This article was a finalist in the 2014 Mayo Clinic Boerhaave History of Medicine Essay Contest.
In the late 1960’s a convergence of fluid
dynamics, laser detecting photodiodes, and high-speed computers with
fluorescent antibody detection allowed for the characterization and
quantification of individual cells in low volumes at a high rate of speed [1, 2]. Prior to flow cytometry (FCM), most clinical
immunology labs and immunophenotyping facilities used fluorescent microscopy to
examine cells. The transition to
clinical cytometry would have remained an uneventfully slow progression if it
weren’t for the advent of acquired immunodeficiency syndrome (AIDS) [3]. This review will discuss the history of flow
cytometry, its role in HIV diagnosis, and conclude with where flow cytometry is
going.
Diagnostic
Flow Cytometry
In the 1600’s van Leeuwenhoek
recorded the first description of the red blood cell (RBC)[4]. By the 1900’s medical scientists, lead by
Paul Ehrlich had developed the triacid stain that allowed for the definition of
the cytoplasm, and nucleus of the cell [4]. And in the 1947, Wallace Coulter working to
standardize paint particle size for the Department of Naval Research developed
the Coulter Principle after passing his own blood through a home made aperture
in his garage and finding that it interrupted an electrical connection to emit
an electric pulse from each cell [5]. This was Coutler’s principle of impedance and
led to the most common diagnostic test used to this day; the complete blood
count (CBC), which even early on separated RBCs from white blood cells (WBCs)
at 6000 cells per second, with a nearly 10 fold reduction in counting errors[5]. The Coulter Corporation,
which Coulter and his brother founded, was later a pioneer in monoclonal
antibody development and flow cytometry as well [5].
In FCM,
cells are passed through plastic tubing to a flow chamber, where the specimen
is surrounded by a saline fluid (sheath), and measured in single file as lasers
of different color light trigger antibody stained cells to fluoresce. Then florescent and light scatter information
is detected via optical sensors and converted by the computer to form a digital
file [6, 7]. This data provides the clinician with
information about the relative and absolute population of immune subtypes in
the blood, aiding in clinical diagnosis.
The first reported coupling of antibodies to fluorescent molecules
occurred in 1942 at Harvard Medical School with the work of Albert Coons, MD,
whereby he labeled antibody with fluorescein isocyanate (FITC) and used it to
stain antigens in tissue sections visualized by fluorescence microscopy [8, 9],
as such, the development of fluorescent technology occurred in parallel to the
work of Coulter in cell counting.
The first patent of a
multiparameter flow cytometric data analysis system occurred with Dittrich
& Gohde in 1968 [10]. Before that however, Mack Fulwyler, having
run out of nuclear fallout to monitor at Los Alamos Scientific Laboratory,
turned his attention to the Coulter Counter and published for the first time
the ability to separate cells electronically based on volume using a Coulter
aperture, effectively proving flow sorting as a viable method [11, 12].
Within a few years, lasers; via tubes
filled with inert gases such as argon, neon, and helium, originally designed
for microscopy [12] were being incorporated by
all the major medical instrument companies into the manufacture of flow
cytometry instruments [2]. These first flow cytometers,
were full of fluidic systems and hand-blown glass vessels, but the growth of
computer electronics lead to advancements in cytometer detection, measurement,
and computation [13]. By combining electrical cell counting with forward
scatter (FSC) and side scatter (SSC) detection of size and complexity and fluorochrome
associated cell markers, flow cytometry would eventually provide a superior
method for cell type specification in the clinical laboratory [2]. Thirty-nine articles on automatic cytology
appeared in the Journal of Histochemistry
and Cytochemistry in 1973, the first compilation of its kind [1], however, it wasn’t until strange
cases of Karposi’s Sarcoma began sprouting up in the early 1980’s, heralding
the beginning of the AIDS pandemic, that the full clinical utility of flow
cytometry was eventually realized [7].
At the same time many labs were
researching the markers on the surface of cells and specifically, through the
creation of immortalized plasma cells, began producing antibodies against
antigens on the surface of T-cells [14]. This would be extended to markers of all
immune components. Since the 1980’s,
over 500 antigens and their representative monoclonal antibodies have been
defined on the surface of human leukocytes and have been designated via a
cluster of differentiation (CD) nomenclature [15] leading to their broad use in
the examination of human peripheral blood mononuclear cells (PBMCs) and other
immune components [16]. These CD markers are commonly used in the
detection and designation of cells in the clinical lab, for example the family
of T-cell specific receptor (TCR) components are collectively known as CD3 (originally designated OKT3 [14]) under this
nomenclature. Another marker, CD4 (OKT4),
is a coreceptor specific for MHC class II T-helper cell activation and is the
primary receptor for HIV infection [15]. The conjugation of these antibodies to fluorochromes
would allow scientists to mark these cells using flow cytometry. Today, multiple analytes are typically tested
at the same time, via cocktails of antigen specific fluorescent antibodies,
therefore detailed cell type specification can be achieved clinically [10, 16].
Historic Uses in AIDS
Research and Diagnosis
Even
before the virus that caused AIDS was discovered, flow cytometry was vital to
the detection of changes in the one known hallmark of the disease at the time,
a decrease in CD4+ T-cell population [7]. Early in the disease course,
cytopenia is seen in the patient’s blood [17]. Along with viral load tests, HIV activity was
first detected via a count of the CD4+ cells, which decline by
30-100 cells/ml per year depending on
viral load [17]. Human Immunodeficiency Virus (HIV) infection,
the virus that causes AIDS, is clinically verifiable even during the early
asymptomatic phase using quantifiable laboratory methods, specifically the CBC
and CD4+ and CD8+ cell counts [17, 18]. Over 2500 publications have utilized FCM in
all stages of HIV research and diagnosis since the mid 1980s [19]. It has become the gold standard in estimating
CD4 counts, which can easily be derived from the FSC and SSC gating of
lymphocytes [17]. Furthermore, FCM detected markers such as
CD3, CD4, CD8, and CD45 are far more accurate then indirect markets widely
tested in the early to mid 1990’s such as b-2
microglobulin, elevated in the seropositive homosexual men, and Neopterin,
elevated in both the urine and serum of AIDS patients [17, 18]. In the late 1980s and early 90s experiments
using FCM confirmed that subsets of CD4+ T cells were targeted by
HIV [18]. The main difference in lymphoid phenotypes seen
in the analysis of flow data was a reduction by 40% in the CD4+ T
helper population and a 45% increase in the CD8+ cytotoxic T cell
population [18]. Since the initial methods of CD4+ detection by flow, more
advanced cell separation techniques have been employed using flow cytometric
marker analysis such as three color (CD3/CD4/CD45) or four color
(CD3/CD4/CD8/CD45) immunotyping and advanced platforms for CD4/CD8 detection in
whole blood [17]. Not only was the detection and progression
of the disease clarified by flow cytometric analysis, but the underlying
mechanism for HIV entry into the cells was aided by advancements in flow
cytometry and antigenic marker profiling.
Specifically, the initial binding of the HIV viral particle glycoprotein
gp120 to T cells occurs through binding CD4 and the coreceptor CXCR4 (CD184,
fusin), which leads to viral infection [20, 21]. In contrast, HIV infection in macrophages and
monocytes requires the coreceptor CCR5 (CD195, RANTES receptor) [21]. These distinctions in HIV entry, allowed for
the separation of three variants of the virus originally classed as HIV-1; X4,
R5X4, and R5, based on the two coreceptors involved in viral entry [21]. Therapies aimed at interfering with CXCR4 and
CCR5 activities have been investigated [21] and examination of the cell
type most diminished can be indicative of the HIV-1 variant present. However, both CXCR4 and CCR5 are important in
normal cellular functions as well, complicating therapeutic targeting [22]. Like these instances, FCM is used clinically
to provide evidence of treatment efficacy for antiviral therapy [17, 19]. A common use of FCM in HIV therapy is in the
examination of cellular apoptosis using propidium iodide (PI) and 7-AAD [10] [23]. Flow cytometric apoptosis assays have shown
clinical utility in elucidating differences in response to antiretroviral
therapy [24]
and determining the mechanisms of cell death via HIV [25]. Due to the rapid dissemination of AIDS, FCM
has gained ground in the clinical lab and has since been used to study the
immunology of patients after vaccine therapies, during cancer treatment, and in
myeloproliferative disorders [7, 26].
Novel
and Current Uses of Flow Cytometry
The
rapid advancements in flow-cell technology are not just a thing of the
past. A brief look at new devices from top
cytometry companies indicates advancements in technologies are making their way
to market and another look at the current literature suggests a proliferation
of innovative usage and technical modifications that will undoubtedly play a
role in medical advances and diagnosis in the future.
Fluorescent bead based assays, for
example, have long been used to quantify analytes traditionally as a
replacement for radioactive immuno-assays (RIA) and enzyme linked immunosorbant
assays (ELISA) [27-29]. A Mayo Clinic assay to detect IgG against IgA
is one example of this used in clinical diagnosis today [28]. Another technique includes
the lysis of cells followed by the addition of antibody conjugated fluorescent
beads to look for subsurface protein concentrations [27,
30]. This technique alone can be clinically useful
for determining the protein level of cells at different stages of development,
or after long term therapy [27]. Cytometric bead array (CBAs) kits examine
multiple phosphorylated proteins simultaneously, downstream of TCR and growth
factor signaling for example, greatly decreasing data collection times and
allowing for quantification of activity [29]. When a second antibody is added,
protein-protein interactions (PPIs) can be detected. Using this so-called IP-FCM, another group at
the Mayo Clinic was able to show the basal and stimulated increase in Zap70
association to the TCR in OT1 cells [31], thus suggesting a more
specific examination of subsurface molecular interactions, then previously allowable
via FCM.
The revolution in flow cytometry that
occurred as a result of AIDS has retrospectively been a boon to many other
areas of clinical diagnostic research as well, leading to extensive ontogeny
determinations for T and B cell development and activation [2, 22, 32]. Flow cytometry of bone marrow and peripheral
blood leukocytes can be used to define heritable and pathological differences
seen with diseases such as acute myelogenous leukemia, acute lymphoblastic
leukemia, and myelodysplastic syndromes as a result of these extensive marker
trees [2, 32-34].
The advance in FCM and the novel use
of fluorometric analysis has opened the doors to new ways of examining the
surface and subsurface interactions of proteins at the cellular level. These techniques, so far commonly employed in
the examination of immunobiological functions, have been expanded into
examinations of other tissues and cell types [35, 36]. Methods of cellular dissociations will allow
more whole cell experiments in previously understudied or hard-to-obtain
specimens. Bead based assays, mentioned
above, will allow for both in vivo
and in vitro diagnostic and
experimental testing, which has otherwise been performed by gel electropheresis
based western blotting techniques.
Specifically, a combination of these approaches will allow the
elucidation of cell specific mechanisms in nervous and tumor tissues in a
similar manner to that employed in the immune system. Combining high-tech flow cytometric
techniques already available, such as those described above, will allow for a
more thorough investigation of signaling cascades within many more understudied
cell populations and greatly expand our understanding of the mechanistic
distinctions inherent between cells.
Thus, flow cytometry, will propel the scientific discovery vital for
determining cell specific individualized therapies of the future.
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