Reading...
Play button
Play button
Use LEFT and RIGHT arrow keys to navigate between flashcards;
Use UP and DOWN arrow keys to flip the card;
H to show hint;
A reads text to speech;
57 Cards in this Set
- Front
- Back
|
Explain the principle of absorption photospectrometry
|
the number of light absorbing molecules in solution are proportional to the amount of color of that solution.
absorbance is proportional to analyte concentration. |
|
|
Describe the six major components of a spectrophotometer
|
1. light source
2. Monochromater 3. Primary exit slit 4. Cuvette 5. Photomultiplier tube 6. Readout device |
|
|
Distinguish the different types of monochromaters, and rank them on their efficiency
|
Prisms- very inefficient
Diffraction gratings- most efficient Interference filters- not true monochromaters because they don't break up white light into a spectrum of colors |
|
|
Spectrophotometer
|
Uses a true monochromator like a prism or diffraction grating
|
|
|
Colorimeter
|
Does not use a true monochromator instead it uses an interference filter
|
|
|
A= abc
|
A- absorbance at a specific wavelength
a - molar absorptivity constant b- cuvette light path in cm c- molarity of light absorbing product |
|
|
Absorbance curves
|
-run at least 6 standards
- plot absorbance vs concentration of each standard - look to see if the method follows beers law - if not linear then this graph can be used as a calibration curve |
|
|
Beers law proportionality calculation
|
(abs. unknown/abs. Standard) x conc. of standard= conc. unknown
- must include the measurement of one standard with the controls and patients |
|
|
%T to absorbance
|
Absorbance= 2.000 - log%T
|
|
|
Absorbance to %T
|
Log %T= 2.000 - abs.
- must take the anti log of (2-abs) to find T |
|
|
0.000 absorbance = ?%T
1.000 absorbance = ?%T |
0.000 abs= 100%T
1.000 abs= 10%T |
|
|
Use of absorbance curves in selecting wavelengths
|
The broader peak has the least chance of a large error
|
|
|
Electromagnetic spectrum energies
|
infrared(-above 750 nm) is low energy
ultraviolet(100-380 nm) is high energy Yellow is medium energy |
|
|
When do you use water blanks.
|
Used to zero the spectriophotometer when the reagent does not absorb light
|
|
|
When do you use specimen or serum blanks?
|
Used when a specimen has color due to hemolysis lipemia or icterus
|
|
|
When do you use reagent blanks?
|
Used to zero the spectrophotometer when the reagent does absorb light
|
|
|
What wavelengths will hemolysis interfere with?
|
Above 600nm no effect
Below 600 nm will interfere Hgb absorbs light from 400-600nm |
|
|
What wavelengths will icteric specimens interfere with?
|
Bilirubin absorbs light from 400-530 nm
Below 530nm will interfere |
|
|
What wavelengths will lipemic specimens interfere with?
|
Chylomicrons scatter light at any wavelength. Results in falsely high absorbances
|
|
|
What wavelengths will turbid specimens interfere with?
|
Triglycerides scatter light at any wavelength. Results in falsely high absorbances
|
|
|
Explain kinetic or multiple point assays
|
Kinetic assays take 2 or more absorbance readings while the color reaction is going on.
-measures absorbance change per unit of time - rate of reaction is proportional to analyze concentration |
|
|
Explain end point assay
|
-add specimen to reagent mix it well, incubate, and read the final absorbance after the reaction stops
-biuret total protein assay is an end point |
|
|
Preferred light source for 180-340 nm
|
Deuterium, hydrogen, or mercury arc
|
|
|
Preferred light source for 340-700
|
Tungsten ir tungsten halide
|
|
|
What is a bandpass? what is considered a narrow and broad bandpass?
|
Bandpass- the actual range of wavelength transmitted by a monochromator.
Narrow- <10nm Broad- >10nm |
|
|
What is the wavelength calibration accuracy, and how is it documented?
|
The actual median wavelength entering your cuvette.
Documented by 1. Holmium Oxide for narrow bandpass 2. Didymium for broad bandpass |
|
|
What is "Stray light" and how is it documented?
|
Light not passing through the cuvette but hitting the photomultiplier.
Documented using cut-off filters 380 and 680 nm |
|
|
Daily spectrophotometer QC tasks.
|
Document wavelength calibration accuracy using holmium oxide or didiymium.
Document Photometric accuracy using SRM-930, 931 neutral gray filters. |
|
|
Weekly spectrophotometer QC tasks.
|
Document- photometric linearity, stray light, and spectral bandwidth.
|
|
|
What is Bichromatic analysis and its advantages
|
Reading the absorbance of each test soulution at 2 or more wavelengths.
Helps minimize chromophore interference effects from hemolysis, bilirubin, etc. |
|
|
Explain principles, advantages, and limitations of Turbidimetric analysis.
|
Based on the scattering of light b precipitate particle in the cuvette.
Common method for urine and CSF protein. Under tightly controlled conditions the amount of precipitate formed is proportional to the protein concentration. Mixing and timing is critical Works best with a braoad bandpass instrument. |
|
|
Explain principles, advantages, and limitations of Nephlometric analysis.
|
Based on light scattering by precipitate particles. Light is detected at an angle to the light source.
1000x more sensitive than Turbidimetric analysis. Can also quantitate immune complexes using an antigen-antibody complex. The amount of light scattering is proportional to the amount of antigen. |
|
|
Explain principles, advantages, and limitations of Fluorimetric analysis.
|
1000x more sensitive than turbidimetric and Spectrophotometric methods.
Very prone to interferences, called Quenching. pH, temperature, and contaminants can cause Quenching. Instrument can measure either a naturally florescent molecule or can utilize IgG antibodies that are tagged with a probe. |
|
|
Explain principles, advantages, and limitations of Flourescence Polarization.
|
Fluorimeter with polarizers that excite precipitate in the cuvette.
Has a filter that will only let light through that is polarized in the original plane. This reduces the amount of Quenching. Abbott Diagnostics developed FPIA. Fluorescence Polarization Immuno Assay (TDx and AxSym) |
|
|
Explain principles, advantages, and limitations of Reflectance Photometry.
|
Principle used by Vitros analyzers.
- The amount of light reflected is proportional to the amount of analyte |
|
|
Explain principles, advantages, and limitations of Chemoluminescence
|
Special instrument similar to nephlometer that detects a "flash of light" given off by certain chemicals when they become oxidized.
- 2,000x more sensitive than spectrophotometry |
|
|
Explain principles, advantages, and limitations of Biochemiluminescence
|
Special form of Chemoluminescence wherein the reaction is mediated by an enzyme.
|
|
|
What is a Control?
|
-A biological solution which has been previously assayed many times, and the target value and acceptable range is known.
- Used to assess the accuracy and validity of the assay. - Must be a biological with a similar matrix to the patient samples. |
|
|
What is a Standard?
|
-A solution with an exact known concentration of analyte.
- Used as a reference point. - Also known as a calibrator -May or may not be biological |
|
|
Reason for 2 controls.
|
If you can assay 2 levels of controls and get acceptable values for both, then we can assume the assay is accurate and patient values are reliable.
- if control values aren't acceptable something is wrong with the assay |
|
|
Protocol if one of two controls is out.
|
DO NOT EVER report patient values when a control is out. It is a violation of CLIA
|
|
|
How do you discern a bad standard?
|
If the standard is bad both controls move in the same direction.
- diluted/low standard= high values - concentrated/high standard= low values |
|
|
When do you run controls on a Batch assay?
|
Two controls each and every run, unless otherwise specified.
|
|
|
When do you run controls on TDx and IMx
|
Three levels of control once per shift, and then one level of control per each assay run.
|
|
|
When do you run controls of a Random Analysis Analyzer?
|
Two levels of controls once per each 8 hour shift, or more often if specified by instrument manufacturer.
|
|
|
Chance of a random analytical error.
|
1 out of every 20 times or 4.5% probability
|
|
|
List things that would cause an assay to be out-of-control.
|
1. Bad controls
2. Bad standards 3. Calibration failure 4. Bad reagent 5. wrong reagent 6. incorrect pipettor 7. incorrect temp 8. incorrect calculations 9. sloppy technique 10. contaminate glassware |
|
|
What to do if assay is out-of-control
|
-immediately record in QC chart
- double check instrument settings, calculations, and check to see if you used the correct reagents, standard, and techniques. -repeat all patient tests with new standards, and controls. - if test is within range record in QC chart and report out patient results |
|
|
what would cause a shift?
|
New lot # of reagents, wrong controls, new spectrophotometer lamp, new tech, new standard
|
|
|
What will cause a trend?
|
Degrading reagents, degrading standards, spectrophotometer lamp burning out, room temp changing, controls degrading or evaporating
|
|
|
What QC charts are used to identify shifts and trends?
|
Shewart and Levy-Jennings
|
|
|
Difference between accuracy and precision
|
Accuracy is the ability to obtain the correct value. Precision is the ability to reproduced the same value over and over
|
|
|
What percentage lie within 1, 2, & 3 SD's on the Gaussian.
|
1- 68.3%
2- 95.5% 3- 99.7% |
|
|
What is proficiency testing?
|
A validation of your lab, instruments, and that your methods can produce quality results.
-3x a year -5 unknowns challenge per each analyte -minimum passing score is 80% |
|
|
what is competency testing?
|
Documents that each individual can perfomr the test accurately
-Includes one on one instruction and demonstration, rading SOP, passing a written quiz, and correctly assaying several unknowns under observation. -Done annually |
|
|
SDI
|
Standard Deviation Index
-Used as a predictor of how your lab will do on the next PT challenge. = (your value - peer value)/ StDev of peer group - SDI of 2.0 or greater is a strong predictor of a future PT failure |
|
|
How do you report patient values that are either below or above the reportable range?
|
If below report "less than Xmg/dl, analyte below assay range"
-If above dilute the specimen with an appropriate diluent, correct for the dilution factor and report that final value, and add a comment thatthe value represents a dilution of the original specimen |
