|
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 (CUSUM) |
V-mask Earliest indication of
systematic errors (trend) |
|
Youden/Twin Plot |
Compare results obtained from
diff. lab |
|
Shewhart Levey-Jennings Chart |
Graphic representation of the
acceptable limits of variation |
|
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 (Imprecision; Indeterminate) |
Causes: -Mislabeling -Pipetting error -Improper mixing of sample and
reagents -Voltage/Temperature
fluctuation -Dirty optics Parameters: SD and CV |
|
Systematic error (Inaccuracy/Determinate) |
Causes: -Improper calibration -Deterioration of reagents -Contaminated solution -Sample instability/unstable
reagent blanks -Diminishing lamp power -Incorrect sample and reagent
volume Parameter: Mean |
|
Multirule Shewhart procedure |
Control rules + Control chart |
|
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 Range/ Reference
Values |
At least 120 individuals should
be tested in each age and sex category |
|
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/ photocell/ photovoltaic
cell |
Simplest detector No external voltage For filter photometers |
|
Phototube |
Contains anode and cathode Req external voltage |
|
Photomultiplier tube |
Most common type 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 à sample One beam à 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 à 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 à atoms |
|
Chopper |
Modulate the light source |
|
Lanthanum/Strontium chloride |
Complex with phosphate 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 |
|
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 à 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 chromatography |
Separation depends on the sign
and ionic charge density |
|
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 chromatography |
Based on differences between
the adsorption and desorption of solutes at the surfaces of a solid particle |
|
Fluorometry/Molecular Luminescence Spectro. |
Det. amt. of lt. emitted by a
molecule after excitation by electromagnetic 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 |
|
Instrumentation |
|
|
Borosilicate glasswares |
For heating and sterilization Ex: Pyrex and Kimax |
|
Boron-free/Soft glasswares |
High resistance to alkali |
|
Corex (Corning) |
Special alumina-silicate glass Strengthened chemically than
thermally 6x stronger than borosilicate |
|
Vycor (Corning) |
For high thermal, drastic heat
and shock Can be heated to 900OC |
|
Flint glass |
Soda-lime glass + Calcium,
Silicon, Sodium oxides Easy to melt For making disposable
glasswares |
|
TD: To deliver |
Exact amount |
|
TC: To contain |
Does not disperse the exact
volume |
|
Blowout |
w/ etched rings on top of pipet |
|
Self-draining |
w/ o etched rings Drain by gravity |
|
Transfer pipet |
Volumetric: for non-viscous
fluid; self-draining Ostwald folin: for viscous
fluid; w/ etched ring Pasteur: w/o consideration of a
specific volume Automatic macro-/micropipets |
|
Graduated or measuring pipet |
Serological: w/ graduations to
the tip (blowout) Mohr: w/o graduations to the
tip (self-draining) Bacteriologic Ball, Kolmer and Kahn Micropipettes: <1 mL |
|
Micropipettes |
TC pipets: Sahli-Hellige pipet Lang-Levy pipet RBC and WBC pipets Kirk and Overflow pipets |
|
Air displacement pipet |
Piston: suction Disposable tip |
|
Positive displacement pipet |
Piston à barrel (like a hypodermic
syringe) |
|
Dispenser/Dilutor pipet |
Liquid: common reservoir à dispense repeatedly |
|
Distilled H2O |
Calibrating medium for TD
pipettes |
|
Mercury |
Calibrating medium for TC
pipettes |
|
Acid dichromate (H2SO4 + K2Cr2O4) |
Cleaning solution for
glasswares |
|
Continuous flow analyzer |
Common reaction vessel Air bubbles: separates and
cleans Glass coil: mix Examples: “STS” Simultaneous Multiple Analyzer
(SMA) Technicon Autoanalyzer II SMAC |
|
Centrifugal analyzer |
Acceleration and deceleration
of the rotor Advantage: Batch analysis Examples: “RICC” Cobas-Bio (Roche) IL Monarch CentrifiChem RotoChem |
|
Discrete Analyzer |
Most popular Req. vol: 2-6 μL Uses positive-displacement
pipets Run
multiple-tests-one-sample-at-a-time Random access capability (STAT) Examples: Vitros Dimension Dade Beckman ASTRA System (4 &
8) Hitachi Bayer Advia Roche Cobas Integra 800 Roche Analytics P Module Automated Clinical Analyzer
(ACA) Star (Dade) Dupont ACA Abbott ABA-100 Bichromatic
Analyzer ABA-200 VP Analyzer American Monitor KDA Olympus Demand |
|
Thin-Film Analyzers (Dry slide technology) |
4 or 5 layers: -Spreading layer -Scavenger layer - Ascorbate
Oxidase -Reagent layer -Indicator layer -Support layer Colored reaction à Reflectance spectrophotometry Examples: “KV2(75)” Kodak Ektachem Vitros 750XRC Vitros 550XRC |
|
Carry over |
Transport of quantity of
analyte or rgt from one specimen rxn into another, and contaminating a
subsequent one |
|
Batch testing |
All samples loaded at the same
time Single test is conducted on
each sample |
|
Parallel testing |
One specimen More than one test is analyzed |
|
Random access testing |
Any sample Any test Any sequence STAT |
|
Sequential testing |
Multiple tests analyzed one
after another on a given specimen |
|
Open reagent system |
System other than
manufacturer’s reagents can be utilized for measurement |
|
Closed reagent system |
The operator can only use the
manufacturer’s reagents |
|
Patient
Preparation |
|
|
Exercise |
Increased: GU2FT C2L3A5P2 GH Urea Urinary protein (Proteinuria) Fatty acid Testosterone CPK (muscle) Creatinine (muscle) Lactate LH LD (muscle) ACP Aldolase (muscle) AST ALT Ammonia Pyruvate Prolactin Decreased: Glucose |
|
Fist clenching |
Increased: “LPP” Lactate Potassium Phosphate |
|
Fasting |
8-16 hours: Glucose Lipids Lipoproteins Increased: Bilirubin (48 hours) Triglyceride (72 hours) |
|
Basal state collection |
Glucose Cholesterol Triglyceride Electrolytes |
|
Diet |
Increased: “GLUC2H” Glucose Lipids Urea (High protein diet) Caffeine: increases glucose Catecholamines 5-HIAA (From Serotonin) |
|
Turbidity/Lactescence |
Triglyceride >400mg/dL |
|
Icterisia |
Bilirubin: 25.2 mg/dL |
|
Icteric samples |
Interfere with: "TACGu” Total Protein Albumin Cholesterol Glucose |
|
Upright/supine (lying) position |
Preferred position Patient should be seated/supine
at least 20 mins before blood collection to prevent hemodilution or
hemoconcentration |
|
Supine à Sitting/Standing |
Vasoconstriction à Reduced plasma volume Increased: “ECA” Enzymes Calcium Albumin |
|
Sitting à Supine |
Hemoconcentration Increased: “P(u)BLIC” Proteins BUN Lipids Iron Calcium |
|
Standing à Supine |
Hemodilution Decreased: “TLC” Triglycerides Lipoproteins Cholesterol |
|
Prolonged standing |
Increased: K+ (muscles) |
|
Prolonged bedrest |
Decreased: Albumin (Fluid retention) |
|
Tourniquet |
Recommended: 1 minute
application |
|
Prolonged tourniquet app. |
Hemoconcentration Anaerobiosis Increased: “C2LEA2K” Calcium Cholesterol Lactate Enzymes Ammonia Albumin K+ |
|
Tobacco smoking (Nicotine) |
Increased: “TUNG2C3” Triglycerides Urea Nonesterified fatty acid Glucose GH Catecholamines Cortisol Cholesterol |
|
Alcohol ingestion |
Increased: “THUG” Triglycerides Hypoglycemia (chronic
alcoholism) Uric acid/Urates GGT |
|
Ammonia |
Increases by 100-200μg/L/cigar |
|
Stress (anxiety) |
Increased: “LAGIC” Lactate Albumin Glucose Insulin Cholesterol |
|
Drugs |
Medications affecting plasma
volume can affect protein, BUN, iron, calcium Hepatotoxic drugs: increased
liver function enzymes Diuretics: decreased sodium and
potassium |
|
Diurnal variation |
"CA3PI2TG” Cortisol ACTH ACP Aldosterone Prolactin Iron Insulin Thyroxine GH |
|
Specimen
Collection and Handling |
|
|
Sleeping patients |
Must be awakened before blood
collection |
|
Unconscious patients |
Ask nurse or relative Identification bracelet |
|
Venipuncture |
Median Cubital (1st)
à Cephalic (2nd) à Basilic (3rd) |
|
Tourniquet |
Velcro or Seraket type 3-4 inches above the site Not exceed 1 minute |
|
Needle |
Bevel up 15-30O angle Length: 1 or 1.5 inch
(Butterfly needle: ½ to ¾ inch) |
|
After blood collection |
Cotton à site Apply pressure for 3-5 minutes |
|
BP cuff as tourniquet |
Inflate to 60 mmHg |
|
Benzalkonium chloride (Zephiran) |
Disinfectant for ethanol
testing Dilution – 1:750 |
|
IV line on both arms |
Discontinue IV for 2 minutes Collect sample below the IV
site Initial sample (5mL) à discard |
|
IV fluid contamination |
Increased: Glucose (10% contam. w/ 5%
dextrose à increased bld glucose by 500
mg/dL) Chloride Potassium Sodium Decreased: Urea Creatinine |
|
Renin blood level |
Collected after a 3-day diet,
from a peripheral vein |
|
Basal state collection |
Early morning blood collection 12 hours after the last
ingestion of food |
|
Lancet |
1.75mm: preferred length to
avoid penetrating the bone |
