MUST TO KNOW IN CLINICAL CHEMISTRY
(From CC by Rodriguez)
Quality Control
Practicability Method is easily repeated
Reliability Maintain accuracy and precision
Intralab/Interlab QC Daily monitoring of accuracy and precision
Interlab/External QC Proficiency testing (Reference lab)
Long-term accuracy
Difference of >2: not in agreement w/ other lab
QC materials Available for a min. of 1 yr
Bovine control materials Preferred (Human: biohazard)
Not for immunochem, dye-binding and bilirubin
Matrix effect Improper product manufacturing
Unpurified analyte
Altered protein
Precision study First step in method evaluation
Nonlab. personnel 29% of errors (lab results)
SD Dispersion of values from the mean
CV Index of precision
Relative magnitude of variability (%)
Variance SD2
Measure of variability
Inferential statistics Compare means or SD of 2 groups of data
T-test Means of 2 groups of data
F-test SD of 2 groups of data
Cumulative Sum Graph V-mask
(CUSUM) Earliest indication of systematic errors (trend)
Youden/Twin Plot Compare results obtained from diff. lab
Shewhart Levey-Jennings Graphic representation of the acceptable limits of variation
Chart
Trend Gradual loss of reliability
Cause: Deterioration of reagents (Systematic error)
Shift Values: one side or either side of the mean
Cause: Improper calibration (Systematic error)
Outliers Values: far from the main set of values
Highly deviating values
Random or systematic errors
Kurtosis Degree of flatness or sharpness
Precision Random error
Accuracy Systematic error
Random error Causes:
(Imprecision; -Mislabeling
Indeterminate) -Pipetting error
-Improper mixing of sample and reagents
-Voltage/Temperature fluctuation
-Dirty optics
Parameters: SD and CV
Systematic error Causes:
(Inaccuracy/Determinate) -Improper calibration
-Deterioration of reagents
-Contaminated solution
-Sample instability/unstable reagent blanks
lec.mt 04 |Page | 1
, -Diminishing lamp power
-Incorrect sample and reagent volume
Parameter: Mean
Multirule Shewhart Control rules + Control chart
procedure
Test method Westgard: at least 40 samples
Reference method Westgard: preferably 100 samples
Analytical Run Control and patient specimens assayed, evaluated, and report together
Physiologic Limit Referred to as absurd value
POCT Performed by nonlab personnel
Quality Assurance Tripod:
Program development
Assessment and monitoring
Quality improvement
Quality Patient Care Test request forms, clear instruction for patient prep., specimen handling…
Reference Range/ Interval At least 120 individuals should be tested in each age and sex category
Range/ Reference Values
Analytical Methods
Wavelength Distance bet 2 successive peaks (nm)
Lower frequency = Longer wavelength (Ex. Red)
Higher frequency = Shorter wavelength (Ex. Violet)
Spectrophotometric meas. Meas. light intensity in a narrower wavelength
Photometric measurement Meas. light intensity w/o consideration of wavelength
Multiple wavelength (uses filter only)
LASER Light Amplification by Stimulated Emission of Radiation
Light source for spectrophotometry
Visible region Tungsten light bulb
Mercury arc
UV Deuterium lamp
Mercury arc
Xenon lamp
Hydrogen lamp
IR Merst glower
Globar (Silicone carbide)
Stray light Wavelength outside the band
Most common cause of loss of linearity
Diffraction gratings Most commonly used monochromator
Cutting grooves
Prisms Rotatable
Nickel sulfate Prevents stray light
Cutoff filter Anti-stray light
Bandpass ½ peak transmittance
Alumina silica glass cuvet Most commonly used cuvet
Quartz/plastic cuvet UV
Borosilicate glass cuvet Strong bases
Photodetector Converts transmitted light into photoelectric energy
Barrier layer cell/ Simplest detector
photocell/ photovoltaic cell No external voltage
For filter photometers
Phototube Contains anode and cathode
Req external voltage
Photomultiplier tube Most common type
lec.mt 04 |Page | 2
, Most sensitive
UV and visible region
Galvanometer/Ammeter Meter or read-out device
Absorbance A = abc (a = absorptivity; b = length of light (1cm); c = concentration)
A = 2 – log%T
Double beam spectro. Splits monochromatic light into two components:
One beam sample
One beam reference soln or blank (corrects for variation in light source
intensity)
Double-beam in space 2 photodetectors (sample beam and reference beam)
Double-beam in time 1 photodetector
Monochromatic light sample cuvet and reference cuvet
Dydimium filter 600 nm
Holmium oxide filter 360 nm
Reagent blank Color of reagents
Sample blank Optical interference (Hgb)
FEP Meas. light emitted by a single atom burned in a flame
Principle: Excitation
Lt. source and cuvette: Flame
For excited ions (Na+, K+)
Cesium and Lithium Internal standards (FEP)
Correct variations in flame
Lithium Preferred internal std
Potent antidepressant
AAS Meas. light absorbed by atoms dissociated by heat
Principle: Dissociation (unionized, unexcited, ground state)
Lt. source: Hollow-cathode lamp
For unexcited trace metals (Ca++ and Mg++)
More sensitive than FEP
Atomizer (nebulizer) Convert ions atoms
Chopper Modulate the light source
Lanthanum/Strontium Complex with phosphate
chloride Avoid calcium interference
Volumetric (Titrimetric) Unknown sample is made to react with a known solution in the presence of an
indicator
Turbidimetry Light blocked
Meas. abundant large particles (Proteins)
Depend on specimen concentration and particle size
Nephelometry Meas. amt of Ag-Ab complexes
Scattered light
Depends on wavelength and particle size
Electrophoresis Migration of charged particles in an electric field
Iontophoresis Migration of small charged ions
Zone electrophoresis Migration of charged macromolecules
Endosmosis Movement of buffer ions and solvent relative to the fixed support
Ex: gamma globulins
Cellulose acetate Molecular size
Agarose gel Electrical charge
Polyacrylamide gel Charge and molecular size
20 fractions (ex. isoenzymes)
Electrophoretic mobility Directly proportional to net charge
Inversely proportional to molecular size & viscosity of the supporting medium
lec.mt 04 |Page | 3
, Isoelectric focusing Molecules migrate through a pH gradient
pH = pI
For isoenzymes: same size, different charge
Densitometry Scan & quantitate electrophoretic pattern
Capillary electrophoresis Electro-osmotic flow
Southern blot DNA
Northern blot RNA
Western blot Proteins
Chromatography Separation by specific differences in physical-chemical characteristics of the
different constituents
Paper chromatography Fractionation of sugar and amino acid
Sorbent: Whatman paper
TLC Screening: Drugs
Retention factor (Rf) value Relative distance of migration from the point of application
Rf = Distance leading edge of component moves
Total distance solvent front moves
Gas chromatography Separation of steroids, barbiturates, blood, alcohol, and lipids
Volatile compounds
Specimens vaporized
Mobile phase: Inert gases
Gas Solid chromatography Differences in absorption at the solid phase surfaces
Gas Liquid chromatography Differences in solute partitioning between the gaseous mobile phase and the
liquid stationary phase
Mass Spectrometry Fragmentation and ionization
GC-MS Gold standard for drug testing
MS/MS Detect 20 inborn errors of metabolism from a single blood spot
HPLC Most widely used liquid chromatography
Fractionation of drugs, hormones, lipids, carbohydrates and proteins
Hydrophilic gel Gel filtration
Separation of enzymes, antibodies and proteins
Ex: Dextran and agarose
Hydrophobic gel Gel permeation
Separation of triglyceride and fatty acid
Ex: Sephadex
Ion exchange Separation depends on the sign and ionic charge density
chromatography
Partition chromatography Based on relative solubility in an organic solvent (nonpolar) and an aqueous
solvent (polar)
Affinity chromatography For lipoproteins, CHO and glycated hemoglobins
Adsorption Based on differences between the adsorption and desorption of solutes at the
chromatography surfaces of a solid particle
Fluorometry/Molecular Det. amt. of lt. emitted by a molecule after excitation by electromagnetic
Luminescence Spectro. radiation
Lt. sources: Mercury arc and Xenon lamp (UV)
Lt. detector: Photomultiplier tubes
2 monochromators:
Primary filter – selects wavelength absorbed by the solution to be measured
Secondary filter – prevents incident light from striking the photodetector
Sensitivity: 1000x than spectro
Quenching Major disadvantage of fluorometry
pH and temperature changes, chemical contaminants, UVL changes
lec.mt 04 |Page | 4
(From CC by Rodriguez)
Quality Control
Practicability Method is easily repeated
Reliability Maintain accuracy and precision
Intralab/Interlab QC Daily monitoring of accuracy and precision
Interlab/External QC Proficiency testing (Reference lab)
Long-term accuracy
Difference of >2: not in agreement w/ other lab
QC materials Available for a min. of 1 yr
Bovine control materials Preferred (Human: biohazard)
Not for immunochem, dye-binding and bilirubin
Matrix effect Improper product manufacturing
Unpurified analyte
Altered protein
Precision study First step in method evaluation
Nonlab. personnel 29% of errors (lab results)
SD Dispersion of values from the mean
CV Index of precision
Relative magnitude of variability (%)
Variance SD2
Measure of variability
Inferential statistics Compare means or SD of 2 groups of data
T-test Means of 2 groups of data
F-test SD of 2 groups of data
Cumulative Sum Graph V-mask
(CUSUM) Earliest indication of systematic errors (trend)
Youden/Twin Plot Compare results obtained from diff. lab
Shewhart Levey-Jennings Graphic representation of the acceptable limits of variation
Chart
Trend Gradual loss of reliability
Cause: Deterioration of reagents (Systematic error)
Shift Values: one side or either side of the mean
Cause: Improper calibration (Systematic error)
Outliers Values: far from the main set of values
Highly deviating values
Random or systematic errors
Kurtosis Degree of flatness or sharpness
Precision Random error
Accuracy Systematic error
Random error Causes:
(Imprecision; -Mislabeling
Indeterminate) -Pipetting error
-Improper mixing of sample and reagents
-Voltage/Temperature fluctuation
-Dirty optics
Parameters: SD and CV
Systematic error Causes:
(Inaccuracy/Determinate) -Improper calibration
-Deterioration of reagents
-Contaminated solution
-Sample instability/unstable reagent blanks
lec.mt 04 |Page | 1
, -Diminishing lamp power
-Incorrect sample and reagent volume
Parameter: Mean
Multirule Shewhart Control rules + Control chart
procedure
Test method Westgard: at least 40 samples
Reference method Westgard: preferably 100 samples
Analytical Run Control and patient specimens assayed, evaluated, and report together
Physiologic Limit Referred to as absurd value
POCT Performed by nonlab personnel
Quality Assurance Tripod:
Program development
Assessment and monitoring
Quality improvement
Quality Patient Care Test request forms, clear instruction for patient prep., specimen handling…
Reference Range/ Interval At least 120 individuals should be tested in each age and sex category
Range/ Reference Values
Analytical Methods
Wavelength Distance bet 2 successive peaks (nm)
Lower frequency = Longer wavelength (Ex. Red)
Higher frequency = Shorter wavelength (Ex. Violet)
Spectrophotometric meas. Meas. light intensity in a narrower wavelength
Photometric measurement Meas. light intensity w/o consideration of wavelength
Multiple wavelength (uses filter only)
LASER Light Amplification by Stimulated Emission of Radiation
Light source for spectrophotometry
Visible region Tungsten light bulb
Mercury arc
UV Deuterium lamp
Mercury arc
Xenon lamp
Hydrogen lamp
IR Merst glower
Globar (Silicone carbide)
Stray light Wavelength outside the band
Most common cause of loss of linearity
Diffraction gratings Most commonly used monochromator
Cutting grooves
Prisms Rotatable
Nickel sulfate Prevents stray light
Cutoff filter Anti-stray light
Bandpass ½ peak transmittance
Alumina silica glass cuvet Most commonly used cuvet
Quartz/plastic cuvet UV
Borosilicate glass cuvet Strong bases
Photodetector Converts transmitted light into photoelectric energy
Barrier layer cell/ Simplest detector
photocell/ photovoltaic cell No external voltage
For filter photometers
Phototube Contains anode and cathode
Req external voltage
Photomultiplier tube Most common type
lec.mt 04 |Page | 2
, Most sensitive
UV and visible region
Galvanometer/Ammeter Meter or read-out device
Absorbance A = abc (a = absorptivity; b = length of light (1cm); c = concentration)
A = 2 – log%T
Double beam spectro. Splits monochromatic light into two components:
One beam sample
One beam reference soln or blank (corrects for variation in light source
intensity)
Double-beam in space 2 photodetectors (sample beam and reference beam)
Double-beam in time 1 photodetector
Monochromatic light sample cuvet and reference cuvet
Dydimium filter 600 nm
Holmium oxide filter 360 nm
Reagent blank Color of reagents
Sample blank Optical interference (Hgb)
FEP Meas. light emitted by a single atom burned in a flame
Principle: Excitation
Lt. source and cuvette: Flame
For excited ions (Na+, K+)
Cesium and Lithium Internal standards (FEP)
Correct variations in flame
Lithium Preferred internal std
Potent antidepressant
AAS Meas. light absorbed by atoms dissociated by heat
Principle: Dissociation (unionized, unexcited, ground state)
Lt. source: Hollow-cathode lamp
For unexcited trace metals (Ca++ and Mg++)
More sensitive than FEP
Atomizer (nebulizer) Convert ions atoms
Chopper Modulate the light source
Lanthanum/Strontium Complex with phosphate
chloride Avoid calcium interference
Volumetric (Titrimetric) Unknown sample is made to react with a known solution in the presence of an
indicator
Turbidimetry Light blocked
Meas. abundant large particles (Proteins)
Depend on specimen concentration and particle size
Nephelometry Meas. amt of Ag-Ab complexes
Scattered light
Depends on wavelength and particle size
Electrophoresis Migration of charged particles in an electric field
Iontophoresis Migration of small charged ions
Zone electrophoresis Migration of charged macromolecules
Endosmosis Movement of buffer ions and solvent relative to the fixed support
Ex: gamma globulins
Cellulose acetate Molecular size
Agarose gel Electrical charge
Polyacrylamide gel Charge and molecular size
20 fractions (ex. isoenzymes)
Electrophoretic mobility Directly proportional to net charge
Inversely proportional to molecular size & viscosity of the supporting medium
lec.mt 04 |Page | 3
, Isoelectric focusing Molecules migrate through a pH gradient
pH = pI
For isoenzymes: same size, different charge
Densitometry Scan & quantitate electrophoretic pattern
Capillary electrophoresis Electro-osmotic flow
Southern blot DNA
Northern blot RNA
Western blot Proteins
Chromatography Separation by specific differences in physical-chemical characteristics of the
different constituents
Paper chromatography Fractionation of sugar and amino acid
Sorbent: Whatman paper
TLC Screening: Drugs
Retention factor (Rf) value Relative distance of migration from the point of application
Rf = Distance leading edge of component moves
Total distance solvent front moves
Gas chromatography Separation of steroids, barbiturates, blood, alcohol, and lipids
Volatile compounds
Specimens vaporized
Mobile phase: Inert gases
Gas Solid chromatography Differences in absorption at the solid phase surfaces
Gas Liquid chromatography Differences in solute partitioning between the gaseous mobile phase and the
liquid stationary phase
Mass Spectrometry Fragmentation and ionization
GC-MS Gold standard for drug testing
MS/MS Detect 20 inborn errors of metabolism from a single blood spot
HPLC Most widely used liquid chromatography
Fractionation of drugs, hormones, lipids, carbohydrates and proteins
Hydrophilic gel Gel filtration
Separation of enzymes, antibodies and proteins
Ex: Dextran and agarose
Hydrophobic gel Gel permeation
Separation of triglyceride and fatty acid
Ex: Sephadex
Ion exchange Separation depends on the sign and ionic charge density
chromatography
Partition chromatography Based on relative solubility in an organic solvent (nonpolar) and an aqueous
solvent (polar)
Affinity chromatography For lipoproteins, CHO and glycated hemoglobins
Adsorption Based on differences between the adsorption and desorption of solutes at the
chromatography surfaces of a solid particle
Fluorometry/Molecular Det. amt. of lt. emitted by a molecule after excitation by electromagnetic
Luminescence Spectro. radiation
Lt. sources: Mercury arc and Xenon lamp (UV)
Lt. detector: Photomultiplier tubes
2 monochromators:
Primary filter – selects wavelength absorbed by the solution to be measured
Secondary filter – prevents incident light from striking the photodetector
Sensitivity: 1000x than spectro
Quenching Major disadvantage of fluorometry
pH and temperature changes, chemical contaminants, UVL changes
lec.mt 04 |Page | 4
