CLSChem - 01 - Instrumentation

  1. How do enzymes function?
    Lower the activation energy of a reaction
  2. Phases of Enzyme reaction with brief explanation.  Which is clinically relevant?
    • Lag Phase: secondary reactions may occur
    • Linear Phase: [S] >> [E] (enzymes saturated)
    • zero order (rate independent of [S])
    • Measurements made in this area
    • Depletion/Inhibition: [S] ~[E] 
    • first order (rate depends on [S])
    • Product accumulates or substrate is no longer readily available
  3. What is Km?
    • Km = [S] at 1/2Vmax
    • Low Km = high affinity = less concentration will produce results
  4. Velocity of enzyme rxn equation
    V = Vmax[S]/Km[S]
  5. What happens in competitive enzyme inhibition?
    • Inhibitor binds at active site (competes)
    • Can be overcome by increased [S]
    • Vmax does not change, but Km is increased
  6. Isoenzymes vs Isoforms
    • Isoenzymes: multiple forms of the same enzyme (same catalytic f(x)) derived from distinct genes
    • Isoforms: post transcriptional (or translational) modification of a single gene product (including alternative splicing)
  7. What units are used for enzyme measurements?
    • International Units
    • 1 IU = 1umol/minute substrate consumed or product formed
    • **Determining enzyme activity, but actually measuring product formation/substrate removal
  8. NAD(P)+ vs NAD(P)H absorbance
    • 340nm
    • NAD+ gives no abs
    • NADH has peak abs
    • Used frequently due to this distinct differentiation
  9. Enzyme measurement types - equilibrium vs kinetic (compare/contrast)
    • Equlibrium (end-point): substrate is completely consumed
    • May need to trap product to approach completion
    • Less sensitive to variables (rate is irrelevant as long as we complete the rxn)
    • Kinetic: measures at specific times, the more points the better
    • Uncommon, but used for decreasing absorbance rxn (starting w/ NADH -> NAD+)
    • Requires highly-controlled conditions (temp will alter the rate of rxn)
  10. Spectrophotometer instrument overview w/ brief explanation
    • Source: Polychromatic light
    • tungsten lamp (320nm+), deuterium (UV-Vis)
    • Monochromator: Prism (old), Diffraction grating (common), filters
    • Cuvets: plastic (not UV), glass (both), quartz (UV, rare)
    • Photodetector: Photomultiplier tube, Photodiode, Diode Array (must common, best)
  11. Formula for % transmittance
    • %T = Is/Io x 100
    • Is = intensity of light transmitted through sample
    • Io = intesntiy of incident light
  12. Beer's Law (equations)
    • A = 2 - log(%T) = abc
    • A = absorbance
    • a = absorptivity (constant)
    • b = path length (constant)
    • c = concentration
    • *NOTE - If A = 0, %T = 100; if A = 2 %T = 1 (logarithmic)
  13. List what must occur for Beer's law to be followed
    • Incident radiation is monochromatic
    • Solvent absorption insignificant
    • Solute concentration is within certain limits
    • No optical interferent is present
    • No chemical reaction occurs within solute
    • No stray light exists
  14. What is the optimal range for measurement's that use Beer's Law?
    0.1-0.7 A (20-80 %T) has least amount of error
  15. Describe the reflectance method
    • Dry reagant slide is hydrated by sample to initiate reaction
    • Reaction occurs on a thin film matrix w/ highly relfective surface
    • Monochromatic light is reflected to detector
  16. Atomic absorption instrument overview w/ brief explanation
    • Hollow cathode: element-specific lamp
    • Flame/graphite tube: heat generates ground-state atoms from rest of sample
    • Monochromator: isolates flame light (stray) from HC (desired)
    • Detector
    • **Typically used for metals, one element at a time
    • **NOTE - this works by detecting a decrease in light from the source, because it is absorbed by the atom in question
  17. Fluorescence theory, basics of instrumentation
    • Absorbance of monochromatic light is followed by emission of a longer wavelength (lower energy)
    • Two monochromators required (excitation, emission) which increases sens and spec
    • Measured at a 90 degree angle to reduce incident light
  18. Turbidimetry/Nephelometry compare/contrast
    • Both measure scattered light by large particles (eg immune complexes)
    • Scattered light is the same wavelength as incident light
    • Turbidimetry: measures decrease in transmitted light
    • Same geometry as spectophotometer (can use typical analyzer)
    • S:N ratio may be an issue (head-on geometry)
    • Nephelometry: measure increase in off-axis light
  19. Potentiometry - theory, common tests, water analogy
    • Measures electrical potential difference between two electrodes (half cells)
    • unknown cell compared to reference cell (AgCl)
    • Used for pH, pCO2, Ion-selective electrodes (Na+, K+, Cl-, Li+, Ca2+)
    • Analogous to water pressure (PSI)
  20. How is pCO2 measured?
    • Severinghaus electrode (potentiometry)
    • pH electrode in HCO3- buffered solution separated from sample by CO2 permeable membrane
    • CO2 crosses membrane, causes H+ release, lowers pH
    • *NOTE - this test always contains an internal reference electrode
  21. Amperometry - theory, common tests, water analogy
    • Measures current at a fixed applied potential
    • Ox/Red potential applied to solid electrode produces current
    • pO2 (Clark electrode)
    • Analagous to instantaneous water flow (L/min)
  22. Coulometry - theory, common tests, water analogy
    • Measures total charge between electrodes over time
    • "titration where the titrant is electically generated" eg. Ag+ produced until all Cl- is bound in AgCl.  Stops when free Ag+ is detected
    • Chloride meter
    • Analagous to cumulative flow (L)
  23. What are colligative properties? How can they me measured?
    • Properties dependent upon number, not nature
    • Freezing Point depression
    • Vapor pressure depression
    • Osmotic pressure (not used)
    • Boiling point (not practical for clinical)
  24. Molarity vs Molality
    • Molarity = mol/L
    • Molality = mol/kg solvent (measured in lab)
  25. Freezing point depression theory
    • One mol /kg lowers fp 1.86C
    • Lower temp below 0 (supercool)
    • Induce crystallization (vibration)
    • Monitor temp as it slowly rises
    • Temp plateus when sample begins to melt until completely thawed
  26. Vapor point depression theory/Osmalal gap
    • Paper disc soaked w/ sample placed in closed chamber
    • Temp measured when dew point is reached
    • PROBLEM - assumes all vapor is water
    • Osmolal gap: calculated osmolality as a surrogate test for ethanol, isoprop, etc
    • gap = measured (freezing point) - calculated
    • gap > 15 is considered significant
    • VAPOR PRESSURE UNDERESTIMATES osmolality in the presence of alcohols and is not recommended
  27. Why is equivalence so important in an AgAb rxn?
    • Ab excess: all antigenic sites are covered, lattice formation inhibited
    • Equivalence: 2-3 Ab per antigen, producing a lattice (ppt)
    • Ag excess: all antibody sites are saturated (2 ag per Ab), lattice formation inhibited
  28. What analytes are typically measured using immunochemistry?
    Proteins, hormones, drugs
  29. Competitive immunoassay theory, adv/dis
    • Patient sample (Ag) competes with labeled compound (Ag*)
    • Decreasing signal corresponds with increasing analyte
    • Adv: can measure large or small molecules
    • Dis: low [Ab] required, less specific than sandwich, limited dynamic range
  30. Heterogenious vs Homogenous Immunoassays
    • Hetero: free-labeled Ag must be removed
    • RIA, ELISA, coated beads, etc
    • Homo: Separation not required, binding of AgAb alters the label in some way
    • EMIT, CEDIA, etc
  31. Homogeneous Immunoassays (EMIT and CEDIA) basic theory
    • EMIT: hapten is bound to enzyme, Ab is added
    • drug present = Ab binds to free drug and enzyme activity occurs
    • drug absent = Ab binds to enzyme and sterically prevents activity
    • CEDIA: enzyme in 2 recombinable fragments, one fragment has bound hapten, Ab added
    • drug present = Ab binds to free drug, enzyme combines, activity occurs
    • drug absent = Ab binds to one fragment, enzyme cannot recombine
  32. Fluorescence polarization theory and basic instrumentation
    • Excitation light is polarized
    • Free fluorescent ligands rotate quickly and depolarize light
    • Bound fluorescent ligands rotate slowly and continue the polarized emission
    • Emission polarizer only allows detection of polarized light
    • 90 degree angle reading
  33. Sandwich immunometric assay theory, adv/dis
    • Capture Ab bound to solid phase, labeled (free) tracer Ab recognized by system
    • Must wash away unbound tracer Ab
    • Adv: increased specificity, no limit to [Ab] (can drive rxn by increasing), greater dynamic range
    • Dis: can only measure proteins (bivalent Ag), hook effect, interfering Ab, Monovalent epitope
    • hook-effect = antigen excess causes underestimation of [ ]
    • Interfering Abs (mouse or other animal) in sample may give false pos or false neg
    • monovalent epitope - multiple subunits may have varying epitopes and this may alter results depending on which epitope is being monitored (eg. hCG from tumors vs preg)
  34. Chemiluminescence theory, adv
    • Emission of light by chemical rxn (wavelength and # photons per label very specific)
    • Adv: no light source OR monochromator required
    • Dominant automated IA label
  35. Immunometric vs Competitive Later Flow Immunoassays
    • Immunometric: color at test line = positive
    • Test line = anti-analyte Ab
    • Control line - anti-IgG Ab
    • Au conjugate = anti-analyte
    • Competitive: Color at test line = negative
    • Positive sample binds probe Ab preventing color at test line
  36. General chromatography theory
    • Separation using mobile and stationary phases
    • Mobile phase can be altered during test
    • More interaction with solid phase = slower elution
    • More interaction with solid phase = faster elution
  37. Basic explanation of the following types of chromatography - partition, adsorption, ion-exchange, size exclusion, affinity
    • Partition: stationary phase = nonpolar bonded "liquid", mobile phase = solvent or heated gas
    • eg. gas chromatography or HPLC
    • Adsorption: stationary phase = solid w/ non-specific adsorption (eg H-bonding), mobile phase = liquid or gas
    • eg. TLC
    • Ion-Exchange: stationary phase = cationic/anionic, mobile phase = altering pH or ionic strength
    • eg. HPLC (abn hgb, HgbA1c)
    • Size exclusion: stationary phase = polymer "beads" w/ controlled pore size, mobile = ? smaller particles enter and are significantly slowed
    • not used in clinical lab (protein purification)
    • Affinity: stationary phase = f(x) group that specifically attracks coumpound of interest, mobile = take everything except compound of interest (binary)
    • eg. HbA1c, LSD purification
  38. Thin Layer Chromatography mobility calculation
    • mobility defined by Rf
    • Rf = spot migration / solvent migration
    • Rf is characteristic, but not necessarily unique
  39. Gas chromatography instrumentation layout, mobility calculation
    • Injection port (volitilizes sample), column oven (controls temp/affinity for mobile), column, detector (FID = organics, NPD = N & P, MS
    • Separates samples based on volatility as temp increases
    • mobility defined by retention time relative to internal standard (RRT)
    • RRT = RT / RTstd
    • RRT is characteristic, but not necessarily unique
  40. HPLC basics
    • Smaller particles, higher pressure, higher performance
    • Reversed phases: nonpolar stationary, polar mobile
    • Gradient elution by increasing solvent strength (MeOH, MeCN)
    • Detection by UV-Vis, Electrochem, MS
    • *NOTE - besides MS most detectors are highly specific
  41. Automated Analytical Steps
    • Sample ID
    • Sample and reagent pipetting
    • Reaction mixture mixing
    • Incubation/Timing
    • Signal Detection
    • Reaction vessel washing
    • Result computation
    • Result entry
  42. Reagant-Blanked Endpoint vs Sample-Blanked Endpoint vs Blanked Rate rxn
    • RB: Single blank for all samples
    • does not correct for any sample abs (lipemia, ichterus, etc)
    • Only a reading at the endpoint
    • SB: Requires 2 reagents, rgt1 is identical to rgt2 except missing a crucial component
    • rgt1 is added to allow "side rxns" to complete
    • blank is taken at this time (with all components present)
    • rgt2 is added and rxn occurs
    • second abs is taken at the completion of this rxn and compared to the blank
    • Rate: blank is same as SB, except several abs are taken as rxn progresses (linear phase)
    • should be a line, and is ΔA/ΔT (slope is related to concentration)
  43. Linear assay calibration vs non-linear assay calibration w/ examples of each type of assay
    • Linear: typically 2-point (zero and midpoint)
    • eg. photometric, electrochemical
    • Non-linear: multi-point calibration w/ curve-fitting algorithsms
    • master curve shape determined by mfgr
    • 2 calibrator points assign position of [ ] / response plot
    • eg. immunoassays, turbidimetric
  44. Determine unknown concentration using linear calibration curve
    • slope = (y2 - y1)/(x2 - x1)
    • [ ] = (Abs - Abs0)/slope
Card Set
CLSChem - 01 - Instrumentation
CLSChem - 01 - Instrumentation