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OCR for page 47
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
Representative terms from entire chapter:
flow cytometers
