clin chem flow cytometry

Flow CytometryFlow Cytometry

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.

ComponentsComponents

Pinch valvePinch valve

Controls whether or not the sample is being introduced

Flow ChamberFlow Chamber

Where cells are passed through

Light SourceLight Source

Can be a laser, an arc lamp or even an LED

Lasers are the most widely used

Collecting LensCollecting Lens

Produces light focused at infinity so that the photomultipliers (PMTs) can be placed at any distance from the flow chamber.

Dichroic FiltersDichroic Filters

Selects light of a particular wavelength.

Barrier FilterBarrier Filter

Blocks any reflected excitation light.

Further filters the light.

Photomultiplier Tubes (PMTs)Photomultiplier Tubes (PMTs)

For measuring fluorescence and refracted light

Converts light energy to electrical energy

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.

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.

Each suspended particle from 0.2 to 150 micrometers passing through the beam scatters the ray.

Refracted light passes through the Collecting lens.

PrinciplePrinciple

Light is filtered in the Dichroic Filters

Light is further filtered by the Barrier Filters

Light reaches the PMTs

PrinciplePrinciple

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

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

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.

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

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

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

ImpedanceImpedance

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

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

PrinciplePrinciple

The pulse is proportional to the size of the particle

The number of pulses is used to determine the number of particles present


Aperture Tube

Electrodes

Counting Unit

FactorsFactors

Multiple particles passing through the aperture

Particles that act as insulators

ApplicationsApplications

Used to determine cell size

Used to determine number of cells present

ElectrochemistryElectrochemistry

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

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

PrinciplePrinciple

a species undergoes redox reaction

REFERENCE ELECTRODE INDICATOR ELECTRODE POTENTIAL DIFFERENCE

A Reference Electrode is used to introduce a potential

An Indicator Electrode is used to measure the potential

ElectrodesElectrodes

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

Ion-Selective Ion-Selective Electrode: TypesElectrode: Types

Glass MembranesGlass Membranes

Made of either Silicate or Chalcogenide

Silicate: single-charged cations (H+, Na+, Ag+)

Chalcogenide: double-charged metal ions (Pb2+, Cd2+)

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.

Liquid Membrane ElectrodeLiquid Membrane Electrode

membrane is an organic polymer saturated with a liquid ion exchanger

selectivity is to Ca2+

Gas ElectrodeGas Electrode

allows small gas molecules to pass and dissolve into internal solution

O2, NH3/NH4+, and

CO2/HCO3-

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

2. pH Electrode2. pH Electrode

measures Hydrogen Ion Activity

pH ElectrodepH Electrode

small bulb containing a Chloride Buffer solution

Internal Electrode usually Ag/ AgCl

External Electrode--Saturated Calomel 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.


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.

3. pC02 Electrode3. pC02 Electrode

a pH electrode contained within a plastic jacket filled with sodium bicarbonate buffer solution

has a gas-permeable membrane

pC02 ElectrodepC02 Electrode

C02 diffuses into the buffer

it reacts with water to form Bicarbonate

Change in pH

AmperometryAmperometry

measurement of the current flow produced by a redox reaction

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

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

SILVER ELECTRODES

Cl-

Cl- Cl-

Cl- Cl-

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


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.

FactorsFactors

TEMPERATURE EFFECTS

LIGHT

CONTAMINATION EFFECTS

ApplicationsApplications

Used in the measurement of blood gases and pH

Used in measuring serum and urine electrolytes (Ion-selective electrode)

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

DisadvantagesDisadvantages

Costly

The time for instrumental equilibration result in slower measurements than visual indicators, which respond immediately

EndEnd

clin chem flow cytometry

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