Flow cytometry Subject

Flow cytometer cell counts are much more precise and more accurate than hemocytometer counts. Hemocytometer cell counts are subject both to distributional (13) and sampling (14—16) errors. The distribution of cells across the surface of a hemocytometer is sensitive to the technique used to charge the hemocytometer, and nonuniform cell distribution causes counting errors. In contrast, flow cytometer counts are free of distributional errors. Statistically, count precision improves as the square root of the number of cells counted increases. Flow cytometer counts usually involve 100 times as many cells per sample as hemocytometer counts. Therefore, flow cytometry sampling imprecision is one-tenth that of hemocytometry. 

A similar method is used for beads with defined fluorochrome content (e g, Quantum beads, Flow Cytometry Standards Corporation), except that the beads are not subjected to the antibody-staining procedure The number of fluorochrome molecules per cell is deduced from the standard curve, and is converted to bound antibody molecules from knowledge of the fluorochrome protein ratio of the conjugate. 

A special issue of Cytometry (Vol. 10, No. 5, 1989) is devoted to Cytometry in Aquatic Sciences. A more recent issue of Scientia Marina (Vol. 64, No. 2, 2000) is also devoted to the same subject Aquatic Flow Cytometry Achievements and Prospects. In addition, Chapter 31 of Melamed et al. discusses the application of flow cytometry to higher plant systems. 

Although books on flow cytometry abound and articles on flow cytometry can be found throughout a great range of publications, the following is a limited list of references that I have found particularly useful for general information on the theoretical basis of flow analysis and as routes into the literature on particular subjects and techniques. 

Van Dilla MA, Dean PN, Laerum OD, Melamed MR, eds. (1985). Flow Cytometry Instrumentation and Data Analysis. Academic Press, London. A venerable, but still current book with an emphasis on the physics and mathematics of flow systems and data analysis. It has some excellent (and readable) articles on some theoretical subjects. 

Acquisition In flow cytometry, acquisition refers to the process of recording the intensity of the photodetector signals from a particle in the transient memory of a computer. Once acquired, the data from a group of particles can be stored permanently on a storage medium from which it can be subjected to analysis. Acquisition and then analysis (in that order) are the two central steps in the flow cytometric procedure. 

Fig. 2. Representative dot plots (PE anti-IL-4 vs. FITC anti-IFN-y) for the analysis of intracellular Th cytokines of CD4+ lymphocytes using the Fastlmmune Cytokine System (BD Pharmingen) by flow cytometry (a) a control subject and (b) an allergic asthmatic patient. The numbers in the quadrants denote the percentages of Thl and Th2 cells (W16). Reproduced with permission from C. K. Wong, C. Y. Ho, F. W. S. Ko, C. H. S. Chan, A. S. S. Ho, D. S. C. Hui, and C. W. K. Lam. Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-y, IL-4, IL-10 and IL-13) in patients with allergic asthma. Clin. Exp. Immunol. 125 177-183, Copyright Blackwell Science Ltd., 2001.

Figure 9.3 Mean total lymphocyte count after one course of treatment. Peripheral blood was collected from individuals administered test article (circles) or placebo (squares) on a weekly basis, and subject to flow cytometry analysis to determine mean lymphocyte counts. A standard panel of fluorochrome-conjugated antibodies was used to identify the various lymphocyte sub populations. The solid bar indicates the dosing interval.

Fig. 1. Quantification of shear-induced platelet aggregation by flow cytometry. Panel A corresponds to an unsheared blood specimen. Panel B corresponds to a blood specimen that has been subjected to a pathologically high level of shear stress for 30 sec. As can be seen in the figure there are three distinct cell populations. The upper population consists of platelets and platelet aggregates. The rbcs-plts population corresponds to platelets associated with erythrocytes and leukocytes. The wbcs population consists of some leukocytes that have elevated levels of FITC autofluorescence. The left vertical line separates single platelets (<4.5 xm in diameter) from platelet aggregates, whereas the right vertical line separates small from large platelet aggregates. The latter were defined to be larger than 10 xm in equivalent sphere diameter.

A wart is a skin lesion caused by infection with human papillomavirus (HPV). Contact immunotherapy is one of the many therapeutic options that has been used to treat warts however, the effectiveness of contact immunotherapy differs from patient to patient, and the cause of this variaHon in clinical response is unclear. To assess cytokine changes in patients after contact immunotherapy with squaric acid dibutylester (SADBE), a total of 21 patients with warts and nine healthy control subjects were enrolled in this study [Ib ]. The frequencies of CD3+ T cells expressing interleukin (IL)-4 IL-10, lL-12, tumour necrosis factor-alpha (TNF-a) and interferon-gamma were measured by flow cytometry analysis of peripheral blood at baseline in both patients and controls, and after SADBE treatment in patients. 

Chapter 5 in Shapiro, Chapter 30 in Volume I of Weir, Section 10 in Current Protocols in Cytometry, Chapter 7 in Darzynkiewicz, and Chapter 22 in Melamed et al. are all good discussions of various aspects of flow data analysis. In addition, an entire book by Watson (1992) is devoted to this subject. 

Darzynkiewicz Z, Robinson JP, Crissman HA, eds. (2001). Methods in Cell Biology, Yols 63A and 63B. Cytometry, 3rd edition. Academic Press, San Diego. An up-to-date compilation covering theory and many practical aspects of flow and image cytometry. The previous edition of this book (Vols. 41A and 41B, 1994) contains many articles on many subjects not covered in the third edition and is also worth reading. 

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