Principles and Applications of Flow Cytometry - A Review
1Professor, Department of microbiology, Kamineni Academy of Medical Sciences and Research Centre, Hyderabad
Flow cytometry is a technique of quantitation of single cell analysis. The flow cytometry uses the principle of light scattering, light excitation and emission of fluorochrome molecules to generate multi-parameter data from particles and cells in the size ranging from 0.2 μm to 150 μm. The cells are suspended in a stream of fluid and passed through electronic system which allows the simultaneous analysis of physical, chemical or both characteristics of up to thousands of cells per second.
Flow cytometry is routinely used in the diagnosis of blood cancers. A variation of this technique enables physical sorting of cells or particles based on their properties so as to purify the population of interest. Wallace.H.Coulter in 1953 patented the first impedance–based flow cytometry device developed using coulter principle. Wolfgang Göhde from the University of űnster developed the fluorescence-based flow cytometers in 1968 and obtained patent for the same. The first FACS (Fluorescence activated cell sorting) instrument was introduced by Becton and Dickinson in 1974. The original name for flow cytometry technology was ‘Pulse cytophotometry’ (German: impulszytophotometrie) based on first patented application of fluorescence-based flow cytometry.1Eight years after the introduction of the first fluorescence-based flow cytometry, the name flow cytometry became popular and the name was decided at the 5th American Engineering foundation conference on automated cytology in Pensacola (Florida) in 1976. Flow cytometers use the principle of hydrodynamic focusing for presenting the cells to the laser or any other exciting light source.
Modern flow cytometers are able to analyze several thousands of particles or cells per second in real-time and can actively separate and isolate particles or cells of specified properties. A flow cytometer is similar to microscope except that instead of producing the images of cells, it offers high through put automated quantification of set parameters. Flow cytometers are essentially made of five components
- Fluidic system which transports the sample to the interrogation point and hydro dynamically focuses it on to a mono dispersed stream for single event measurement.
- The optics which include lasers of various wave length, mirrors and lenses to focus the laser on the particle stream which allows the fluorescent dyes tagged to the cells or particles to be excited and emit light at longer wave lengths.
- The electronic system which collects the emitted or scattered light and converts it into proportional electronic signals. These signals are then digitized and processed for analysis
- An analysis and amplification system
- Computers with suitable software for the analysis of the signals.
The sample is initially placed in the sample chamber where the air pressure forces the cells or the particles through a probe and tubing to the nozzle assembly. At the same time the sheath fluid which is usually a phosphate buffer saline is flowing into the nozzle at a pressure slightly less than the sample pressure. This creates laminar flow and a vortex within the assembly which hydro dynamically focuses the sample so that it exits the nozzle vertically separated in the stream of sheath fluid. Essentially there is a narrow, faster moving single-file stream of the sample within a slower moving stream of sheath fluid. This allows excitation of sample particles one-by-one.
The laser beams are focused just below the nozzle. This is called the ‘interrogation point’. As the particle or the cell passes through the beam of interrogation point it generates multiple optical signals.
The primary laser beam is 488 nm and is used to gather the following information.
Forward scatter is a light signal proportional to the physical size of the particle. The forward scatter light is collected by the photo detector placed in line with the lasers, sent to the electric system and is normally used as a trigger to begin the data acquisition sequence as well as for the primary purpose of describing the cell diameter.
As the particle or the cell passes through the laser beam, depending upon the internal composition of the particle or the cell the light bounces around and refracted in all the directions. Side scatter parameter is the indication of the internal complexity of the cell and the scattered light is collected perpendicular to the incident beam. An erythrocyte with no nucleus and with very few organelles will show very small side scatter signal compared to a complex structured cell like lymphocyte which generates a high side scattered signal.2
The laser beam simultaneously excites the fluorophores tagged to the cells or the particles which causes them to emit the light of a different wave length than that of the lasers. These signals are collected in the same direction as the side scatter, but pass through a series of long-pass, short-pass and band-pass filters to allow only certain wave length to reach the appropriate detector. These detectors and photomultiplier tubes which generate electrical signals based on the magnitude of the collected light at its assigned wave length as determined by the filter set up. These electrical pulses are subsequently digitized and sent to the computer for analysis.
The process of collecting data from sample using flow cytometers is called data acquisition which is mediated by a computer physically connected to the flow cytometers. The software used handles the digital interface with the flow cytometers. The current record for a commercial instrument is four lasers and 18 fluorescent detectors. Increasing the number of lasers and detectors enables multiple antibody labeling and can more precisely identify the target population by their phenotypic markers.
Fluorescence-activated Cell Sorting (FACS):
Flow cytometers are capable of sorting the cells or particles of specified properties. Using the information gathered from the sample through forward scatter, side scatter and fluorescent signals one can identify the subsets of cells or particles that may be useful for further study or culturing.
A wide range of fluorophores can be used as labels in flow cytometry. Fluorophores are typically attached to antibodies that recognize the target feature on or in the cell. They may also be attached to a chemical moiety with affinity for cell membrane or another cellular structure. Each fluorophore has characteristic excitation and emission wave length and the emission spectra often overlap. This property should be kept in mind when combination of fluorophores are used which depends on the wave lengths of the lamps used, the lasers used to excite the fluorophores and the detectors available. Currently available commercial flow cytometers allow as many as 18 fluorophores and the level of complexity requires laborious optimization to limit artifacts as well as a complex deconvolution algorithm to separate the overlapping spectra. Quantum dots are used sometimes in place of fluorophores because of their narrow emission peaks. To overcome the fluorophore labeling limit, lanthanide isotopes are attached to antibodies. This method theoretically can allow the use of 40-60 distinguishable labels. Cells are introduced into a plasma ionizing them and allowing time of- flight mass spectrometry to identify the associated isotope labels. This technique permits the use of large number of labels but it currently has low through put capacity. It also destroys the analyzed cells precluding their recovery by sorting.3
Acquisition of Data:
Acquisition of data depends upon the parameter selected. In flow cytometry the data is acquired from forward scatter (FSC), side scatter (SSC) which is also known as orthogonal or 90◦ scatter. The FSC is proportional to the cross sectional area of the particle or the cell and provides the measurement of size. The SSC is sensitive to the cell granularity.
The intensity of the fluorescence is also measured orthogonal to the cell stream.
Five fluorophores that are commonly used and conjugated to secondary antibodies, avidin or monoclonal antibodies are Fluorescein isothiocyanate (FITC), Pyoerythrin (PE), Phycoerythrin-texas red tandem complex (PETR), Phycoerythrin-cyanin 5 tandem complex (PECy5) and Perdinin chlorophyll-P (PerCP). All these fluorophores can be excited at 488 nm and the fluorescein emission for FITC = 530 nm, for PE = 575 nm, for PETR = 613 nm and for both PECy5 and PerCP = 670 nm. Up to four fluorescent antibodies can be combined because the colors can be measured separately. However, in general only three colors are considered. To accomplish color separation suitable spectral filters and dichroic mirrors are used that reflect or transmit specific colors. Because the emission wave lengths of the fluorophores can be very broad, it is necessary to remove the unwanted overlapping colors by electronic compensation. This is achieved by appropriately adjusting the compensation circuits of the instrument. To perform this task compensation standards are used and these are available commercially for FITC and PE. For other fluorochromes compensation standards are to be prepared by the user.
- The associated computer with each flow cytometer controls the cytometer during the data acquisition. It is used to
- Select the parameter for measurement
- Select the area, width or height or different parameter
- Adjust the voltage on the photomultiplier tube Adjust the gain settings on the amplifiers Select linear or logarithmic amplification Select and adjust the threshold settings
- Adjust the settings for the color compensation
- Select the Histogram or Cytogram for the display
- Draw regions or set gates to be used during the data acquisition
- Control the cell sorting process if the cytometer can sort the cells
To display the data from single parameter one can use a univariable histogram. Using bivariate histogram, the correlation between two parameters can be determined in the form of a dot, contour or density plot (See Figure).To visualize the multi-parameter data a different strategy known as ‘regions or gates’ is adopted . Regions are the shapes that are drawn around population of interest on a one or two parameter plot. When a region is used to limit the cells that are drawn on a plot it is termed as gate.
The figure shows two dot plots from some four parameters derived from human peripheral blood leukocytes. The cells were labeled with anti-CD4-FITC and anti-CD8-PE. CD4 or CD8 proteins are expressed on T-lymphocytes. The light scatter defines three distinct populations of cells: granulocytes, monocytes and lymphocytes (G, M and L). In the plot of CD4 and CD8 there are at least four sub populations but one cannot immediately make the link between the different populations depicted in the dot plot. A region R1 has been drawn around the lymphocyte population in the light scatter Cytogram. The computer has colored the, cells in the region red. Any cell fitting in this region is also colored red in all other dot plots created from this data. The color identifies the lymphocytes in the plot of CD4 versus CD8 and this approach is called color gating. A similar procedure is followed to show the fluorescence of monocytes. Monocytes primarily express CD4 where as lymphocytes can express CD4m 0r CD8 but not both on the same cell.
Gates can be combined with each other using Boolean logic (AND, OR, NOT). The most common combination is to use the gates sequentially. If a cell is in gate 1 or gate 2 something has to be done. The given figure shows data from human peripheral blood leukocytes stained with anti-CD20-FITC (B-cell marker) and CD2-PE (a T-cell marker) and CD8-ECD (a marker for cytotoxic T-cells). A region is drawn around the lymphocytes on the scatter plot (A) and this is used to set a gate on a plot of CD2 versus CD20 fluorescence. A further region is set on the CD2+ and CD20–ve cells. Both the regions are used to gate a display of CD2 versus CD20 fluorescence. The cells displayed were in region (lymphs) and region (T-cells). All other cells are excluded. The CD8+ cells and CD2+ cells are shown in the plot pointing out with an arrow.
Determination of Positives:
Detection of Rare Events:
Storage of Data: Clinical Applications of Flowcytometry:
Immunophenotyping or the identification and quantitation of cellular antigens with fluorochrome labeled antibodies is an important application of flow cytometry. Immunophenotyping is critical to the initial diagnosis and classification of acute leukemias, chronic lymphoproliferative disorders and malignant lymphomas, since the treatment strategy largely depends up on the antigenic parameters. Flow cytometry is also a sensitive method to monitor the prognosis of a patient after chemotherapy and bone marrow transplantation. The acute type leukemias are further sub classified as lymphoblastic type (ALL) and myeloid type (AML) based on morphological, cytochemical and immunophenotypic features. Several types of monoclonal antibodies against the cellular antigens are available for immunophenotypic analysis of leukemias or malignancy. In order to establish the B-cell or T-cell clonality, a panel of antibodies are used. The pan-B-cell panel comprises of CD19, CD20 and CD22 and Pan- T-cell panel would include CD2, CD3, CD4and or CD7. Monoclonal antibody against leukocytic common antigen (CD45) is often included in the panel to differentiate the haematological malignancies from other neoplasms and to help detect the population of blast cells, since almost all leukemic cell population show decreased CD45 expression compared to the normal leukocytes. CD34 and HLA-DR antigens are the markers for haemopoietic stem cells used for the diagnosis of acute leukemia and quality assurance of bone marrow transplantation.4
Precursor B-ALL is the most common sub type of ALL and comprises of 75-85% of ALL cases. These cases originate from B-lymphocyte at relatively early stage of development. The diagnosis of B-ALL primarily relies on the reaction of the CD10 and CD19 monoclonal antibodies. Leukemias of T-cell lineage (T-ALL) comprise 15-25% of ALL cases. The flow cytometry diagnosis of T-ALL is more difficult than that of B-ALL, since monoclonality is not easy to demonstrate and markers that are detected only in the early phase of maturation of cell are absent in matured T-cells are a few and occur uncommonly in T-ALL. The most sensitive marker for T-ALL seems to be Pan-T-40 kd antigen defined by anti-leu-9 (CD7). Leukemia of myeloid lineage expresses CD13, CD33 or CD117 and the monocytic leukemias are positive for CD4, CD11b, CD11c, CD14, CD36 and CD66or CD68. CD41 and CD61 are useful in establishing megakaryocytic lineage for an acute leukemia and the erythro- leukemias express CD235 (Glycophorin-A).
Flow cytometry is a simple rapid method for the detection of minimal residual disease (MRD). The persistence of malignant cells within the bone marrow or other tissues of patients with haematological malignancies after remission at levels below the limits of detection by conventional morphological assessment are believed to be the source of disease relapse in many patients. Researchers are actively evaluating the significance of minimal residual disease (MRD) as elimination does not alter the survival rate.
Laboratory criteria for the detection of MRD must meet sensitivity (detection limit of at least 10–3 cells), specificity (ability to differentiate both normal and malignant cells), reproducibility and applicability (easy standardization and rapid collection of the results). However, immunophenotypic analysis, cytogenetics, fluorescent in situ hybridization (FISH), Southern blotting, PCR and other technologies with detection limit of 10–2 to 10–4 cells have been applied as well as the clonogenic assay which has the detection limit of ≤ 0–4.
The detection of CD10, TdT+ or CD34+ cells in cerebrospinal fluid is diagnostic of MRD since immature leukocytes with these markers are not normally present in CSF. The expression of TdT, CD3, CD1a or the dual phenotype CD4+ / CD8+ by bone marrow cells is diagnostic of residual MRD in T-ALL since cells with this phenotype are normally confined to Thymus. The detection of B-ALL MRD is more difficult because small numbers of immature B-cells are normally present within the bone marrow. 5
Flow cytometry is useful in the enumeration of lymphocyte sub population CD4+ T-cell and CD8+ T-cell levels. Since the enumeration of the absolute CD4+ T-cells by flow cytometry and HIV_ RNA levels by RT-PCR has proven critical for the diagnosis and prognostication of HIV infection and management of patients receiving anti-retroviral therapy. Presently most laboratories use 3 or 4 colored immunophenotypic analysis for lymphocyte sub set enumeration with a CD45 side scatter gate to identify the lymphocyte population and to eliminate dead cells, debris and degranulated granulocytes for analysis. Three-color analysis is performed with two labeled specimens (i.e. CD45-CD3-CD4 and CD45-CD3-CD8) while four- color analysis is performed with single labeled specimen (i.e. CD45-CD3-CD4-CD8). Earlier the absolute lymphocyte count was determined by a Haematology analyzer and then used in conjunction with flow cytometric data to calculate CD4+ and CD8+ counts. Presently some flow cytometers perform the absolute lymphocyte counts also.
Studies on HIV-infected patients in research laboratories have led to a number of discoveries awaiting wide spread clinical utilization. One of the most useful observations is the quantitative determination of CD38 expression on CD8+ T-cells which in some studies found to be superior to the measurement of viral load by HIV-RNA quantification for predicting the disease progression and survival of HIV-infected patients. Other flow cytometry assays under study for the management of HIV-infected patients include measurement of frequency of antigen-specific immune response, measurement of cell turn over, programmed cell death and HIV-viral burden.
Flow cytometry is commonly used for diagnosis and management of some of the primary (congenital) immunodeficiency diseases, a heterogeneous group of diseases of host’s defense system which commonly present in childhood as chronic or recurrent infection, failure to thrive, unusual infections, allergic disorders and leukocyte adhesion disorders.
In oncology selection of optimal chemotherapeutic agent is one of the major problems that is encountered quite often. A major cause for failure of many natural products used as chemotherapeutic drugs is development of multi-drug resistance (MDR) is because of the individual tumor variability. The over expression of P-glycoprotein and other proteins involved in cellular transport is a frequent cause for the MDR. In conjunction with Immunocytochemistry and molecular techniques, flow cytometry has been essential for measuring the expression of cell surface and intracellular markers for MDR, assessing the intracellular accumulation and efflux of chemotherapeutic agents and studying the other mechanisms leading to MDR. The identification of intrinsic or acquired MDR is potentially significant clinical value in planning the chemotherapy and several clinical trials of drug efflux blockers that are under study.
The long-sought goal of Oncologists is the reliable in vitro prediction of tumour cell sensitivity to radiation or chemotherapy prior to starting the treatment in individual cancer patients. Flow cytometry measurement of cell viability or apoptosis has been used to design drug treatment protocols and improve the accuracy and reliability of conventional clonogenic assays.
Ligands, antigens or molecule targeted biological therapy using monoclonal antibodies is the most rapidly emerging area of Pharmacology for a wide variety of human diseases. These agents work through the same mechanism such as direct disruption of cell proliferation, anti-apoptosis by blocking the cell membrane receptors and circulating ligand associated with signal transduction, while others serve as target system for other cytotoxic products. The first of these new class Pharmaceutical agents made available is anti-CD3 (OKT3) developed for immunosuppressive therapy of solid organ transplantation rejection. More recently monoclonal antibodies for CD2, CD33, CD25, CD45 and CD12 are made available. Prior to the treatment flow cytometry analysis is critical for confirming that the antigens are produced by aberrant cells.
The enumeration of peripheral blood reticulocytes is required to know about the functional integrity of bone marrow. Reticulocytes increase in cases of anemia with functional bone marrow Reticulocytopaenia occurs in anaemic patients with dysfunctional bone marrow. Reticulocyte counts are useful in the evaluation of bone marrow regenerative activity after chemotherapy or bone marrow transplantation apart from evaluating the anaemic patients. However, reticulocyte count by flow cytometer is more accurate, precise and cost effective than manual counting and presently many clinical laboratories use flow cytometry for the enumeration of reticulocytes. Apart from reticulocyte counts, flow cytometry provides a variety of additional reticulocyte parameters such as reticulocyte maturation index (RMI), immature reticulocyte fraction (IRF) which are not available with manual method. These indices appear valuable in the clinical diagnosis and monitoring of anaemia and other diseases.
The development of technology to perform reticulocyte counts by optical light scatter is a major advancement in clinical Haematology analyzers where they make use of nucleic acid binding dyes and fluorescent dyes for counting the reticulocytes.
Flow cytometry can be applied for the analysis of structure and function of platelets in the research laboratories. Flow cytometric analysis of platelets has inherent difficulties because of its small physical size and bio variability. Nevertheless, several clinical assays are performed in flow cytometry laboratories. These assays have been classified by Bode and Hickerson to include platelet surface receptor quantification and distribution for the diagnosis of congenital platelet disorders, platelet associated IgG quantitation for the diagnosis of immune thrombocytopaenia, platelet cross- matching in transfusion, reticulate platelet assay to detect ‘stress’ platelets, fibrinogen receptor occupancy studies for monitoring the clinical efficacy of platelet –directed anti-coagulation in thrombosis, detection of activated platelet surface markers, cytoplasmic calcium ion measurement and platelet micro particles for assessment of hyper coagulable states.
Flow cytometry is also a valuable tool in transfusion medicine. Detection and accurate quantitation of feto-maternal haemorrhage can be of great value since it can be prevented by appropriate intra-partum and post-partum administration of Rh immunoglobulin to prevent such immune sensitization because immune sensitization is a dreaded consequence of feto-maternal haemorrhage in Rh negative woman with a Rh positive fetus. The flow cytometer can be used for fetal cell detection in maternal blood samples easily. Flow cytometry is simple, more reliable and precise alternative to the manual Kleihauer-Betke technique especially in massive feto-maternal haemorrhage. Flow cytometry does accurate quantitation and helps in the reduction of clinical usage of anti-D immuneglobulin. Well performed Kleihauer-Betke test is still useful as a screening test for the detection of feto-maternal haemorrhage. It offers additional potential application for the study of fetal haemoglobin (HbF) levels or frequency of adult red cells with low levels of HbF in individuals with haemoglobinopathies and medical evaluation of anaemic patients including sickle cell and thalassemic patients. 6,7
The development of DNA analysis of neoplasia by flow cytometry became possible due to significant understanding of human cell cycle in combination with flow cytometric technology. A defined number of cells are stained with a known saturating amount of DNA-specific fluorescent dye under the controlled conditions of temperature, PH, and ionic strength. The cells are then analyzed using flow cytometer and the amount and the intensity of the dye bound to DNA is measured based on a statistically significant number of cells (≥ 0,000 cells) within few minutes.
The relative total DNA content in the unknown cell population is determined by comparing to the cells analyzed with known and constant DNA content. In addition small proportion of cells can be detected in a heterogeneous mixture and cell population with small variation in DNA content (~ 4% with a CV of 2%) can be detected.
The analysis of cell function such as expression of surface antigens is particularly important in Transplantation medicine and diseases of immune system.
Hence many investigations focus on functional analysis of the lymphocyte.
Virtually every event that occurs during the process of lymphocyte activation and their proteins can be measured by flow cytometry, particularly the determination of tyrosine phosphorylation, calcium flux etc., Intracellular calcium flux is measured using multiplex bead technology with radiometric calcium indicators whose spectral characteristics change with Ca+ binding. Calcium flux has been used to study platelet activation in response to different agonists and lymphocyte activation in viral infections and other diseases. Flow cytometric measurement of oxidative burst in neutrophils has been used as a screening test for chronic granulomatous diseases. The most common technique for this purpose uses a non-fluorescing dye (dihydrorhodamine-123) that selectively gets concentrated in mitochondria and is oxidized to a brightly fluorescing compound (rhodamine-123) during the normal oxidative burst. Lymphocyte neoantigens such as CD11b / CD18 and CD154 are up-regulated during lymphocyte activation. Detection of these proteins can be done by flow cytometry. Clinical applications of flow cytometry in solid organ transplantation include pre-transplant cross-matching, HLA-antibody screening and post-transplantation antibody monitoring. In bone marrow transplantation the enumeration of CD34+ haemopoietic stem cells in peripheral blood and bone marrow correlates with engraftment success and the length of haemopoietic recovery following stem cell transplantation including pre- transplantation determination of the efficacy of ex vivo T-cell depletion and post-transplantation evaluation of immune recovery, graft rejection, graft-versus-host disease and graft-versus- leukaemia effect. The detection of bacteria and Yeast cells in the blood and body fluids is important for the diagnosis of a number of different diseases. In clinical infection such as bacteremia the concentration of contaminants may be in the order of 10 bacteria per ml of blood, while the number of erythrocytes is greater than 109 per ml. However, methods are available for selective lysing of erythrocytes in a given blood sample leaving sufficiently low concentration of erythrocytes to allow rapid sample through put capabilities of flow cytometer to be utilized for the detection of bacteria.8
Table I: Fluorescent dyes used for determining the viability by Flow Cytometry
|Stain||Mode of Action||Result|
|Baclight kit: Molecular probes||Propidium iodide excluded
by intact membrane.
All cells take up SYT09
|Live cells green Dead cells red|
|Bis-(1-3-dibutyl barbituric acid)
trimethine Oxanol (DIBA C4(3)
|Up take by dead cells||Dead cells appear green or yellow|
|Calcifluor white||Up take by dead cells||Dead cells appear blue|
|5-cyano-2,3-ditolyl tetrazolium chloride (CTC)||Respiratory activity||Live cells appear red|
|Fluorescein diacetate /
|Enzymic activity||Live cells appear green|
|Rhodamine 123||Up take by live cells||Live cells appear green|
|TO-PRO-3 /Propidium iodide||Excluded by intact membrane||Dead cells appear red|
The detection of specific pathogens has been much improved with flow cytometry with the availability of monoclonal antibodies which can easily be tagged with a fluorescent dye like Fluorescein isothiocyanate (FITC) and can be excited by the 488 nm Argon ion laser.9
Antibiotic susceptibility is another important area for the clinical application of flow cytometric methods. A variety of fluorescent stains for assessing the viability of the microorganisms have been identified (See Table) and they are particularly useful for determining the efficacy of various antimicrobial compounds (either in vitro or in vivo). Microorganisms are exposed to antibiotics or antifungal agents are compared to control samples (untreated with antimicrobial compounds) and appropriate stains are used to identify the changes in the nucleic acids, proteins and membranes etc. Antibiotic induced damage to the cell membrane can be determined by the entry of a fluorescent compound which is normally prevented from entry into the cell by the intact cell membrane. The response of the cells to an antibiotic that microorganism to be identified without the need of observing for the microbial growth. Flow cytometric susceptibility testing can be performed within a few hours and consequently this method has the potential to contribute to the decision of which combination of antimicrobial drugs would most appropriate for a particular patient.
The flow cytometer is a versatile tool with enormous potential for the study of cells or particles. However, many diagnostic laboratories limit the use of flow cytometry to immunophenotypic analysis and lymphocyte sub set analysis. Flow cytometer provides multi-parametric analysis at the single cell level. The development of new fluorochromes including UV-excited, complex dyes (tandem dyes), and nanocrystals as well as new generation of modular flow cytometers using small solid state lasers, robotics and advance. HTPS designed for automated high throughput analysis of novel bio- response modifying drugs permit analysis of 9-10 cell samples per minute from 96- well micro plate. Another interesting development of flow cytometer is the laser scanning cytometer (LSC), a microscopic slide based technology capable of acquiring multi-parametric data from selected cells from a heterogeneous population which is proving particularly useful for the analysis of fine needle aspirates and body fluid specimens.10
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