Week 3 Flashcards
what is reflectance photometry
- measure the light reflected not transmitted by a colored product on a reflective surface
-the light that is not absorbed by the colored product is reflected and measured
-Intensity of reflected light is inversely proportional to concentration of analyte (the higher the concentration the more light is absorbed)
what does reflectance photometry use
-specular reflectance where light is shone on an opaque surface and is reflected in parallel beams and measured with reflectometer
-white surface - zero concentration max reflectance
-black surface - zero reflection - highest concentration
as you have more reagents the color will be darker. Higher the concentration lower the transmittance
these are used to set the limits of the reflectometer
how is reflectance photometery graphed
-as negative slope
-reflectance and concentration are in an inverse relationship
-the higher the reflectance the lower the concentration
-non linear
-a math algorithm is needed to linearize and determine specimen concentration
what are the components of a reflectance photometer and what is it used for
light source: tungsten or quartz halogen lamp where light hits the sample at 90 degree angle
Wavelength selector: selects the wavelength at maximum absorbance
Photodetector: the reflected light is made parellel and measured at 45 degree angle
Dry slide tech: routine chem
Reagent stick test: urine dipstik
Glucose meters for home reading
What is light scatter and what factors affect the scattering of light
-when radiant energy collides with molecules in a solution and gets reflected or scattered
-some light is transmitted
-the scatter does not have the same intensity in all directions - most is forward scatter which is opposite of incident
factors affecting scattering of light
-particle size and weight
-particle concentration
-wavelength
what is radiation scattering photometry and what are the two types
-light passing through a suspension. Some light is absorbed and some is transmitted
Turbidimetry:
measures intensity of transmitted light
Nephelometry:
measures intensity of scattered light
measured at right angles to incident light beam
what is turbidimetry
-turbid solutions can decrease the intensity of the incident beam caused by scattering, reflectance and absorption?
-apparent increase in absorbance
-light blocked by the suspension will depend on particle size, number and shape
-timing of turbid reactions is critical
-temperature and pH must be maintained for precision
transmitted light measured as absorbance is directly proportional to analyte concentration
what is a turbidimeter used for and what are its parts?
used for:
-Micro Analyzer- bacteria causes turbidity, AST, G/NG
-Coagulation Analyzer: PT, APTT and factor assays. Fibrin strands from clots block transmittance of radiant energy
-Chemistry - proteins in urine and CSF
The turbidimeter is composed of:
-Light source: tungsten or some visible radiation
-Monochromator: providing light of non absorbable wavelength (otherwise it would measure both turbidity and absorption). a shorter wavelength has increased sensitivity while longer wavelengths help minimize absorption in coloured samples like hemolysis or bilirubin)
-Photodetector- responds to transmitted light
what is nephelometery
-measures scattered light at a fixed 30-90 degree angle from incident light
-detects light scattered or reflected towards a detector that is not in the direct path of the transmitted light
-the scatter is dependent on wavelength and particle size
-it is more sensitive than turbidimetry in low concentrations
amount of scattered light is directly proportional to analyte concentration
what are the components and uses of a nephelometer
light source:
-high intensity
-helium neon laser, halogen , xenon lamp
Photodetector
-angled 30-90 degree from cuvetter
-must not measure in same direction as transmitted light
Sources of error: substances in the sample that can scatter light like lipemia
use
-Cell counter, flow cytometer, coagulation
-Combined with immunological tests- AG/AB complexes can change light scatter as they form
-measuring serum proteins: IgG, C3, C4, transferrin, haptoglobin, RF, CRP
What is fluorometry
- when a molecule absorbs light at one wavelength and emits light at a longer wavelength
-when electrons get excited they each a higher energy level and because they are unstable the energy will lower to ground state
-on ground state molecules will emit energy as radiation which is different and longer wavelength than that absorbed - Stokes Shift (difference between excitation and emission)
what are the components of fluorometer and what are its uses
light source: high energy UV lamp- mercury vapor, xenon arc
Monochromator - two filters primary and secondary
Detector- place 90 degrees to cuvette to prevent excitation light from striking detector
use
-measure ca, mg, hormones, b12, blood typing
-immunoassay analyzers
-fluorescent microscopy
-binding assays
what are the two filters in a flourometer
primary:
-between light source and sample
-selects for excitation wavelength
-light shines on the fluorescent molecules in the cuvette
Secondary :
-in front of the detector
-selects emission wavelength that the fluorescent molecules from the cuvette emit
-emission wavelength is longer than excitation wavelength
what are the advantages and disadvantages to a fluorometer
Advantage
-more sensitive and specific than routine
-secondary filter blocks interference
Disadvantages
-sensitive to temp, pH, solvent changes
-self quenching - fluorescence reduction when concentration is high
What is chemiluminescence and what is it used for
-measuring light emission as a result of chemical reaction - AG/AB reaction
-mostly a curve not linear - needs alot of calibrators
-reaction intermediates will decay to ground when light is emitted in reaction - as these intermediates go from a high energy to low energy level they emit the light
-light measure is directly or inversely proportional to analyte concentration
use:
-forensic science with luminol and hydrogen peroxide producing a blue light
-Beckman coulter
what are the dis/advantages of chemiluminescence and how it is different from fluorometry
Advantage:
-easy to use and simple
-highest light intensity
-can measure a low concentration down to picomoles
-flash type reaction very fast
-no excitation radiation or monochromator needed
Disadvantages
-impurities can cause background signaling which can over time degrade sensitivity and specificity
it is different from fluorometry because a)no excitation b)no monochromator. Radiation comes from one molecule only
what is flame photometry
-exciting Na, K, and Li with hot flame to emit energy at specific wavelength
-emitted light can have different wavelengths but the lien spectrum is different for each element Na makes orange flame
-intensity related to analyte concentration
What does a flame photometer contain
Atomizer:
sprays diluent into the flame
monochromator- likes spectrophotometer
Burner/flame- heat excites atoms being measure
-propane and air used for flame
-temperature of flame is vital
What is refractormetry
-when light changes direction going from one transparent medium to another of a different density
-the greater the density the larger the refraction
what is a refractometer used for
-measures specific gravity/relative density of urine or protein serum
-has a scale that corresponds to the RI of the solution
-relative density = density of urine/density of H2O
how is the refractive index measured
-once measure it can be related to density of a specific medium
=speed of light in a vacuum/sol in a medium
-affected by wavelength of radiation must be constant
-as the concentration of solute increases so the density and refractive index
Where would you use lasers in a lab
-when radiant energy interacts with excited atoms to produce intense radiation
-can be energy source in spec or nephle
-used for structure determination, sample ID
-clinically in flow for WBC diff
what is primary container sampling
when instruments sample right from the tube eliminates aliquotting
what is closed tube primary sampling
when the probe pierces vacutainer stopper
how do Dry chemistry systems remove protein/interferents
Vitros 350
the spreading layer separates substance from lmw and hmw
what are the different types of sample delivery
Discrete Analysis
-sample separated into separate compartments for specific tests with specific reagents being added
Vitros 350 detects sample volume and clot detection
Centrifugal analysis
-using centrifugal force to mix sample and reagent
Random Access Analysis
-any sample in any order can be analyzed
Batch Analysis
-samples processed in sample run for same analyte
Profile Analysis (Panels)
card, liver,
how can photo degradation be a sample handling error
some substances are sensitive to UV light
-bilirubin use containers made of dark glass
What is an open system
-when CPU allows you to use other manufacturers reagents
-help if you are trying to develop a new procedure R&D Au480
how can evaporation occur in sample handling
-humidity air flow- caps that can be pierced should be used
how can temperature degradation cause sample handling error
ensure you load in temperature control zone
what occurs in the reaction phase
-mixing samples and reagents in an homogenous mixture
-not in Vitros but in AU
Incubation
-need time for the chemical/enzymatic reaction to occur
-no photometric readings taken at this time
What is a closed system
when analyzers only take reagents from their manufacturer
-unable to modify test parameters must run assay as it exists Vitros 350, Beckman
how to minimize carryover
aspirate wash fluid
back flush probe with saline
use disposable sample probes
in Au480- probes washed between sample by dipping in wash cup
in Vitros 350- use disposable pipette tips
where does the measurement phase occur
-in a reaction vessel like a cuvette
-permanent ones need to be washed with DI water
-the optical system (light source, monochromator, and detector) needs to be in the proper position for measurement to occur accurately
-readings can be single end point or multi end point (kinetic or rate) or continuous
-polychromatic measurement where you measure the primary wavelength of max A and secondary wavelength of min A and calculate delta A
how is the signal processed/ data managed in an instrument
-calculate result from signal
Analog- comes from the detector through voltage or electrical energy in comparison to reference signal - spec
Digital - date in units, needs analog to digital convertor
Instrument readout - visual test results
LED, CRT monitor
Data management
-sending results to printer
how are test results reported
-transcriptions - subject to human error
-results directly uploaded to HIS/LIS with specimen number
what is dwell time
minimum time for instrument to obtain result
what are working standards made from
dilutions from a concentrated stock
-analyze concentrations from low to high to prepare a standard graph Abs vs concentration
-each method has a linearity limit
what does calibration do and when is it needed
establishes a relationship between signal (Absorbance, reflectance and fluorescence) and concentration
must calibrate when:
-new reagent lot
-QC out of Westgard rule range
-after major maintenance - part replaced, shutdown
-calibration period ended
