TRAINING and EDUCATION
Demystifying the Lab
ASH Clinical News takes a look at the complex scientific techniques that
hematologists/oncologists hear about every day, with practical information
for the practicing clinician.
DEMYSTIFYING
Multicolor Flow Cytometry
Flow cytometry has come a long way in the
80 years since its inception. The now highly
sophisticated technique is an indispensable tool
for the diagnosis, prognosis, and monitoring
of patients with hematologic malignancies
and a range of other disorders. Yet, its use
began during the 1930s as a relatively simple,
automated way to count red blood cells: A
red blood cell suspension is passed through
a capillary tube and each cell is accounted
for by a photoelectric apparatus attached to a
microscope. 1
In 1949, engineer and inventor Wallace
Coulter discovered a method for detecting
and counting cells or particles in suspension as
each passed through an aperture in an electric
current, which eventually became known as
the Coulter Principle. In 1953, he patented an
apparatus that incorporated this principle, the
Coulter Counter, that still is used today for
characterizing human blood cells. 2
German scientist Wolfgang Göhde, PhD,
further refined the technique and added new
detection capabilities such as measurement of
size and granularity by light scatter and fluores-
cence. He is credited with developing the first
commercial fluorescence-based flow cytometry
device in 1968. 3
Today’s state-of-the-art clinical flow cytom-
eters are multicolor flow cytometry instruments
that go well beyond the ability to count cells,
allowing clinicians and researchers to routinely
detect a combination of as many as eight to 10
cell surface and cytoplasmic antigens – all on
the same cell from a patient’s blood sample.
“For the field of hematologic malignancies,
multicolor flow cytometry is one of the best
tools we have for detecting and subtyping these
cancers,” says David L. Jaye, MD, a pathologist
specializing in lymphoid malignancies and an
assistant professor in the department of pathol-
ogy and laboratory medicine at the Emory
University School of Medicine in Atlanta.
ASH Clinical News spoke with Dr. Jaye and
other researchers about the principles behind
multicolor flow cytometry, its clinical applica-
tions in hematology and oncology, and the
evolving role of this powerful diagnostic tool.
Flow Cytometry 101
Flow cytometry is performed on a laboratory
machine called a flow cytometer, which counts
cells and particles, measures their forward
and side scatter properties, excites the cell-
associated fluorochromes with lasers, and then
detects the fluorescence linked to these cells.
The dyes, called fluorochromes or fluorophores,
are fluorescent, chemical compounds that can
re-emit light upon excitation.
ASHClinicalNews.org
Following the discovery of monoclonal anti-
bodies in the 1970s, these dyes are conjugated
to monoclonal antibodies that bind to antigens
on the surface of cells or in the cell’s cytoplasm. 4
Flow cytometers are used to identify and count
the cells present in a patient sample, but it also
can be used to physically sort cells for further
analyses.
Only a single fluorochrome was used to
label the antibodies initially, but today, there
are tens of fluorochromes in use. Each emits
light at different wavelengths, which allows
researchers to incubate cell samples with mul-
tiple fluorochrome-antibody combinations – as
many as 10 for clinical applications and even
more for research applications – facilitating the
detailed characterization of subpopulations of
cells within a sample.
“We can look at
multiple antigens
on the same cell
and the process
is automated,
removing the
chance of human
error.”
like “whether the patient had leukemia or lym-
phoma cells present.”
Two decades later, “most laboratories are
doing 10-color flow cytometry and labeling
a single cell with up to eight antibodies, plus
forward light scatter and side light scatter,” she
continued. “Eight- and 10-color flow cytom-
etry are reliable tools and are now considered
the standard in the industry.”
To prepare a patient cell sample for multi-
color flow cytometry, the sample can be
processed to make the cells permeable, allowing
the added antibodies to both bind to antigens
on the cell surface and within the cytoplasm and
nucleus – all on the same cell. At the same time,
this allows scientists to assess the cell’s physical
properties, including relative size. “We’re get-
ting information on the size of the cell, as well
as its internal complexity, so that you can tell a
malignant lymphocyte from a neutrophil, for
example,” said Dr. George.
Smaller Samples, More Information
“The power of flow cytometry is that it gives us
information about the cell lineage – lymphoid
or myeloid, B cell or T cell,” Dr. George com-
mented. “We also can figure out the cells’ state
of differentiation, whether these are imma-
ture cells, like leukemic blasts from the bone
marrow, or more fully differentiated cells, like
a mature B-cell lymphoma.” The antibodies
used to detect certain cell markers also provide
information for clinicians about the presence
of either wild-type or abnormal or malignant
cells.
Pathologists use the flow cytometry–generated
data to assess whether the populations of cells
—DAVID L. JAYE, MD
from the patient contain malignant cells. To-
gether with histopathology data, this informa-
With the use of multiple fluorochromes,
tion informs a diagnosis.
the process and analysis become more chal-
“Flow cytometry is often ordered upfront
lenging. Ideally, there will be minimal spectral to make a diagnosis when a hematologic
overlap among the wavelengths emitted by
malignancy is suspected clinically,” Dr. Jaye
each fluorochrome, which avoids confounding said. Various institutions, including the World
the instrument’s ability to distinguish among
Health Organization, have specified criteria
cell populations. But there is some room for
for the diagnosis of hematologic malignan-
spectral overlap, because today’s machines
cies using distinct markers. 5 For example, for
can correct for this wavelength overlap, called
acute myeloid leukemia, pathologists look for
“spillover.”
cytoplasmic myeloperoxidase, which points to
“We’ve been doing flow cytometry for he-
a myeloid lineage. For T-cell lymphomas, they
matopathology for decades and, 20 years ago,
look for the CD3 surface antigen, and for B-
we were only able to label cells from a patient
cell malignancies, they look for surface CD19
sample with a few different colors,” explained
and cytoplasmic CD22 and CD79a.
Tracy I. George, MD, professor of pathology
The other important technique for immuno-
at the University of Utah School of Medicine
phenotyping a patient’s biopsy or bone mar-
and a medical director of hematopathology at
row sample is immunohistochemistry, which
ARUP Laboratories in Salt Lake City, Utah.
also uses antibodies to detect specific cell-
The limited number of colors meant that pa-
associated antigens within a sample to identify
thologists could answer only simple questions,
Continued on page 118
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