Differences in doublet analysis have the potential to alter DNA cell-cycle measurements. The techniques for doublet determination are often used interchangeably without regard for the complexity in cell shapes and sizes of biological specimens.G 0/1 doublets were identified and quantitated using fluorescence height versus area and fluorescence width versus area pulse measurements, by enumerating the proportion of G 2 ؉ M cells that lack cyclin B1 immunoreactivity, and modeled in the DNA histograms by software algorithms. These techniques were tested on propidium iodide-stained whole epithelial cells or nuclei from asynchronous cultures, or after exposure to chemotherapeutic agents that induced cell-cycle arrest and were extended to human breast tumor specimens having DNA diploid patterns.G 0/1 doublets were easily discernible from G 2 ؉ M singlets in cells or nuclei that are generally homogenous and spherical in shape. Doublet discrimination based on pulse processing or cyclin B1 measurements was nonconcordant in some nonspherical cell types and in cells following cell cycle arrest. Significant differences in G 0/1 doublet estimates were observed in breast tumor specimens (n ‫؍‬ 50), with estimates based on pulse width twice those of pulse height and nearly five times greater than computer estimates. Differences between techniques are attributed to difficulties in the separation of the boundaries between G 0/1 doublets and G 2 ؉ M singlet populations in biologically heterogeneous specimens.To improve reproducibility and enhance standardization among laboratories performing cell cycle analysis in experimental cell systems and in human breast tumors, doublet discrimination analysis should best be accomplished by computer modeling. Shape and size heterogeneity of tumor and arrested cells using pulse-processing can lead to errors and make interlaboratory comparison difficult. Cytometry (Comm. Clin. Debris and aggregates can be prominent components of DNA histograms, affecting the accuracy and reproducibility of cell-cycle estimates (1-4). Debris originate from the damage and disintegration of cells following apoptosis or the fragmentation associated with the slicing of cells or nuclei during mechanical disaggregation. Clumping may be due to the incomplete disruption of tissues by mechanical or enzymatic means into single-cell or nuclear suspensions, by the use of alcohol-based fixatives that yield DNA histograms with low coefficients of variation of the G 0/1 peak but induce clumping, or by centrifugation. In addition, clumping may be an inherent attribute of some cell types, e.g., keratinocytes.Aggregates can be composed of large clusters of cells or nuclei or two or more G 0/1 (2N) events adhered together (G 0/1 doublets) that are indistinguishable from particles with 4N, 6N, or 8N DNA content. Large clumps can be removed by nylon mesh filtration (typically 35-53 m), sheared apart by passage through small gauge needles, or identified on the basis of forward-angle light scattering (2). Strategies to separate overla...

We identified eight cases of T-cell lymphoma with evidence of a gamma delta phenotype over a 13-year period. Seven of these cases conformed to a distinct clinicopathologic entity of hepatosplenic gamma delta T- cell lymphoma. Nearly all of these patients were young adult males (five of seven), with a median age at presentation of 20 years. They presented with marked hepatosplenomegaly, without lymphadenopathy or significant peripheral blood lymphocytosis. Thrombocytopenia was seen in all patients, and five of seven were mildly anemic. The clinical course was aggressive, and despite multiagent chemotherapy, the median survival duration was less than 1 year. The morphologic findings were uniform; a monomorphic population of medium-sized lymphoid cells with moderately clumped chromatin and a rim of pale cytoplasm infiltrated the sinusoids of the spleen, liver, and bone marrow. The cells had a characteristic immunophenotype: CD2+, CD3+, CD4-, CD5-, CD7+, CD16+, CD57-, CD25-, T-cell receptor (TCR)delta +, beta F1-. CD8 was positive in four of seven cases tested, and CD56 was positive in five of six. All cases expressed the cytotoxic granule-associated protein, TIA1, but perforin was detected in only one case. All cases with assessable DNA had a TCR gamma gene rearrangement, and lacked Epstein-Barr virus sequences. Isochromosome 7q was identified in two cases with cytogenetic information. The one case of cutaneous gamma delta T-cell lymphoma differed in its clinical manifestations, histologic appearance, and immunophenotype. We conclude that hepatosplenic gamma delta T-cell lymphoma is a distinct clinicopathologic entity derived from cytotoxic gamma delta T cells, and should be distinguished from other lymphomas of T-cell and natural-killer cell (NK)-like T-cell derivation.

Background: Among all hematologic malignancies, B-cell chronic lymphocytic leukemia (BCLL) has the highest familial clustering (three-to sevenfold increase), strongly suggesting a genetic component to its etiology. Familial BCLL can be used as a model to study the early pathogenesis of this disease.Methods: We examined nine kindreds from the National Cancer Institute's Familial BCLL Registry, consisting of 19 affected members with BCLL and 33 clinically unaffected first-degree relatives. Flow cytometric immunophenotyping to detect a B-cell monoclonal lymphocytosis (BCML) was performed. Monoclonality was confirmed by polymerase chain reaction analysis of whole blood DNA. Cell cycle analysis for aneuploidy was conducted.Results: In all affected individuals, we observed the classic BCLL CD5/CD19/CD20/CD23 immunophenotypic patterns. Six of the 33 unaffected individuals (18%) had evidence of BCML. Additional individuals (13/33, 39%) showed some other abnormality, whereas 14 individuals (42%) were normal. Based on an estimated prevalence of 0.7% for BCML in the general population, the finding of six subjects (18%) with clonal abnormalities in this relatively modest sample was significantly greater than expected (i.e., 18% vs. 0.7%, P < 5.7 ؋ 10