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Clinical medicine By T. Vincent Shankey, Advanced Technology Center, Beckman Coulter, Inc. Dr. Shankey (Ph.D., University of Florida School of Medicine, 1977) studied the structure and function of IgM antibodies for his dissertation. Before joining the Advanced Technology Center at Beckman Coulter, Inc., in 2001, he was the director of research for the Urology Department and scientific director of the Clinical Flow Cytometry Laboratory at Loyola University Medical Center near Chicago, Illinois, for more than 13 years. His research has utilized both flow and image cytometry and focused on genomic instability and patterns of evolutionary changes in bladder and prostate cancers. His clinical experience in cytometry included DNA content analyses of human tumors. Since joining the Advanced Technology Center, Dr. Shankey has worked on signal transduction pathways in human cancers, focusing on the development of unique biomarkers for molecularly targeted therapeutics. hen I was a graduate student, I spent many hours in front of a centrifuge built W by Spinco, the centrifuge company acquired by Arnold O. Beckman in 1954. When we have been in science for a while we tend to forget how science evolved to the point where it is today. We forget that before the analytical techniques were devel- oped to study protein-protein interactions, the gold standard was the analytical centrifuge. Another key instrument developed in the 1940s and 1950s was the flow cytometer (Figure 23). At that time, researchers and physicians wishing to do counts of blood cells had to rely on manual counts in counting chambers, with differential blood smears to assess white cell populations. These counts were statistically hard to reproduce and relied on the technical ability of the person using the microscope. A significant development was the development of a cell counter by Walter Coulter in Chicago. He and Arnold Beckman shared a number of characteristics. They were both curious about how ideas worked. They also were inspired by technological challenges. As Coulter is reported to have said, "Challenges are good, and we sure Tabletop centrifuge. Courtesy of Wikipedia. had our share of good." INSTRUMENTATION FOR A BETTER TOMORROW 47

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12,000 10,000 8,000 6,000 4,000 2,000 0 1975-1976 1976-1977 1977-1978 1978-1979 1979-1980 1980-1981 1981-1982 1982-1983 1983-1984 1984-1985 1985-1986 1986-1987 1987-1988 1988-1989 1989-1990 1990-1991 1991-1992 1992-1993 1993-1994 1994-1995 1995-1996 1996-1997 1997-1998 1998-1999 1999-2000 2000-2001 2001-2002 FIGURE 23 Publications using the keyword "flow cytometry" from PubMed; shown are 62,496 references from 1975 to 2002. Important work on flow cytometry was also done at Los Alamos National Laboratory in New Mexico, which remains the home today of the National Flow Cytometry Resource. Since the 1950s, a research group there has worked on many of the technologies underly- ing flow cytometry, such as hydrodynamic focusing of cells being carried in a fluid stream (Figure 24). The addition of laser detectors to flow cytometers in 1972 marked another critical advance. The combination of multiple lasers and detectors made possible several com- mercially successful machines marketed by Becton, Dickinson and Company and by Coulter during this period (Figure 25). In flow cytometry, individual cells travel in suspension past excitation sources, usually a laser, in a liquid medium. As it passes by the excitation source, each cell scatters some of the source light but also often emits light as fluorescence. Physical and chemical charac- teristics such as cell structure, cell size, and particle morphology can be measured from the scattering. While fluorescence may occur naturally, cells are usually stained with fluores- cent dyes that bind specifically to cellular constituents. The intensity of the resulting fluo- rescence emission is measured at several wavelengths simultaneously to identify the quantities of specific components of the cells. The outputs of this method are frequency histograms and dot plots. Frequency histograms display relative fluorescence or scattered light signals plotted against the number of events, whereas dot plots show one dot or point related to the amount of two parameters (identified on the x- and y-axis of the plot) for each cell that passed through the instrument. 48 INSTRUMENTATION FOR A BETTER TOMORROW

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32 104 42.4% 24.1% 104 38% M1 103 103 ents PE 102 APC Ev 102 19.4% 14.3% CD3101 CD34101 0 100 100 101 102 103 104 100 101 102 103 104 100 0 1023 CD8 FITC CD8 FITC Side Scatter FIGURE 24 Flow cytometry data analysis. Left-hand plot shows a one-color histogram plot of CD8 expression by peripheral blood (PB) lymphocytes. Approximately 38 percent of events fall between the marker boundaries and are therefore regarded as CD8 positive. The center plot also shows CD8 expression on PB lymphocytes, but depicts the relationship between CD8 and the T-cell marker CD3. It is apparent that the CD8 positive population depicted by the histogram contains two CD3-defined subpopulations (CD3 positive and CD3 negative), and that the CD3 negative fraction (lower right quadrant) expresses CD8 at a lower intensity than the CD3 positive fraction (upper right quadrant). The right-hand plot shows a population of CD34 ositive stem cells plotted against side scatter. Today flow cytometers are used in a wide variety of applications. One of the most impor- tant is known as immunophenotyping--detecting the types and numbers of immune sys- tem cells in a blood sample. For example, one hallmark of HIV infection is the loss of a distinct subpopulation of T cells called CD4 cells. This particular disease, I've been told, has sold more flow cytometers than any other disease. Flow cytometers also are used in many other applications. For example, when a patient comes to a doctor's office with what appears to be leukemia or lymphoma, the disease is characterized using multiple sets of cell surface markers. Treatments then can be tailored to the particular form of the disease. If we characterize people, we know the first line of treatment to use. This has been one of the first instances of personalized medicine. Flow cytometry also has been critically important in detecting what is called minimal residual disease. After a patient completes a course of therapy, a biological sample is taken from the patient to see how many tumor cells remain. The number of diseased cells remaining suggests how likely the patient is to respond to continued treatment. You get a predictive value out of this technology (Figure 26). The continued development of flow cytometry offers great potential. The first step is to define what the goals of an instrument are. The users of an instrument need to talk to peo- ple who are building an instrument and say,"This is what we need. Build us an instrument that will answer those questions." Otherwise, companies frequently build machines to answer questions that you don't have. INSTRUMENTATION FOR A BETTER TOMORROW 49

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Ease of use is often as important as the capabil- ities of an instrument. For example, as HIV infection becomes a critical problem around the world, the use of instruments in many dif- ferent settings must be considered. We need to monitor biomarkers to know when to start giv- ing people therapies, and then monitor when to increase or decrease those therapies. To achieve this goal, instruments need to be straightfor- ward and robust, with relatively automatic data FIGURE 25 A color photograph of a flow cytometer showing the analysis and interpretation. Also needed are multiple colors of lasers. strict standards and controls, so that the instru- ment will function as desired. While the instrument itself may appear to be relatively unso- phisticated, the package itself needs to be very sophisticated. For research purposes, technological capabilities are important, and instrument develop- ers are building machines with greatly enhanced capabilities. Also, flow technologies and imaging technologies are being merged to allow researchers to examine subcellular components. Such analyses can be very complex, which is very useful in the trans- lational effort to discover new things. FIGURE 26 The EPICS ALTRA flow cytometer is a powerful and flexible cell sorter. Courtesy of the Beckman Coulter, Inc. 50 INSTRUMENTATION FOR A BETTER TOMORROW