clin chem flow cytometry
TRANSCRIPT
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Flow CytometryFlow Cytometry
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Flow CytometryFlow Cytometry
a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus.
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ComponentsComponents
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Pinch valvePinch valve
Controls whether or not the sample is being introduced
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Flow ChamberFlow Chamber
Where cells are passed through
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Light SourceLight Source
Can be a laser, an arc lamp or even an LED
Lasers are the most widely used
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Collecting LensCollecting Lens
Produces light focused at infinity so that the photomultipliers (PMTs) can be placed at any distance from the flow chamber.
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Dichroic FiltersDichroic Filters
Selects light of a particular wavelength.
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Barrier FilterBarrier Filter
Blocks any reflected excitation light.
Further filters the light.
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Photomultiplier Tubes (PMTs)Photomultiplier Tubes (PMTs)
For measuring fluorescence and refracted light
Converts light energy to electrical energy
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PrinciplePrinciple
A beam of light of a single wavelength is directed onto a hydrodynamically-focused stream of fluid.
After the sample is introduced, it passes through a sheath fluid so that the cells align singly.
As each cell passes through the laser, a characteristic pattern of light is produced.
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PrinciplePrinciple
The sheath fluid is driven through the flow chamber by air pressure supplied by a compressor.
A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam(FSC, Forward Scatter) and several perpendicular to it (SSC, Side Scatter) and one or more fluorescent detectors.
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Each suspended particle from 0.2 to 150 micrometers passing through the beam scatters the ray.
Refracted light passes through the Collecting lens.
PrinciplePrinciple
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Light is filtered in the Dichroic Filters
Light is further filtered by the Barrier Filters
Light reaches the PMTs
PrinciplePrinciple
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Information about the physical and chemical structure of each individual particle are then derived.
*FSC correlates with the cell volume and SSC depends on the inner complexity of the particle (i.e., shape of the nucleus, the amount and type of cytoplasm granules or the membrane roughness).
PrinciplePrinciple
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Fluorescence-activated Sorting Fluorescence-activated Sorting (FACS)(FACS)
provides a method for sorting a heterogeneous mixture of biological cells into two or more containers.
based upon the specific light scattering and fluorescent characteristics of each cell
provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest
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PrinciplePrinciple
The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid.
A vibrating mechanism causes the stream of cells to break into individual droplets.
The flow passes through a light measuring station where the character of interest of each cell is measured.
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An electrical charging ring is placed just at the point where the stream breaks into droplets.
Particles passing at this point become charged.
Particles of different charges separate to opposite deflector plates.
Particles are diverted into containers based on their charges.
PrinciplePrinciple
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AdvantagesAdvantages
Its fluidic system delivers cells to the measuring point one at a time so data are collected for individual cells
It is rapid (100-500 cells/s)
Makes use of very sensitive sensors so measurements are more quantitative
Can be used on several different characteristics (protein content, DNA content, lipid content) of cell allowing you to define subpopulations and/or distinguish between different cell types
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ApplicationsApplicationsMeasurement of Apoptosis
Detection of unknown organisms
Detection of spores
Measurements on red blood cells (RBCs): Detection and quantitation of RBC-bound proteins, quantitation of RBC-bound immunoglobulins, detection and quantitation of RBC antigens and antibodies, detection and quantitation of minor RBC populations.ion
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ImpedanceImpedance
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Impedance
describes a measure of opposition to alternating current (AC).
based on the change in electrical resistance across an aperture when a particle in a conductive liquid passes through this aperture
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PrinciplePrinciple
There exists an electrical resistance across an aperture
Once a particle passes through this aperture, there is a change in the resistance
That change produces a voltage pulse
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PrinciplePrinciple
The pulse is proportional to the size of the particle
The number of pulses is used to determine the number of particles present
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ComponentsComponents
Aperture Tube
Electrodes
Counting Unit
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FactorsFactors
Multiple particles passing through the aperture
Particles that act as insulators
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ApplicationsApplications
Used to determine cell size
Used to determine number of cells present
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ElectrochemistryElectrochemistry
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ElectrochemistryElectrochemistry
It involves measurement of voltage or current generated by activity of specific ions.
It includes Potentiometry, Amperometry and Coulometry.
It deals with the chemical change produced by electric current and with production of electricity by chemical reactions
It provides an accurate measurement of the amount of electrical energy consumed or produced
Involves Redox Reactions
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potentials are produced between the interface of the metal and its ions
the potential between 2 electrodes in a solution is measured, and is proportional to the concentration of the solute
PotentiometryPotentiometry
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PrinciplePrinciple
a species undergoes redox reaction
REFERENCE ELECTRODE INDICATOR ELECTRODE POTENTIAL DIFFERENCE
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A Reference Electrode is used to introduce a potential
An Indicator Electrode is used to measure the potential
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ElectrodesElectrodes
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1. Ion-Selective Electrode1. Ion-Selective Electrode
an electrochemical transducer
only responds to a specific ion
converts the activity of a specific ion into an electrical potential measured by a voltmeter or pH meter
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Ion-Selective Ion-Selective Electrode: TypesElectrode: Types
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Glass MembranesGlass Membranes
Made of either Silicate or Chalcogenide
Silicate: single-charged cations (H+, Na+, Ag+)
Chalcogenide: double-charged metal ions (Pb2+, Cd2+)
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Crystalline membranes
made from mono- or polycrystallites of a single substance.
Selectivity of crystalline membranes can be for both cation and anion of the membrane-forming substance.
An example is the fluoride selective electrode based on LaF3 crystals.
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Liquid Membrane ElectrodeLiquid Membrane Electrode
membrane is an organic polymer saturated with a liquid ion exchanger
selectivity is to Ca2+
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Gas ElectrodeGas Electrode
allows small gas molecules to pass and dissolve into internal solution
O2, NH3/NH4+, and
CO2/HCO3-
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Enzyme ElectrodeEnzyme Electrode
Immobilized enzyme binds to gas permeable membrane
Catalytic enzyme reaction produces small gaseous molecule (H+, NH3, CO2)
Gas sensing probe measures change in gas concentration in internal solution
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2. pH Electrode2. pH Electrode
measures Hydrogen Ion Activity
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pH ElectrodepH Electrode
small bulb containing a Chloride Buffer solution
Internal Electrode usually Ag/ AgCl
External Electrode--Saturated Calomel Electrode
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pH ElectrodepH Electrode
The glass bulb on the bottom, is coated both inside and out with a ~10 nm layer of a hydrated gel.
The two layers are separated by a layer of dry glass.
The silica glass structure (its atomic structure) is shaped in such a way that it allows Na+ ions some mobility.
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pH ElectrodepH Electrode
The metal cations (Na+) in the hydrated gel diffuse out of the glass and into solution while H+ from solution can diffuse into the hydrated gel.
The change in free energy is measured by the electrodes.
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3. pC02 Electrode3. pC02 Electrode
a pH electrode contained within a plastic jacket filled with sodium bicarbonate buffer solution
has a gas-permeable membrane
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pC02 ElectrodepC02 Electrode
C02 diffuses into the buffer
it reacts with water to form Bicarbonate
Change in pH
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AmperometryAmperometry
measurement of the current flow produced by a redox reaction
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CoulometryCoulometry
measures the quantity of electricity needed to convert an analyte to a different oxidation state
used to measure Chloride ion serum, plasma, CSF and sweat samples
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CoulometryCoulometry
a constant current is applied across 2 silver electrodes
Silver ions are liberated into the specimen at a constant rate
Chlorides combine with the Silver ions to form AgCl
Electrodes sense excess Silver and stop titration
Number of Ions released by Ionization = No. of Cl
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SILVER ELECTRODES
Cl-
Cl- Cl-
Cl- Cl-
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P02 Gas-Sensing ElectrodeP02 Gas-Sensing Electrode
uses a gas-permeable membrane, usually Polypropylene
Oxygen passes through the membrane
Oxygen reacts with the platinum cathode, and reduced to Water
Change in current in the cell
Change in current is directly proportional to P02
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ComponentsComponents
1. A bulb made from a specific glass 2. Internal electrode, usually silver chloride electrode or calomel electrode3. Internal solution (buffered solution)4. AgCl precipitate5. Reference electrode 6. Reference internal solution7. Junction8. Body of electrode, made from non-conductive glass or plastics.
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FactorsFactors
TEMPERATURE EFFECTS
LIGHT
CONTAMINATION EFFECTS
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ApplicationsApplications
Used in the measurement of blood gases and pH
Used in measuring serum and urine electrolytes (Ion-selective electrode)
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AdvantagesAdvantagesCan be used for Multiple Analyte Detection
Allows the determination of the concentrations multiple species that may be present in the analyte.
Can be performed even if the sample has many particulates or is deeply colored
No additive is required
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DisadvantagesDisadvantages
Costly
The time for instrumental equilibration result in slower measurements than visual indicators, which respond immediately
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EndEnd