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What is analytical specificity?
ability to measure substance of interest and nothing else that's chemically similar
What is functional sensitivity? Analytical sensitivity?
lowest value for which clinicians find have acceptable reproducibility, esp for troponins, CRP, d-dimers, etc.; detection limit (lowest value distinguishable from 0 = analytical sensitivity) or slope of line for determining concentration
What are calibrators?
samples similar to the patient, conc. related to stds
What is matrix effect?
Results that vary due to sample makeup (like aqueous)
What is an isobestic point?
Wavelength at which two compounds have same absorbance
How is absorbance measured in the presence of two compounds using the isobestic point?
- A (cmpd 1) = [Absorbance at λ2 - Absorbance at λ1]
- λ2 = wavelength at which absorbance of interferent is Ax and cmpd of interest has A=0
- λ1 = isobetric point
- *Subtract out absorbance from interferent to get absorbance of cmpd of interest
In spectrophotometry, how is the light source selected?
Light source should cover all wavelengths you want to measure: continuous spectrum source (qrtz halogen, tungsten), discrete spectrum source (mercury, deuterium, xenon) or high intensity single λ (laser)
In spectrophotometry, how is the monochromator selected?
Narrow range if using continuous spectrum for specificity
How does a photomultiplier tube work as a spectrophotometer detector? Diode array?
- Photomultiplier - multiple stages amplify # electrons released by light
- Diode array - strip of semiconductor cells release electrons in resp certain λs, sends electrical signal (can measure multiple λs at once)
How does nephelometry differ from turbidimetry?
- Nephelometer ("cloud meter") measures light dispersion when hits particles in solution (detects at an angle to light source) → ↑# particles with ↑ light
- Turbidimetry measures decrease in light (A) by particles (how much light cut off by particles) → ↓# particles with ↓light
What two instruments are used in detection of Ag-Ab complexes? What is a major interference for both instruments?
Lipemia - causes light scatter
In fluorometry, two monochromators are required to select the exciting light and the emitting light. What do the terms quenching and inner filter mean?
- Quenching - when emitted light is absorbed, like in drugs of abuse
- Inner filter - when exciting light absorbed, so get falsely low result
How does atomic absorption measure concentration?
Element absorbs light emitted by same element in cathode tube and ↓light intensity as passing through vaporized element proportional to concen
How does chromatography separate compounds? What technique is used for urine samples?
Based on differences in solubility in stationary and mobile phases; "reversed phase" chromatography where have stationary non-polar phase & polar mobile phase
In chromatography, what is elution? Resolution?
Removing compounds from stationary to mobile phase; ability to separate closely related cmpds - depends on surface area & diff in polarity btw two phases
Name three ways resolution can be improved in chromatography?
- 1) ↑length column
- 2) alter mobile phase/diff in polarity btw mobile & stationary phase
- 3) ↓particle size
All of the following do not improve chromatography resolution except:
A) Rate of flow in mobile phase
D) ↑length of column
D - all other choices can improve separation time but not resolution
Typical boards Q scenario: you want to separate 4 compds using chromatography. Three of them come off at 1.2, 1.4 and 1.6 minutes, the 4th in 20 mins. How can you increase the rate of analysis? What can you do?
Alter temp, rate of flow in mobile phase, or pressure after the first three cmpds come off to get the 4th one quicker
How does ion exchange separate compounds?
Charged stationary phase with opposite charged cmpds attached, then use increasing ionic strength in mobile phase to elute
How does gas chromatography separate compounds? What is used in the stationary & mobile phases?
Based on volatility by producing cmpd derivatives; stationary ph - high boiling point liq or solid, mobile ph - inert gas
Name three methods to identify/detect compounds after separation with chromatography?
- 1) Thin layer chrom - look at distance cmpd traveled relative to solvent
- 2) Column chrom - look at elution time
- 3) Use mass spec as detector
How are compounds separated by electrophoresis? What affects rate of migration?
Charge density; voltage potential
What is an isoelectric point (pI)? When pH>pI, what is the charge? When pH<pI?
Point where no net charge; Negative charge (as ↑pH, give off more H+ ions); Positive charge
What dyes are used to stain DNA in electrophoresis? Proteins?
Ethidium bromide; Coomassie blue or Ponceau red S
How does electrophoresis move proteins through the medium and what end do they move towards (cathode or anode)?
Proteins are net (-) so electromotive force pulls them towards the anode except for gamma globulins, which have a weak net(-), so the buffer's flow/endosmotic force pulls them towards the cathode.
How is the electromotive force created in electrophoresis?
Buffer flow/endosmosis carries substances as it moves towards the cathode(-) & solid support(-) is drawn towards anode(+) → net electromotive force pulls (-) charged molecules towards anode & endosmotic force pulls weakly (-) or (+) charged molecules towards cathode
How can capillary electrophoresis be used to identify monoclonal Abs since you can't do immunofixation?
- Immunosubtraction electrophoresis:
- 1) take 5 test tubes, put gel with antisera in each test tube (one for each monoclonal Ab)
- 2) add patient sample, let Abs bind
- 3) spin down gel
- 4) run the sample to see what peaks remain and which Ab stayed behind with the gel. The missing band is the monoclonal Ab of interest
How can gamma globulins be separated out into oligoclonal bands in CSF electrophoresis?
Isotopes emit 3 types of radioactivity. Which radioactive particle is high energy? Which is composed of an electron or positron and has mod energy? Which one has low energy?
Gamma rays; beta; alpha
How do you calculate time for radioactive isotopes to fall 50% of baseline?
0.693/λ (# is natural log of 2)
How are beta particles measured? Gamma?
Scintillation counters - photon or positron hits crystal (NaI) or liquid (2,5 phenyl oxazole/PPO), photon of light emitted, amount light directly proportional to concen; current is measured when radiation ionizes gas molecules