TRENDS IN LABORATORY TESTING

Slides:



Advertisements
Similar presentations
Research Techniques Made Simple: Flow Cytometry Richard R
Advertisements

Absorption of photon elevates chromophore to excited state.
Cell Culture Facilities and Equipment Presenter: Dr. R. Rahbarghazi.
1 2 3 * Flow = cells in motion * Cyto = cell * Metry = measure * Measuring properties of cells while in a fluid stream * Flow Sorting * Sorting (separating)
What is Flow Cytometry? Introduction to Flow Cytometry
Issues in Multicolor Flow Cytometry: Beyond 6 Colors
Fluorescence-Activated Cell Sorting/Flow Cytometry Shared Resource Laboratory FACS/FCMSRL Core B Smooth Muscle Plasticity COBRE Director: Doug Redelman,
Quality Control in Immunophenotyping Dr. N. Varma Prof. & Head – Hematology Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh.
Welcome IQAC at DHVI CD4 Immunophenotyping for HIV Monitoring Flow Cytometry.
Center of Excellence in Cancer Research Principles of flow cytometry
CHAPTER 37 MLAB 1415 HEMATOLOGY JOANNA ELLIS, MLS(ASCP) Optical Light Scatter and Flow Cytometry.
Introduction To Flow Cytometry:
Advancements in FACS analyzers optical design leads to greater functionality and a smaller footprint. INTRODUCTION In the past decade, instrumentation.
FLOW CYTOMETRY Dr. MOHAMMED H SAIEMA LDAHR KAAU FACULTY OF APPLIED MEDICAL SCIENCES MEDICAL TECHNOLOGY DEPT. 2 ND YEAR MT INSTROMINTATION EXT
FLOW CYTOMETRY Dr. MOHAMMED H SAIEMA LDAHR KAAU FACULTY OF APPLIED MEDICAL SCIENCES MEDICAL TECHNOLOGY DEPT. 2 ND YEAR MT INSTROMINTATION EXT
King Saud University Riyadh Saudi Arabia
Flow Cytometry. Allows For Detection Of Surface Markers Of Cells Allows For Detection Of Intracellular Factors Allows Detection Of Secreted Factors By.
Overview What is flow cytometry? Development of flow cytometry Components of Flow Typical applications Flow data.
Basics of Flow Cytometry Holden Maecker. Outline Definitions, what can be measured by flow cytometry Fluidics: Sheath and sample streams, flow cells,
Pharos university Faculty of Allied Medical SCIENCE Clinical Laboratory Instrumentation (MELI-201) Dr. Tarek El Sewedy.
Applications of flow cytometry in basic immunology Generation and characterization of DC Assays for T cell activation –Cell proliferation – Cell division.
Department of Biotechnology and Microbiology
Introduction to Flow Cytometry
Antigen antibody reactions
Practical molecular biology PD Dr. Alexei Gratchev Prof. Dr. Julia Kzhyshkowska Prof. Dr. Wolfgang Kaminski.
Cell viability studies Sepideh Khoshnevis. The Goal To distinguish live cells from dead and apoptotic cells in order to calculate the the percentage of.
Flow Cytometry at Boston University Medical Campus Introduction to some methods that we offer Yan Deng (X4-5225), Gerald Denis (X4-1371),
Introduction To Flow Cytometry By Noha Kamel. Flow cytometry is a method of measuring multiple physical and chemical characteristics of particles by optical.
Dr Gihan Gawish Hydrodynamic focusing is a technique used to provide more accurate results from flow cytometers or Coulter counters for determining the.
Basic Principles in Flow Cytometry
 Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid.
Automation in haematology
Immunology Assays for Clinical Research Yan Ge, Ph.D. University of Virginia
Basic Principles, Instrumentation, and Practices
Flow Cytometry Principles & practice of “Fluorescence Spectroscopy in Biological Diagnosis & Research” Dr.Hekmatimoghaddam Assistant professor of pathology.
FLOW CYTOMETRY  Definition: Measuring properties of cell as they flow in a fluid suspension across an illuminated light path.
Flow Cytometry Basic Training. What Is Flow Cytometry? Flow ~ cells in motion Cyto ~ cell Metry ~ measure Measuring properties of cells while in a fluid.
Flow Cytometry (FCM) Yun-Ju Lee September 13, 2002.
1 Flow Cytometry in the Clinical Laboratory Patricia Aoun, M.D., M. P. H. Jean Bailey, MT-ASCP Kellie Neth, MT-ASCP The Nebraska Medical Center.
2015 היחידה להפרדת תאים ד"ר דבי איצקוביץ'
Flow Cytometry Becton Dickinson Asia Limited Company.
Quality Controls: Get your instruments under control!
Antigen-Antibody Interactions: Selected Tests (Contd.)
Medical Laboratory Instrumentation (Blood cell counter) Third Year Dr Fadhl Alakwa UST-Yemen Biomedical Department.
Flow Cytometry. Applications FRET- protein interaction Membrane protein expression Intracellular protein expression Cell viability Ca 2+
Human cytomegalovirus (HCMV) is a member of the herpesvirus family. About 70-90% of the population is infected with HCMV. In healthy.
Immunofluorescence Lab. 10.
Identification of Cancer Stem Cells using Flow Cytometry Analysis تعيين الخلايا الجذعية السرطانية باستخدام تحليل التدفق الخلوي Dr. Ayat Al-Ghafari Biochemistry.
Multi-parameter Flow Cytometry: available dyes and combined usage Martin R. Goodier Department of Immunology Imperial College London.
ICC Immunocytochemistry
1 SOFTWARE FOR VERIFICATION OF HOMOGENEITY RESULTS FROM AUTOMATED URINE ANALYZERS IN REAL TIME Waldemar Volanski Ademir Luiz do Prado Geraldo Picheth et.
Indirect methods of cell counting
Multiplex analysis of cytokines Russell Garland KWS BioTest
Medical Laboratory Instrumentation
Showdown at the FLOW-K Corral
Flow Cytometry Halima Moncrieffe, University College London, UK IL-17
Flowcytometry.
Flow Cytometry FACS (Fluorescence-Activated Cell Sorter)
FLOWCYTOMETRY.
Techniques for measuring minimal residual disease in leukemia
Lymphocytes.
COMPENSATION + FITC - PE - + PE - FITC - FITC detector FITC PE
Transplant: immunology and treatment of rejection
Laboratory Techniques in Immunology
IMMUNOPHENOTYPING LEUKEMIAS AND LYMPHOMAS
Optical Design Laser Sample Dichroic Filters Flow cell Bandpass
Flow Cytometry and Sorting Part 3
The Art of Flow Cytometry
Volume 36, Issue 1, Pages (January 2012)
Presentation transcript:

TRENDS IN LABORATORY TESTING FLOW CYTOMETRY Suzanne Kamel-Mohamed PhD, MBA, MT (ASCP) Associate Clinical Laboratories Consultants Sanford, Florida www.clinicallabconsultants.com

Flow Cytometry Flow cytometry is a technique used to measure the physical and chemical properties of cells or cellular components. Cells are measured individually, but in large numbers. Synonymous with FACS (fluorescence-activated cell sorter). Also, simply referred to as “Flow.”

History In the clinical lab, mixed cell populations of the blood were evaluated manually by microscope. In the 1950’s, the Coulter counter automated cell counting based on size. Eosinophil Neutrophil By the 1970’s, a method was needed to automatically separate living cells into subpopulations for further study. Lymphocyte Basophil Monocyte

First FACS In 1974, Dr. Leonard Herzenberg of Stanford patented a device that sorted living cells into collection vessels for further use in biological analyses – the first FACS.

Eosinophil Neutrophil Then… …and Now.

Flow Cytometer Instrumentation There are four general components of a flow cytometer: Fluidics Optics Detectors Electronics Understanding how a flow cytometer operates is critical to the design and execution of flow cytometry experiments.

Lasers and Fluorescence In modern flow cytometers, more than one laser is focused on the sample stream. In this way, not only can cells be measured based on their size and internal complexity, but they can also be measured based on their fluorescent signal intensity. Fluorescence is typically giving by cells through the use of fluorescent dyes called fluorochromes.

Differentiating Among Cell Types The introduction of fluorochromes into flow cytometry converted this method of cell detection into a powerful tool for the rapid differentiation of cells.

Fluorochrome Emission The laser beam excites the fluorochrome at a specific wavelength (absorption) and the fluorochrome emits light at a separate wavelength (emission). Note that absorption color differs from emission color.

Flow Cytometer Electronics The voltage pulse height, width, and area are determined by the particle’s size, speed, and fluorescence intensity. The pulse parameters are then acquired and analyzed in real-time by a computer.

Flow Cytometer Instrumentation Graphical Summary

Different Fluorescence-activated Cell Sorters BD FACSVantage BD FACSAria

Benchtop Flow Cytometers BD FACSCalibur BD LSR II

Fluorochrome-conjugated Antibodies Initially, fluorescent dyes commonly employed in microscopy were used to stain whole cells. However, dye uptake by cells was unreliable and led to problems with data reproducibility. Subsequently, antibodies were covalently bound to fluorochromes as a means of specifically and reliably labeling cells.

Polyclonal vs. Monoclonal Antibodies Polyclonal antibodies bind to multiple aspects of the same antigen. Their heterogeneity causes problems with standardization when used in flow cytometry. Homogeneous monoclonal antibodies bind to only one aspect of an antigen and will reproducibly label cells. polyclonal antibodies monoclonal antibodies

Cell-Surface Markers Monoclonal antibodies are used to recognize specific antigens on the surface of cells. These cell-surface markers characterize different cell types. Fluorochrome-tagged monoclonal antibodies brightly label cells for detection by the flow cytometer.

T Cell Subsets In immunology, the use of fluorochrome-tagged monoclonal antibodies resulted in the discovery of phenotypically diverse T cell subsets. This revolutionary observation made Regulatory T cell flow cytometry the preferred research tool of modern immunology. Dendritic cell Effector T cell

Immunophenotyping Many cell surface features (as well as some internal characteristics) can be simultaneously assessed by employing different combinations of fluorochromes. Several uniquely colored fluorochromes are available to conduct such multicolor (multiparameter) experiments.

Immunophenotyping However, many fluorochromes possess overlapping emission wavelengths.

Compensation When the wavelengths of two fluorochromes overlap, the observed fluorescent signal detected by the flow cytometer may not be the actual signal displayed by the cell. In other words, the cell appears to possess a surface marker or phenotype that it does not actually have.

Data Analysis Flow cytometry is utilized both in the clinical lab and the research lab. Standardization has resulted in data that is reproducible across laboratories. Accurate data representation is key to this reproducibility. This is a 2D dot plot; a commonly used method of data representation.

Data Analysis Flow cytometry computer software can generate data in the form of density plots and contour plots.

Gating gating is performed to isolate cell subpopulations of interest. This step often eliminates the need to physically sort cells for further analysis.

Applications - Clinical Bone marrow cells are evaluated based on SSC and CD45 expression to diagnose acute lymphoblastic leukemia. normal patient patient with ALL Jennings, C. & Foon, K.(1997). Blood, 90(8), 2863-2892.

Applications - Clinical CD4+ T cell counts are used to monitor the progression of AIDS in HIV-infected patients.

Applications - Research A kinetics assay, such as Ca2+ mobilization, can be performed using a fluorochrome, indo-1, that binds to calcium ions. Cells are loaded with indo-1 and then stimulated to mobilize Ca2+. The UV laser excites the indo-1 and a fluorescent pulse is observed over time.

Applications - Research Several fluorochromes (DAPI, propidium iodide, 7-AAD, etc.) bind directly to DNA and are used to estimate the amount of DNA present in a cell. The amount of DNA in a cell determines whether it has entered the cell cycle.

CLINICAL APPLICATIONS Immuno-phenotyping: T-Cell subset analysis AIDS & Auto-Immune diseases. Multi-color fluorescence CD45/CD8/CD4/CD3 in single tube 2. Lymphocyte Activation: Tissue typing and Organ matching for transplantation Calcium ion flux, pH change, membrane potential changes, appearance of activation antigens (CD69, CD98, CD25, CD71, HLA-DR) insulin receptors, RNA Synthesis, DNA Synthesis, Cell division 3. Leukemia phenotyping: Identification of CD34 and CD11b and CD15 ( AM)

CLINICAL APPLICATIONS CONTINUED… B. DNA content analysis C. Hematology: 1.Functional and viability Bone marrow transplantation (CD34, CD45/R/RO, CD33, HLA-DR 2. Reticulocyte enumeration Bone marrow transplantation and bone marrow recovery 3. Organ transplantation

CLINCAL APPLICATIONS CONTINUED… D. Sperm Analysis Clinical microbiology Microbial Identification Susceptibility F.Vaccines  

Summary Flow cytometers measure cells based on their size, internal complexity, and fluorescence. Qualitative and quantitative analyses of cell populations have clinical and research applications. Successful experimental design depends on an understanding of flow cytometer instrumentation and basic immunological principles. “Flow Cytometry will have a great impact on the future of clinical testing”