5. Automation

Automation Overview

  • Clinical laboratories generate about 80% of the information physicians rely on to make crucial treatment decisions.

  • Patients, physicians, and healthcare workers expect test results that are accurate and done quickly.

  • Expectations of turnaround times (TAT) have been key to the development of lab equipment as well as Point of Care Testing (POCT) devices.

  • Major advances in clinical lab:

    • Miniaturization

    • Linking patient results across provinces (Electronic Medical Record shared results)

    • Automated instruments of all sizes (both in robotics and IT)

Benefits of Automation

  • Reduction in Medical Errors: Critical errors still occur in mislabeling or misidentification.

  • Reduced Specimen Sample Volume: 200 µL or less.

  • Increase in Accuracy and Precision: Automation enhances test precision.

  • Improved Safety for Lab Staff: Reduces risks from sample handling, including eliminating the need for cap removal.

  • Faster Turnaround Time: Automation allows for quicker processing of test results.

  • Alleviation of Shortages: Automation helps counteract the impending shortage of skilled laboratory staff.

Automation Processes

  • Automation processes include special design, compatibility, and integration of automated clinical systems.

  • Standardized analyzers include devices for sampling patient specimens or other samples to be tested (such as blanks, controls, and standard solutions).

  • Analyzers possess features to add specimens to reagents in the correct sequence and incubation modules for specific reactions.

  • Measurement devices: Often utilize photometric technology to quantify the extent of reactions.

  • Recording mechanism: Provides a final reading or permanent record of results and interfaces with a computer.

  • Most analyzers can process a variety of specimens, increasing efficiency and reducing human error related to repetition and manipulations (e.g., pipetting errors).

Automated Analyzers

  • Choice of instrument depends on:

    • Volume of determination in the lab

    • Type of data profile generated

    • Level of staffing

    • Initial cost and maintenance

    • Operational costs and time required for each analysis.

  • Designed to perform frequently ordered tests. In large volume hospitals and reference centers, completely automated lab systems may be employed.

  • Each automated instrument can function separately or be integrated with other lab instruments.

  • Instruments can link in continuous operations, including robotic specimen processing.

  • Highly automated systems are used predominantly in larger clinical chemistry and hematology labs, while semi-automation is present in urinalysis, blood banks, and microbiology laboratories.

Three Phases in Testing

  1. Pre-analytical Phase:

    • Involves specimen labeling, centrifuging, and sorting.

    • Errors often occur, such as labeling the wrong tube.

  2. Analytical Phase:

    • Entails chemical reactions and measurement resulting in data generation.

  3. Post-analytical Phase:

    • Data storage, reflex testing, and electronic reporting occur here.

  • Most laboratory errors (up to 70%) occur in the pre-analytical phase.

Major Types of Analyzers

  • Continuous Flow:

    • Liquids are pumped through a system of tubing, separating samples with air bubbles.

  • Centrifugal Analysis:

    • Uses centrifugal force for mixing reagents and samples, allowing multiple samples to run simultaneously.

  • Discrete Analysis:

    • Each sample is contained in its own reaction container (cuvette), and this is the current industry standard.

Components of a Discrete Analyzer

  • Sample Loader:

    • Utilizes circular carousels or continuous racks.

  • Sampling Probe:

    • Aspires precise microlitre volumes and includes liquid level sensing.

  • Reagents Station:

    • Refrigerated compartments for open or closed systems.

  • Mixing Unit:

    • Incorporates stirring paddles or ultrasonic vibrations.

  • Incubation Chamber:

    • Maintains a constant temperature of 37°C.

Measurement Technologies

  • The “brain” of the analyzer:

    • Spectrophotometry: Measures light absorbance to determine concentration.

    • Ion Selective Electrodes (ISE): Specifically for electrolytes (Na, K, Cl).

    • Chemiluminescence: Utilizes light-emitting chemical reactions for immunoassay testing.

    • Turbidimetry/Nephelometry: Measures light scatter for protein analysis.

Total Laboratory Automation

  • The Track System: Conveyor belts that transport tubes between modules.

  • Decappers: Automated removal of tube stoppers.

  • Onboard Storage: Automated retrieval of samples for repeat or reflex testing.

Quality Control and Calibrations

  • Westgard Rules: Criteria used to determine the acceptance or rejection of analytical runs.

  • Quality Control (QC): Involves running stable materials at different levels (normal/abnormal) to ensure systems are in statistical control regarding accuracy and precision.

  • Calibration: The process of setting a ruler for the instrument using known standards.

Troubleshooting and Maintenance

  • Daily Maintenance: Includes cleaning probes, checking temperatures, and replenishing reagents (e.g., replacing ERF daily).

  • Common Issues:

    • Fibrin clots in probes.

    • Reagent depletion.

    • Lamp failure.

  • Instructor’s Rule:

    • If QC is out, the sequence of checks should involve checking the reagent, then the calibrator, then the instrument, and lastly the patient’s sample.

Steps in Automated Analysis

  • Major steps designed by manufacturers to replicate manual techniques:

    1. Specimen collection and processing

    2. Measurement and delivery of specimen and reagents

    3. Chemical reaction phase

    4. Measurement phase

    5. Signal processing and data handling

Specimen Collections and Processing

  • Proper specimen collection, labeling, and transport to the lab are critical for accurate analysis.

  • Automation of specimen preparation includes:

    • The use of barcoded labels to eliminate clerical errors related to patient data entry.

    • Automation allows for efficient storage and retrieval processes.

  • Ultimately, proper identification is the responsibility of the staff member collecting the sample.

Specimen and Reagent Measurement and Delivery

  • Automated instruments combine reagents with measured specimen amounts:

    • Reagents must be introduced correctly for accurate analysis.

    • Random-access analyzers test specimens sequentially based on selected tests.

    • A microprocessor controls the addition of diluents and reagents, initiating spectrophotometric measurements of varied samples.

    • Some analyzers employ a circular or parallel configuration for testing.

Chemical Reaction Phase

  • Reagents can be classified as liquid or dry chemistry, with varying handling based on instrument capabilities.

  • The chemical reaction phase entails mixing, separation, incubation, and reaction time:

    • Continuous flow analyzers utilize air bubbles between sample plugs.

    • Discrete analyzers maintain reactants in individual containers, functioning as cuvettes for optical analysis.

  • Incubation allows time for reaction at controlled temperatures set by the analyzer.

Measurement Phase

  • Traditionally relies on photometry and spectrophotometry to measure absorbance.

  • Alternate methods include nephelometry, chemiluminescence, enzyme immunoassays, and ion-selective electrodes (ISE).

  • Regular standardization and calibration are essential for accurate results, with QC specimens needing to be analyzed frequently.

Signal Processing and Data Handling

  • Results are visualized on a readout using LEDs or monitors, convertible to hard copy or electronic formats.

  • Data management systems interface with analyzers and host Laboratory Information Systems (LIS).

    • Automated QC data management with evaluation against acceptable limits.

  • The LIS can auto-verify results within predefined parameters, sending them directly to patient files, while technologists review all data that falls outside these limits.

Automation in Clinical Chemistry

  • Automated analyzers replicate standard manual reactions.

  • Unique methods, such as chemiluminescence, are specifically developed for immunoassays.

Analyzer Methods - Spectrophotometry & Photometry

  • Photometric Instruments: Measure light intensity; often use filters or prisms to isolate wavelength ranges.

  • Beer’s Law: States that the concentration of a substance is directly proportional to the extent of light absorbed or inversely proportional to the logarithm of transmitted light.

  • Spectrophotometric Instruments: Evaluate light transmitted by a solution to ascertain concentrations of light-absorbing substances.

Radiant Energy

  • Radiant energy passing through an object is partially reflected, absorbed, or transmitted.

  • Absorbance (A): Defined as the amount of light absorbed, derived from %T using the formula: Extra open brace or missing close braceExtra open brace or missing close brace.

  • Absorbance depends on molecular and ionic types present and can vary with concentration, pH, or temperature.

  • Deviations from linearity can occur at high absorbances (around 2.0 units).

Components of the Spectrophotometer

  • Key components include:

    • Light Source: Tungsten or deuterium/mercury arc lamp (UV).

    • Monochromators: Wavelength isolators such as filters and prisms.

    • Sample Cell: Typically plastic or quartz, sensitive to scratches and contaminants.

    • Photodetectors: Convert radiant energy to electrical energy (e.g., phototube, photocell, photomultiplier tube).

Wavelength Selectors

  • Various types:

    • Filters

    • Prisms

    • Grating monochromators

    • Holographic gratings

  • Quality described by nominal wavelength, effective bandwidths, and bandpass.

Wavelengths

  • Nominal Wavelength: Peak transmittance in nanometers.

  • Spectral Bandwidth: Range of wavelengths halfway between the baseline and peak.

  • Bandpass: Total range of wavelengths transmitted.

Photomultiplier Tubes

  • Used in low radiant power situations (low analyte concentrations).

  • Similar to phototubes but output is amplified significantly (up to one million-fold).

  • Highly sensitive to UV and visible radiation with fast response times.

Signal Processors and Readout

  • Signal processing involves several steps:

    • Amplification of the signal.

    • Current rectification and direction.

    • Phase alteration of the signal.

    • Filtering of unwanted components and mathematical computations.

Quality Assurance in Spectrophotometry

  • Wavelength Accuracy: Measured at specific wavelengths, e.g., protein readings at 540 nm.

  • Assessed with special glass optical filters (e.g., didymium, holmium oxide).

  • Linearity: Checked with colored solutions that may be diluted; deviations can arise from issues pertaining to light source and monochromator performance.

Absorbance

  • Ideal: Linear relationship according to Beer’s Law.

  • Deviation: Caused by electronic limitations or instrument malfunctioning.

Reflectometry

  • Principle: A filter photometer measures the quantity of light reflected from a liquid sample on a non-polished surface.

  • Reflectance correlates nonlinearly with concentration:

    • Ideal reflectance for a pure white standard equals total light reflection.

    • Pure black material represents zero reflectance, absorbing all light.

  • Instrumentation involves tungsten-halide lamps, photodiodes, and filters for wavelength isolation, converted by computers into concentration units.

Fluorometry

  • Basic instrumentation involves filter fluorometers that assess concentrations of solutions with fluorescing molecules.

  • Design: Source emits short-wavelength excitation light, with mechanical attenuators to adjust intensity.

  • Advantages: Enhanced specificity and sensitivity.

  • Disadvantages: Susceptible to environmental changes (e.g., chemicals, UV light can lead to quenching).

Turbidimetry

  • Measures light transmission reduction due to particle formation within a sample.

  • Sensitivity is generally higher compared to nephelometry and is applied in microbiology and coagulation studies.

Chemiluminescence

  • Involves chemical energy producing excited intermediates that return to ground states with photon emission.

  • No requirement for excitation radiation.

  • Utilized in various oxidation reactions and measures turbidity or protein concentrations.

Ion-Selective Electrodes (ISE)

  • Designed for sensitivity towards specific ions.

  • Types:

    • pH electrodes for detecting hydrogen ion activity.

    • Gas-sensing electrodes detect specific gases; distinct in chemical mechanisms.

    • Enzyme electrodes incorporate immobilized enzymes catalyzing reactions.

Potentiometry

  • Measures potential voltage between two electrodes in a solution; requires a constant voltage reference.

  • Relates electrode potential to ionic concentration via the Nernst equation.

Reference Electrode

  • Common types:

    • Calomel electrode

    • Silver/silver chloride electrode.

  • Provide analytical qualities necessary for reliable measurements in conjunction with ISEs.

Advantages of ISE Over Wet Chemistry

  • Direct measurement without reagent prep or standard curves.

  • Cost-effective, swift, sensitive, easily maintained, and adapted to automation.

pH Electrode

  • Glass electrodes measure hydrogen ion activity, using a chloride buffer and silver/silver chloride reference.

  • Hydrogen ions replace sodium ions, affecting potential difference and thus pH reading.

pCO2 Electrode (Gas Analyzers)

  • Comprises a plastic jacket containing sodium bicarbonate buffer with a gas-permeable membrane.

  • When CO2 from blood contacts the membrane, reacts with the buffer, shifting pH levels indicative of concentration.

    • Reaction: e⁢x⁢t⁢C⁢O2+e⁢x⁢t⁢H2⁢e⁢x⁢t⁢O⁢i⁢g⁢h⁢t⁢l⁢e⁢f⁢t⁢h⁢a⁢r⁢p⁢o⁢o⁢n⁢s⁢e⁢x⁢t⁢H2⁢e⁢x⁢t⁢C⁢O3⁢i⁢g⁢h⁢t⁢l⁢e⁢f⁢t⁢h⁢a⁢r⁢p⁢o⁢o⁢n⁢s⁢e⁢x⁢t⁢H⁢C⁢O3−+e⁢x⁢t⁢H+

Amperometry

  • Measures current flow produced during oxidation-reduction reactions, applicable in various electrode designs.

pO2 Gas Sensing Electrode

  • The Clark PO2 electrode permits dissolved oxygen passage through a membrane, reacting with a platinum cathode, producing electron flow proportional to oxygen levels.

Osmometry

  • Measures osmolarity of solutions (serum, plasma, urine) using techniques like freezing-point depression, vapor pressure, and osmotic pressure.

  • Osmolarity: Concentration expressed per volume of solution (osmol/L), while Osmolality: Concentration per kilogram of water (mOsmol/kg) is superior as it remains temperature-independent.

Freezing Point Osmometer

  • Utilizes rapid supercooling and agitates samples to establish equilibrium at freezing point, measured by thermistors and converted to milliosmoles/kg.

Conclusion

  • Automation enhances laboratory processes but does not replace Medical Laboratory Technologists (MLTs).

  • It shifts the role of MLTs toward data management and problem-solving, underscoring the importance of validation of analyzer results.