Wednesday, May 28, 2014

Diagnostic Flow Cytometry and the AIDS Pandemic

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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