficients (CV) of frequency distributions of G1/G0 and G2/M phases, the modes ratio of these distributions and doublets generation are at least comparable to those obtained with ethanol treatment. The assay developed in the present study, that we called flow acetone-staining technique (FAST), accurately analyzes cell cycle, physical parameters and immunophenotypes in heterogenous cell populations, and thus provides a useful tool for cytomics. ' 2008 International Society for Analytical CytologyKey terms acetone; multiparametric flow cytometry; DNA content; conservative fixation; T and B blasts ANALYTICAL multiparametric flow cytometry including the measurement of the DNA content is accomplished by different procedures, sometimes complex and requiring specific reagents. Among several published protocols, the most variable step is fixation: the kind of fixatives used (1), the variations of the protocol steps (2,3) and the step in which the cells are fixed during the procedure (4). Cross-linking fixatives (e.g., paraformaldehyde) yield the best preservation of physical parameters and of surface and internal antigens, as well as good cell recovery. However, crosslinked cell samples do not allow a precise and linear measurement of DNA content (CV 3 of G1-G0 distribution; G2/G1 modes ratio 5 2 AE 0.02) even when

The reviewed FF protocol appeared at least as suitable as the TRF method. Measures obtained by TRF can be affected by chromosome end variability, DNA fragmentation, incomplete digestion and unsuitable electrophoresis. In contrast, the FF technique analyses telomeric sequences confined to preserved nuclei thus overcome most previous limitations. As yet, however, the FF telomere measure cannot be performed together with immunophenotyping and/or generation study by the dye dilution method.