MLT 101 Test Preview

Methodology Overview

  • Emphasis on the importance of methodology in chemistry automation and analyzer techniques, particularly in serology and immunochemistry.

  • Discussion on information systems, specifically the role of middleware or software in laboratory settings.

Analytical Techniques Focus

  • Percent Transmittance vs. Absorbance:

    • Percent Transmittance: The amount of light that passes through a sample.

    • Absorbance: The amount of light absorbed by the sample, which is the opposite of percent transmittance.

    • Key Equation:

    • Absorbance (A) is defined by the equation: A=2−log⁡%TA=2-\log_{\%T}

Color Wheel and Wavelengths

  • Understanding that the color of the solution influences the wavelength of light to be used to measure the solutions concentration:

    • Always select the opposite color from the color wheel:

    • Green Solution: Use red light;

    • Blue Solution: Use orange light;

    • Purple Solution: Use yellow light;

    • Red Solution: Use green light.

  • Why? Because for each color we see can absorb a maximum amount, which is usually the opposite of them on the color wheel. This is called the wavelength of maximum absorbance. When we are calculating the concentration of an unknown solution, we want to use the wavelength of the color that is most absorbed, which is the color opposite of what we see (or what is transmitted) according to the color wheel.

  • Various examples associated with sports teams to help memorize opposite colors:

    • Purple and yellow -> Minnesota Vikings/LSU;

    • Red and green -> Christmas

    • Blue and orange -> Florida Gators/Miami Dolphins.

Equations and Beer's Law

  • The relationship between absorbance and concentration:

    • Beer's Law Equation:
      CsAs=CuAu\frac{Cs}{As}=\frac{Cu}{Au}

  • Reminder that absorbance is directly related to concentration. Absorbance must be converted into concentration units in order for the measurement to have meaning. Whatever the amount of Absorbance the colored solution has, the amount of concentration it has.

  • Light can hit a solution 3 different ways:

    • Transmitted (passes through);

    • Absorbed (energy is taken up);

    • Reflected (bounces back).

  • Monochromator: This is the heart of the Spectrophotometer. Used to select specific wavelengths in a spectrometer, isolating it

  • This has 3 components:

  1. Entrance Slit -This is where the light is measured

  2. Dispersing Element -usually a grating that disperses light once it is hit

  3. Exit Slit- Where light exits and is also measured (this and the Entrance Slit must be very narrow

  • Photodetector : Detects the wavelength of the light from the Exit Slight and converts into electrical energy and sends it to the readout device in the spectrophotometer

Spectrophotometry Measurement Definitions

  • Define Wavelength- Distance between 2 crests of a wave (measured in nm); we use this measurement unit when it comes to absorbance.

  • Define Frequency- Number of oscillations per second

  • Define Oscillations- The time frame it takes to get from middle of upward line to Crest to trough and back to line before another crest

  • Atomic Absorption Spectrometry:

    • Measures concentration via the absorption of electromagnetic radiation by atoms, not molecules.

  • Flame Photometry:

    • Measures light emitted by excited atoms.

Turbidity and Nephelometry

  • Turbidity: Measures concentration of particulate matter in a straight line (90 degree angle)

  • Nephelometry: measures light much like Turbidity, but measures light of cloudy matter causing light scattering at different angles (typically 45 and 90 degrees).


Electrochemistry and Ion Selective Electrodes

  • Electrochemistry: The spontaneous flow of electrons from electrodes with lower electron affinity.

  • Ion Selective Electrodes (ISE): • This Electrochemistry is designed to be sensitive toward individual ions. This is the replacement for traditional titrations (e.g. coulometric titrations) in analyzers.

LASER Applications

  • LASER stands for:

-Light

-Amplification by

-Stimulated

-Emission of

-Radiation

  • Laser spectrometry can be used for determination of structure, identification of samples, and diagnosis.

  • Used in Flow Cytometry to identify leukocytes

  • flow cytometry uses laser light scatter to determine cell size, nucleus size, presence of granules, and other cellular characteristics.

  • while laser applications are limited in chemistry, they are extensively used in hematology and microbiology.

Electrophoresis Overview

  • Electrophoresis Definition: The migration of charged particles or solids in an electric field, commonly discussing proteins.

Osmometry Overview

  • Osmometry- Measures concentration of solute particles in a solution

Fluorometry

  • Fluorometry- Measures concentration of solutions that contain fluorescing molecules.

Chemiluminescence

  • Chemiluminescence- Part of chemical energy generated produces excited intermediates that decay to a ground state with emission of photons.



Chromatography Methods

  • Chromatography- Group of techniques used to separate complex mixtures on basis of different physical interactions


  • Types of Chromatography Used (There are 3):

    • Thin Layer Chromatography (TLC); A variant of column chromatography.


    • High Performance Liquid Chromatography (HPLC); Uses pressure for fast separations, controlled temperature, in-line detectors, and gradient elution techniques

      Components include: pumps, columns, sample injectors, detectors, recorders


    • Gas Chromatography (GC); Used to separate mixtures of compounds that are volatile or can be made volatile

      Components include: columns, detectors

  • Key applications for chromatography techniques include forensic analysis and major research laboratories.

Mass Spectrometry Techniques

  • Mass Spectrometry- Used as a detector to identify samples eluting from gas chromatographic or HPLC columns

  • Analyzers used:

  • Quadrupole Analyzers: Provides high sensitivity and resolution for quantitative analysis. -Most commonly used

  • Iron trap: a modified quadrupole

  • MS/MS: tandem use of mass spectrometers for greater selectivity and lower detection limits

  • Ion Trap Analyzers: Capable of trapping ions for high-resolution mass analysis.

  • High-resolution MS


  • MALDI-TOF: Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry.

    • Provides bacterial fingerprints; faster than traditional methods. Widely used now in clinical laboratories, MALDI-TOF enables rapid identification of microorganisms, significantly improving diagnostic workflows and patient care.

  • Operation Mechanism:

    • Two-phase process involving a laser to vaporize and ionize samples that generate high-resolution mass results via time-of-flight.

    • the samples with higher mass will arrive later than those with lighter mass. Starting from the Ionization process, the device creates a print-out of the bacterial profile based on their time of flight.

  • Why do we use MALDI-TOF now? Significantly cheaper and faster than traditional automated biochemical identification techniques.

Bacterial Growth and Testing

  • Growth of bacteria requires a minimum of 24 hours.

    • Some strains (e.g., Strep pyogenes) may show growth in 18-19 hours if conditions are optimal (e.g., slight CO2 increase).

    • Traditional culture practices and needing pure colonies for accurate microanalysis highlighted.

Clinical Chemistry Automation

What are the driving forces to Chemistry Automation?

  1. Higher volume of testing, faster turnaround time

  2. Fewer, more centralized core labs

  3. Regulatory standards requiring greater accuracy and precision

  4. Intense competition among instrument manufacturers

  5. Decreased operating budgets for labs, ability to “walk away”


How did we get to Automation?

  • Before 1957- Manual testing previously required 1-2 hours per test

  • 1957- First Automated Analyzer: “AutoAnalyzer”

    ;-Capable of providing a single test result on about 40 samples/h

  • Second Generation: Simultaneous Multiple Analyze -this allow for techs to work multiple channels simultaneously

-This produced 6 to 12 test results simultaneously at rate of 360 to 720 tests/h

  • 1970 - First Commercial Centrifugal Analyzer introduced

-An alternative to continuous flow technology

-Spin-off technology from NASA space research

  • 1970 - Automatic Clinical Analyzer Introduced

-First noncontinuous flow, discrete analyzer

-First instrument to have random access capabilities

-Unique features: plastic test packs, positive patient identification, infrequent calibration

  • 1976 - Thin film analysis technology introduced

  • 1978 - Kodak Ektachem Analyzer produced

-First instrument to use microsample volumes and reagents on slides for dry chemistry analysis

-First instrument to incorporate computer technology extensively into its design and use

  • 1980 - Discrete Analyzers

-Ion-selective electrodes, fiberoptics, polychromatic analysis

-Sophisticated computer hardware and software for data handling

-Larger test menus

  • Today’s Time-Most Recent Advances:

  1. Point-of-Care Benchtop Analyzers

    -Small, portable, easy to operate

    -Used in primary in physician office laboratories, surgical and critical care units

  2. Immunochemistry Analyzers

    -Assaying drugs, specific proteins, tumor markers, hormones

    -Instruments using fluorescence polarization immunoassay, nephelometry, and immunoassay with chemiluminescent detection

Modular Analyzers

-Combination of chemistry and immunoassay


What are the 3 basic approaches to Automation?

  1. Continuous Flow

-Liquids are pumped through system of continuous tubing.

-Samples are introduced in a sequential manner

  1. Centrifugal Analysis

-Force of centrifugation transfers and contains liquids.

-Capable of batch analysis

  1. Discrete Analysis

-Separation of each sample and reagent in a separate container

-Most popular type; can run multiple tests on one sample at a time or multiple samples one test at a time

Clinical Industry Automation Steps (Basic)

  1. Specimen Preparation and Identification

    *Specimen preparation

    -Can be automated by robotics.

    -Can be skipped, if whole blood is used for analysis.

    -Plasma separator tube can be used and primary tube sampling performed with heparin plasm

  2. Specimen identification

    -Bar code label affixed to primary collection tube.

  3. Specimen Measurement and Delivery

    -Circular carousels or rectangular racks hold specimen containers.

    -Primary collection tubes or microsample tubes are placed in carousels or racks.

    -Aliquot is measured through aspiration of sample into a probe.

    -Probe and tubing are cleaned after each dispensing to minimize carryover, unless disposable probes or tips are used.

  4. Reagent Systems and Delivery

-Liquid: available in bulk volume containers or unit doses

-Dry bottled as lyophilized powder, requiring reconstitution

-Multilayered dry chemistry slide

-Preservation: refrigeration, reconstitution of dry tablet, or combination of two stable components

-Dispensed via tubing from bulk containers, syringes that pipette reagents into reaction containers, piston-driven pumps connected by tubing, or pressurized reagent bottles

  1. Chemical Reaction Phase

    -Mixing: reagents and sample

    -Coiled tubing (continuous flow analyzers)

    -Rapid start–stop of reaction tray (RA1000)

    -Rapid start–stop of rotation or bubbling of air (centrifugal)

    -Separation: separating undesirable substances from sample

    -Incubation: heating bath (water or air) to maintain required temperature of reaction mixture

    -Reaction time: depends on rate of transport through system and timed reagent additions

  2. Measurement Phase

    *Systems for measurement

    -Ultraviolet, fluorescent, and flame photometry

    -Ion-specific electrodes

    -Gamma counters

    -Luminometers

    -Visible and ultraviolet light spectrophotometry (most common)

    -Fluorescence polarization, chemiluminescence, bioluminescence

Signal Processing and Data Handling

-Accurate calibration is essential to obtaining accurate information.

-Multiple instruments that measure the same constituent in a lab should be calibrated so that results are compatible.

-Automated instruments, once calibrated, provide long-term stability of standard curve; require only monitoring.

-Some instruments are self-calibrating.

-Advanced automated instruments have method of reporting printed results.

Computerized monitoring is available for many parameters.

  1. Send result(s) to laboratory information system (LIS)

Total Laboratory Automation Analytical Phases

Data Management in Clinical Labs

  • Hospital Information System (HIS) vs. Laboratory Information System (LIS):

    • Middleware serves as an interface connecting lab computers to instruments, ensuring data sequence and accuracy in patient results.

Immunoassays Techniques

  • Competitive Immunoassay:

    • The amount of color intensity is inversely proportional to the titer of the antibody; a higher titer results in lower color.

  • Sandwich Immunoassays:

    • Antigen binds between two antibodies (solid phase and labeled); used for antigen detection.

  • Common enzymes in enzyme-linked immunosorbent assays (ELISA) include horseradish peroxidase and alkaline phosphatase.

Importance of Calibration and Training in Point-of-Care Testing

  • Point-of-care results must always be supported by laboratory tests.

  • Responsibilities include ensuring proper calibration, training of personnel, and annual evaluations.

Conclusion

  • Summary of lessons covering methodology, clinical automation, clinical testing principles, and application of analytical techniques in laboratory settings.