March, 2002

RECENTLY PUBLISHED:

UNIT 3.5 Visualization of Aerosol Contamination (A. Oberyszyn, Ohio State University, Columbus, Ohio). The routine operation of a flow cytometer generates aerosols, and most commercially available instrument are not designed to be 100% effective in their elimination. Aerosols are a concern for operators as well as for others in the facility because of the potential for exposure to contamination, especially during sorting of biohazardous samples. This unit describes a visual method for examining aerosol containment using a commercial resin that fluoresces under UV or black light illumination. The method is rapid, inexpensive, and simple. Results are immediate and potentially quantitative

UNIT 6.10 Immunotypic Analysis of Platelets (L.A. Krueger, M.A. Barnard, A.L. Frelinger III, M.I. Furman, and A.D. Michelson, University of Massachusetts Medical School, Worcester, Mass.). Platelets are the smallest cellular component in the peripheral circulation. Their primary role is maintenance of hemostasis. The evaluation of platelets by flow cytometry has proven beneficial in the investigation of many disease states, including inherited defects, cardiovascular disease, stroke, and many other inflammatory processes. This unit describes protocols to evaluate platelet surface glycoproteins, platelet activation status, and platelet responsiveness to agonist. Detailed discussion of appropriate sample handling, reagent preparation, flow cytometric setup, and data interpretation are provided for three independent assays.

UNIT 9.18 Stem Cell Identification and Sorting Using the Hoechst 33342 Side Population (SP) (M.A. Goodell, Baylor College of Medicine, Houston, Tex.). This unit describes the use of Hoechst 33342 to identify and purify murine hematopoietic stem cells, the so-called side population. Three properties of the dye contribute to the ability to distinguish stem cells in this way. Hoechst is a DNA-binding dye. It has at least two binding modes that result in different spectral properties, allowing resolution of multiple populations by viewing fluorescence at two wavelengths simultaneously. The ability to discriminate SP cells is based on the differential efflux of the dye by a multi-drug-like transporter. Particular attention is given to the critical aspects of Hoechst concentration, cell concentration, staining time, and staining temperature.

UNIT 10.12 Dial-In Flow Cytometry Data Analysis (F.L. Battye, The Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia). As listmode data files continue to grow larger, access via any kind of network connections becomes more and more trouble because of the enormous traffic generated. The limited speed of transmission via modem makes analysis almost impossible. This unit presents a solution to these problems, one that involves installation at the central storage facility of a small computer program called a Web servlet. Operating in concert with a Web server, the servlet assists the analysis by extracting the display array from the data file and organizing its transmission over the network to a remote client program that creates the data display. The author discusses a recent implementation of this solution and the results for model transmission of two typical data files. The system greatly speeds access to remotely stored data, yet retains the flexibility of manipulation expected with local access.

UNIT 8.12 Comparative Genomic Hybridization (CGH): A Molecular Cytogenetic Method to Detect Unbalanced Genetic Aberrations (E. Schröck, Institute for Molecular Biotechnology, Jena, Germany; Z. Weaver, National Cancer Institute, Bethesda, Md.; and D. Albertson, University of California, San Francisco, Calif.). This unit presents comparative genomic hybridization (CGH), a genome-wide screening technique for genetic aberrations in tumor samples. Specific emphasis is placed on recent applications to the analysis of murine model systems for human cancer. CGH is an invaluable tool for identifying the characteristic genetic rearrangements in these models. The authors discuss an exciting new method currently being developed, array CGH, which results in a tremendous increase in resolution. Oncogene amplifications and deletions of tumor-suppressor genes are detected on a single-gene level. Detailed protocols are supplied for CGH analysis of both human and mouse chromosomes.

UNIT 8.11 Principles and Applications of PRINS in Cytogenetics (J. Koch, Danish Cancer Society, Aarhus, Denmark). A flexible, low-cost alternative to FISH, primed in situ labeling (PRINS), has traditionally been used to detect tandemly repeated target sequences in chromosomes and nuclei. The technique is capable of discriminating among closely related DNA sequences in situ and has the advantage of using very small probes which easily penetrate to almost any target. This unit describes basic PRINS and the alternative version, dideoxy-PRINS, which can increase the sensitivity of the reaction by an order of magnitude. New material on multicolor PRINS and quantitative PRINS has been added. Protocols for detection of single-copy sequences and for application to the study of in-vivo activity of DNA-modifying enzymes are planned.

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UNIT 1.18 Dynamic Thermoregulation of the Sample in Flow Cytometry (S.W. Graves, R.C. Habbersett, and J.P. Nolan, Los Alamos National Laboratory, Los Alamos, N. Mex.). Fine control of temperature is an important capability for any analytical platform. A circulating water bath has been the traditional means of maintaining constant temperature in the sample chamber of a flow cytometer, but this approach does not permit rapid changes in sample temperature. This unit explains the use of Peltier modules for regulation of sample temperature. The heat pumping generated by the passage of current through properly matched semiconductors, known as the Peltier effect, makes it possible for these thermoelectric modules to both heat and cool. The authors describe the construction of a Peltier module–based thermoregulation unit in step-by-step detail and present a demonstration of flow cytometry measurements as a function of temperature.

UNIT 3.4 Safe Use of Hazardous Chemicals (G. Lunn, Baltimore, Md, and G. Lawler, Purdue University, West Lafayette, Ind.). Many of the reagents listed in Current Protocols in Cytomtery are hazardous to the user: toxic, oncogenic, mutagenic, teratogenic, allergenic, or just plain irritating. This informative unit discusses the basic minimum safety requirements for handling, storage, and disposal of chemicals in the laboratory. Extensive tables provide detailed information on types of hazards, chemical incompatibilities, chemical resistance of glove types, and biological stains. The authors provide a number of protocols for the decontamination and disposal of some commonly encountered hazardous chemicals, as well as analytical detection procedures for verifying their effectiveness. Also included are an extensive reference bibliography and a list of useful Web sites. This unit has been revised to included additional protocols on enzyme prohibitors.

UNIT 6.11 Immunophenotypic Analysis of PNH Cells (S.J. Richards and P. Hillmen, Leeds Infirmary, Leeds, United Kingdom). Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem-cell disorder in which a gene mutation results in cellular inability to synthesize the glycosylphosphatidylinositol (GPI) anchors needed to attach various proteins to the cell membrane. Traditional clinical laboratory PNH screening and diagnosis are based upon demonstration of increased erythrocyte susceptibility to lysis by activated complement. These methods are neither very specific nor very sensitive, and have largely been superseded by the rapid, sensitive, and specific flow cytometric analysis of GPI-linked antigen expression. The protocols in this unit cover immunophenotypic analysis of red blood cells, peripheral blood granulocytes, and peripheral blood monocytes, as well as a four-color modification for simultaneous analysis of peripheral blood granulocytes and monocytes.

UNIT 10.13 The Application of Data Mining to Flow Cytometry (A. Nguyen, University of Texas, Houston, Tex.). Data mining is the process of automating information discovery to detect useful patterns, correlations, and trends. Existing data must be fitted into a representative model from which useful information can be derived through a variety of algorithms. The routine generation of vast amounts of data make flow cytometry a logical target for the application of data mining. This informative unit discusses the steps of the data-mining process using the immunophenotyping of hematologic neoplasms to demonstrate the application. The author describes several types of algorithms and provides a useful resource list of commercially available tools.

UNIT 10.14 Calibration and Shading Correction for Fluorescence Microscopes (M. Model, Chagrin Falls, Ohio). Standardization in image cytometry involves intensity calibration and shading correction. This unit presents a method using concentrated solutions of fluorophores. A drop of highly concentrated fluorescein or rhodamine placed between a slide and a coverslip produces a spatially uniform fluorescent sample with reproducible quantum yield and resistance to photobleaching. The technique has a number of practical features making it inexpensive, reproducible, and straightforward