Flow acetone-staining technique: a highly efficient procedure for the simultaneous analysis of DNA content, cell morphology, and immunophenotype by flow cytometry.

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 8 degrees C. 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.

Hepatitis C virus drives the unconstrained monoclonal expansion of VH1-69-expressing memory B cells in type II cryoglobulinemia: a model of infection-driven lymphomagenesis.

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 V(H)1-69 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 V(H)1-69(+) cells in patients with type II mixed cryoglobulinemia. By immunophenotyping and cell size analysis, we could define three discrete stages of differentiation of V(H)1-69(+) B cells: naive (small, IgM(high)IgD(high)CD38(+)CD27(-)CD21(high)CD95(-)CD5(-)), "early memory" (medium-sized, IgM(high)IgD(low)CD38(-)CD27(+)CD21(low)CD95(+)CD5(+)), and "late memory" (large-sized, IgM(low)IgD(low-neg)CD38(-)CD27(low)CD21(low-neg)CD5(-)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 V(H)1-69(+) 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 V(H)1-69(+) 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 V(H)1-69(+) natural Ab escapes control mechanisms and subverts B cell homeostasis. Genetic alterations may provide a further growth advantage leading to an overt lymphoproliferative disorder.

Human peripheral B cells: a different cytometric point of view.

BACKGROUND: Human peripheral B lymphocytes, analyzed by current flow cytometers, frequently show complex patterns of morphological and fluorescence signals. However, fluorescence intensity values are commonly reported without any correlation to the cell surface area. We propose a different approach, based on the evaluation of the ratio of phenotype fluorescence intensity to forward scatter intensity, to determine the apparent fluorescence density of surface molecules. METHODS: Starting from list mode acquired data, and after logical gating of live B cells, the analytical procedure suggests a serial scanning of the FSC versus SSC plot to obtain apparent fluorescence density of progressively larger cells. RESULTS: This method, applied to normal human peripheral B lymphocytes, was able to detect the presence of steady and modulated (with respect to cell size) fluorescence densities for a variety of surface molecules. B cells from patients with B cell disorders displayed interesting alterations of the phenotype density values and distributions. CONCLUSIONS: Our preliminary data show that, in human B cell cytometry, the apparent fluorescence density based method allows one to recognize variations in fluorescence intensities solely due to cell size differences and to disclose patterns of expression not detectable by the conventional intensity based approach.