Alice L. Givan’s research while affiliated with Geisel School of Medicine at Dartmouth and other places

A flow cytometer is an instrument that illuminates cells (or other particles) as they flow individually in front of a light source and then detects and correlates the signals from those cells that result from the illumination. In this chapter, each of the aspects of that definition will be described: the characteristics of cells suitable for flow cytometry, methods to illuminate cells, the use of fluidics to guide the cells individually past the illuminating beam, the types of signals emitted by the cells and the detection of those signals, the conversion of light signals to digital data, and the use of computers to correlate and analyze the data after they are stored in a data file. The final section of the chapter will discuss the use of a flow cytometer to sort cells. This chapter can be read as a brief, self-contained survey. It can also be read as a gateway with signposts into the field. Other chapters in this book will provide more details, more references, and even an intriguing view of the future of cytometry.

A flow cytometer is an instrument that illuminates cells (or other particles) as they flow individually in front of a light source and then detects and correlates the signals from those cells that result from the illumination. In this chapter, each of the aspects of that definition will be described: the characteristics of cells suitable for flow cytometry, methods to illuminate cells, the use of fluidics to guide the cells individually past the illuminating beam, the types of signals emitted by the cells and the detection of those signals, the conversion of light signals to digital data, and the use of computers to correlate and analyze the data after they are stored in a data file. The final section of the chapter will discuss the use of a flow cytometer to sort cells. This chapter can be read as a brief, self-contained survey. It can also be read as a gateway with signposts into the field. Other chapters in this book will provide more details, more references, and even some controversy about specific topics. Key WordsFlow cytometry–fluidics–fluorescence–laser

A better understanding of immune effector and regulatory pathways has led to innovative, and complex, immunotherapy strategies. CD8(+) cytolytic T lymphocytes (CTL) provide one common pathway of tumor cell destruction. The peripheral blood CTL compartment typically comprises a minority of anti-tumor CD8(+) lymphocytes and the determination of their number during clinical trials is the focus of various laboratory methods. We have monitored tumor specific CD8(+) as well as CD4(+) lymphocyte precursor frequencies in the peripheral blood using a Dye Dilution Proliferation Assay (DDPA). We summarize our experience applying DDPA in a multi-parameter, antigen-specific assay, detailing some of its complexities and advantages. We provide examples of our clinical trial results showing tumor-specific CD8(+) and CD4(+) precursor frequency (PF) data in patients being treated on novel immunotherapy trials.

Cell-tracking dyes stain cells with bright fluorescence which is partitioned between daughter cells after each cell division, so that the daughter cells have closely half the intensity of the parent cells from which they were derived. Therefore, the intensity of a cell, relative to its intensity at the time of staining, provides information about how many divisions have occurred. Knowing the number of division cycles that have occurred, one can calculate the number of cells in the original population (before culture) that were going to go on to proliferate. In this way, cell-tracking dyes provide a flow cytometric method for determining the proportion of cells in a population that will go on to proliferate in response to stimulation. This dye-dilution methodology can, therefore, detect proliferation of rare antigen-specific cells that increase in number during division and, importantly, can be used to back-calculate the precursor frequency of these rare cells. Simultaneously, the phenotype of these cells can be determined, as well as their ability to synthesize cytokines, and to express or not relevant antigen-binding receptors and activation markers.

To test the effect in normal human volunteers of transient systemic inflammation on the variability in time-series behaviors of widely divergent physiologic measures of the human inflammatory response. Prospective study of human volunteers who were tested on 2 consecutive days, a control day and a treatment day. Each participant served as his or her own control. Critical care facility of a university medical center. Subjects were eight healthy human volunteers. Participant subjects were tested on both a baseline day with no intervention and on a treatment day when they received 4 ng/kg intravenous Escherichia coli endotoxin. Continuous electrocardiographic recordings and serial blood sampling (performed every 5 mins) were used to create time-series of heart rate (R-R intervals), neutrophil function (phagocytosis), and plasma cortisol concentrations. For each primary measure, we recorded a significant increase in the regularity (decreased variability) of the functional measurement as assessed by the statistical entity, approximate entropy. Increased regularity, or decreased variability, of organ functions is a generalized response to systemic inflammation that occurs in widely divergent systems during endotoxemia.

The T-cell receptor provides T cells with specificity for antigens of particular molecular structure (the "epitope"); the T-cell pool in an individual responds to the presence of many different antigenic epitopes, but any particular T cell will respond preferentially to one defined epitope. After stimulation of a T cell by the binding of its receptor to its cognate antigen in the context of a major histocompatibility complex (MHC) molecule on an antigen-presenting cell, the T cell will begin to proliferate and synthesize cytokines. Tetramer binding and the enzyme-linked immunospot (ELISPOT) method have been used to determine what proportion of cells in the T-cell pool can bind to a defined antigenic peptide or will secrete cytokines in response to a particular antigenic stimulation. The method described here uses tracking dyes to determine what proportion of T cells will proliferate in response to stimulation. As a flow cytometric "single-cell" method, it can be combined with tetramer and cytokine staining to determine the precursor frequencies of cells in the T-cell pool able to bind tetramer, to synthesize cytokines, and to proliferate in response to antigen.

Triggering of the T-cell receptor by cognate antigen induces a variety of cellular events leading to cell proliferation and differentiation. While the plasticity and diversity of T-cell responses have been recognized for a long time, few quantitative studies have been conducted to measure what proportion of specific T cells will enter a given differentiation program after antigen stimulation. In the present study, we analyzed human T cells cultured with influenza-peptide-loaded dendritic cells. We compared three individual methods for assaying the frequency of antigen-specific T cells: ELISPOT, tetramer-binding, and proliferation. The three methods yielded similar but not identical results. In order to study these differences at the single cell level, we developed a multiparameter flow cytometric method, which allows simultaneous analysis of antigen-specific tetramer binding, T-cell proliferation, and cytokine production. Based on these data, we used flow precursor frequency analysis to calculate the proportion of eight different precursor subsets in the original, resting population. We conclude that approximately half of the cells that bound specific tetramers actually proliferated and synthesized IFNgamma in response to antigen. In addition, similar numbers of cells that did not bind tetramer proliferated (but did not synthesize IFNgamma). The method allows for an estimate of the precursor frequency of each functional subset within the initial population. It could be applied to additional markers of function and differentiation, combining all parameters into a description of the complex response potential of a T-cell pool.

CD163, a monocyte and macrophage-specific surface glycoprotein, which is increased by interleukin-10 and glucocorticoids, is a scavenger receptor for hemoglobin/haptoglobin complexes. We report a rapid and highly reproducible rise in soluble CD163 in the plasma of human volunteers given intravenous lipopolysaccharide (LPS). We also show that LPS induces shedding of CD163 from the surface of isolated monocytes, identifying shedding from monocytes and macrophages as a likely mechanism for the endotoxemia-associated rise in plasma CD163 in vivo. Studies using the inhibitor TAPI-0 indicate that a metalloproteinase is responsible for LPS-mediated shedding of CD163. Finally, we demonstrate a marked increase in surface CD163 expression on circulating monocytes 24 h following experimental endotoxemia. These findings show that CD163 is rapidly mobilized in response to bacterial endotoxin. As hemoglobin can bind LPS and enhance its toxicity, it will be important to determine how cell surface and soluble CD163 influence inflammatory processes during sepsis.

Unlabelled: We previously reported that a continuous insulin infusion improves neutrophil phagocytic function after cardiac surgery in diabetic patients. These data suggested that hyperglycemia impairs neutrophil function, and because nondiabetic patients also experience hyperglycemia during cardiac surgery, we hypothesized that a continuous insulin infusion would improve glucose control and neutrophil function in nondiabetic cardiac surgical patients. Patients were randomized to receive either no insulin (Control group) or a continuous insulin infusion (Insulin group), with glucose measurements every 10 min during cardiopulmonary bypass (CPB). Blood glucose was significantly lower in the Insulin group immediately after surgery but not during surgery. When assessed as the percentage of phagocytic cells, neutrophil function was similar in the Control and Insulin groups at baseline (55% and 57%, respectively) and after CPB (38% and 43%, respectively). However, a quantitative determination of neutrophil phagocytic activity showed that whole blood neutrophil phagocytic capacity increased significantly in both groups at 60 min after CPB when compared with their respective baseline values and that the increase in total neutrophil phagocytic capacity was significantly more in the Insulin group compared with the Control group (P = 0.036). This observation was primarily due to a larger increase in the peripheral blood neutrophil count and not to increased activation of neutrophils. Implications: IV insulin, as used in this study, had effects on blood glucose only after cardiac surgery, when it was associated with an increased neutrophil count and a greater total capacity of peripheral blood neutrophils to ingest foreign particles.