Neal A. Benson

Flow cytometric analysis of DNA in bone and soft‐tissue tumors using nuclear suspensions

Ninety‐four bone and soft tissue tumors were analyzed for their DNA content using flow cytometry (FCM). A simple, rapid method for preparing isolated nuclear suspensions was used. Tissues, minced in a hypotonic solution containing detergent and propidium iodide as a fluorescent probe for DNA, provided in most instances high nuclear yields from only 0.02 to 0.03 g of solid tumor. Whereas all nonneoplastic samples had a diploid DNA content, various degrees of abnormal DNA distributions were detected in 90% of the neoplastic samples and were present in benign as well as malignant tumors. Our findings demonstrate that FCM DNA analysis is practical in most musculoskeletal tumors and support the observations of others that abnormal DNA content may serve as a general neoplastic marker in these tumors.

Heterogeneity of aldehyde dehydrogenase expression in lung cancer cell lines is revealed by Aldefluor flow cytometry‐based assay

We have been interested in studying the roles of two aldehyde dehydrogenases in the biology of lung cancer. In this study, we seek to apply Aldefluor flow cytometry‐based assay for the measurement of aldehyde dehydrogenase (ALDH) activity in lung cancer cell lines, which may become a new tool that will facilitate our continued research in this field.

Progress toward analysis of D8/17 binding to B cells in children with obsessive compulsive disorder and/or chronic tic disorder

BACKGROUND Previous research has suggested that a subgroup of children with obsessive compulsive disorder (OCD) have neuropsychiatric sequelae of streptococcal pharyngitis, similar to that seen in the neurological manifestation of rheumatic fever (RF). Monoclonal antibody D8/17 demonstrates increased binding to B cells in patients with RF and in patients with neuropsychiatric disorders using immunofluorescent microscopy. OBJECTIVE The aim of this study was to determine if an earlier immunofluorescent microscopy study of monoclonal antibody D8/17 in childhood-onset OCD and/or chronic tic disorder (CTD) could be replicated using the more objective method of flow cytometric analysis. METHOD D8/17 binding to B cells was determined in patients with OCD and or CTD (N=32), and healthy controls (N=12) by flow cytometric analysis. RESULTS Subjects with OCD/CTD showed increased mean cell binding (26.0%) of monoclonal antibody compared with healthy controls (9.1%) (p<0.001). When using the threshold of greater than 19% binding (95% upper confidence interval) as a measure of positivity, 65.6% of patients compared with 8.3% of controls showed increased antibody binding to B cells (p=0.01). CONCLUSIONS Although this study reports positive results, many methodological issues will need to be addressed before generalized use of assay for diagnostic purposes.

L-selectin expression enhances clonogenesis of CD34+ cord blood progenitors.

L-selectin, a surface adhesion glycoprotein expressed on leukocytes, has a well-established role in mediating inflammation and lymphocyte recirculation. Recent evidence suggests that L-selectin may also influence hematopoiesis. We observed that a greater proportion of CD34+ cells express L-selectin in cord blood compared with adult bone marrow, and we hypothesized that L-selectin expression is associated with enhanced clonogenic properties. To test this, we compared CD34+/L-selectin+ cells with CD34+/L-selectin- cells in hematopoietic clonogenic assays. From CD34+/L-selectin+ cell cultures, we observed a 3-fold increase of d 12-14 colony-forming unit-granulocyte/macrophage and multipotent progenitor cells, and a 5-fold enhancement of primitive d 21 high proliferative potential colony-forming cells compared with the progeny of CD34+/L-selectin-cells. We conclude that CD34+ cord blood cells expressing L-selectin are enriched in their clonogenic activity compared with cell fractions lacking L-selectin expression.

Real-time adaptive clustering of flow cytometric data

Abstract In dealing with massive flow cytometric data, a real-time adaptive clustering technique referred to as RTAC has been developed. This technique adopts the brain metaphor for information processing. The problem-solving structure is configured as a connectionist network. Information is encoded in the form of connection weights. The structure evolves as more data are seen by adjusting its weights, governed by a learning equation. The number of clusters need not be predefined. The algorithm is fast and robust. The results are reported from the domain of measuring the antigenic properties of blood samples. Its relations to other clustering alternatives are discussed. The technique has been validated statistically with respect to self-consistency.

Tissue Dynamics of CD8 Lymphocytes That Suppress Viral Replication in Cats Infected Neonatally with Feline Immunodeficiency Virus

The purpose of this study was to determine the tissue distribution and antiviral activity of the CD8 lymphocytes that suppress the replication of feline immunodeficiency virus (FIV). Cell-associated FIV load, CD8alpha(+)beta(low) cells, and CD8 cell-mediated suppression of FIV were measured serially in the blood, thymus, and peripheral lymph nodes after neonatal inoculation. Between 6 and 10 weeks, relative numbers of CD8alpha(+)beta(low) cells increased, whereas CD8alpha(+)beta(high) cells declined in the thymus and blood of infected cats. By 12-16 weeks, the lymph nodes were enlarged because of an absolute expansion of all CD8beta subpopulations. The strength of CD8 cell-mediated FIV suppression in vitro, but not CD8alpha(+)beta(low) cell content, was correlated inversely with virus load in the thymus and blood. Thus, after neonatal FIV inoculation, CD8alpha(+)beta(low) cells first occupy the thymus and blood, where strong CD8 cell-mediated antiviral activity is linked to reduced virus load in multiple lymphoid tissues.

Effect of Methotrexate on Cell Cycle and DNA Synthesis of Astrocytes in Primary Culture: Flow Cytometric Studies

Relatively pure population of astrocytes in primary culture were examined by flow cytometry at various time intervals after exposure to 1, 10 and 100 iiM of methotrexate (MTX). The percentage population of cells in different phases of the cell cycle was determined using propidium iodide (PI) to measure cellular DNA content. DNA synthesis was assessed by measuring the fluorescence intensity of F1TCconjugated antibody to bromodeoxyuridine (BrdUrd). The two parameters were correlated to determine the location of BrdUrd-incorporating cells within the cell cycle. Exposure of astrocytes to MTX caused a consistent increase in number of cells in S-phase which correlated with the increase in BrdUrd incorporation. These studies provide supportive evidence that the astrogliosis seen in MTX encephalopathy may be due to a primary involvement of astrocytes.

Cell cycle status of CD34+ cells in human fetal bone marrow

We used flow cytometric analysis to determine the cell cycle characteristics of human CD34+ cells from fetal bone marrow (BM), adult BM, and umbilical cord blood (UCB) samples. Fetal BM had three-fold more cells in the S-phase than did adult BM or UCB.

Analysis of neoplastic leukocyte surface antigens in unfractionated blood.

The detection of leukemic cells in peripheral blood is based on cytologic and cytochemical methods. Recently, the characterization of leukemic cells has been improved by the analysis of cell surface antigens. Abundant leukemic cells are relatively easy to identify. Small numbers of circulating leukemic cells, however, may be difficult to recognize by conventional or immunological techniques. This is of particular importance in treated patients in whom the presence of low levels of circulating leukemic cells may have considerable clinical relevance. We used multiparameter correlated flow cytometric analysis to detect and characterize human leukemia/lymphoma cells in small samples of unfractionated peripheral blood. Leukocyte surface antigens were labeled with monoclonal antibodies and simultaneous forward and right-angle light scatter and fluorescence signals were measured from each cell. An interactive computer program was written that permitted the two light scatter measurements to be displayed on the screen on a cell-by-cell basis (dot-plot). Subpopulations of interest could then be selected on the dot-plot for analysis of their fluorescence distribution. On the basis of their dual scatter properties and their antigenic profile, neoplastic leukocytes were recognized even in cases in which leukemic cells were infrequent and not detectable by conventional cytologic methods.

Distinctive expression of the T4 antigen in normal and stimulated lymphocytes.

Correlated light scatter and fluorescence flow cytometric analysis of human peripheral blood lymphocytes showed that the expression of the T4 antigen was higher in the larger lymphocytes than in the smaller lymphocytes. A similar expression pattern was observed for HLA Class I antigens but not for T3 and T8, whose expression was independent of cell size. Results with lymphocytes from spleen, lymph node, and tonsil were comparable to those of peripheral blood. Thymocytes, however, were smaller and expressed less T4 and T8 than peripheral lymphocytes. In studies of lymphocytes stimulated in vitro with allogeneic cells or pokeweed mitogen, two populations of T4-positive cells were observed: one of large cells expressing high amounts of T4 and one of small cells expressing low amounts of T4. Similar patterns were seen with T8, although less consistently. In contrast, the expression of T3 was the same in both large and small cells. The large cells expressing high amounts of T4 were not restricted to cells engaged in DNA synthesis or mitosis. This was established by selectively analyzing cells in the G0G1 phases of the cell cycle and by studying stimulated lymphocytes no longer undergoing proliferation. Taken together, these results suggest that immature T lymphocytes are small and express low amounts of T4 and T8. We postulate that as they differentiate, cell size and T4 expression increase proportionally, both parameters increasing even further after antigenic or mitogenic stimulation. The quantitative expression of T4, and probably of T8 but not of T3, is therefore intimately related to maturation and activation of lymphocytes, a fact that may conceivably be related to a functional role of these surface molecules.