Principles of Instrumentation

• Laboratories employ diverse methodologies for testing various analytes, with understanding these methods providing crucial context for interpreting lab results.
• Lab methods are rooted in biology, chemistry, and physics, involving step-by-step procedures.

Principles of Instrumentation

• Chemical tests rely on the chemical reaction of substrates, enzymes, and cofactors.
• Methodology is used for detecting the endpoint of the reaction.
• Automation synchronizes every aspect of testing.

Spectrophotometry

• Spectrophotometry is a cornerstone of automated clinical chemistry, relying on two key principles: substances absorb light at unique wavelengths, and the amount of light absorbed is proportional to the substance present.
• The spectrophotometer gauges light absorbance in chemical reactions, such as endpoint colorimetric, endpoint enzymatic, and kinetic reactions.

Endpoint Colorimetric Spectrophotometry

• Involves reactions where a colored product (chromogen) forms and is measured by its light absorbance.
• The Jaffe reaction for creatinine analysis exemplifies this method.

Endpoint Enzymatic Spectrophotometry

• Employs an enzyme to catalyze reactions, often resulting in a coenzyme that strongly absorbs light at lower wavelengths.
• The hexokinase method for glucose measurement is an example.

Kinetic Spectrophotometry

• Measures an active enzyme's ability to catalyze or accelerate a chemical reaction, converting substrate to product, the enzyme isn't consumed
• Measuring enzyme levels provides clinically relevant results and is preferred for enzyme testing.
• Enzyme activity is indicated by the change in absorbance per minute.
• Multiple absorbance measurements are taken to determine the average change in absorbance per minute.

Zero-Order Kinetics

• Provides the most accurate measure of enzyme activity because all active sites are filled with substrate.
• Substrate concentration is in excess, and conditions are optimal.
• Confirmed when the change in absorbance per minute is constant, despite a consistent linear increase or decrease in product absorbance.

Analysis with Electrochemistry

• It measures ions in solution via their electrical properties, and these ions are called electrolytes.
• Electrolytes include ionized sodium (Na), potassium (K), chloride (Cl–), bicarbonate (HCO3–), and calcium (Ca2).
• It also involves producing electrons or ions in a chemical reaction from nonionic analytes, such as oxygen or urea nitrogen.

Electrophoresis

• Electrophoresis is the movement of charged particles in an electrical field.
• Separation of charged particles occurs at different rates.
• Employed to separate molecules before identification.
• Serum proteins at pH 8.6 become negatively charged and migrate to an anode in a buffer.

Chromatography

• Chromatography is a technique for separating molecules based on their distribution between two phases.
• Separation occurs on a stationary phase influenced by a mobile phase; molecules are separated based on their solubility and interaction with these phases.
• Two main types exist: plane and column chromatography.

Immunological Techniques

• Immunoassay is an analytical method using antibodies or antigens to measure specific chemicals or analytes.
• Immunoassay results are crucial in health decisions, including virus detection, drug monitoring, and screening for drugs of abuse.

Enzyme-Labeled Immunoassay (ELISA)

• An enzyme commonly labels the immunoassay reaction.
• After the antibody-antigen reaction, the product is determined by measuring the enzyme label.
• Substrate is added, and the product of the enzyme-activated reaction is measured via spectrophotometry.

Chemical Analyzer's Automation

• Automation involves using machines to perform lab testing steps with minimal human intervention.
• Automation handles specimen identification, volume measuring, pretreatment, reagent volume measuring, mixing, incubation, reaction timing, analysis, calculations, and result presentation.
• It may also include specimen transport and reagent storage.

Calibration

• Calibration is essential for ensuring accurate reporting of patient results.
• It links the analytical signal with the analyte concentration in body fluids.
• Calibration uses a series of calibrators with known analyte concentrations.
• The instrument is programmed with each analyte's concentration and adjusts the signal to match the given values.
• Plotting signal versus analyte concentration creates a calibration curve, establishing the relationship between the analyte and the signal.
• A calibration curve shows a linear signal increase with concentration from the limit of detection (LOD) to the limit of linearity (LOL), defining the analytical measurement range (AMR). Beyond LOL, the line becomes non-linear.