This document provides an overview of the applications of flow cytometry to clinical microbiology. It discusses how flow cytometry can be used for the direct detection of bacteria, fungi, parasites and viruses through detection of antigens or nucleic acids. It also describes how flow cytometry can be applied to serological diagnosis and antimicrobial susceptibility testing. The document outlines how flow cytometry allows for rapid analysis and monitoring of infections and antimicrobial therapy. In conclusion, it discusses the benefits flow cytometry provides for clinical microbiology and its future potential.
Flow cytometry is a technique that measures and analyzes physical and chemical characteristics of cells or particles as they flow in a fluid stream through a beam of light. It works by using lasers to illuminate cells and detectors to measure light scattering and fluorescent emissions. The main components are a fluidics system to transport cells, an optics system with lasers and detectors, and an electronics system to convert signals into data. Flow cytometry allows identification and quantification of cell populations and has many applications in research, clinical diagnosis, and environmental monitoring.
Flow cytometry for cell componenet analysisRAJA GOPAL
Flow cytometry is a technique that uses lasers and fluorescence to analyze physical and chemical characteristics of cells as they flow in a fluid stream. It allows simultaneous analysis of thousands of cells per second based on parameters like cell size, granularity, and detection of cell surface antigens using specific antibodies labeled with fluorochromes of different colors. Specimens suitable for analysis include blood, bone marrow, body fluids, and cell suspensions generated from tissues. Flow cytometry has various applications like immunophenotyping, DNA analysis, diagnosis of conditions like PNH, reticulated cell counting, and blood bank testing.
Flow cytometry is a technique that uses lasers and fluorescence to count and examine microscopic particles like cells. It can measure multiple parameters of individual cells as they flow in a liquid stream past the laser beam at thousands of cells per second. Components include a flow cell to arrange cells in a stream, optical systems to generate light signals, detectors to convert light signals to electrical signals, and read-out devices to analyze the results. Flow cytometry is used widely in clinical laboratories for applications like immunophenotyping, DNA analysis for malignancy, detecting enzymatic deficiencies, genetic diseases, and hematology analysis.
The document provides an overview of flow cytometry, including its history, principles, and applications. It discusses how flow cytometry allows for the measurement of cellular characteristics like fluorescence and light scattering at high speeds. Key developments include the first apparatus for detecting bacteria in a fluid stream in 1947 and the first cell sorter in 1965. The term "fluorescence activated cell sorter" or FACS was coined in 1972. Flow cytometry integrates technologies like lasers, optics, fluidics, and electronics to analyze individual cells and measure parameters such as cell size, granularity, and receptor expression. It has various applications in fields like immunology, genetics, and microbiology.
Flow cytometry is a process that can quantify and categorize cells by measuring their physical and chemical characteristics. Cells pass through a flow cytometer one by one where they are hit by a laser beam, scattering light. Detectors then measure the light and determine characteristics like size, shape, and protein expression. This allows cells to be separated into subpopulations and quantified. Flow cytometry is useful for clinical and research applications like performing complete blood counts and identifying T cell subtypes.
Flow cytometry is a laser-based technique used to analyze physical and chemical properties of cells/particles as they flow in a fluid stream through a laser beam. It allows for rapid, multiparameter analysis of individual cells. The key components of a flow cytometer are fluidics, optics, and electronics systems. Lasers illuminate cells and light is scattered and detected to analyze properties like size, granularity, and fluorescence. Applications in food microbiology include quantifying bacteria in foods and beverages, monitoring dairy starters, and studying probiotics under stress conditions.
Flow cytometry is a technique that measures and analyzes physical and chemical characteristics of cells or particles as they flow in a fluid stream through a beam of light. It works by using lasers to illuminate cells and detectors to measure light scattering and fluorescent emissions. The main components are a fluidics system to transport cells, an optics system with lasers and detectors, and an electronics system to convert signals into data. Flow cytometry allows identification and quantification of cell populations and has many applications in research, clinical diagnosis, and environmental monitoring.
Flow cytometry for cell componenet analysisRAJA GOPAL
Flow cytometry is a technique that uses lasers and fluorescence to analyze physical and chemical characteristics of cells as they flow in a fluid stream. It allows simultaneous analysis of thousands of cells per second based on parameters like cell size, granularity, and detection of cell surface antigens using specific antibodies labeled with fluorochromes of different colors. Specimens suitable for analysis include blood, bone marrow, body fluids, and cell suspensions generated from tissues. Flow cytometry has various applications like immunophenotyping, DNA analysis, diagnosis of conditions like PNH, reticulated cell counting, and blood bank testing.
Flow cytometry is a technique that uses lasers and fluorescence to count and examine microscopic particles like cells. It can measure multiple parameters of individual cells as they flow in a liquid stream past the laser beam at thousands of cells per second. Components include a flow cell to arrange cells in a stream, optical systems to generate light signals, detectors to convert light signals to electrical signals, and read-out devices to analyze the results. Flow cytometry is used widely in clinical laboratories for applications like immunophenotyping, DNA analysis for malignancy, detecting enzymatic deficiencies, genetic diseases, and hematology analysis.
The document provides an overview of flow cytometry, including its history, principles, and applications. It discusses how flow cytometry allows for the measurement of cellular characteristics like fluorescence and light scattering at high speeds. Key developments include the first apparatus for detecting bacteria in a fluid stream in 1947 and the first cell sorter in 1965. The term "fluorescence activated cell sorter" or FACS was coined in 1972. Flow cytometry integrates technologies like lasers, optics, fluidics, and electronics to analyze individual cells and measure parameters such as cell size, granularity, and receptor expression. It has various applications in fields like immunology, genetics, and microbiology.
Flow cytometry is a process that can quantify and categorize cells by measuring their physical and chemical characteristics. Cells pass through a flow cytometer one by one where they are hit by a laser beam, scattering light. Detectors then measure the light and determine characteristics like size, shape, and protein expression. This allows cells to be separated into subpopulations and quantified. Flow cytometry is useful for clinical and research applications like performing complete blood counts and identifying T cell subtypes.
Flow cytometry is a laser-based technique used to analyze physical and chemical properties of cells/particles as they flow in a fluid stream through a laser beam. It allows for rapid, multiparameter analysis of individual cells. The key components of a flow cytometer are fluidics, optics, and electronics systems. Lasers illuminate cells and light is scattered and detected to analyze properties like size, granularity, and fluorescence. Applications in food microbiology include quantifying bacteria in foods and beverages, monitoring dairy starters, and studying probiotics under stress conditions.
Application of Fluorescence Activated-Cell Sorting (FACS) in separation of di...Goh Mei Ying
Fluorescence activated cell sorting (FACS) enables the separation of different cell populations from a mixed community by sorting individual cells based on their light scattering and fluorescent characteristics. Cells are tagged with fluorescent dyes and passed through a stream that is broken into droplets and charged before cells are deflected into collection tubes. FACS has applications in separating cell types like γδ T cells, screening proteins, and determining biomarker levels. While powerful, FACS also has limitations such as slow speed, high cost, and a need for specialized equipment.
This document discusses flow cytometry, which allows detection of surface markers, intracellular factors, and secreted factors of cells. It also allows detection of DNA content. The document explains that flow cytometry works by passing cells in a fluid stream through a laser beam, which scatters light and excites fluorescent markers to identify cell characteristics. Antibodies tagged with fluorescent dyes bind to specific markers on cells and allow distinguishing between cell types.
This document provides an introduction to flow cytometry. It defines flow cytometry as a method for sensing individual cells in a fluid stream as they pass through a laser beam, measuring light scattering and fluorescence. Key aspects of flow cytometry systems and methodology are described, including hydrodynamic focusing of cells, light scattering measurements, use of fluorescent markers, optical and electronic components, data acquisition and analysis techniques like gating and compensation. The history of technological developments in flow cytometry is also summarized.
“Cell Memory Mechanism Discovered” And “Scientists Visualize How Cancer Chr...Carito Mora Sánchez
This document summarizes two scientific articles about recent discoveries related to DNA and cell function. The first article discusses the discovery of a "cell memory mechanism" involving transcription factors and cohesin proteins that help transcription factors correctly relocate on DNA after cell division. The second article describes research visualizing how chromosome translocations, which are linked to cancer, form in living cells through advanced imaging technology, providing insight into DNA alterations. Both studies provide new understanding that could help address diseases.
Flow Cytometry Training talks - part 1
This forms the first session of the Garvan Flow , Flow Cytometry Training course. this is a 1 1/2 day training course aimed at giving new and experienced researchers a better understanding of cytometry in medical and biological research.
Flow cytometry allows for multiparametric analysis of physical and chemical characteristics of particles like cells. It works by passing particles in a fluid stream through a laser beam, detecting light scattering and fluorescence. Main applications include diagnosis of hematological malignancies through immunophenotyping, analysis of DNA content and cell cycle, and organ transplant monitoring. New developments aim to improve sensitivity and integrate imaging capabilities.
Flow cytometry can be used for a variety of clinical applications including analysis of leukemia and lymphoma through immunophenotyping of cells. It is used to monitor transplant rejection through detection of antibodies and T cell counts. Other uses include stem cell enumeration, detection of autoantibodies, monitoring HIV infection through CD4 counts, analysis of fetal-maternal hemorrhage, evaluation of immunodeficiency diseases, analysis of paroxysmal nocturnal hemoglobinuria, measurement of contaminating leukocytes in blood transfusions, and platelet counting and functional analysis. The principles of flow cytometry involve light scattering and fluorescence detection of cells in a hydrodynamically focused stream that are then separated electrostatically for sorting based on multivariate data analysis.
This document provides an overview of flow cytometry including:
- An introduction to flow cytometry techniques and applications from multiple speakers
- Descriptions of key components and parameters measured in flow cytometry like scatter, fluorescence, and fluorochromes
- Examples of flow cytometry applications in fields like cell viability, proliferation, and surface marker analysis
- A discussion of antibody conjugation methods and considerations for multi-color flow cytometry experiments
Cell sorting is a technique used to separate specific cell populations from tissues. The first step involves disrupting connections between cells using enzymes or calcium-chelating agents. This converts the tissue into single cells. Flow cytometry can then identify and separate different cell types by measuring light scattering or fluorescence emitted from individually labeled cells as they pass through a laser. Specific cells are labeled with fluorescent antibody markers and can then be isolated from unlabeled cells using a fluorescence activated cell sorter.
The document provides an overview of the basic principles and components of flow cytometry. It discusses how flow cytometry works by measuring the properties of cells in fluid flow, using a combination of fluidics to introduce cells, optics to generate and collect light signals, and electronics to convert signals to digital data. Key aspects summarized include how cells are hydrodynamically focused and interrogated by light scatter and fluorescence to derive information on their size, granularity, and marker expression that can be analyzed using software.
Flow cytometry is a technique that allows for the analysis of physical and fluorescent characteristics of single cells as they flow in a liquid stream past a laser. It involves using antibodies tagged with fluorescent dyes to detect cell antigens. This allows for the identification of cell types, lineages, and maturation stages. Flow cytometry provides sensitive, quantitative, and multiparameter analysis of cells and is widely used for immunophenotyping in hematological malignancies and other disorders.
Flow cytometry is a technique used to quantify and characterize cells by suspending them in a fluid and passing them through an electronic detection apparatus. It allows cells to be categorized based on their physical properties like size, granularity, and presence of cell surface markers or intracellular proteins. This document provides an overview of how flow cytometry works and its applications, including performing white blood cell differentials and distinguishing T cell subpopulations.
Flow cytometry and fluorescence activated cell sorting (FACS)Abu Sufiyan Chhipa
Flow cytometry is a technology that analyzes physical and chemical characteristics of particles in fluid as they pass through a laser. It is used for cell counting, sorting, biomarker detection, and protein engineering. The basic principle is passing cells in single file past a laser for detection, counting, and sorting. It has applications in leukocyte analysis, DNA analysis, detecting enzymatic deficiencies, minimal residual disease, detecting autoantibodies, fetal-maternal hemorrhage quantification, and reticulocyte analysis.
This document summarizes flow cytometry, a technique used to count and examine microscopic particles suspended in fluid. It describes key components of modern flow cytometers including lasers, detectors, and computer systems that can analyze thousands of particles per second. The document outlines the principles of how each cell passes through a laser, scatters and emits light, which is detected and analyzed by software. Common applications like cell sorting, fluorescence detection using labeled antibodies, and measurable parameters are discussed. Terminology related to instrumentation, optical systems, data analysis and compensation are also introduced.
Flow cytometry measures properties of cells in a fluid stream by staining cells with fluorescent antibodies and analyzing them with a flow cytometer instrument. It allows for the characterization of cell populations based on cell surface and intracellular markers. Flow cytometry is useful for immunophenotyping in diseases like leukemia where it can identify the cell lineage involved (lymphoid vs myeloid) and detect minimal residual disease during treatment. In this case, the 5-year old boy presenting with fever, flow cytometry immunophenotyping should be performed to diagnose causes like acute lymphoblastic leukemia by examining markers like CD19, CD5, CD10 on lymphocytes.
FACS and MACS with their applications in biological research.Deepak Agarwal
Flow cytometry (FACS) and magnetic activated cell sorting (MACS) are techniques used to analyze and separate cells based on their physical and chemical characteristics. FACS uses lasers to detect cell properties and sort cells into containers one by one, while MACS uses magnetic microbeads attached to cells to separate them in high gradient magnetic fields. These techniques have various applications in research including identifying stem cells, characterizing cancer cells, studying cell cycles, and isolating cell populations for further analysis.
This document provides an overview of flow cytometry and fluorescence-activated cell sorting (FACS). It describes flow cytometry as a technique for measuring physical and chemical characteristics of cells as they flow in a fluid stream, allowing for single cell analysis. FACS extends this by using fluorescence to identify cell characteristics and sort cells into separate collections based on these characteristics. The key components of a flow cytometer are described as lasers, optics including filters and detectors, fluidics to hydrodynamically focus cells, and electronics to convert optical signals to digital data. Applications including cell phenotyping, apoptosis analysis, and cell cycle analysis are discussed. Cell sorting and quantitative analysis of cell cycle phases are also summarized.
Flow cytometry definition, principle, parts, steps, types, usesGayathri Devi S
Flow cytometry is a technique that uses lasers to detect and measure physical and chemical characteristics of cells or particles in fluid suspension. Cells pass through a laser beam, which scatters light and causes fluorescence that is detected by sensors. Measurements of scattered and fluorescent light provide information about cell size, granularity, and expression of targeted proteins or nucleic acids. Flow cytometry allows rapid multi-parameter analysis of individual cells in heterogeneous populations and is widely used for clinical, research, and industrial applications.
This document provides an introduction and table of contents for a book on advanced laboratory techniques in avian medicine. The introduction gives an overview of traditional diagnostic methods and emerging molecular biological techniques. The table of contents outlines two main sections - traditional diagnostic methods, and molecular biological techniques - covering topics like isolation and identification of microorganisms, serological procedures, and nucleic acid and protein methods.
Application of Fluorescence Activated-Cell Sorting (FACS) in separation of di...Goh Mei Ying
Fluorescence activated cell sorting (FACS) enables the separation of different cell populations from a mixed community by sorting individual cells based on their light scattering and fluorescent characteristics. Cells are tagged with fluorescent dyes and passed through a stream that is broken into droplets and charged before cells are deflected into collection tubes. FACS has applications in separating cell types like γδ T cells, screening proteins, and determining biomarker levels. While powerful, FACS also has limitations such as slow speed, high cost, and a need for specialized equipment.
This document discusses flow cytometry, which allows detection of surface markers, intracellular factors, and secreted factors of cells. It also allows detection of DNA content. The document explains that flow cytometry works by passing cells in a fluid stream through a laser beam, which scatters light and excites fluorescent markers to identify cell characteristics. Antibodies tagged with fluorescent dyes bind to specific markers on cells and allow distinguishing between cell types.
This document provides an introduction to flow cytometry. It defines flow cytometry as a method for sensing individual cells in a fluid stream as they pass through a laser beam, measuring light scattering and fluorescence. Key aspects of flow cytometry systems and methodology are described, including hydrodynamic focusing of cells, light scattering measurements, use of fluorescent markers, optical and electronic components, data acquisition and analysis techniques like gating and compensation. The history of technological developments in flow cytometry is also summarized.
“Cell Memory Mechanism Discovered” And “Scientists Visualize How Cancer Chr...Carito Mora Sánchez
This document summarizes two scientific articles about recent discoveries related to DNA and cell function. The first article discusses the discovery of a "cell memory mechanism" involving transcription factors and cohesin proteins that help transcription factors correctly relocate on DNA after cell division. The second article describes research visualizing how chromosome translocations, which are linked to cancer, form in living cells through advanced imaging technology, providing insight into DNA alterations. Both studies provide new understanding that could help address diseases.
Flow Cytometry Training talks - part 1
This forms the first session of the Garvan Flow , Flow Cytometry Training course. this is a 1 1/2 day training course aimed at giving new and experienced researchers a better understanding of cytometry in medical and biological research.
Flow cytometry allows for multiparametric analysis of physical and chemical characteristics of particles like cells. It works by passing particles in a fluid stream through a laser beam, detecting light scattering and fluorescence. Main applications include diagnosis of hematological malignancies through immunophenotyping, analysis of DNA content and cell cycle, and organ transplant monitoring. New developments aim to improve sensitivity and integrate imaging capabilities.
Flow cytometry can be used for a variety of clinical applications including analysis of leukemia and lymphoma through immunophenotyping of cells. It is used to monitor transplant rejection through detection of antibodies and T cell counts. Other uses include stem cell enumeration, detection of autoantibodies, monitoring HIV infection through CD4 counts, analysis of fetal-maternal hemorrhage, evaluation of immunodeficiency diseases, analysis of paroxysmal nocturnal hemoglobinuria, measurement of contaminating leukocytes in blood transfusions, and platelet counting and functional analysis. The principles of flow cytometry involve light scattering and fluorescence detection of cells in a hydrodynamically focused stream that are then separated electrostatically for sorting based on multivariate data analysis.
This document provides an overview of flow cytometry including:
- An introduction to flow cytometry techniques and applications from multiple speakers
- Descriptions of key components and parameters measured in flow cytometry like scatter, fluorescence, and fluorochromes
- Examples of flow cytometry applications in fields like cell viability, proliferation, and surface marker analysis
- A discussion of antibody conjugation methods and considerations for multi-color flow cytometry experiments
Cell sorting is a technique used to separate specific cell populations from tissues. The first step involves disrupting connections between cells using enzymes or calcium-chelating agents. This converts the tissue into single cells. Flow cytometry can then identify and separate different cell types by measuring light scattering or fluorescence emitted from individually labeled cells as they pass through a laser. Specific cells are labeled with fluorescent antibody markers and can then be isolated from unlabeled cells using a fluorescence activated cell sorter.
The document provides an overview of the basic principles and components of flow cytometry. It discusses how flow cytometry works by measuring the properties of cells in fluid flow, using a combination of fluidics to introduce cells, optics to generate and collect light signals, and electronics to convert signals to digital data. Key aspects summarized include how cells are hydrodynamically focused and interrogated by light scatter and fluorescence to derive information on their size, granularity, and marker expression that can be analyzed using software.
Flow cytometry is a technique that allows for the analysis of physical and fluorescent characteristics of single cells as they flow in a liquid stream past a laser. It involves using antibodies tagged with fluorescent dyes to detect cell antigens. This allows for the identification of cell types, lineages, and maturation stages. Flow cytometry provides sensitive, quantitative, and multiparameter analysis of cells and is widely used for immunophenotyping in hematological malignancies and other disorders.
Flow cytometry is a technique used to quantify and characterize cells by suspending them in a fluid and passing them through an electronic detection apparatus. It allows cells to be categorized based on their physical properties like size, granularity, and presence of cell surface markers or intracellular proteins. This document provides an overview of how flow cytometry works and its applications, including performing white blood cell differentials and distinguishing T cell subpopulations.
Flow cytometry and fluorescence activated cell sorting (FACS)Abu Sufiyan Chhipa
Flow cytometry is a technology that analyzes physical and chemical characteristics of particles in fluid as they pass through a laser. It is used for cell counting, sorting, biomarker detection, and protein engineering. The basic principle is passing cells in single file past a laser for detection, counting, and sorting. It has applications in leukocyte analysis, DNA analysis, detecting enzymatic deficiencies, minimal residual disease, detecting autoantibodies, fetal-maternal hemorrhage quantification, and reticulocyte analysis.
This document summarizes flow cytometry, a technique used to count and examine microscopic particles suspended in fluid. It describes key components of modern flow cytometers including lasers, detectors, and computer systems that can analyze thousands of particles per second. The document outlines the principles of how each cell passes through a laser, scatters and emits light, which is detected and analyzed by software. Common applications like cell sorting, fluorescence detection using labeled antibodies, and measurable parameters are discussed. Terminology related to instrumentation, optical systems, data analysis and compensation are also introduced.
Flow cytometry measures properties of cells in a fluid stream by staining cells with fluorescent antibodies and analyzing them with a flow cytometer instrument. It allows for the characterization of cell populations based on cell surface and intracellular markers. Flow cytometry is useful for immunophenotyping in diseases like leukemia where it can identify the cell lineage involved (lymphoid vs myeloid) and detect minimal residual disease during treatment. In this case, the 5-year old boy presenting with fever, flow cytometry immunophenotyping should be performed to diagnose causes like acute lymphoblastic leukemia by examining markers like CD19, CD5, CD10 on lymphocytes.
FACS and MACS with their applications in biological research.Deepak Agarwal
Flow cytometry (FACS) and magnetic activated cell sorting (MACS) are techniques used to analyze and separate cells based on their physical and chemical characteristics. FACS uses lasers to detect cell properties and sort cells into containers one by one, while MACS uses magnetic microbeads attached to cells to separate them in high gradient magnetic fields. These techniques have various applications in research including identifying stem cells, characterizing cancer cells, studying cell cycles, and isolating cell populations for further analysis.
This document provides an overview of flow cytometry and fluorescence-activated cell sorting (FACS). It describes flow cytometry as a technique for measuring physical and chemical characteristics of cells as they flow in a fluid stream, allowing for single cell analysis. FACS extends this by using fluorescence to identify cell characteristics and sort cells into separate collections based on these characteristics. The key components of a flow cytometer are described as lasers, optics including filters and detectors, fluidics to hydrodynamically focus cells, and electronics to convert optical signals to digital data. Applications including cell phenotyping, apoptosis analysis, and cell cycle analysis are discussed. Cell sorting and quantitative analysis of cell cycle phases are also summarized.
Flow cytometry definition, principle, parts, steps, types, usesGayathri Devi S
Flow cytometry is a technique that uses lasers to detect and measure physical and chemical characteristics of cells or particles in fluid suspension. Cells pass through a laser beam, which scatters light and causes fluorescence that is detected by sensors. Measurements of scattered and fluorescent light provide information about cell size, granularity, and expression of targeted proteins or nucleic acids. Flow cytometry allows rapid multi-parameter analysis of individual cells in heterogeneous populations and is widely used for clinical, research, and industrial applications.
This document provides an introduction and table of contents for a book on advanced laboratory techniques in avian medicine. The introduction gives an overview of traditional diagnostic methods and emerging molecular biological techniques. The table of contents outlines two main sections - traditional diagnostic methods, and molecular biological techniques - covering topics like isolation and identification of microorganisms, serological procedures, and nucleic acid and protein methods.
This document discusses molecular diagnostic methods for detecting drug resistance in gram-positive bacteria. It covers several key points:
1) Traditional phenotypic methods for antimicrobial susceptibility testing require pure culture and take several days for results, while genotypic methods can provide results within hours directly from patient specimens without culturing.
2) Important resistance mechanisms in clinically relevant gram-positive pathogens include beta-lactamases in Staphylococcus and penicillin-binding protein mutations conferring methicillin resistance.
3) Newer molecular techniques for antimicrobial susceptibility testing include PCR, real-time PCR, and next-generation sequencing, which can more rapidly detect resistance genes compared to traditional phenotypic methods.
Advanced Next Generation DNA sequencing can more accurately diagnose infections by identifying bacteria, fungi, and viruses compared to traditional culture techniques. This allows for better treatment decisions. PathoGenius Laboratory uses Next Generation Sequencing to identify microbes in samples, providing physicians with diagnostic results to inform customized treatment. Previous methods of relying only on culture were found to significantly underrepresent the microbes present. Molecular diagnostic techniques provide more comprehensive information about biofilms and chronic infections compared to traditional approaches.
This document discusses advanced next-generation DNA sequencing techniques for identifying bacteria, fungi, and viruses. It highlights how DNA sequencing can identify over 13,000 pathogen DNA codes and is not affected by transport or culture issues. Examples are given where DNA sequencing identified chronic infections that cultures failed to detect. The document emphasizes how biofilms are difficult to culture but can be identified by DNA sequencing, and are responsible for 80% of infections. It provides examples comparing DNA sequencing to traditional cultures for identifying pathogens in chronic wounds and urine samples. The document also discusses using DNA diagnostic tests to provide treatment recommendations like customized topical therapies and antibiograms.
This document provides guidelines for antimicrobial susceptibility testing. It discusses the principle of determining antimicrobial susceptibility and factors that influence the testing such as pH, moisture, and divalent cations. It describes different methods for testing including disk diffusion, dilution, and diffusion/dilution. It provides details on reagents, inoculum preparation, and procedures for performing disk diffusion testing. The document is intended to standardize antimicrobial susceptibility testing methods.
This document provides guidelines for antimicrobial susceptibility testing. It discusses the principle of determining antimicrobial susceptibility and factors that influence the testing such as pH, moisture, and divalent cations. It describes different methods for testing including disk diffusion, dilution, and diffusion/dilution. It provides details on reagents, inoculum preparation, and procedures for performing disk diffusion testing. The document is intended to standardize antimicrobial susceptibility testing methods.
This document discusses a study that used next-generation DNA sequencing to analyze the microbiome of neuropathic diabetic foot ulcers. The study found that DNA sequencing identified more bacterial diversity in the ulcers than traditional cultures. DNA sequencing also showed higher microbial loads in the ulcers compared to cultures. This demonstrates that cultures underrepresent the true bacteria present in chronic wounds like diabetic foot ulcers. The study provides support for using molecular diagnostic approaches like DNA sequencing rather than solely relying on traditional cultures to identify bacteria in chronic wounds.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
The document summarizes a study conducted over six months to identify common bacterial contaminants in a microbiology laboratory. Samples were collected from surfaces, air, hands, and clothing of laboratory personnel using culture methods and identified using biochemical and molecular tests. The most common contaminants isolated were Micrococcus (52.94% of cultures) and Bacillus subtilis (23.52% of cultures), primarily from surfaces and air, respectively. The study concludes that proper disinfection, sterilization, and personal hygiene are needed to reduce laboratory contaminants and prevent false positive culture reports and laboratory-acquired infections.
This document outlines procedures for detecting various contaminants in human cell cultures, including microbes, fungi, viruses, and mycoplasma. It describes microscopic and macroscopic checks for initial detection of bacterial or fungal contamination. Detection of viral contamination involves using PCR or other techniques to test for pathogens like HIV, HBV, and EBV. Mycoplasma contamination is detected using one of two methods - a PCR-based test or a luminometric assay that detects mycoplasma enzyme activity. Periodic screening is important to ensure cell cultures are free of contamination.
The document discusses general principles for diagnosing infectious diseases, including:
1. Physical examination, clinical diagnosis, and epidemiological assessment help identify possible pathogens.
2. Laboratory tests are needed to confirm the causative agent, including microscopic examination, culture-based methods, and immunological or molecular detection techniques.
3. Proper specimen collection, transport, and timing are important for accurate diagnostic results.
This document provides an overview of the book "Veterinary Mycology" by Indranil Samanta. It begins with a foreword by Dr. Ricardo Negroni praising the author's qualifications and the comprehensive nature of the book. The preface written by Dr. Samanta outlines the importance of studying mycology in veterinary medicine and his intentions for the book. The book is divided into three parts, with the first discussing the history and general characteristics of fungi. The second part describes specific fungal pathogens, and the third covers laboratory diagnosis of fungal infections.
This document provides an overview of global regulatory guidance for ensuring viral safety in biologics production. It discusses three key approaches: preventing contamination through high quality raw materials; detecting contamination through testing cell banks, raw materials, and process intermediates; and evaluating viral clearance in the production process. The summary discusses the types of regulatory documents that provide guidelines on raw materials and cell lines, as well as strategies for preventing contamination, detecting contamination through a variety of assay methods, and limitations of detection assays.
Viral Risk Mitigation - A Global Regulatory PerspectiveMilliporeSigma
Looking for insights into current global regulatory expectations for viral safety? Read the special report from BioProcess International, in collaboration with Martin Wisher, Senior Regulatory Consultant focusing on BioReliance biosafety® services.
This document discusses biological markers for predicting response to Bacillus Calmette–Guerin (BCG) treatment in patients with non-muscle invasive bladder cancer (NMIBC). It identifies several promising markers based on a literature review, including tumor associated macrophages (TAMs), human leukocyte antigen (HLA) class I, a combination of Ki-67/CK20 proliferation markers, and certain cytokine levels like IL-2, IL-8, and the IL-6/IL-10 ratio. The document advocates using mathematical models along with molecular/cellular biology and clinical data to better understand these complex markers and their relationship to individual patient responses to BCG therapy for NMIBC.
This document discusses microbial diagnostic tests and their applications. It begins by introducing the importance of microbial diagnostics in identifying pathogens that cause disease. It then describes various diagnostic test types including culture techniques, molecular diagnostics like PCR, immunodiagnostic tests, and biosensors. The document also discusses laboratory diagnosis of bacteria, fungi, and viruses through techniques like microscopy, culturing, serology, antigen detection, and nucleic acid detection. It emphasizes the role of diagnostic tests in facilitating appropriate treatment and antimicrobial stewardship.
Undergraduate Research Conference Poster 2015Kendra Liu
This study developed a method to quantify and detect the physiological state of bacterial and eukaryotic cells using fluorescent dyes and microchip-based flow cytometry on the Agilent 2100 Bioanalyzer System. Salmonella enterica and Madin-Darby Canine Kidney cells were stained with SYTO62 and SYTOX dyes to label live and dead cells, respectively, and counted on the instrument. Cell numbers determined with this method matched counts from traditional plating and microscopy methods. The assay takes 30 minutes to analyze 6 samples and distinguishes live from dead cells rapidly with low sample and reagent use.
Isolation of bacteria is a significant step in diagnosing and managing bacterial infections. It involves collecting specimens, preserving and transporting them to the lab, examining samples microscopically, and using various culture and non-culture methods to isolate bacteria. Culture methods include using solid or liquid media, and automated systems, to allow bacterial colonies to grow. Non-culture methods involve molecular techniques like PCR. Proper specimen handling and use of appropriate culture conditions and media allow isolation of pathogenic bacteria to enable treatment and control of infections.
This seminar report discusses biosensors used in agriculture. It provides an overview of different types of biosensors including electrochemical, potentiometric, amperometric, calorimetric and optical biosensors. It discusses the principle of signal transduction that biosensors use to convert biological reactions into electrical signals. The report also examines the role of biosensors in agriculture for detecting crop diseases and pathogens in plants. Some advantages of biosensors include high sensitivity, selectivity and rapid response times. Potential disadvantages include susceptibility to interference and limited lifespan.
This document provides information on the laboratory diagnosis of tuberculosis. It discusses the classification of mycobacteria, specimen collection, and the various diagnostic methods used which include smear microscopy, culture, and molecular tests. Smear microscopy has limitations but is used in low-resource settings due to its low cost. Culture is the gold standard but is complex and requires biosafety. Liquid culture systems allow for faster results than solid media. Drug sensitivity testing determines resistance and is important for treatment. Molecular tests like line probe assays and GeneXpert can rapidly detect M. tuberculosis and resistance, with GeneXpert suitable for both pulmonary and extrapulmonary samples. Microcare Laboratory in Surat, India provides various tuberculosis diagnostic services and
Similar to Applications of flow cytometry to clinical microbiology (20)
This document summarizes new concepts in hemostasis, which is the process of blood clotting and wound healing. It discusses three phases of coagulation - initiation, amplification, and propagation. The initiation phase begins when tissue factor is exposed by damaged blood vessels, forming a complex with factor VIIa that activates small amounts of factors IX and X. This generates low levels of thrombin, starting the amplification phase where feedback loops dramatically increase coagulation factor activity through activation of factors V and VIII. High levels of thrombin then propagate fibrin clot formation. The document also reviews platelet activation and interactions with endothelial cells that regulate thrombus formation and coagulation.
Hougie 2004-journal of-thrombosis_and_haemostasisLAB IDEA
The document provides historical context on the development of the cascade/waterfall hypothesis of blood coagulation. It discusses the landmark 1964 papers by Macfarlane and Davie & Ratnoff that independently proposed the cascade model. Macfarlane described coagulation as a "cascade" and "photochemical amplifier", while Davie & Ratnoff termed it the "waterfall" concept. Both proposed coagulation occurs through a series of enzymatic reactions between clotting factors. The collaboration between clinical researcher Ratnoff and biochemist Davie led to the influential "waterfall" paper.
El documento resume el sistema de coagulación, incluyendo la hemostasia secundaria, los componentes como plaquetas y factores de coagulación, y los mecanismos como la cascada de coagulación. También describe la regulación de la hemostasia a través de sistemas anticoagulantes como la antitrombina III y la proteína C, y la evaluación por laboratorio incluyendo pruebas como el tiempo de sangrado, recuento de plaquetas, y tiempos de protrombina y tromboplastina parcial activada.
Este documento resume información sobre parasitosis intestinales. Explica que los parásitos intestinales incluyen protozoos, helmintos y pueden causar cuadros clínicos variables, desde infecciones asintomáticas hasta afectar uno o más órganos. Describe en detalle la amibiasis intestinal causada por Entamoeba histolytica, incluyendo su ciclo de vida, manifestaciones clínicas, diagnóstico y tratamiento. También menciona brevemente la balantidiasis.
CLASE ASCITIS
DR. RAUL CARRILLO ORTIZ
MODULO MEDICINA INTERNA
ROTACIÓN PATOLOGÍA CLÍNICA
DEPARTAMENTO DE MEDICINA Y NUTRICIÓN
UNIVERSIDAD DE GUANAJUATO
Las enfermedades musculares esqueléticas incluyen una variedad de condiciones que causan debilidad muscular. El diagnóstico adecuado requiere historia clínica, marcadores musculares como enzimas y proteínas, electromiografía y biopsia muscular para diferenciar alteraciones musculares de las neurológicas. Existen diferentes mecanismos fisiopatológicos involucrados como fenómenos autoinmunes, y condiciones pueden ser congénitas o adquiridas. El laboratorio juega un papel importante en el
Este documento describe la enfermedad hepática grasa no alcohólica (EHGNA), también conocida como esteatosis hepática. Define la EHGNA como la acumulación de grasa en el hígado en ausencia de consumo excesivo de alcohol. Explica que la EHGNA puede evolucionar a esteatohepatitis no alcohólica, cirrosis y hepatocarcinoma si no se corrigen los factores de riesgo subyacentes. Finalmente, discute las opciones de tratamiento, que incluyen cambios en el estilo de
Historical perspective and future direction of coagulation researchLAB IDEA
This document provides a historical perspective on the advances made in coagulation research over the past 100 years. Remarkable progress has been achieved, starting from early clinical observations of rare bleeding disorders to current understanding involving complex reaction pathways. Key developments include the discovery of coagulation factors and inhibitors through study of patient plasma, development of laboratory tests enabling factor assays, and evolving cascade models incorporating new knowledge. Recent technologies like gene targeting and stem cells promise further insights. Close collaboration between basic scientists and clinicians has been important to research progress and clinical applications. Future areas may include gene and cell-based therapies building on past successes translating basic research into improved patient care.
Lipoprotein (a) [Lp(a)] levels are strongly associated with cardiovascular disease risk. Lp(a) consists of an LDL-like particle bound to apolipoprotein(a) [apo(a)]. Apo(a) is produced in the liver and its production rate is the main determinant of plasma Lp(a) levels. Recent studies show that ligands of the farnesoid X receptor can significantly lower Lp(a) levels by inhibiting apo(a) transcription. While lowering Lp(a) may reduce cardiovascular risk, consensus reports have been cautious about routinely measuring Lp(a) due to lack of intervention studies demonstrating that lowering Lp(a) reduces hard endpoints and lack of effective medications to lower Lp(
This document summarizes the pathophysiology, diagnosis, and management of metabolic acidosis. It describes how metabolic acidosis is characterized by a reduction in serum bicarbonate and secondary decreases in arterial carbon dioxide partial pressure and blood pH. Acute metabolic acidosis lasting minutes to days is more common and often due to organic acid overproduction, while chronic metabolic acidosis lasting weeks to years usually reflects bicarbonate wasting or impaired renal acidification. Diagnosing the cause involves calculating the serum anion gap to classify disorders as normal or elevated. Adverse effects differ between acute and chronic forms. Treatment of acute metabolic acidosis with base administration is controversial due to lack of benefit and potential complications, while chronic metabolic acidosis treatment
El documento describe la alcalosis metabólica. Explica que es un trastorno metabólico frecuente en pacientes hospitalizados que causa un aumento de los niveles de bicarbonato en la sangre. Las causas incluyen la infusión de bicarbonato, pérdidas gástricas o renales de ácidos, y el uso de diuréticos. El tratamiento se enfoca en corregir la causa subyacente, restaurar los niveles de cloro y tratar las complicaciones como la hipopotasemia.
El documento describe los tres sistemas principales que regulan la concentración de iones de hidrógeno (H+) en el cuerpo: 1) los sistemas amortiguadores químicos de los líquidos orgánicos, 2) el centro respiratorio, y 3) los riñones. Los sistemas amortiguadores químicos reaccionan en segundos, el aparato respiratorio actúa en minutos, y los riñones tienen la respuesta más lenta en horas o días. Los riñones juegan un papel clave al secretar iones de hid
El documento resume los conceptos clave de acidosis y alcalosis, incluyendo las definiciones de acidosis y alcalosis, las causas de acidosis metabólica, y formulas y medidas utilizadas para evaluar trastornos del equilibrio ácido-base como el anión gap, gap osmolal urinario y brecha delta.
La lesión renal aguda (LRA) se define como la pérdida rápida de la función renal dentro de las 48 horas, lo que resulta en la retención de desechos en la sangre como la creatinina y la urea. La LRA puede ser prerrenal, causada por una perfusión renal inadecuada; intrínseca, causada por daño al riñón; u obstructiva posrenal, causada por una obstrucción del tracto urinario. El diagnóstico incluye exámenes clínicos y de laboratorio como la medición de creatin
El documento resume varios artículos y libros sobre lesión aguda renal, incluyendo Waikar S.S., Bonventre J.V. (2016) que discute la lesión aguda renal en el libro Harrison. Principios de Medicina Interna, 19e. También se mencionan artículos de Kidney International Supplements (2012) y Lancet (2012) que tratan sobre este tema.
TRASTORNOS DE LA COAGULACION EN HEPATOPATIASLAB IDEA
Este documento describe los trastornos de la coagulación que pueden ocurrir en pacientes con enfermedad hepática, incluyendo alteraciones plaquetarias, disminución en la síntesis de factores de coagulación, disfibrinogenemia e hiperfibrinólisis. Explica que estos trastornos ocurren cuando los niveles plasmáticos de los factores de coagulación están por debajo del 30-40% de los niveles normales. Finalmente, resume los tratamientos utilizados para corregir estos trastornos, como
El documento describe la hepatitis B, un virus que causa inflamación del hígado. El virus de la hepatitis B es un DNA envuelto que puede causar infección aguda o crónica. La transmisión ocurre principalmente a través del contacto con sangre o fluidos corporales. La infección aguda generalmente se resuelve espontáneamente, mientras que la infección crónica puede conducir a cirrosis o cáncer de hígado. El diagnóstico se realiza mediante pruebas serológicas y la carga viral. No existe un tratamiento
Fasting is not routinely required for determination of a lipid profileLAB IDEA
This document provides a joint consensus statement from the European Atherosclerosis Society and European Federation of Clinical Chemistry and Laboratory Medicine regarding fasting vs. non-fasting lipid profiles. It finds that observational data indicate maximal mean changes after meals are small and not clinically significant. It recommends the routine use of non-fasting lipid profiles to improve patient compliance, while fasting may be considered if non-fasting triglycerides are over 5 mmol/L. Abnormal non-fasting concentrations that should be flagged are outlined.
La trombocitopenia idiopática es un trastorno caracterizado por la producción de autoanticuerpos contra las plaquetas. Puede presentarse en niños de 2 a 4 años después de infecciones respiratorias o en adultos, especialmente mujeres entre 15 y 40 años. Los síntomas incluyen petequias, equimosis y sangrado de las encías. El tratamiento incluye corticoesteroides, inmunoglobulina o esplenectomía.
HCL Notes und Domino Lizenzkostenreduzierung in der Welt von DLAUpanagenda
Webinar Recording: https://www.panagenda.com/webinars/hcl-notes-und-domino-lizenzkostenreduzierung-in-der-welt-von-dlau/
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Wir erklären Ihnen, wie Sie häufige Konfigurationsprobleme lösen können, die dazu führen können, dass mehr Benutzer gezählt werden als nötig, und wie Sie überflüssige oder ungenutzte Konten identifizieren und entfernen können, um Geld zu sparen. Es gibt auch einige Ansätze, die zu unnötigen Ausgaben führen können, z. B. wenn ein Personendokument anstelle eines Mail-Ins für geteilte Mailboxen verwendet wird. Wir zeigen Ihnen solche Fälle und deren Lösungen. Und natürlich erklären wir Ihnen das neue Lizenzmodell.
Nehmen Sie an diesem Webinar teil, bei dem HCL-Ambassador Marc Thomas und Gastredner Franz Walder Ihnen diese neue Welt näherbringen. Es vermittelt Ihnen die Tools und das Know-how, um den Überblick zu bewahren. Sie werden in der Lage sein, Ihre Kosten durch eine optimierte Domino-Konfiguration zu reduzieren und auch in Zukunft gering zu halten.
Diese Themen werden behandelt
- Reduzierung der Lizenzkosten durch Auffinden und Beheben von Fehlkonfigurationen und überflüssigen Konten
- Wie funktionieren CCB- und CCX-Lizenzen wirklich?
- Verstehen des DLAU-Tools und wie man es am besten nutzt
- Tipps für häufige Problembereiche, wie z. B. Team-Postfächer, Funktions-/Testbenutzer usw.
- Praxisbeispiele und Best Practices zum sofortigen Umsetzen
Essentials of Automations: The Art of Triggers and Actions in FMESafe Software
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In the rapidly evolving landscape of technologies, XML continues to play a vital role in structuring, storing, and transporting data across diverse systems. The recent advancements in artificial intelligence (AI) present new methodologies for enhancing XML development workflows, introducing efficiency, automation, and intelligent capabilities. This presentation will outline the scope and perspective of utilizing AI in XML development. The potential benefits and the possible pitfalls will be highlighted, providing a balanced view of the subject.
We will explore the capabilities of AI in understanding XML markup languages and autonomously creating structured XML content. Additionally, we will examine the capacity of AI to enrich plain text with appropriate XML markup. Practical examples and methodological guidelines will be provided to elucidate how AI can be effectively prompted to interpret and generate accurate XML markup.
Further emphasis will be placed on the role of AI in developing XSLT, or schemas such as XSD and Schematron. We will address the techniques and strategies adopted to create prompts for generating code, explaining code, or refactoring the code, and the results achieved.
The discussion will extend to how AI can be used to transform XML content. In particular, the focus will be on the use of AI XPath extension functions in XSLT, Schematron, Schematron Quick Fixes, or for XML content refactoring.
The presentation aims to deliver a comprehensive overview of AI usage in XML development, providing attendees with the necessary knowledge to make informed decisions. Whether you’re at the early stages of adopting AI or considering integrating it in advanced XML development, this presentation will cover all levels of expertise.
By highlighting the potential advantages and challenges of integrating AI with XML development tools and languages, the presentation seeks to inspire thoughtful conversation around the future of XML development. We’ll not only delve into the technical aspects of AI-powered XML development but also discuss practical implications and possible future directions.
HCL Notes and Domino License Cost Reduction in the World of DLAUpanagenda
Webinar Recording: https://www.panagenda.com/webinars/hcl-notes-and-domino-license-cost-reduction-in-the-world-of-dlau/
The introduction of DLAU and the CCB & CCX licensing model caused quite a stir in the HCL community. As a Notes and Domino customer, you may have faced challenges with unexpected user counts and license costs. You probably have questions on how this new licensing approach works and how to benefit from it. Most importantly, you likely have budget constraints and want to save money where possible. Don’t worry, we can help with all of this!
We’ll show you how to fix common misconfigurations that cause higher-than-expected user counts, and how to identify accounts which you can deactivate to save money. There are also frequent patterns that can cause unnecessary cost, like using a person document instead of a mail-in for shared mailboxes. We’ll provide examples and solutions for those as well. And naturally we’ll explain the new licensing model.
Join HCL Ambassador Marc Thomas in this webinar with a special guest appearance from Franz Walder. It will give you the tools and know-how to stay on top of what is going on with Domino licensing. You will be able lower your cost through an optimized configuration and keep it low going forward.
These topics will be covered
- Reducing license cost by finding and fixing misconfigurations and superfluous accounts
- How do CCB and CCX licenses really work?
- Understanding the DLAU tool and how to best utilize it
- Tips for common problem areas, like team mailboxes, functional/test users, etc
- Practical examples and best practices to implement right away
Pushing the limits of ePRTC: 100ns holdover for 100 daysAdtran
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2. 168 ´
ALVAREZ-BARRIENTOS ET AL. CLIN. MICROBIOL. REV.
of these situations. In bacteremia and bacteriuria, FCM would
not only rapidly detect organisms responsible for the infection
but would also initially identify the type of microorganism on
the basis of its cytometric characteristics. Although FCM offers
a broad range of potential applications for susceptibility test-
ing, a major contribution would be in testing for slow-growing
microorganisms, such as mycobacteria and fungi (108, 163,
262). Results are obtained rapidly, frequently in less than 4 h;
when appropriately combined with the classical techniques,
FCM may offer susceptibility results even before the microor-
ganism has been identified. The most outstanding contribution
offered by FCM is the detection of mixed populations, which
may respond to antimicrobial agents in different ways (331).
This technique could also be applied to study the immune
response in patients, detect specific antibodies (27, 133), and
FIG. 1. Light-scattering and fluorescence signal production at the flow cell
analysis point of the flow cytometer. From Purdue Cytometry CD-ROM vol. 1 monitor clinical status after antimicrobial treatments (58, 244).
(adapted with permission of the publisher). Moreover, when properly applied, FCM can be adjusted to use
defined parameters that avoid subjectivity and aid the clinical
microbiologist in the interpretation of specific results, partic-
ularly in the field of rapid diagnosis.
in clinical microbiology laboratories. Results are offered
quickly, the diagnosis of emerging infections has become eas-
ier, and unculturable pathogens have been identified (109). TECHNICAL BASIS OF FLOW CYTOMETRY
On the other hand, the current organization of clinical mi- FCM is an analytical method that allows the rapid measure-
crobiology laboratories is now subject to automation and com- ment of light scattered and fluorescence emission produced by
petition, both overshadowed by increasing costs (282, 339). suitably illuminated cells. The cells, or particles, are suspended
Increased use of automation in clinical microbiology laborato- in liquid and produce signals when they pass individually
ries is best exemplified by systems used for detecting bactere- through a beam of light (Fig. 1). Since measurements of each
mia, screening of urinary tract infections, antimicrobial suscep- particle or cell are made separately, the results represent cu-
tibility testing, and antibody detection. To obtain better mulative individual cytometric characteristics. An important
sensitivity and speed, manufacturers continuously modify all analytical feature of flow cytometers is their ability to measure
these systems. Nevertheless, the equipment needed for all multiple cellular parameters (analytical flow cytometers).
these approaches is different, and therefore the initial costs, Some flow cytometers are able to physically separate cell sub-
both in equipment and materials, are high. sets (sorting) based on their cytometric characteristics (cell
Flow cytometry (FCM) could be successfully applied to most sorters) (Fig. 2). The scattered light (intrinsic parameters) and
FIG. 2. Scheme of optic (dichroic mirrors and bandpass filters) and illumination (laser) systems of a flow cytometer with six parameters detected (size, granularity,
and four fluorescences) by separate photomultiplier tubes (except size, which can be detected by photodiode or a PMT tube) and sorting capacity. From Purdue
Cytometry CD-ROM vol. 1 (adapted with permission of the publisher).
3. VOL. 13, 2000 FLOW CYTOMETRY IN CLINICAL MICROBIOLOGY 169
fluorescence emissions of each particle are collected by detec- that allows the analysis of the huge amount of information
tors and sent to a computer, where the distribution of the produced by multiparameter data acquisition. The analytical
population with respect to the different parameters is repre- software permits the study and independent analysis of a par-
sented. Scattered light collected in the same direction as the ticular subpopulation. Besides all the statistical information,
incident light is related to cell size, and scattered light collected the data can be represented in several different ways: mono-
at an angle of 90° gives an idea of the particle complexity. This parametric histograms, biparametric histograms, and three-
parameter is related to cell surface roughness and the number dimensional representations (Fig. 3). There is a growing mar-
of organelles present in the cell. Size and complexity are con- ket of commercial FCM software. Free software can also be
sidered intrinsic parameters since they can be obtained without downloaded from the Internet, where it is possible to find
having to stain the sample. To obtain additional information, information about all the fields related to FCM (cytometry
samples can be stained using different fluorochromes. Fluoro- network sites, http://nucleus.immunol.washington.edu/ISAC
chromes can be classified according to their mechanism of /network_sites.html; JCSMR flow cytometry software, http://
action (127): those whose fluorescence increases with binding jcsmr.anu.edu.au/facslab/facs.html; ISAC WWW home page,
to specific cell compounds such as proteins (fluorescein iso- http://www10.uniovi.es/ISAC.html).
thiocyanate [FITC]), nucleic acids (propidium iodide [PI]),
and lipids (Nile Red); those whose fluorescence depends on FLOW CYTOMETRY AND MICROBIOLOGY,
cellular physiological parameters (pH, membrane potential, A LONG TIME TOGETHER
etc.); and those whose fluorescence depends on enzymatic
activity (fluorogenic substrates) such as esterases, peroxidases, From the beginning of FCM (68), the ancestor of modern
and peptidases (Table 1). Fluorochromes can also be conju- flow cytometers has been identified with an aerosol particle
gated to antibodies or nucleotide probes to directly detect counter designed to analyze mine dust (124). This apparatus
microbial antigens or DNA and RNA sequences. was used in World War II by the U.S. Army in experiments
A typical flow cytometer has several parts. (i) The hydraulic for the detection of bacteria and spores. Gucker et al. (124)
system produces the fluid stream, with a liquid sheath sur- reported that the instrument could be used with biological
rounding the cell suspension (hydrodynamic focusing). This samples (bacteria), as well as particles in air suspension or
sheath is responsible for the passage of the particles through aerosols. Thus, FCM with an application to microbiology orig-
the sensing point at a constant velocity. (ii) The illumination inated many years before the use of flow cytometry as a tool for
system consists of the light that produces the scatter signals studying mammalian cells. The original device incorporated a
and fluorescence emission when the particles pass through it. sheath of filtered air to limit the air sample stream to the
There are two types of flow cytometers, depending on the central portion of the flow chamber. The detector used was a
illumination source: those with a laser light source, and those then recently developed device called a photomultiplier tube.
with an arc lamp source. Each has it own advantages and Particle counters based on the Coulter orifice principle, in
disadvantages, but the main difference lies in their fields of which the difference in electrical conductivity between the cells
application. Arc lamp cytometers are frequently used in mi- and the medium in which they are suspended is measured by
crobiological applications due to their better scatter resolution the change in electrical impedance produced as they pass
and versatility. In contrast, laser flow cytometers have wider through an orifice, were later developed. These instruments
applications in immunology and hematology because they ex- were widely applied in hematology studies. However, the first
cite fluorochromes associated with cells. Studies comparing the real flow cytometer was built by Kamentsky et al. (154), using
two types of cytometers have concluded that the selection of spectophotometric techniques to detect and measure nucleic
one rather than the other depends mainly on the range of acids and light scattering of unstained cervical cells in a flow
wavelengths required for the excitation of the selected fluores- stream. At the same time, Fulwyler, working at the Los Alamos
cent stains (13, 161). Our personal experience supports this Scientific Laboratory, described the first flow cytometer with
opinion and work should aim at developing protocols accord- sorting capability (104). This machine worked by measuring
ing to the type of cytometer available. (iii) The optic system cell volumes obtained by the Coulter orifice principle. Fulwyler
focuses incident light on the crossing particles, recovers the adapted the ink jet printer principle, using electrostatic deflec-
scattered light and the fluorescence produced by the fluoro- tion of charged droplets, as a cell-sorting mechanism. In fact,
chromes present in the cells, and directs both to the appropri- sorting capability was introduced to demonstrate the accuracy
ate photomultiplier tubes (Fig. 2). (iv) The electronic system of the signals obtained by the machine and to ascribe a given
transforms the incident light from fluorescence and light scat- distribution of cell volume detected by an electronic signal to a
tered into electric pulses (analogic). The magnitudes of these specific cell type. During the 1970s, applications of FCM to
pulses are distributed electronically into channels, permitting research into mammalian cells advanced rapidly, but at that
the display of histograms of the number of cells plotted against time few instruments were developed for microbiological stud-
the channel numbers (digital) (68). If the instrument has the ies. The subsequent applications to microbiology of FCM tech-
capacity to do so, it also controls the cell-sorting process. Flu- niques that were initially developed to study mammalian cells
orescence-activated cell sorting refers to the ability to select a were due to optical improvements in flow cytometers and
subpopulation from the whole population, following cytomet- newly developed fluorochromes. The development of an arc
ric classification, and to physically separate this particular pop- lamp-based instrument by Steen’s group in 1979 (301, 303)
ulation. To do this, the machine produces a uniform stream of allowed the use of FCM for basic research on bacteria. Be-
droplets; a particular droplet containing a cell can be charged, cause of the design of the flow chamber and the use of pho-
permitting selection of the droplet when it passes through an tomultiplier tubes for detecting scattered light, this instrument
electrical field produced by deflection plates (Fig. 2). In this was ideal for studying microorganisms (7, 37). The promising
way, two populations can be sorted at the same time (positively tool described by Boye and Steen in 1983 became a “potent
and negatively charged droplets). A new high-speed sorter illuminating light” in the 1990s (38), as was stated in the book
machine has been developed with the possibility of sorting four edited by David Lloyd, Flow Cytometry in Microbiology (186a),
populations at the same time (MoFlo; Cytomation, Freiburg, from which most microbiological cytometrists have learned
Germany). (v) The data analysis system consists of software their trade. In the last years of the 1990s, the applications of
4. 170
´
TABLE 1. Some of the fluorescent molecules used to study microorganisms by flow cytometry
Excitation wavelength Emission wavelength
Dye Ligand or substrate Applications
( max) (nm) ( max) (nm)
TOTO-3 642 660 DNA, RNA DNA quantification, cell cycle studies
SYTOX Green 504 525 DNA, RNA Viability, DNA quantification
PI 536 625 DNA, RNA Viability, DNA quantification, cell
cycle studies
Ethidium bromide 510 595 DNA, RNA DNA quantification, cell cycle studies
ALVAREZ-BARRIENTOS ET AL.
Hoechst 33258/33342 340 450 DNA (GC pairs) Cell cycle studies
SYTO 13 488 509 DNA, RNA Viability, DNA quantification, cell
cycle studies
Mithramycin 425 550 DNA Cell cycle studies
Pyronine Y 497 563 RNA RNA quantification
FITC 495 525 Protein Microbe detection
Texas Red (sulforhodamine isothiocyanate) 580 620 Protein Microbe detection
Oregon Green isothiocyanate 496 526 Protein Microbe detection
Indo-1 340 398–485 Ca2 Ca2 mobilization
Fura-2 340 549 Ca2 Ca2 mobilization
Fluor-3 469 545 Ca2 Ca2 mobilization
BCECF 460–510 520–610 pH Metabolic variations
SNARF-1 510 587–635 pH Metabolic variations
DIOC6(3) 484 501 Membrane potential Antibiotic susceptibility, metabolic
variations
Oxonol [DiBAC4(3)] 488 525 Membrane potential Antibiotic susceptibility, metabolic
variations
Rhodamine 123 507 529 Membrane potential Antibiotic susceptibility, metabolic
(mitochondria) variations
Fun-1 508 525–590 Yeast vacuolar enzyme activity Yeast metabolic state
Nile Red 490–550 540–630 Lipids
Lectins Depends on fluorochrome Depends on fluorochrome Membrane oligosaccharides Cell wall composition, microbe
conjugated conjugated detection
Fluorescently labeled oligonucleotides Depends on fluorochrome Depends on fluorochrome Nucleotide sequences Microbe identification
conjugated conjugated
Calcofluor white 347 436 Chitin and other carbohydrate Fungal detection
polymers
Substrates linked to fluorochromes Enzyme activities Metabolic activity
Antibodies labeled with flurochormes Antigens Microbe detection
CLIN. MICROBIOL. REV.
5. VOL. 13, 2000 FLOW CYTOMETRY IN CLINICAL MICROBIOLOGY 171
FIG. 3. The data obtained from a flow cytometer can be displayed in several ways. The most common are the mono- and biparametric histograms (A and B), which
usually include a statistical analysis of the results. (A) Monoparametric histogram showing the selected parameter on the x axis and the relative cell number on the y
axis. (B) Biparametric histogram showing cells distributed as a function of their signal intensity with respect to each parameter. Cells located in the upper left quadrant
are positive for the parameter represented on the y axis, cells located in the upper right quadrant are positive for both parameters, cells located in the lower left quadrant
are double negative, while cells in the lower right panel are positive for the parameters on the x axis. (C and D) Three-dimensional representations. The z axis can
represent the relative number of cells (C) or a third parameter (D), such as scattered light on the x and y axes and fluorescence signals on the z axis.
FCM in microbiology have significantly increased (9, 28, 103, teria in the same sample (301, 302, 306). One parameter was
148, 291). light scattered (size), and the other was either fluorescence
emission from fluorochromes coupled to cellular components
General Applications of Flow Cytometry (protein and DNA) or autofluorescence (Fig. 4), or light scat-
to Microbiology tered acquired from another angle (42, 151, 277, 293, 301, 302,
306, 316). However, the use of several fluorochromes for direct
The applications of FCM to microbiology have been so staining or through antibody or oligonucleotide conjugates
widespread that discussion of all of them is beyond the scope plus size detection is the simplest way to visualize or identify
of this review. For more information, see the excellent reviews microorganisms by FCM (6, 7, 10, 11, 257, 317, 332).
by Davey and Kell (68), Porter et al. (263), and McSharry (211) The simple and rapid assessment of the viability of a micro-
and the “Bible” of flow cytometry by Howard Shapiro, Practi- organism is another important aspect of FCM. The effect of
cal Flow Cytometry (291). Below, we briefly describe some of environmental stress or starvation on the membrane potential
the applications of FCM in the field of microbiology, focusing of bacteria has been studied by several groups using fluoro-
on present or future applications in clinical microbiology. chromes that distinguish among nonviable, viable, and dor-
Earlier works by Steen had demonstrated the applicability of mant cells (155–157, 188; see references 68, 71, 78, 89, 135,
dual-parameter analysis to discriminate among different bac- 150, 200–203, 265, and 328 for reviews). Other authors have
6. 172 ´
ALVAREZ-BARRIENTOS ET AL. CLIN. MICROBIOL. REV.
7. VOL. 13, 2000 FLOW CYTOMETRY IN CLINICAL MICROBIOLOGY 173
demonstrated the use of PI as a viability marker in yeasts (72), with viral infection, including HSV (144, 146), Epstein-Barr
Pneumocystis carinii (177), and bacteria (89, 230, 264). virus (EBV) (4), influenza virus (267), measles virus (93), pap-
FCM has also been used in metabolic studies of microor- illomavirus (340), and HIV-1 (129, 194) infections. Further-
ganisms. This was first accomplished by Thorell (316), using more, by means of biotinylated or directly FITC-labeled virus,
autofluorescence due to NADPH and flavins as metabolic sta- interactions of EBV (142, 159, 182, 335), echovirus (206), ad-
tus markers. Other authors studied DNA, protein, peroxide enovirus (217), influenza virus (228), simian virus 40 (SV40)
production, and intracellular pH (3, 5, 39, 140, 324, 345). Re- (20), human T-cell leukemia virus type 1 (HTLV-1) (110),
cently developed fluorochromes and kits (Sytox Green and measles virus (226, 232), bovine herpesvirus (326), papilloma-
Live/Dead kits; Molecular Probes, Eugene, Oreg.) have been virus (268), bunyaviruses (249), poliovirus (102), and HIV-1
used for the FCM-based counting of live and dead bacteria and (14, 308, 318) with their putative cell receptors have been
yeasts (176, 178, 311), simplifying staining protocols and mak- described. These investigations show that FCM is able to pro-
ing data interpretation easier. Other kits are available for de- vide solutions to problems arising when working with micro-
tecting gram-positive and gram-negative bacteria or for study- organisms.
ing yeast organelles (127). Recently, Mason et al. (204)
described a method which enables the discrimination of gram- APPLICATIONS OF FLOW CYTOMETRY TO
positive from gram-negative bacteria on the basis of the fluo- CLINICAL MICROBIOLOGY
rescence emitted when the organisms are stained with two
fluorochromes. These authors correctly predicted the Gram The isolation of microbes and their identification, the detec-
stain reaction of 45 strains of clinically relevant organisms, tion of increased levels of antibodies to a particular pathogen
including several known to be gram variable. In addition, rep- in the course of an illness, and direct detection of microbial
resentative strains of gram-positive anaerobic organisms, components (nucleic acids and proteins) in clinical samples
which are normally decolorized during the traditional Gram obtained from different tissues or body fluids are the main
stain procedure, were classified correctly by this method. tools for laboratory diagnoses of microbial infections. Effective
FCM also offers the possibility of studying gene expression antimicrobial therapies have indeed been developed because
using reporter genes in yeasts (56, 258, 297, 338) and bacteria early treatment is crucial in many cases; therefore, rapid diag-
(8, 55). The development of gene expression systems based on nosis is essential in the fight against infection.
green fluorescent proteins facilitates this kind of study due to
the simplicity of the technique (60, 77, 229, 320). Direct Detection
The sensitivity of FCM allowed Philips and Martin (255) to
detect Bacillus spores (254). Using a similar approach, Griffiths Bacteria. Antibodies are currently changing the way in which
et al. (121) and Challier et al. (49) were able to sort spores we identify microbes, making it easier and faster. Their spec-
from Dictyostelium discoideum and Enterocytozoon bieneusi, re- ificity and the possibility of using fluorochrome-labeled anti-
spectively. These examples show the potential of FCM in the bodies to specific antigens render them one of the most pow-
investigation of small microbes. erful tools in the identification of pathogens. The main
The interaction between pathogens and phagocytic cells has disadvantage of this method is still the limited availability of
also been studied by FCM (22, 23). The development of fluo- antibodies directed against particular microbes. Other advan-
rochromes to detect oxidative bursts due to phagocytosis (17, tages of using antibodies are that the cells do not need to be
251, 281) increased the number of studies with different mi- cultivable and that the method is simple and fast. In an early
crobes such as Borrelia burgdorferi (17), Staphylococcus spp. work from 1983, Groschel (122) explained somewhat prophet-
(128, 196), Escherichia coli (70, 271), Bordetella pertussis (299), ically the use of antibodies in clinical microbiology. FCM in
Cryptococcus neoformans (48), Salmonella (272), and yeasts conjunction with fluorescent antibodies has been used to de-
(90, 96, 114). tect surface antigens in Haemophilus (298), Salmonella (57,
FCM has been extensively used for studying virus-cell inter- 207), Mycobacterium (238), Brucella (35), Branhamella ca-
actions (172, 180, 334). This topic was reviewed in depth by tarrhalis (29), Mycoplasma fermentans (50), Pseudomonas
McSharry in 1994 (211). Modulation of the expression of cel- aeruginosa (134), Bacteroides fragilis (191, 239) and Legionella
lular proteins due to viral infection has been studied by FCM (143), among other microorganisms. These examples illustrate
for cytomegalovirus (CMV) (116), herpes simplex virus (HSV) the sensitivity and specificity of using antibodies that allow the
(149), adenovirus (168), human immunodeficiency virus (HIV) detection of particular cell types (of as few as 100 cells per ml
(53), and hepatitis B virus (HBV) (346). Perturbation of the in 30 min) in heterogeneous populations (57).
cell cycle and DNA replication in virus-infected cells have also The first study detecting of microbes in blood by using FCM
been studied by FCM for papillomavirus (25), CMV (80) and was done with ethidium bromide as the detecting fluoro-
human HIV-1 (273). This technique has been also used to chrome (195). Blood cells were lysed, and the remaining bac-
study the effect of viral infection on intracellular Ca2 levels teria were stained with ethidium bromide; as few as 10 E. coli
([Ca2 ]i) by Irurzun et al. (145) and by Miller et al. (221). FCM cells/ml were detected. Using ethidium bromide fluorescence
has also permitted the demonstration of apoptosis associated and light-scattered signals, Cohen et al. (58) were able to
FIG. 4. (A) Dual-parameter analysis of forward light scatter (size) and red fluorescence signals allowed the discrimination between two species of Candida, based
on different fluorochrome staining backgrounds. These yeast species are indistinguishable by monoparametric analysis of forward light scatter or red autofluorescence.
However, after addition of PI, they show different basal levels, and if this is plotted against size, it is possible to discriminate them. This kind of analysis permits
quantification of both species in mixed cultures. (B) Quantification of different protein amounts (measured as FITC fluorescence) can be used to distinguish different
microorganisms such as those represented in the histogram (from Purdue Cytometry CD-ROM, vol 2., ISSN 1091-2037, provided by Hazel M. Davey [adapted with
permission of the publisher]). (C) Dual-fluorescence discrimination of fungal spores. Spores from Aspergillus, Mucor, Cladosporium, and Fusarium were fixed and
stained with Calcofluor, which binds to chitin in the spore wall, and PI, which stains nucleic acids. As shown, the spores have different amounts of chitin and nucleic
acids, permitting their segregation by FCM. Samples shown in panel A were run on a FACScan (Becton-Dickinson) flow cytometer, the ones shown in panel B were
run on an EPICS Elite (Coulter) flow cytometer, and those shown in panel C were run on a Bryte-HS (Bio-Rad) flow cytometer.
8. 174 ´
ALVAREZ-BARRIENTOS ET AL. CLIN. MICROBIOL. REV.
detect bacteria in 43 clinical specimens from several sources, strated that mixed infections occur, and hence the treatment
such as wound exudates, bile, serous-cavity fluids, and bron- for such circumstances can be established.
chial-lavage fluids, in less than 2 h, although they were unable Parasites. The first applications of FCM to parasites in-
to identify them. volved a study of the cell cycle and the amounts of DNA of
An FCM method for the direct detection of anaerobic bac- Physarum polycephalum myxamoebae (319) and the character-
teria in human feces was described by van der Waaij et al. ization of monoclonal antibodies against membrane antigens
(322), using PI for discriminating the patient’s cells and ex- from Leishmania (337). Specific clinical applications came
cluding large particles by forward light scatter. At the same later, when Flores et al. (99) used monoclonal antibodies,
time, fluoresceinated antibodies against human immunoglob- FCM, and immunofluorescence microscopy for the direct iden-
ulin A (IgA) were added to detect IgA-coated bacteria. This tification of Naegleria fowleri and Acanthamoeba spp. in clinical
method allows the rapid and highly sensitive assessment of specimens.
fecal flora by specific IgA-FITC fluorescence without the need Several approaches have been developed in the last few
to culture the samples. years to detect intracellular parasites, such as Plasmodium
Another way in which FCM can achieve direct diagnosis is (147, 153, 240–242, 250, 319, 325). Such work took advantage
by fluorescent-oligonucleotide detection. By combining rRNA- of the absence of DNA in erythrocytes. Thus, if the parasite is
targeted fluorescent probes and 4 ,6-diamidino-2-phenylindole inside the cell, its DNA can be stained with specific fluoro-
(DAPI) for nucleic acid staining, Wallner et al. (333) showed chromes and detected by FCM. The multiparameter analysis
that it was possible to detect Acinetobacter spp. by FCM. To permitted by FCM can be used to study other characteristics,
date, this approach has not been used with clinical samples, such as parasite antigens expressed by the erythrocyte (which
perhaps owing to its methodological complexity. Nevertheless, can be detected by antibodies conjugated with fluorochromes)
the specificity provided by the oligonucleotide probe to identify (54, 153) or the viability state of the parasitised cell. Further-
the putative infectious agent can be taken advantage of (315), more, the technique can be used with either fresh or fixed cells
thus promising many future applications. (241, 242). An important study showing the benefits of flow
The use of different-sized fluorescent microspheres coated cytometry is that of Dixon et al. (81), who compared normal
with antibodies against microbes is a new application of flow light microscopy, immunofluorescence microscopy, and FCM
cytometry for direct diagnosis (169). This method detects the in the detection of Giardia lamblia cysts. They showed that
binding of specific microbes to antibody-coated microspheres when FCM is used in combination with immunofluorescence, a
by measuring the decrease in the fluorescence emission of the larger number of samples can be analyzed in a relatively short
microspheres due to the shading effect of microbes on both the period and, more important, that this technique affords more
consistent results than either conventional or immunofluores-
exciting and emitting light. With different-sized fluorescent
cence microscopy when samples containing small number of
microspheres, several pathogens can be detected simulta-
cysts are analyzed.
neously in the same sample. This approach could also be used
Viruses. FCM allows both detection and quantification of
with fungi, parasites, and viruses, as well as in infections pro-
infected cells directly in clinical samples or after inoculation
duced by combinations of these. In fact, as discussed below, a
and culture of the virus in cell culture.
similar approach has been used for the simultaneous detection
(i) Detection and quantification of viral antigens. FCM can
of plant viruses (137). detect viral antigens either on the surface of or within infected
Fungi. With regard to yeasts, the work by Groshen et al. cells (172). It can rapidly detect and quantify virus-infected
(122), Chaffin et al. (47), and Han et al. (126) has shown that cells using antibodies that specifically recognize surface or in-
surface antigens of Candida albicans can be detected by flow ternal antigens (91, 211, 213, 334); in the latter case, perme-
cytometry in conjunction with available specific antibodies. As abilization of the cells is required. A thorough review of dif-
discussed below, this approach can be used for clinical samples. ferent permeabilization methods, including the advantages and
The possibility of serotyping Candida isolated from clinical disadvantages of each, for viral antigen and nucleic acid de-
samples emerged from the work of Chaffin et al. in 1988 (47) tection has been written by McSharry (211). Direct and indi-
and Brawner and Cutler in 1989 (40). However, it was not until rect fluorescent-antibody methods are used. Direct detection
1996 that Mercure et al. (219) validated the FCM serotyping involves the use of fluorescently labeled antibody (labeled with
procedure, using serotype A-specific antisera. According to FITC or phycoerythrin). In the indirect fluorescent-antibody
Mercure et al., the most striking feature of this method is its method, unlabeled antibody is bound to infected cells, which
reliability. Ninety-four strains isolated from patients were an- are then incubated with fluorescence-labeled anti-Ig that binds
alyzed by a slide immunofluorescence assay and FCM. FCM to the first viral antibody.
was able to detect the presence of two different strains in a As previously stated, FCM is carried out on single cells, and
culture that was assumed to be pure and serotyped four strains therefore FCM analysis of virus-infected cells is best suited to
whose serotypes could not be determined by slide immunoflu- blood, bronchoalveolar lavage fluid, and urine samples (172).
orescence. Again, it was acknowledged that when a cytometer However, it is also possible to analyze cells from tissues that
is available, the procedure is probably more cost-effective than have previously been treated enzymatically (211).
a commercially available kit for Candida serotype determina- Based on the potential of FCM for multiparametric analysis,
tion. Since the origin of the infecting strain(s) is often ques- there are two key advantages to its use in studying viral infec-
tioned when clinicians encounter patients with repeated epi- tion: (i) its ability to analyze several parameters in single-
sodes of Candida infections, FCM can help to discriminate infected cells at the same time and (ii) its ability not only to
among strains, as demonstrated in this work (219). detect but also to quantify infected cells. These parameters
The diagnosis of onychomycosis based on clinical presenta- may be related to particular components or events of the
tion, culture, and microscopy is hampered by false-negative infected cell or components (proteins or nucleic acids) of the
and false-positive results that confuse treatment outcomes. virus. For this reason, FCM has been a powerful tool to char-
Using FCM and antibodies directed against yeasts, Pierard et acterize the mechanisms of viral pathogenesis. Furthermore,
al. (256) identified fungal pathogens and differentiated them FCM allows simultaneous detection of several viruses in a
from nonpathogenic ones. Furthermore, the authors demon- sample by using (i) antibodies to different viral antigens con-
9. VOL. 13, 2000 FLOW CYTOMETRY IN CLINICAL MICROBIOLOGY 175
jugated to different fluorochromes, or (ii) specific viral anti- sensitivity of the method was somewhat lower than that of the
bodies conjugated to latex particles of different sizes. As stated slide method, it might be sufficient to predict the disease.
above, the presence of different viral antigens is detected by Honda et al. (132) also identified specific CMV-infected cell
differences in the forward-scattered light as a consequence of populations in peripheral blood lymphocytes from CMV-in-
the different sized particle used for each antibody. For exam- fected patients by FCM. Using monoclonal antibodies directed
ple, Iannelli et al. (137) simultaneously detected cucumber against immediate-early CMV antigen (see above) or against
mosaic, tomato, and potato viruses by using 3-, 6-, and 10- m- several cell membrane markers to phenotype infected periph-
diameter latex particles, respectively. Although this method eral blood cells from bone marrow transplant recipients, these
was aimed at the detection of plant viruses, its basis could be authors developed a rapid and quantitative FCM method for
applied to the detection of animal or human viruses in any the detection of immediate-early CMV antigen. The detection
clinical sample, such as the simultaneous detection of CMV, of CMV antigens specifically in the polymorphonuclear leuko-
HSV, and HBV in organs destined for transplantation as well cytes from transplanted patients with CMV pneumonia sug-
as in transplanted patients and coinfections in HIV-infected gests that the FCM antigenemia assay would be useful for
individuals. predicting CMV-associated disease in transplant recipients
Flow cytometric analysis has allowed the detection and (132). In summary, FCM offers a rapid and suitable quantifi-
quantification of SV40 T antigen in infected cells and moni- cation of the CMV viral load. Although systematic compara-
toring the kinetics of T-antigen expression. By means of mul- tive evaluations of the CMV viral load using this method are
tiparametric analysis, using PI for the measurement of cellular needed, current data are promising.
DNA and FITC-labeled antibody for the detection of viral The detection of cytomegalic endothelial cells in peripheral
antigens, Lehman et al. (171, 179) related high levels of SV40 blood of patients is another means of monitoring active CMV
T antigen to the appearance of cells with tetraploid DNA infection. Through enrichment of endothelial cells in the
content due to a cell cycle block at G2/M. mononuclear fraction by density centrifugation, endothelial
Detection of immediate-early, early, and late CMV antigens cell-specific staining, and fluorescence-activated cell sorting of
by monoclonal antibodies permits direct diagnosis and quan- these cells, a method with 10-fold greater sensitivity than cy-
tification of CMV infection. This is a frequent complication in tocentrifugation of the mononuclear cell fraction alone has
immunosuppressed patients, including transplant recipients recently been developed for quantification of cytomegalic en-
and AIDS patients. In 1988, by means of FCM, Elmendorf et dothelial cells by FCM (158). Belles-Isles et al. (24) have also
al. (91) detected early CMV antigen 30 min after virus adsorp- suggested using FCM to monitor CMV infections by monitor-
tion to fibroblasts. Thus, FCM permits the detection of viral ing the CD8 CD38 T-cell subset in kidney transplant recip-
ients; this T-cell subpopulation usually increases during active
infection earlier than does conventional immunofluorescence
viral infections. Quantification of CD8 CD38 T cells by
microscopy or the detection of cytopathic effects in cell culture
dual-color FCM in 77 kidney transplant recipients during the
(91, 289, 310).
posttransplantation period detected high levels of CD8
During active infection, CMV disseminates in the blood, and
CD38 subsets in all patients with CMV disease. Belles-Isles et
viremia has been described as a major risk factor for the pro-
al. (24) therefore concluded that the percentage of CD8
gression to clinical disease, particularly in allogeneic bone mar-
CD38 T cells constitutes an immunologic marker that can
row transplant recipients (31). Accordingly, quantification of serve as a tool for early detection of viral diseases.
the viral load in persistently infected hosts may provide a Viral antigens have also been detected by FCM for the
method to predict the development of CMV disease and help diagnosis of hepatitis and herpesvirus infections. Quantitative
to differentiate symptomatic infection from asymptomatic and dynamic analyses of hepatitis virus markers are important
shedding. Preventive strategies increasingly use the CMV load in the follow-up of antiviral treatments (32). HBV surface
as a surrogate marker for disease and initiate antiviral treat- (HBsAg) and HBV core (HbcAg) antigens in peripheral blood
ment based on the systemic viral load (31). Sensitive tech- mononuclear cells (PBMCs) from HBV patients have been
niques, such as the pp65 antigenemia assay or the quantitative detected by FCM using antibodies (52, 278). In one study, 35
PCR assay, allow the detection and quantification of systemic patients with HBV chronic active hepatitis and 38 out of 60
CMV. Both assays provide a good estimation of the systemic patients with acute hepatitis B (63%) expressed HbsAg in
CMV burden. Owing to the high sensitivity of these assays, PBMC. In another work, Chemin et al. demonstrated the se-
CMV is also detectable in patients with asymptomatic infec- lective detection of HBsAg and HBcAg on B lymphocytes and
tions. However, patients with disease often have a higher viral natural killer cells from chronically HBV-infected patients
load and can therefore be discriminated (31, 314). (52). Hepatitis C virus (HCV), woodchuck hepatitis virus, and
The pp65 antigenemia assay determines the systemic CMV varicella-zoster virus were also detected by FCM in PBMCs
load and consists of direct staining of polymorphonuclear leu- using monoclonal antibodies to HCV core antigen (34), poly-
kocytes with monoclonal antibodies against the lower matrix clonal antibodies to woodchuck hepatitis virus (51), and anti-
protein pp65 (31, 112, 314). This determination has classically bodies to the gpI glycoprotein from varicella-zoster virus (296),
been made by very difficult and time-consuming microscopic respectively. Thus, FCM detection of viral antigens offers a
observation of immunostained cells (112). Recent studies have potentially useful automated assay for the clinical diagnosis of
evaluated FCM for the direct detection and quantification of multiple blood-borne viruses.
CMV antigens in polymorphonuclear leukocytes from trans- HSV antigens have also been detected in HSV-1, HSV-2,
plant recipients (76, 132, 141). Measurement of pp65 CMV and human herpesvirus 8 (HHV-8)-infected cells by FCM
antigenemia by FCM overcame these problems owing to its (117, 213, 300, 347). After overnight amplification of clinical
speed and automation and showed it to be a specific and samples suspected to contain HSV, FCM detected virus 1 to 3
reproducible method, especially when the paraformaldehyde- days before cytopathic effects were detected in cell culture
methanol permeabilization-fixation method and antibody 1C3 (213). Sensitive FCM assays have also been recently developed
(to late antigens) were used (141). Good agreement was found to quantitate rotavirus in clinical and environmental samples
between the degree of DNA load and the level of antigenemia (2, 19).
detected by FCM in renal transplant recipients. Although the Finally, FCM has also been extremely useful in the study of
10. 176 ´
ALVAREZ-BARRIENTOS ET AL. CLIN. MICROBIOL. REV.
HIV infection (174, 211, 213). By studying HIV-infected cell while only 0.9% were found when CMV antigens were ana-
lines by FCM in 1987, Cory et al. (61) determined the percent- lyzed by immunoenzymatic labeling (184). Identification of
age of infected cells and the relative amount of p24 antigen per specific CMV-DNA or RNA by this method, with the possi-
cell. These and other authors used the same assay to detect and bility of phenotyping cells by FCM, should permit latency stud-
quantify HIV-infected cells in cell cultures by monitoring p24, ies in CMV infections through the identification of specific
p17, nef, gp120, gp41, and gp160/gp41 expression (61, 62, 130). cells actively replicating the virus and cells that harbor the virus
This method proved to be more sensitive and accurate for in a latent state, acting as a reservoir for infection. Further-
quantitative studies and faster than other methods of HIV-1 more, FCM permits these cells to be sorted for the character-
detection, such as the reverse transcriptase (RT) assay or de- ization of latency and reactivation mechanisms.
termination of syncytium formation. Two fluorescence in situ hybridization-FCM assays have also
Detection of HIV antigens on peripheral blood mononu- been developed for monitoring EBV-infected cells in blood
clear cells by FCM is a useful method for monitoring HIV (66). Crouch et al. were able to quantify EBV-infected cells in
replication in vivo by monitoring the number of circulating suspension for both the latent and replicative phases of the
CD4 cells positive for p24 (63, 131, 235), p24 and nef (213), virus, using in situ hybridization with two different fluores-
or p18 and p24 (107). In all these works, the percentage of cells cently labeled probes (specific for each phase of EBV replica-
expressing these antigens was statistically correlated with the tion) and FCM (66). This in situ hybridization-FCM assay
clinical status of the patient. Furthermore, the authors re- detected one positive cell out of 9,000, which is sufficient for a
ported an inverse correlation of HIV antigen-positive mono- diagnosis of EBV-infected cells in transplant recipients with
nuclear cells and the number of CD4 cells. Therefore, these lymphoproliferative disease.
assays are useful for rapidly monitoring disease progression in As an alternative to conventional PCR radioactive methods,
HIV-seropositive individuals and for monitoring the effect of nonradioisotope FCM detection of viral PCR products was
antiviral therapy. Some studies found a lack of correlation developed (342–344). Following virus-specific PCR amplifica-
between cell-associated antigen detection by FCM and the tion incorporating digoxigenin-labeled dUTP, labeled ampli-
detection of HIV antigens in sera from HIV-seropositive indi- cons are hybridized with biotinylated probes, the hybrid DNA
viduals by the standard antigen capture enzyme-linked immu- is captured using streptavidin-coated beads, and FCM analysis
nosorbent assay (ELISA). As explained by McSharry et al. of the binding of FITC-labeled anti-digoxigenin antibodies is
(213), the lack of correlation is due to a masked antigenemia in performed (342–344). This PCR immunoreactive bead (PCR-
the presence of immunocomplexes, which could underestimate IRB) assay has been used for the detection and quantification
the amount of antigen present in peripheral blood detected by of HIV-1 (342, 343) and HBV (344) viral genomes. As few as
ELISA. However, in spite of being a good assay for evaluating two or three copies of HIV-1 proviral DNA sequences were
disease progression, FCM detection of HIV antigens is not rapidly detected in PBMC from HIV-1-infected blood donors,
sensitive enough to detect the low levels of HIV-infected pe- a sensitivity comparable to that of the conventional radioactive
ripheral blood cells in asymtomatic HIV-1-seropositive indi- detection of PCR products (342, 343). The PCR-IRB assay is
viduals (213). This lack of sensitivity can be overcome, as a very simple, specific, sensitive, and automatic assay for the
discussed below, by coupling in situ PCR and FCM for the detection of specific HIV-1 amplicons. Yang et al. (343), test-
detection of small numbers of peripheral blood cells infected ing a panel of 20 pedigreed PBMC specimens, demonstrated a
with low levels of HIV in asymptomatic individuals. perfect correlation with the results from conventional radioac-
(ii) Detection and quantification of viral nucleic acids. The tive assays. By this method, Dorenbaum et al. (84) detected
emergence of PCR and rPCR RT-PCR techniques has allowed about three copies of proviral HIV DNA using primers for the
the highly sensitive detection of specific viral nucleic acids long terminal repeat sequences. In a double-blind study of
(DNA or RNA) in virus-infected cells. These methods are blood samples from 14 mother-infant pairs using the PCR-IRB
indeed the most sensitive for the detection and characteriza- assay, these authors obtained similar results to those found
tion of viral genomes, especially in the case of rare target viral with the commercial Amplicor HIV-1 PCR kit. On testing 20
sequences (123). However, the association between the viral specimens of blood donors, with or without markers of HBV
nucleic acid and an individual cell is lost, and therefore no infection, PCR-IRB detected HBV DNA in a 1,000-fold-
information about productively infected cell populations is ob- higher dilution than the infectious dose needed to produce
tained by this method. FCM analysis of fluorescent in situ infection in chimpanzees (344). The PCR-IRB assay proved to
hybridization in cell suspension overcomes this problem (33, be specific and more sensitive than the PCR analyses involving
173), since this assay can be coupled with simultaneous cell hybridization with radioactive probes for the detection of HBV
phenotyping (by using specific antibodies to different cell in blood. Importantly, the FCM assay avoids the use of radio-
markers). isotopes.
FCM detection of in situ hybridization has been used to FCM and RT-PCR have detected gene expression in indi-
analyze rare virus-producing cells in peripheral blood samples. vidual cells (111). Muratori et al. (225) developed an in situ
HIV-1 RNA in infected cell lines was detected by fixation of RT-PCR technique using fluorescein-labeled HCV specific
the cells in suspension, hybridization with HIV-1 genomic primers detected by FCM for the quantification and pheno-
probes labeled with digoxigenin–11-dUTP, detection with flu- typing of HCV-infected cells in clinical blood samples. Al-
orescent anti-digoxigenin antibody, and FCM analysis of the though HCV infects PBMC, the small proportion of circulating
fluorescence signals thus generated (33). Link et al. (184) de- infected cells is not easily detectable by conventional RT-PCR.
tected CMV antigen pp65 with immunoenzymatic labeling by These authors detected HCV in PBMC cells of 50% of patients
day 4, whereas CMV-DNA was detected by PCR coupled to with chronic hepatitis C tested; the proportion of HCV-in-
FCM detection of in situ hybridization 4 h postinfection on fected cells ranged from 0.2 to 8.1%.
T-lymphoblastoid cells (MOLT-4). This method also detected Recently, a very sensitive and powerful PCR-driven in situ
and quantified mononuclear peripheral blood leukocytes in a hybridization assay has been developed (115). This method
patient with active CMV infection (184). Of CMV-DNA-pos- combines the sensitivity of PCR with the specificity of in situ
itive mononuclear peripheral blood leukocytes from patients hybridization, allowing rapid and reproducible detection of
with active CMV infection, 15% were detected by this method, single-copy proviral DNA or low-abundance viral mRNA in
11. VOL. 13, 2000 FLOW CYTOMETRY IN CLINICAL MICROBIOLOGY 177
subsets of cells in suspension. This assay employs PCR- or conventional PCR, nucleic acid expression is not analyzed in-
RT-PCR-driven in situ amplification of viral sequences in fixed dependently in each cell). Multiparametric analysis of infected
cells in suspension with sequence-specific primers and digoxi- cells allows the detection of single-copy proviral DNA or low-
genin-linked dUTP. The product DNA is hybridized with a abundance viral mRNA (225, 244, 245) in specific subsets of
fluorescein-labeled oligonucleotide probe, and the cell suspen- cells that can be phenotyped at the same time. Moreover, FCM
sion is then analyzed by FCM (245). This method has been is a very useful tool to study the mechanisms of viral latency by
used for the detection of HIV-1 DNA and mRNA sequences in association of different stages of the virus cycle and disease
individual cells in both cell lines and cells from HIV-1-infected progression with the location of the virus in specific cell pop-
patients (243–245). The sensitivity and specificity of this tech- ulations. This can be achieved by using probes to specific
nique revealed a linear relationship for the detection of a mRNAs related to different viral replication stages (66).
single copy of intracellular proviral DNA over a wide range of Knowledge of cell populations in which the virus is either
HIV-1-infected cell concentrations (245). Re et al. (274) ana- replicating or in a latent state has important implications for
lyzed the presence of HIV-1 proviral DNA in PBMC from our understanding of virus replication in vivo and progression
HIV-infected patients at different stages of the disease by a to disease and hence for therapeutic treatments. Furthermore,
PCR-in situ hybridization FCM assay and correlated the data it should be possible to sort these cells for further analysis.
with p24 antigenemia and virus isolation. p24 antigenemia Double staining of viral nucleic acids together with viral pro-
correlated with the number of CD4-positive cells but was de- teins or surface markers is also possible (33, 66). In comparison
tected in only a very low percentage of patients with a cell with the detection of the PCR-driven in situ hybridization by
count greater than 200 CD4 T cells per liter. As stated above, fluorescence microscopy (92), in which a large number of mi-
detection of HIV antigens is not sensitive enough to detect the croscopic fields must be studied, FCM allows the analysis of
low levels of HIV-infected peripheral blood cells in asym- thousands of cells in a few seconds. The speed and automation
tomatic HIV-seropositive individuals. The virus was isolated in of these assays make them optimal for the rapid diagnosis of
most patients with a T-cell count below 500 per liter but only viral infections. Since these assays can also determine the rel-
in 4 of 14 patients with a cell count higher than 500 CD4 T ative number of cells bearing viral genomes and the viral load,
cells per liter. In contrast, the PCR-in situ hybridization FCM they could be used to evaluate and monitor antiviral treat-
assay revealed detectable levels of proviral DNA in all the ments. Amplification of nucleic acid sequences of viruses from
HIV-1-positive subjects studied, even those with a cell count cerebrospinal fluid, blood, or tissues, which are difficult to
higher than 500 CD4 T cells per liter. These data underscore isolate by conventional diagnostic techniques, together with
the potential of this assay for detecting small numbers of the detection of such nucleic acids by FCM open new possi-
PBMC infected with low levels of HIV in asymptomatic indi- bilities in the diagnosis of viral infections and the character-
viduals. ization of viral pathogenesis.
In HIV-1-infected patients, plasma viral RNA levels (viral
load) correlate with disease progression (105, 216, 327). Ac- Serological Diagnosis
cordingly, evaluation of this marker by RT-PCR is extensively
used to monitor the kinetics of HIV infection and the effects of Bacteria. The identification of pathogens by microsphere
antiretroviral treatments (12). However, the RT-PCR assay immunoassays using FCM offers the specificity provided by
does not characterize the cell populations contributing to the antibodies coupled with the speed and multiparametric analy-
plasma viral load. The PCR-driven in situ hybridization sis provided by FCM. Although these assays can be used to
method coupled to the FCM assay described above allows directly detect microbes, they are more useful for detecting
simultaneous phenotyping of infected cells and hence quanti- antibodies against microbes in sera obtained from patients.
fication of HIV-1 proviral DNA or RNA molecules in specific Generally, either a bacterial antigen preparation or the whole
cell populations. Patterson et al. (244) identified and quanti- organism is attached to polystyrene microspheres with a uni-
fied cell subsets in the peripheral blood of HIV-1 patients form diameter. The antigen-coated microspheres are incu-
expressing HIV-1 RNA by using PCR-driven in situ hybridiza- bated with the sera, the putative human antibodies recognize
tion coupled to FCM. They found a good correlation between the antigen, and, in a second step, a fluorescence-conjugated
the FCM-determined percentage of HIV RNA-positive cells antibody against human Igs is used for detection. FCM allows
and the expected percentage of HIV RNA-positive cells on the this assay to be completed in a short time with excellent sen-
basis of plasma viral load, with sensitivities of less than 30 sitivity and reliability. Using this approach, Best et al. (27)
copies of RNA per cell and a detection limit of 3.5% HIV-1 detected the presence of antibodies against Helicobacter pylori
RNA-positive cells within a heterogeneous population. Simul- in sera from 55 patients. These authors demonstrated that this
taneous immunophenotyping by FCM showed that a signifi- method was as sensitive and reliable as ELISA but faster and
cantly higher fraction of patients with a high plasma viral load cheaper. The simplicity of the technique and the stability of the
(more than 20,000 copies/ml) harbored HIV RNA-positive coated microspheres make the FCM immunofluorescence as-
monocytes than did those with a low plasma viral load. Fur- say highly practical for serodiagnosis.
thermore, the PCR-driven in situ hybridization and FCM assay The characteristics of FCM allow the detection of more than
permits the determination of the presence in HIV-1-infected one antigen at the same time. Thus, simultaneous detection of
patients of both latent and transcriptionally active viral infec- multiple antibodies using different-sized particles coated with
tion by detection of proviral DNA or viral mRNA. Patterson et different antigens or microbes would make it possible to detect
al. (245), testing nine HIV-1-infected patients, observed a sig- multi-infection diseases. Furthermore, using several fluoro-
nificant proportion of PBMCs infected with HIV-1, with most chrome-conjugated antibodies against human Igs and differ-
of the cells having viruses in a latent state (the percentage of ently sized microspheres, FCM can gather information from
PBMCs with proviral DNA varied from 4 to 15% whereas the each different-sized microsphere and particular fluorescence
percentage of PBMCs with tat mRNA varied from 1 to 8%). signals. Using Brucella abortus-coated microspheres and Staph-
The above FCM nucleic acid detection techniques have sim- ylococcus aureus fixed cells, dual antibody detection in sera and
ilar sensitivity to conventional PCR, but with the added bene- milk from cows has been reported by Iannelli et al. (138). In
fits derived from expression analysis in individual cells (in this assay, antibodies against the two bacteria are identified on
12. 178 ´
ALVAREZ-BARRIENTOS ET AL. CLIN. MICROBIOL. REV.
the basis of the altered size of B. abortus cells due to the when a large number of tests is used) compared with routine
microspheres. immunofluorescence assays by fluorescence microscopy and
Another use of the microsphere fluoroimmunoassay by further stressed that it could be fully automated.
FCM is to detect bacterial toxins. In cases where the suspicion Viruses. Some methods have been routinely used to detect
of Clostridium difficile infection is high, it is necessary to con- specific antibodies to viral antigens. Among these techniques
firm the presence of toxin A in patient samples. Renner (275) are ELISA, complement fixation, indirect immunofluores-
reported a microsphere fluoroimmunoassay for C. difficile cence microscopy, and Western blotting. In addition, the de-
toxin A using microspheres of two different sizes. The largest tection and quantification of antibodies to viral antigens can be
one was coated with polyclonal antibody against toxin A, and carried out by FCM. This technique has been used to detect
the smaller one, which was fluorescent, was coated with mono- and quantify antibodies to CMV (209), HSV-1 and HSV-2 (46,
clonal antibody against toxin A. In the first step, large micro- 209), HCV (187, 210, 279), and HIV-1 (100, 118, 133, 290,
spheres were added to the stool samples, and after incubation, 294).
smaller fluorescent microspheres were added. FCM measure- Most of these viral antibody quantifications use a micro-
ment allowed the separation and washing steps to be omitted sphere-based immunoassay and FCM. In this assay, polysty-
by gating the light scattered by the larger microspheres and rene microspheres attached to viral antigens are used as a
measuring only the associated fluorescence from the smaller support for viral antibody detection by FCM. For the simulta-
particles. Renner compared this method with the cytotoxin neous detection of two or more viruses, different-sized micro-
assay and culture of the organism from patients with C. diffi- spheres, each coated with a specific viral antigen, are used. The
cile-associated gastrointestinal disease. The results showed that assay has the advantage of simultaneous detection of multiple
the fluoroimmunoassay was less sensitive than the cytotoxin antibodies with high analytic sensitivity. Simultaneous detec-
assay and culture but had the same specificity, with the advan- tion and quantification of antibodies to CMV and HSV was
tage of being rapid, and, as the author stated “in laboratories achieved by McHugh et al. (209) using this method. Using
with a flow cytometer, this offers an alternative method for the particles of different sizes coated with p31, gp120, p24, and
laboratory diagnosis of C. difficile-associated gastrointestinal gp41 antigens from HIV-1, Scillian et al. (290) were able to
disease.” Tapp and Stotzky (313), using a similar approach to detect and quantify the specific antibodies. An FCM immuno-
detect and track the fate of toxins from Bacillus thuringiensis, fluorescence assay (FIFA) with high sensitivity and specificity
concluded that FCM is more sensitive and rapid than dot blot was developed by Sligh et al. (294) to detect antibodies to
ELISA and that it is possible to process many samples easily. HIV-1 by using HIV-1-infected cell lines. The cells are incu-
The detection of antibodies with borreliacidal activity in sera bated with the sera to be tested, and incubation with an FITC-
from patients with Lyme disease can help in both early and late conjugated anti-human Ig and FCM allows the quantification
serodiagnosis. Using FCM, it is easy to detect the loss of of HIV-1 antibodies in the sera. Based on this assay, Folghera
viability of Borrelia burgdorferi incubated with sera from pa- et al. (100) developed a FIFA for the quantitative determina-
tients with Lyme disease, using fluorochromes that detect the tion of HIV p24 in HIV-1-infected cells and used the reduction
damage caused by antibodies. Callister et al. (43) used acridine in HIV-1 p24 antigen expression in these cells to determine the
orange to demonstrate this effect. neutralizing-antibody titers in human sera (100). This method
The above work demonstrates the potential of FCM as a also allowed the rapid detection and monitoring of antibodies
routine technique in clinical microbiology laboratories for de- to native and recombinant human HIV-1 envelope glycopro-
tecting the presence of antibodies against microbes in patient teins following gp160 immunization (118). A new serological
sera and for reliably checking the presence of toxins in clinical assay, the recombinant FIFA, was later described (133) for the
samples. early detection of HIV-1 antibodies. In this assay, antibodies in
Fungi. The use of FCM and the antibodies present in patient sera are evaluated by FCM for binding to the HIV-1 recom-
sera to detect fungal pathogens was first described by Libertin binant insoluble forms of proteins Gag-p45, Gag-gp41, and
et al. (183) in 1984. Pneumocystis carinii cysts in lung homog- gp160 expressed in insect cells by a baculovirus expression
enates from biopsy specimens were detected by these authors system. The sensitivity of this method permits earlier detection
using sera from patients and experimentally infected rats. of antibodies after initial infection than for enzyme immuno-
Bergbrant (26), using FCM to monitor antibodies against Pity- assays, with a reduction in the “window” period, i.e., the time
rosporum ovale in sera from patients with seborrheic dermati- between initial infection and the time of seroconversion, a
tis, demonstrated that there was no relationship between this parameter which is critical in infection from blood transfusions
microorganism and the illness. (133).
Although the tools to directly detect antibodies against fungi The humoral immune response to HCV has been evaluated
in patient sera do exist, no work validating the FCM procedure in patients with chronic hepatitis (187). Antibodies to HCV
has yet been published. core and NS3 antigens have been quantified using immunoas-
Parasites. The presence in patient sera of antibodies to any say beads and FCM (210) in blood donors. The microsphere
particular parasite can permit an FCM-based diagnosis of par- assay resulted in increased sensitivity (fivefold higher than that
asitism. Martins-Filho et al. (199), using FCM on serum from of reference methods) of HCV detection and resolved a sig-
patients chronically infected with Trypanosoma cruzi, devel- nificant proportion of indeterminate samples. A fast FCM as-
oped a sensitive method for the immunodetection of anti- say that measures the neutralization of the binding of recom-
trypomastigote membrane-bound antibodies. They were also binant HCV E2 envelope protein by antibodies to human cells
able to monitor the treatment in order to establish its effec- has also been described (279). This method permits study of
tiveness. A similar assay was developed by Cozon et al. (64) the natural immunity to HCV and should be useful in the
with Toxoplasma gondii, using fixed tachyzoites and specific development and validation of vaccination protocols.
conjugates for different human Ig heavy chains. They were able To conclude, the investigations of Best et al. (27) and Ian-
to quantify the amounts of IgM, IgG, and IgA antibodies in nelli et al. (138), among others, offer the possibility of perform-
patient sera by measuring the amount of fluorescence bound to ing FCM serodiagnosis in an elegant, rapid, cheap, and precise
tachyzoites. The authors stated that the method might offer a manner. The technique is simple and can be used for many
major improvement in cost-effectiveness per sample (especially pathogens (including viruses), with an additional possible ad-
13. VOL. 13, 2000 FLOW CYTOMETRY IN CLINICAL MICROBIOLOGY 179
vantage of detecting more than one microorganism in a single concentration effects (106), which offer pharmacodynamic data
sample. The use of FCM in clinical microbiology laboratories based on the antimicrobial activity, are rarely determined in
would allow detection times and costs to be reduced. At clinical laboratories since these determinations (190) are te-
present, however, it is not in general use and the setting up of dious and time-consuming.
such protocols can be fairly time-consuming. FCM has proved to be very useful for studying the physio-
logical effects of antimicrobial agents on bacterial cells due to
ANTIMICROBIAL EFFECTS AND SUSCEPTIBILITY their effect on certain metabolic parameters (membrane po-
TESTING BY FLOW CYTOMETRY tential, cell size, and amount of DNA). In addition, FCM is a
reliable approach for susceptibility testing, offering results in
The first experiments in which FCM was used to study the terms of the bactericidal or bacteriostatic effect (86, 108, 198,
effects of antimicrobial agents in prokaryotes were carried out 262, 331).
at the beginning of the 1980s (136, 302, 304, 306). In the 1990s, The studies by Martinez et al. (198) and Steen et al. (302) in
there were interesting advances in this field from microbiology 1982 on the antimicrobial effects on bacteria assayed by FCM
laboratories, and the number of scientific articles addressing are now considered classic. They demonstrated that the effect
the antimicrobial responses of bacteria (including mycobacte- of -lactam antibiotics on E. coli can be detected by measure-
ria), fungi, and parasites to antimicrobial agents increased con- ments of light scattering and DNA content after 10 min of
siderably. The development of FCM in combination with fluo- incubation with the drug. Using FCM, Steen et al. (302)
rochromes permits the assessment of individual viability and showed the effect of several antibiotics that inhibit protein
functional capacity (membrane potential and metabolic path- synthesis (chloramphenicol, erythromycin, doxycycline, and
ways) within microbial populations. This allows investigators to streptomycin) on E. coli with the fluorescent DNA probe mith-
explore the possibility of performing susceptibility testing ramycin (305). Since then, FCM has been used to measure the
within the applications of FCM. Also, the introduction of this effects of different antimicrobial agents on bacteria (Table 2).
technique in clinical laboratories for routine susceptibility test- Realizing the potential of FCM in this field, several compa-
ing has been proposed, since this approach can be performed nies have developed new products to rapidly perform antibiotic
reliably in just a few hours. Several examples of the study of the susceptibility testing in clinical microbiology laboratories. We
antimicrobial effect and susceptibility testing by FCM are have used one of these products (Bac-light; Molecular Probes)
shown in Table 2. to analyze the reliability of this test in clinical microbiology.
Available data clearly demonstrate the utility of this tech- We worked with two strains of Enterococcus, one sensitive to
nique to also assay viral susceptibility. Since the pioneer works vancomycin (E. faecalis ATCC 29212) and other resistant (E.
of Rosenthal et al. (280) and Pauwels et al. (247) on the use of faecium U2A1), with a vanA element responsible for its van-
FCM to study the effect of antiviral agents on herpesvirus and comycin resistance phenotype. In addition, as a control we
HIV, FCM has been used by several groups, particularly those included an E. coli isolate showing intrinsic resistance to van-
studying these viruses and CMV. The detection and quantifi- comycin. The above-mentioned products are kits with two fluo-
cation of viral antigens and viral nucleic acids in combination rochromes that permit the detection of live and dead cells.
with antibodies or nucleic acid probes, respectively, or any Bacterial cells were incubated with and without vancomycin for
other cellular parameter associated with viral infection have 2 h at the respective MICs of this drug (Fig. 5). Antimicrobial
been used in FCM protocols for the in vitro evaluation of effects were detected by measuring the variations in fluores-
antiviral-drug activities. In addition, the possibility of on-line cence due to dead cells within 3 h of incubation with the
monitoring of the antiviral treatments ex vivo has also been antibiotic. Another advantage of FCM (Fig. 5), as mentioned
explored. The latter approach has also been applied to bacte- above, is the visualization of the heterogeneity of the response
rial and fungal infections; rapid and sensitive protocols for the of the cells to the antimicrobial agent. This heterogeneity is
evaluation of microbial responses to antimicrobial agents are detected by determining the presence of subpopulations that
also available. are less susceptible to the antimicrobial agent treatment. Da-
In the following sections, the possible uses of FMC in anti- vies and Hixson have recently reported similar results (69).
microbial susceptibility testing are discussed according to the Therefore, it is feasible to use FCM and fluorescence probes to
type of pathogen involved. perform rapid testing of the susceptibility of bacteria to a panel
of antimicrobial agents before choosing the treatment. More-
Antibacterial Agents over, resistant subpopulations can be effectively detected, rep-
resenting an interesting advantage of FCM that can be suitably
Standardized methods for performing in vitro susceptibility exploited in clinical studies (331).
testing of bacteria in clinical microbiology laboratories are Measurement of bacterial susceptibility. By using fluores-
widespread (98). Qualitative and/or quantitative results are cence probes, FCM measures the light scattering of cells in
given on the basis of the size of the zone of inhibition or MIC. suspension and fluorescence of cellular contents or metabolic
Despite the automation of MIC-based broth microdilution sys- activity. Thus, the activity of antimicrobial agents can also be
tems, an 18- to 24-h incubation period is usually needed before described in terms of changes in cell shape and in fluorescence
antimicrobial activity can be quantified. Recently, fluorogenic, (262, 329, 330). In this sense, Walberg et al. (329, 330), and
turbidometric, and colorimetric technology has reduced sus- others (108, 200) showed that light scattering is a useful pa-
ceptibility testing to 4 to 6 h (83, 98). However, unless the rameter of antimicrobial effects, irrespective of their mecha-
bacterial inoculum is quantified, only the bacteriostatic effect, nisms of action. This was demonstrated with two different
i.e., the MIC, is generally tested by clinical laboratories. The drugs: ceftazidime, which acts on the bacterial envelope, and
MBC, which reflects the bactericidal effect of antimicrobial ciprofloxacin, whose target is a gyrase. Nevertheless, the same
drugs, is rarely determined. This requires calculation of bacte- authors (329, 330) used both fluorescence detection and light-
rial counts, generally expressed as CFU, which involves bacte- scattering analysis when susceptibility testing was performed
rial culture dilutions and subcultures. In addition, neither MIC directly in clinical samples in order to distinguish bacterial cells
nor MBC determinations consider the heterogeneity of bacte- from debris and other components.
rial populations. Similarly, postantibiotic and subinhibitory Two major categories of fluorochromes have been used to