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246 Cards in this Set
- Front
- Back
8 steps in obtaining a laboratory test
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1. test order placed
2. patient identified/sample collected and labeled 3. specimen transported to lab 4. specimen accessioned 5. specimen processed 6. specimen analyzed 7. result reviewed and verified 8. result released |
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Types of samples for chemical analysis
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whole blood, serum, plasma, urine, other fluids (i.e. CSF, amniotic fluid, saliva, etc.)
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Serum
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supernatant obtain if blood sample is allowed to clot and then centrifuged
free of fibrinogen |
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Plasma
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clear yellow supernatant obtained by centrifuging a blood sample that has been treated with an anticoagulant
does have fibrinogen has more protein than serum |
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Which sample is usually preferred in the industry - plasma or serum?
|
plasma, mostly due to the time it takes serum to clot before processing
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Anticoagulant
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substance the suppresses, delays or prevents coagulation of blood by preventing the formation of fibrin
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Preservative
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chemical that prevents a change in the concentration of analytes in a sample of blood, urine or other body fluid
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Additive
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chemical added to a specimen that changes one or more of its physical or chemical properties
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Red-top collection tube
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no anticoagulant or preservative, used for collecting serum, need 20 minutes for clotting before centrifugation, mostly used for blood bank speciments, some chemistries (drugs, trace metals)
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Gold-top and tiger-top collection tubes
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SST, contains a gel that forms a physical barrier between the serum and cells after centrifugation, no other additives, can be used for most chemistry analysis but gel affects some lab tests
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Gray-top collection tube
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sodium fluoride and potassium oxalate OR sodium iodoacetate, cannot use for electrolytes (iodoacetate has less of an effect), stabilizes glucose in plasma
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Sodium fluoride and potassium oxalate
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inhibits enolase
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Sodium iodoacetate
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inhibits G-3-P dehydrogenase
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Green-top collection tube
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sodium, ammonium or lithium salt of heparin, anti-coagulant
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Lithium heparin
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Most common anti-coagulant in chemistry laboratory, inhibits thrombin
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Lavender-top collection tube
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EDTA anti-coagulant, cannot use for any divalent cations or sodium/potassium, only required for hemoglobin analysis
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Light blue-top collection tube
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sodium citrate anti-coagulant, causes problems with almost all chemistry analytes
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Sodium citrate
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anti-coagulant which chelates calcium and inhibits coagulation, used for coagulation studies, not used in chemistry
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Which anticoagulant is pH neutral?
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lithium heparin
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What are some laboratory systems that are utilized to avoid patient and sample identification errors?
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bar code labeling, delta checks, criteria for sample acceptance/rejection
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What type of analyte is required for delta check analysis?
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Analytes that do not change drastically from day to day
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What are some preanalytical errors that occur during blood collection?
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prolonged tourniquet application, improper site of draw (above IV, for example), improper order of draw, improper time of day, use of improper tube, incomplete filling of tubes, improper antiseptic use (i.e. when testing for alcohol)
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What analytes are affect by diurnal variation?
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iron, cortisol and other hormones, glucose, etc.
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What analytes are affected by posture?
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Proteins decrease when standing
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What analytes are affect by diet?
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glucose, lipids, electrolytes, phosphorus, hormones, etc.
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What analytes will be affected by physical training?
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protein, creatinine, lactic acid, uric acid, creatinine kinase
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What analytes will be affected by smoking?
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ammonia, CEA, glucose, GH, urea
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What analytes will be affected by gender?
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hormones, calcium, iron
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What analytes will be affected by age?
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ALP (released from bone during growth), creatinine (renal function)
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What analytes will be affected by alcohol intake?
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GGT, Glucose, Triglyceride
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What are analytical variables that can affect the reliability of results?
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anticoagulant used, sample integrity, instrument functioning, method accuracy and reliability, following SOP correctly
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Lipemic sample
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turbid sample due to increased triglyceride, dilutes plasma or serum analytes, can only be used for lipid analysis until lipids removed by ultracentrifugation, can be confused with turbidity due to protein
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Icteric sample
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yellow sample due to increased bilirubin, affects colorimetric assays
|
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Hemolyzed sample
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red sample due to hemoglobin, falsely increases potassium, phosphorus and decreases glucose, also affects colorimetric assays
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What are postanalytical variables that can affect the reliability of results?
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accurate reporting, accurate transfer of information electronically (from LIS to HIS), timeliness of result verification, appropriate flagging of results
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Photometry
|
Measurement of intensity of light falling on a surface from a source without consideration of wavelength
|
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Spectrophotometry
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Measurement of intensity of light at a selected wavelength
The mostly widely used automated method in the clinical chemistry laboratory. |
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Two principles spectrophotometry is based on
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Substances absorb light at unique wavelengths
Amount of light absorbed is proportional to the concentration |
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Light is electromagnetic radiation or photons of energy and it behaves like a....
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wave
|
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Wavelength of light equals...
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the distance between identical sites on a consecutive wave (i.e. peak to peak or trough to trough)
|
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Relationship between wavelength and energy
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Energy in Ergs = Planck's constant x Frequency of light in cycles per second
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Energy and wavelength are ______ related to each other
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inversely
E = hc / lambda |
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UV has a _____ wavelength and a _____ energy than visible light
|
lower, higher
|
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Wavelength < 190nm
|
X rays and gamma rays
|
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Wavelength 190-390 nm
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UV
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Absorption
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electromagnetic radiation is assimilated by a substance (neither reflected or transmitted)
|
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Wavelength 380 - 750 nm
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Visible light
|
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Wavelength > 750 nm
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Infrared
|
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When an atom or molecule absorbs a photon...
|
valence electrons are raised from a low energy orbital (ground state) to a higher energy orbital (excited state) by absorbing the energy of the photon, changes occur in the vibration and rotation of covalent bonds, excited electron will fall back to ground state by emitting energy (transmittance)
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% Transmittance
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I sample / I incident x 100
OR T/I x 100 |
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Each succuessive layer of solute absorbs a _____ % of light incident on it
|
constant
|
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Beer's law
|
concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light
Absorbance = proportionality constant (L/mol x cm) x length of light path (cm) x concentration (mol/L) |
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Absorbance varies with...
|
concentration, pH and temperature
|
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Relationship between absorbance and %T
|
A = 2 - log %T
|
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Assumptions of Beer's law
|
light striking the solute is only one wavelength
solute being analyzed is the only colored solute present in the solution light being measured comes only from the analytical beam of light |
|
Single calibrator method
|
A unknown / A calibrator = C unknown / C calibrator
|
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Tungsten and Tungsten Halogen lamp
|
320 to >1000 nm (Visible and near IR)
|
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Xenon lamp
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200 to 800 nm (UV and visible)
|
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Deuterium-discharge & hydrogen lamp
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165 to 450 nm (UV)
|
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Mercury-arc lamp
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UV and visible range
|
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Laser
|
Light amplification by stimulated emission of radiation
intense and focused beam of light of very specific wavelength Used commonly for hormones and drugs |
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Monochromator
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system that isolates radiant energy of desired wavelength and excludes other wavelengths
|
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In a monochromator, the degree of wavelength isolation is dependent on.....
|
the width of entrance and exit slits
|
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Spectral bandpass/bandwidth
|
Defines the range of wavelengths that will be transmitted
lower number means less interference calculated as the width in nm of the spectral transmittance curve at a point equal to one-half peak transmittance |
|
A chromagen absorbs the wavelength that corresponds to...
|
the energy level of its electrons
|
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Types of fixed wavelength monochromators
|
colored glass/plastic, interference filter
|
|
How does an interference filter work?
|
Uses constructive interference to create reinforced waves of desired wavelength. The filter transmits the wavelength that is an even multiple of the distance between its sides. (spectral bandwidth between 5 and 15 nm)
|
|
Types of variable wavelength monochromators
|
prisms, diffraction grating
|
|
Prisms are now commonly used in spectrophotometry (T/F)
|
False, they are rarely used anymore
|
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How does diffraction grating work?
|
It is based on the principle that wavelengths bend as they pass through a sharp corner; short wavelengths are diffracted through a small angle while large wavelengths are diffracted through a large angle.
|
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What are the properties of an ideal cuvet?
|
square with plane-parallel optical surfaces and a constant light path, scratch-resistant, transmits essentially all incident light
|
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What do detectors in spectrophotometers do?
|
Convert light energy into electrical energy by allowing light to hit the surface and dislodge electrons, producing a current or potential difference which may then be measured
|
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What are quality assurance procedures that must be performed on spectrophotometers?
|
Checks on wavelength accuracy, stray light, detector function and linearity
|
|
When is bichromatic analysis used?
|
when interfering chromogens are present (hemolysis, lipemia, etc.)
|
|
How does bichromatic analysis work?
|
Light beam is split in two and a different wavelength is selected for each beam.
One wavelength is desired peak of analyte, other wavelength is peak of interfering chromogen. Subtract second wavelength from first to determine concentration of analyte. |
|
Serum blank
|
use serum instead of water for blank, used for colored serum (lipemic, hemolyzed)
|
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Hemolyzed samples are not appropriate for which studies?
|
Electrolytes (especially potassium), LD
|
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Reagent blank
|
If you have a colored reagent, use it for a blank instead of water
|
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Standard deviation
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Quantifies the degree of dispersion of data points about the mean
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Coefficient of variation
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% cv = standard deviation/mean X 100
Measure of variability expressed as a percentage |
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Frequency histogram
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Graphical representation of data, values on x-axis versus number of time the value is obtained on the y-axis
|
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Normal distribution
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equal number of values above and below the mean, Gaussian curve (bell-shaped) where one standard deviation includes 68.2% of the data, two standard deviations include 95.5% of the data and three standard deviations include 99.7% of the data
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Range
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difference between the highest and lowest values in the sample of population
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When are parametric statistics used?
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when variability is known
|
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Inferential statistics
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Summarize data to draw logical conclusions about a population from a sample
Used to compare features of two or more groups of data |
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Method evaluation parameters
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Linearity, method comparison experiment, reference range establishment or validation, analytic sensitivity, specificity, predictive value
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Linearity (Analytical Measurement Range)
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Also known as dynamic range. Concentration range over which measured concentration is equal to actual concentration.
|
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How are linearity studies performed?
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By assaying a set of standards in duplicate or triplicate with concentrations focused at specific points (medical decision points) then graphing expected concentration (x-axis) versus mean of results (y-axis). A 45 degree angle confirms linearity.
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Comparison of two testing methods
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Compare new method and old method by running at least 40 samples using both methods and compare the two sets of results
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When comparing two test methods, analysis must be completed within...
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2 to 4 hours by both methods
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What type of samples must be used when comparing two test methods?
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At least 50% abnormal, with half of those values low and half high
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Total error
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Random error + Systematic error
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Random error
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present in all measurements with no pattern; due to instrument, operator or environment
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Systematic error
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consistent error in one direction, should not be present in a method, can be proportional or constant
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Accuracy
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Obtaining the true value, method comparison study, recovery study or linearity check usually verifies accuracy
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Precision
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Reproducibility, estimated by running a sample of known concentration repeatedly
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Linear regression method comparison analysis
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Plot results of new method (y) versus old method (x), results should agree within 5%
|
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In linear regression method comparison analysis, the y-intercept represents _____ error and the slope represents _____ error
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constant, proportional
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Purpose of recovery experiment
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Used to look for effect of unknown interfering substance,
|
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How is a recovery experiment performed?
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Divide sample in two parts then add known amount of pure analyte to one sample (spiked). Analyze both samples and calculate percent recovery:
amount recovered/amount added x 100 = percent recovery 95-105% = no interference |
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How is the total allowable error determined for each analyte?
|
CLIA 88 (see pages 459-461 in text)
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What are the two sub-types of systematic error?
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constant and proportional
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The correlation coefficient r is utilized for what?
|
to show the strength of the relationship or correlation between x and y variables
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What is indicated by r=0?
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There is no relationship between the x and y variables
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When comparing a new method and old method of testing, what is an acceptable r value?
|
0.95 to 1.05 (positive or negative)
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What is the equation for systematic error?
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SE = constant error + proportional error
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What is the equation for proportional error as obtained from a linear regression model?
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PE = (slope - 1) x 100
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What is the constant error as obtained from a linear regression model?
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CE = y-intercept
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Standard error of the estimate compares...
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the values obtained and the values expected based on the linear regression model
this is a measure of random error/precision |
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Parametric statistics are used when...
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variability is known
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The t-test determines what?
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statistically significant difference between means of two groups, used for accuracy
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The f-test determines what?
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statistically significant difference between standard deviations of two groups, used for precision
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Chi square is a parametric method (T/F)
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False, it is a nonparametric method
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Chi square indicates what?
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the statistical significance if probability of difference between calculated value and critical value is less than 5%
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105% recovery is acceptable in a recovery experiment (T/F)
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True, 95-105% recovery is acceptable and indicates no interference
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Equation for percent recovery
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amount recovered/amount added x 100 = percent recovery
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Why is a percent recovery experiment usually performed?
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to check for interfering substances
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The analytic sensitivity or detection limit
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the smallest concentration of an analyte that can be measured accurately by an instrument or method
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Analytic specificity
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the method's ability to measure the analyte of interest
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Method of developing predictive value
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evaluate analytes on large group of patients with the disease and large group of patients that do not have the disease
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Diagnostic sensitivity
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probability of a positive result if the disease is present
Sensitivity = true positive / (true positive + false negative) x 100 |
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Diagnostic specificity
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probability of negative result if the disease is not present
Specificity = true negative/ (true negative + false positive) x 100 |
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Positive Predictive Value
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true positives/all positives x 100
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Negative Predictive Value
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true negatives/all negatives x 100
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Method of developing predictive value
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evaluate analytes on large group of patients with the disease and large group of patients that do not have the disease
|
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Diagnostic sensitivity
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probability of a positive result if the disease is present
Sensitivity = true positive / (true positive + false negative) x 100 |
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Diagnostic specificity
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probability of negative result if the disease is not present
Specificity = true negative/ (true negative + false positive) x 100 |
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Positive Predictive Value
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true positives/all positives x 100
|
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Negative Predictive Value
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true negatives/all negatives x 100
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Define reference range
|
the acceptable range of an analyte or acceptable result of a lab test for a healthy patient
this range differentiates between sick and well |
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Data must be stratified when determining a reference range (T/F)
|
True, data must initially be stratified by age and gender in order to determine if reference ranges must be separate
|
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How often must a reference range be validated after it is established?
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Every year with at least 20 samples
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Parametric studies can only be used if the frequency distribution is _______
|
Gaussian (normal)
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With a Gaussian distribution, reference range = 95% of the group = ______
|
mean +/- 2 standard deviations
|
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The receiver operating characteristic curve (ROC) plots...
|
sensitivity versus 100% - specificity
greater area under curve indicates better test |
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For a screening test, is sensitivity or specificity more important?
|
Sensitivity
|
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For a confirmatory test, is sensitivity or specificity more important?
|
Specificity
|
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Fluorometer
|
instrument that detects fluorescent emissions, often used in clinical laboratory in highly automated immunoassay systems for measurement of drugs, hormones, etc.
|
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There are two curves that are present on a fluorometer graph of absorbance versus wavelength, what do they represent?
|
the light emitted from the light source (higher energy, lower wavelength), the light emitted from the sample (lower energy, higher wavelength)
|
|
Types of lamps used in fluorometer
|
halogen - least expensive
xenon - most used laser - most expensive |
|
The light source (lamp) and detector in the fluorometer are at what angle from each other?
|
90 degree
|
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Fluorescence and concentration are ______ proportional
|
directly
|
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Advantages of fluorometry
|
High sensitivity and specificity
|
|
Disadvantages of fluorometry
|
very sensitive to environmental changes: pH, temperature, contamination
|
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Chemiluminescence
|
chemical reaction that emits light
|
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Advantages of chemiluminescence
|
quick, very low detection limits, one step reactions, simple
|
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Disadvantages of chemiluminescence
|
impurities can cause background signals and decrease the sensitivity and specificity
|
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Nephelometry
|
amount of light scattered as it passes through the particulate solution
|
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Turbidometry
|
Measures decrease in the amount of light as it passes through a particulate solution
|
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Turbidometry can measure the presence of soluble particles (T/F)
|
False, turbidometry can only measure the presence of insoluble particles
|
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Turbidometry measures at an angle of _______ to the light source, while nephelometry measures at an angle ________ to the light source
|
180 degrees, other than 180 degrees
|
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Limitations of nephelometry and turbidometry
|
lipemic, hemolyzed or contaminated specimens not recommended
improper centrifugation may cause interference sample cuvettes must be clean/free of scratches |
|
Reflectance spectrophotometry
|
spectrophotometric technique in which light is reflected from the surface of a reaction and used to measure the amount of the analyte
|
|
Hemoglobin A is what percentage of hemoglobin in the body?
|
80%
|
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The glucose oxidase-peroxidase method is specific for what type of glucose?
|
Beta-D-glucose
Must allow alpha-D-glucose to be converted to beta-D-glucose before measuring |
|
Oxidating agents produce a falsely _____ glucose result in the glucose oxidase-peroxidase method
|
elevated
|
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Reducing agents produce a falsely _____ glucose result in the glucose oxidase-peroxidase method
|
decreased
|
|
Absorbance for NADH or NADPH is always measured at what wavelength?
|
340 nm
|
|
Benedict's copper reduction reaction measures what?
|
Glucose reducing cupric ions to cuprous ions. This produces a yellow or red cuprous compound.
|
|
What substances would interfere with the Benedict's copper reduction reaction test for glucose?
|
Any other reducing agent (including other reducing sugars)
For this reason, it is only used for babies under the age of one (in order to check for all metabolic sugar disorders) |
|
The reference method for glucose determination is ______
|
hexokinase
|
|
Hb A1c estimates the average blood glucose over ________
|
2-3 months (lifespan of the red blood cell)
|
|
The goal of diabetes therapy is a result of Hb A1c less than ____
|
7%
|
|
Methods to separate glycated hemoglobin
|
ion-exchange or affinity chromatography, electrophoresis, isoelectric focusing, high-pressue liquid chromatography, immunoassay
|
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Ion-exchange chromatography is extremely sensitive to _______
|
temperature
|
|
Affinity chromotagraphy utilizes _____ as a reagent to bind ________
|
boronic acid, glycosylated protein
|
|
EDTA
|
Chelates divalent cations to prevent coagulation
produces falsely low iron, Ca and Mg results |
|
Apoferritin
|
protein that binds iron in storage, called ferritin when iron is bound
|
|
Transferrin
|
glycoprotein that binds iron in circulation for transport
known as apotransferrin when iron is not bound |
|
Hepcidin
|
regulator molecule for iron intake
as hepcidin decreases, iron intake increases and splenic iron is released |
|
Serum iron is sensitive to the ______ stage of iron deficiency
|
mild
|
|
Serum iron levels _____ after body stores are fully depleted but _______ hemoglobin levels drop
|
decrease, before
|
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Serum transferrin is ______ in iron deficiency
|
increased
|
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Each transferrin binds _____ molecules of iron
|
2
|
|
Transferrin saturation is the ratio of _______ to _______
|
serum iron, iron-binding capacity
|
|
Transferrin saturation is the most accurate indicator of ________
|
iron supply to the bone marrow
|
|
The Serum Transferrin Receptor Assay indicates _______. The circulating sTfR becomes _____ in iron deficiency.
|
the cells' need for iron, elevated
|
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The best diagnostic test for iron deficiency is _________
|
serum ferritin because it measures iron stores in the body
|
|
For serum ferritin, a concentration below ______ indicates iron deficiency
|
12 micrograms/L
|
|
The R/F ratio measures what?
|
the sTfR/serum ferritin ratio
It is used to provide an estimate of iron stores. |
|
The R/F ratio test may be adversely affected by?
|
oral contraceptives, inflammation
|
|
Special collection considerations for iron measurement
|
early morning collection, no hemolysis, fasting
|
|
Steps in colorimetric measurement of iron
|
1. Add acid to remove iron from transferrin
2. Add reducing agent to convert Fe3+ to Fe2+ 3. Add an oxidized dye than can be reduced to cause a color change which can be measured colorimetrically |
|
Lead poisoning blocks the heme synthesis pathway at what point(s)?
|
Alpha-ALA dehydratase, Coproporphyrinogen oxidase, Ferrochelatase
|
|
% saturation of transferrin =
|
serum iron/TIBC * 100
|
|
UIBC =
|
TIBC - Serum iron
|
|
Special considerations for collection of lead testing samples
|
Must use lead-free needle, metal free collection tube (royal blue), prevent post-collection contamination
|
|
Best test for detection of lead exposure and reference range
|
whole blood lead measurement
<10 mcg/dL normal in children >10 mcg/dL toxicity indicated >30 mcg/dL in adults indicates significant exposure |
|
Free erythrocyte protoporphyrin assay (EPP) is not useful as a screening test for lead exposure because ________
|
it is not a sensitive indicator of low-level lead (<35 mcg/dL)
|
|
The common feature of all porphyrias
|
excess accumulation of porphyrins or porphyrin precursors in the body
|
|
When testing for plasma glucose, the plasma must be separated from the cells within ______ of collection and refrigerated to prevent ________
|
1 hour, loss of glucose due to cellular metabolism
|
|
Fasting plasma glucose samples should be obtained when?
|
In the morning after an 8 to 10 hour fast
|
|
What preservative is added to the tube used to collect fasting plasma glucose samples? What color is the tube?
|
Sodium fluoride, grey
FYI: lithium heparin tubes are not as preferable but may be used |
|
What reaction does hexokinase catalyze?
|
phosphorylation of glucose to G-6-P
|
|
How does EDTA interfere with the Hexokinase method of measuring glucose?
|
EDTA binds MG2+. This interferes because ATP is stabilized by magnesium and ATP is necessary for the phosphorylation of glucose in the first reaction of the procedure.
|
|
Is the Hexokinase enzyme specific for glucose?
|
No.
This could potentially cause interference in the hexokinase method of glucose measurement as other carbohydrates could use up all of the ATP, limiting the phosphorylation of glucose. |
|
First rxn of hexokinase method of glucose measurement
D-glucose + ________ + hexokinase ⇀ glucose-6-phosphate + ADP |
ATP(Mg2+)
|
|
First rxn of hexokinase method of glucose measurement
D-glucose + ATP(Mg2+) + ______ ⇀ glucose-6-phosphate + ADP |
Hexokinase
|
|
Second rxn of hexokinase method of glucose measurement
G6P + NAD+ + G6PD ⇀ ______ + NADH + H+ |
6-phosphogluconic acid
|
|
Second rxn of hexokinase method of glucose measurement
G6P + NAD+ + _______ ⇀ 6-phosphogluconic acid + NADH + H+ |
G-6-PD (glucose-6-phosphase dehydrogenase)
|
|
Aldose
|
A carbohydrate containing an aldehyde (carbonyl bound to a carbon and a hydrogen) group at the end of the carbon chain
|
|
Ketose
|
A carbohydrate containing a ketone, a carbonyl connecting two internal carbons.
|
|
Simple sugars contain which functional groups?
What is the empirical formula for any carbohydrate? |
One aldehyde and two ore more hydroxyl groups.
Empirical formula = (CH2O)n where n=3 or more |
|
What is the most abundant and important carbohydrate in nature?
|
D-glucose
|
|
Reducing sugar
list examples (gold star for every one you get!) |
Carbohydrate with an aldehyde or ketone group; this allows the sugar to become oxidized/be a reducing agent
ex: glucose, galactose, fructose, lactose, maltose |
|
Oligosaccharides
|
Carohydrate 2 to 10 monosaccharides long; joined by covalent bonds
|
|
three most abundant disaccharides and their substituents
Which ones are capable of reducing? |
Maltose = glucose + glucose
Lactose = glucose + galactose Sucrose = glucose + fructose Maltose and lactose can reduce |
|
Where are monosaccharides absorbed after their breakdown?
|
small intestine
|
|
Describe the functions of pancreatic and salivary amylases
|
Salivary amylase: cleaves starch polymers into dextrins
|
|
Second rxn in Hexokinase method
G6P + ________ + G6PD ⇀ 6-phosphogluconic acid + ________ + H+ |
NAD+, NADH
|
|
At what wavelength is the absorbance measured in the hexokinase method? What does this measure?
|
340 nm, to capture the conversion of NAD+ to NADH
|
|
Glucose oxidase-peroxidase method rxn 1
________ + O2 + glucose oxidase → ________ + H2O2 |
Beta-D-Glucose, gluconic acid
|
|
GOD-POD method rxn 1
B-D-glucose + O2 + _______ → gluconic acid + ________ |
glucose oxidase, H2O2
|
|
GOD-POD method rxn 2
H2O2 + _________ + peroxidase → _________ + H2O |
reduced dye, oxidized dye (color change)
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Peroxidase performs what function in the second reaction of the GOD-POD method?
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The peroxidase catalyzes the oxidation of the dye utilizing oxygen obtained from the hydrogen peroxide molecule
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Explain how the GOD-POD method measures glucose levels.
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Beta-D-Glucose reduces H2O to hydrogen peroxide (glucose oxidase catalyst)
This hydrogen peroxide then oxidizes a color changing dye (peroxidase catalyst) The colored dye is measured spectrophotometrically |
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What are substances that can interfere with the GOD-POD glucose method? How do they interfere?
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Any strong reducing or oxidizing agent.
A reducing agent will use up the hydrogen peroxide in the second reaction, falsely decreasing the result An oxidizing agent will oxidize the dye further resulting in a falsely increased result. |
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Name examples of substances that will interfere with the GOD-POD glucose method
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Bleach (oxidizing agent - will falsely increase result)
Ascorbic acid (reducing agent - will falsely decrease result) |
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Is the copper reduction test specific for glucose?
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No, it is positive for any reducing sugar
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How are monosaccharides other than glucose utilized by the body?
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Non-glucose monosaccharides are converted to glucose by hepatic enzymes
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Before glucose can be metabolized or stored, what reaction takes place?
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The hexokinase reaction wherein the hexokinase enzyme converts glucose into glucose-6-phosphate
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Preproinsulin
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Preproinsulin contains both the C peptide and the signal peptide; converted almost immediately to proinsulin after translation into protein in Beta-cels
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Proinsulin
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- formed from preproinsulin after cleavage of signal peptide
- Stored in Beta-cells of the pancreatic islets of Langerhans - Stimulus to convert to insulin results in the cleavage of C-peptide |
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Describe insulin formation from proinsulin
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Insulin is converted from proinsulin by the cleavage of the C-peptide, leaving only the A and B chain connected by disulfide bridges
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Insulin inhibits which pathways?
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Glycogenolysis (glycogen formation in liver)
Gluconeogenesis (glucose synthesis in liver) Lipolysis in adipose tissue Protein breakdown/amino acid release |
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C-peptide levels are used clinically to...
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Determine how much insulin the patient is producing naturally
in contrast, therapeutic insulin will not cause a change in C-peptide levels |
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Insulin stimulates which physiological processes in the:
liver? adipose tissue? muscle/other cells? |
Liver: glycogenesis and fatty acid synthesis
Adipose tissue: glycerol and fatty acid synthesis Muscle/cells: inc. permeability to glucose, amino acid uptake, protein synthesis |
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Hypoglycemia is seen at ____ glucose levels; symptoms set in at ____ glucose levels (panic value). Describe these symptoms
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Hypoglycemia: <70 mg/dL
Panic/symptoms: <50 mg/dL Symptoms: Confusion, chills, rapid HB, weakness, nausea, trembling |
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Which hormones have the opposite effect of insulin on blood glucose levels?
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Glucagon
Epinepherine Growth hormone Cortisol |
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Which method of glucose measurement is the reference method?
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Hexokinase
the second reaction of this method is very specific for G6P this method is not very susceptible to interference (only EDTA, some other carbohydrates in rxn 1) |
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In the GOD-POD method, why must the sample be incubated?
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To allow all A-D-glucose to convert to B-D-glucose. This ensures that the 1st reaction will measure all glucose present.
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Other than measuring glucose levels, what else is the copper reduction test used for?
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Evaluation of infants for metabolic disorders. This test is sensitive to all reducing sugars so it may be used to detect sugar breakdown defects such as galactosemia.
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How does the copper reduction test for glucose work?
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Glucose (or any reducing sugar) reduces cupric ions to cuprous ions to produce a yellow or red cuprous compound
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What does the glucose oxidase enzyme catalyze in the first reaction of the GOD-POD method?
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B-D-glucose to gluconic acid and oxygen to hydrogen peroxide via redox reaction
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What does the peroxidase enzyme catalyze in the second reaction of the GOD-POD method?
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The oxidation of the dye by hydrogen peroxide via redox reaction
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How is the final product of the GOD-POD measured?
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The oxidized dye is measured by spectrophotometry
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How can a pO2 electrode be used as an alternative to the peroxidase reaction in the glucose oxidase method?
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O2 electrode measures the stoichiometric decrease in pO2 in the glucose oxidase reaction (oxygen is used up to oxidize glucose).
This indicates how much B-D-glucose is being converted to gluconic acid. Therefore, colorimetric peroxidase reaction is unnecessary. |
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4 different methods that can be used to measure glycosylated hemoglobin
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Ion-exchange chromatography method, affinity chromatography method, immunoassay, electrophoresis (including isoelectric focusing)
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What can interfere with the measurement of hemoglobin by ion exchange chromatography?
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Abnormal hemoglobins (have different charge)
increased hemoglobin A level (falsely increased result) decreased RBC survival (falsely decreased result) |
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The resin in ion exchange chromatography adsorbs glycosylated hemoglobins, while non-glycosylated hemoglobins elute (T/F)
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False, the resin in ion exchange chromatography is negatively charged, therefore glycosylated hemoglobins elute (they are less positively charged than nonglycosylated hemoglobins)
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What are some etiologies of hypoglycemia?
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Excessive insulin response
Insulinoma Depleted glycogen storage Rigorous exercise Fasting Liver disease |
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Lab criteria for diagnosis of diabetes mellitus for: CPG, FPG, OGTT, HgbA1C
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First, any diagnosis must be done based on tests on at least two separate days.
CPG: ≥ 200 mg/dL + symptoms FPG: ≥ 126 mg/dL OGTT +2hr: ≥ 200 mg/dL Hgb A1C: ≥ 6.5% |
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Childhood onset, ketoacidosis and FPG that rarely exceeds 500 mg/dL are characteristics of...
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Type I diabetes mellitus
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DM Type 1 is an insulin _____ while DM Type 2 is an insulin ______
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Type 1: insulin deficiency
Type 2: Insulin resistance |
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Very high FPG levels (>1000 mg/dL) are characteristic of
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DM Type 2
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Nonketonic-hyperosmolar coma is associated with...
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DM Type 2
Coma results from severe dehydration due to water following glucose into excreted urine |