Analytical Instrumentation: EasyRA Chemistry Analyzer • Serum Protein Electrophoresis (SPE) • Gas Chromatography (GC)

Automation in Clinical Chemistry

Automation allows large volumes of lab tests to be performed with high precision and minimal manual work. Includes three stages: preanalytic (sample prep), analytic (measurement), and postanalytic (reporting).

Three Stages of Analytical Process

Preanalytic: sample collection and labeling. Analytic: measurement and analysis (most automated). Postanalytic: validation, documentation, reporting.

Advantages of Automation

Increased analytical speed, reduced human error, smaller reagent/sample use, lower cost, consistent turnaround.

Continuous Flow Analyzer

Pumps liquids through continuous tubing separated by air bubbles; rapid, consistent analysis but high reagent use and contamination risk.

Centrifugal Analyzer

Mixes and measures reactions by spinning samples; compact and efficient but performs one test at a time.

Discrete Analyzer

Uses individual cuvettes for isolated reactions; allows random access and multiple tests but higher disposable costs.

Random Access Analyzer

Can perform any test on any sample anytime; supports both STAT and routine testing simultaneously.

Open Reagent System

Compatible with multiple suppliers; more flexible and cost-effective.

Closed Reagent System

Uses manufacturer-specific reagents (EasyRA type); ensures consistency and quality control.

RFID Wedges

Store reagent identity, lot number, expiration, calibration, and remaining tests to prevent misuse and maintain traceability.

Reagent/Sample Area

Holds carousels for organized access; includes RFID reader.

Transfer Arm/Probe

Aspirates, dispenses, mixes (via air injection), and self-cleans after each transfer.

Reaction Area

Contains cuvette carousel and photometer for reaction monitoring.

Photometer

Uses Xenon Flash Lamp and interference filters (340-700 nm) to read absorbance.

Heated Air Bath

Keeps reactions at 37 °C ± 0.25 °C for consistency.

Fluidics Drawer

Holds diluent and waste bottles; manages flushing and cleaning cycles.

Common EasyRA Tests

Glucose, electrolytes (Na⁺, K⁺, Cl⁻, Li⁺), total protein, albumin, cholesterol, triglycerides, and liver enzymes (ALT, AST, ALP).

Acceptable Sample Types

Serum, plasma, diluted whole blood, and urine.

Daily Maintenance

Clean probe/ISE, inspect dilutor and waste lines, prime solutions.

Weekly Maintenance

Check photometer precision and pipette accuracy.

Quality Control Practices

Run two QC levels per analyte daily, use Levey-Jennings plots, and external programs (CAP).

Detection Principles

Photometry: absorbance → concentration; Potentiometry: ion-selective electrodes measure voltage vs. Ag/AgCl reference.

Purpose of Electrophoresis

Separates charged biomolecules (proteins, DNA) by applying an electric field across a gel; smaller, more negative proteins move faster toward the anode.

Gel

Porous matrix (agarose or polyacrylamide) for separation.

Comb

Forms sample wells before gel sets.

Buffer

Maintains pH and conducts current.

Wells

Hold samples for loading.

Factors Affecting Migration

Net charge, size, shape, and isoelectric point (pI).

Albumin Fraction

Most abundant plasma protein; decreases in liver/kidney disease.

α₁-Globulins

Include α₁-antitrypsin and α₁-acid glycoprotein; increase with inflammation.

α₂-Globulins

Include haptoglobin and ceruloplasmin; decrease in hemolysis.

β-Globulins

Include transferrin and complement proteins; increase in iron deficiency.

γ-Globulins

Include immunoglobulins (IgG, IgM); elevated in chronic infections or multiple myeloma (M-spike).

SDS-PAGE Principle

Separates proteins by molecular weight only; SDS gives uniform negative charge, β-mercaptoethanol breaks disulfide bonds.

Stacking Gel (pH 6.8)

Concentrates proteins into tight bands before separation.

Resolving Gel (pH 8.8)

Separates proteins by size during run.

Role of SDS

Denatures proteins and provides uniform negative charge.

Role of β-Mercaptoethanol

Breaks disulfide bonds between subunits.

Appearance of Monoclonal Gammopathy

Narrow, dark γ-region band (M-spike) on SPE gel.

Purpose of GC

Separates volatile compounds by distributing them between a gas mobile phase and stationary phase; separation based on boiling point, vapor pressure, and polarity.

Xenon Lamp Advantage

Provides stable, intense light across wide wavelength range; more reliable than tungsten.

Carryover Prevention

Probe automatically rinses with diluent between each sample transfer.

Cuvette Carousel + Photometer Interaction

Carousel rotates cuvettes past optical sensor for timed absorbance readings (kinetic assays).

Buffer Role in Electrophoresis

Maintains electrical conductivity and pH stability during run.

Stacking vs. Resolving Gel

Stacking compresses bands; resolving separates by size.

Importance of Electrical Contact

Ensures current flows uniformly across gel for consistent migration.

Causes of Smeared Bands

Overloading, degraded samples, buffer/pH issues, or poor contact.

Carrier Gas Purpose in GC

Acts as mobile phase to move analytes through column.

Heated Injection Port Reason

Must be hotter than analyte's boiling point to vaporize sample instantly.

Stationary Phase Role

Determines separation based on analyte interactions (polarity/affinity).

FID vs. TCD Comparison

FID: sensitive to organics but destructive. TCD: universal, non-destructive, lower sensitivity.

Retention Time Variability

Influenced by temperature, flow rate, column length, and stationary phase properties.