Analytical Instrumentation Exam 2

What is the analytical process of automation in clinical chemistry?

• Preanalytical

• Analytic (most automated)

• Postanalytical

What are the advantages of automation?

• Speed of analysis

• Human error is reduced

• Reproducibility increases

• Decreased sample and reagent volume

• Decreased cost of consumables

• Lower cost per test

What are the basic approaches with automated analyzers?

• Continuous flow analyzers

• Centrifugal analysis

• Discrete analysis

What is continuous flow analysis and what are the disadvantages?

• When liquids (reagents, dilutants, and samples) are pumped through a system of continuous tubing

  • A series of air bubbles at regular intervals separate samples

• Disadvantages

  • Does not allow test selection

  • Must run continuously when there are no tests

  • Reagents must be drawn at all times even when there are no tests to perform

  • Must be monitored all the time for carry over problems

What is centrifugal analysis, the process of it, and disadvantages?

• When samples and reagents are added in a specially designed centrifugal type cuvet with 3 compartments:

  • Sample compartment

  • Reagent compartment

  • Reaction mixture compartment

• Process

  • Mixing of sample reagent occurs when the rotor holding the cuvet is spun at high speeds and then suddenly stops

  • Cuvet passes through the optical path of the optical system for spectrophotometric measurement

• Disadvantages

  • Only one test can be performed at a time

What is discrete analysis?

• The separation of each sample and accompanying reagents in a separate container

  • Sample reactions are kept discrete and the sample container is disposed 

  • Uses random access fluid, which is a hydrofluorocarbon liquid, to reduce surface tension between the sample/reagent and their tubing (reduces carry over)

• Each sample is treated differently according to the tests requested and programed by the operator

• Disadvantage

  • Have the capability to run multiple tests on one sample or multiple samples, one test at a time

  • Keeps sample and carryover to a minimum, but increases the cost per test due to disposable products

How is a reagent handled and what are the different reagent systems?

• Reagents are stored on automated analyzers at room temperature and refrigerated compartments

• Different reagent systems

  • Open reagent system

    • An analytical system which allows reagents from sources outside of the reagent manufacturer

  • Closed reagent system

    • An analytical system for which the reagents are provided only by the manufacturer

      • Tends to be more expensive

What are different types of clinical chemistry tests?

• Enzyme levels (liver function tests)

• Ion levels (Na+, K+, Li+, and Cl-)

• Glucose

• Total protein/albumin

• Cholesterol

What are different sample types?

• Serum

• Plasma

• Diluted blood

• Urine

• Diluted urine

In the EasyRA analyzer, what is the purpose of the RFID chips embedded in each reagent wedge label, and how does the instrument use this technology during operation?

• Each reagent wedge contains a Radio Frequency Identification (RFID) chip embedded in its label.

• An RFID reader is located in the base of the reagent/sample area.

  • The reader scans the RFID chips on the wedges.

    • This provides the instrument with:

      • All reagent parameters.

      • The current number of tests remaining on each wedge.

    • This technology allows for:

      • Accurate reagent tracking.

      • Automatic data entry.

        • Efficient test management.

In the Easy RA analyzer, what happens in the reaction area, and how do the cuvette carousel and cuvettes function during testing?

• The reaction area of the Easy RA analyzer includes the cuvette carousel and cuvettes.

  • The carousel holds up to 72 individual cuvettes.

  • A probe dispenses both samples and reagents into the cuvettes for analysis.

  • The cuvette carousel rotates at the appropriate time, allowing each cuvette to move through the testing cycle.

  • As each cuvette passes by the photometer, the instrument measures absorbance to generate and record test results.

In the Easy RA analyzer, what is the function of the photometer in the reaction area, and what are its key features?

• The photometer is located in the reaction area of the Easy RA analyzer.

  • It contains six interference filters.

  • It can make optical measurements at seven wavelengths: 340, 405, 520, 550, 600, 660, and 700 nm.

  • The light source used is a Xenon flash lamp.

  • These features allow the analyzer to accurately measure absorbance for different types of chemical reactions and tests.

In the Easy RA analyzer, what is the purpose of the heated air bath in the reaction area?

• Keeps the air around the cuvettes at 37 C ± 0.25

• Ensures that all reactions take place at a predefined temperature

In the Easy RA analyzer, what are the main functions of the probe located on the transfer arm?

The probe on the transfer arm performs several key functions:

• Picks up reagents and samples and dispenses them into cuvettes.

• Transfers samples into the ISE Module when required.

• Mixes reagents and samples in cuvettes by injecting air into them.

• Receives diluent from the dilutor pump to clean the probe and remove contaminants.

• Delivers the waste and used diluent into the wash cup for disposal.

What daily maintenance procedures are required for the Easy RA analyzer, and what is the purpose of each step?

• Daily Cleaning:

  • Clean the probe to prevent sample contamination.

  • Clean and calibrate the ISE module to ensure accurate electrolyte measurements.

• Daily Inspection:

  • Check the dilutor pump for proper operation.

  • Check the probe for cleanliness and function.

  • Check waste and diluent containers to ensure proper levels and disposal.

  • Check the pump tubes for wear or leaks.

• Priming the Diluent:

  • Brings fluid back into the Easy RA system to prepare it for testing.

• Prime ISE Cal A/B:

  • Brings calibration fluids (Cal A and Cal B) back into the ISE module to prepare it for accurate operation.

What quality control procedures are required for the Easy RA analyzer, and what does each one ensure?

• Daily Use of QC Material:

  • Run two levels of quality control material for each chemistry test reported by the laboratory every day.

  • Ensures accuracy and reliability of test results.

• Weekly Use of the EasyRA Precision Test:

  • Verifies the precision of the photometer and pipetting systems.

  • Ensures the analyzer is dispensing and measuring consistently.

• External Quality Control:

  • Performed through the College of American Pathologists (CAP) program.

  • Provides independent verification of the analyzer’s performance against external standards.

What aspects must be evaluated during method validation for the Easy RA analyzer?

• Laboratories must validate the performance of all methods before reporting patient results.

• The validation process includes checking:

  • Accuracy – ensures test results are close to the true value.

  • Precision – confirms the analyzer produces consistent results.

  • Reportable range – defines the limits within which accurate results can be obtained.

  • Reference intervals – verifies that Medica’s normal values are appropriate for the lab’s specific patient population.

What detection methods are used by the Easy RA analyzer, and how are enzymes and sample ions measured?

• The Easy RA analyzer uses absorbance/transmittance photometry as its main detection principle.

  • The contents of each cuvette are analyzed at specific times and wavelengths, which are unique for each analyte.

• Enzyme Measurements:

  • Measured by the rate at which they convert one colored substance into another, indicating enzyme activity.

• Sample Ion Measurements:

  • Ion-selective electrodes (ISEs) are used, which allow only one type of ion to pass through.

  • The potential of each electrode is measured relative to a stable silver/silver chloride reference electrode.

  • The ISE measures ion concentration in solution by detecting the current flow through the ion-selective membrane.

  • This method is a form of potentiometry.

What is electrophoresis, and how does it separate macromolecules such as DNA or proteins?

• A laboratory technique used to separate macromolecules, such as DNA or proteins, based on their size and charge.

• It uses a gel matrix made of agarose or polyacrylamide, which acts as a molecular sieve to slow the movement of larger molecules.

• The process relies on electric charges—an electric current is applied across the gel:

  • Positively charged molecules move toward the negative electrode (cathode).

  • Negatively charged molecules move toward the positive electrode (anode).

    • The principle of separation is that opposites attract, and smaller or more highly charged molecules migrate faster through the gel.

What are the main components found inside an electrophoresis chamber, and what is the function of each?

• Gel:

  • Held in a casting tray and made of agarose (or sometimes polyacrylamide).

  • Acts as a molecular sieve, allowing particles to move slowly toward the oppositely charged side.

  • Initially poured as a hot liquid that solidifies upon cooling.

• Comb:

  • Placed in the slots of the casting tray before the gel is poured.

  • Once the gel solidifies, the comb is removed, leaving behind small indentations called wells.

• Wells:

  • The small holes formed in the gel after removing the comb.

  • Serve as loading sites where samples are placed before running the electrophoresis.

• Buffer:

  • A conductive solution that allows electric current to pass through the chamber.

  • Poured into the chamber until it covers the top of the gel tray.

  • Enables current to flow from the cathode (negative end) through the buffer to the anode (positive end), allowing the charged molecules to migrate through the gel.

What is protein electrophoresis, and how are proteins separated during the process?

• Protein electrophoresis is a technique that uses an electric field to separate proteins based on their charge and size.

• When an electric current is applied:

  • Positively charged proteins move toward the cathode (negative electrode).

  • Negatively charged proteins move toward the anode (positive electrode).

• The gel matrix slows the movement of larger proteins, allowing smaller or more strongly charged ones to migrate faster, resulting in distinct protein bands that can be analyzed.

What are the functions of proteins?

• Enzyme catalysis (enzymes are proteins)

• Metabolic regulation

• Binding and transport of small molecules (ex. transferrin, ceruloplasmin, and hemoglobin)

• Gene regulation (transcription factors)

• Immunological defense (antibodies)

• Cell structure (structural proteins in cell walls)

What are the basic structural components of an amino acid, and how many different amino acids make up proteins?

• Each amino acid has a:

  • Central carbon atom (α-carbon)

  • Amino group (-NH₂)

  • Carboxylic acid group (-COOH)

  • Hydrogen atom

  • Side chain (R group) that determines its unique properties

• There are 20 different amino acids, each with a distinct R group, which combine to form proteins.

Which amino acids are responsible for giving proteins their positive or negative charge at physiological pH?

• At physiological pH (~7.4):

  • Glutamic acid and aspartic acid give proteins a negative charge.

  • Lysine and arginine give proteins a positive charge.

• The overall charge of a protein depends on the balance between these acidic and basic amino acids.

What is the isoelectric point of a protein, and how does it affect the protein’s movement in an electric field during electrophoresis?

• The isoelectric point (pI) of a protein is the pH at which the protein has no net charge.

• At its isoelectric point, a protein will not migrate in an electric field because it has no net positive or negative charge.

• Proteins with a net charge (positive or negative) will migrate in an electric field, and the extent and direction of migration depend on their charge relative to the applied field.

How can the pH and buffer system influence a protein’s charge and migration rate during electrophoresis?

• The direction and extent of a protein’s migration can be altered by using an acidic, neutral, or alkaline buffer system during electrophoresis.

• A protein’s net charge depends on its amino acid composition and the pH of the surrounding environment.

• Proteins with more net charge (either positive or negative) than others of the same shape and size will travel faster through the gel.

• This variation in net charge allows proteins to be separated by electrophoresis

How does the shape of a protein affect its behavior during electrophoresis, and what factors can cause a protein to become denatured?

• Proteins have varied three-dimensional shapes and complex folding patterns, which are critical to their biological function.

  • Tightly folded proteins appear smaller and move through the gel faster than loosely folded proteins.

• Native proteins are in their normal, biologically active forms.

  • Proteins can become denatured (lose their shape and biological activity) due to:

    • Detergents

    • Extreme pH conditions

    • Organic solvents

  • Denaturation changes a protein’s size and shape, affecting its migration during electrophoresis.

How does the size of a protein affect its properties, and what factors determine a protein’s molecular weight?

• The molecular weight of a protein depends on the number and type of amino acids in its polypeptide chain(s).

• Proteins can be composed of a single polypeptide chain or multiple polypeptide chains.

• Polypeptide chains within a protein can be identical, similar, or completely different from one another.

• The number and nature of polypeptides in a protein significantly affect its mass, size, and three-dimensional shape, which in turn influences its migration during electrophoresis.

What factors influence the electrophoretic migration rates of proteins?

• The migration rate of a protein during electrophoresis is influenced by several factors:

  • Amount of charge – proteins with more net charge migrate faster.

  • Sign of charge – determines the direction of migration toward the anode or cathode.

  • Size of the protein – smaller proteins move through the gel faster than larger ones.

  • Shape of the protein – tightly folded proteins migrate faster than loosely folded proteins.

• All these factors together determine how a native protein moves during electrophoresis.

How do different serum proteins migrate during electrophoresis, and how can electrophoretic patterns be used clinically?

• Human serum contains many different proteins, each with unique migration rates:

  • Albumin: Most abundant serum protein; fastest migration rate; molecular weight ~66.5 kDa.

  • Gamma globulins (antibodies): Slowest migration rate; molecular weight ~146,000–970,000 kDa.

• The electrophoretic pattern of serum proteins can help diagnose certain diseases by revealing abnormal protein levels or distributions.

What is SDS-PAGE, and how does it allow proteins to be separated by molecular weight?

• SDS-PAGE is a type of protein electrophoresis that separates proteins primarily by their molecular weight.

• SDS (sodium dodecyl sulfate):

  • Found in the sample buffer and gel.

  • Unfolds proteins and coats them with a uniform negative charge.

• BME (β-mercaptoethanol):

  • A reducing agent that breaks disulfide bonds within the protein.

• Together, SDS and BME ensure that the charge of the protein is proportional to its size, allowing proteins to migrate according to molecular weight during electrophoresis.

What are the main characteristics of polyacrylamide gels (PAGE), and how do they differ from agarose gels?

• Polyacrylamide gels are chemically inert and do not interact with proteins.

• They can be made at different concentrations, creating varied pore sizes.

• They set chemically through polymerization, unlike agarose gels, which set thermally.

• PAGE gels are typically run vertically, whereas agarose gels are usually run horizontally.

What are the roles of the stacking gel and resolving gel in PAGE?

• Stacking Gel:

  • Poured on top of the resolving gel, pH 6.8, with large pores.

  • Concentrates proteins into a thin starting band before they enter the resolving gel.

• Resolving Gel:

  • pH 8.8, with small pores.

  • Separates proteins based on size, allowing precise determination of molecular weight.

What is a discontinuous buffer system in PAGE, and how does it affect protein migration and resolution?

• A discontinuous buffer system means that the buffer in the tank has a different pH than the buffer used to make the gel.

• This creates a gradient that causes proteins to focus into a single band as they migrate through the large pores of the stacking gel.

• Once in the resolving gel, proteins separate based on size, resulting in high resolution and well-defined bands.

What factors that will affect separation of protein bands?

• The speed that the protein migrates is characteristics for that protein

• Proteins are very sensitive to pH

• Increasing the voltage will cause proteins to migrate faster (may cause melting of the gel and poor band resolution)

• Increasing the ionic concentration of your buffer will decrease the speed of migration

What are the key features and capabilities of the Bio-Rad Mini-PROTEAN® Tetra Cell System?

• The Bio-Rad Mini-PROTEAN® Tetra Cell System is a next-generation mini cell system for 1-D vertical gel electrophoresis.

• It can run precast or hand-cast polyacrylamide gels (SDS-PAGE).

• Supports 1–4 mini gels (each 7 cm × 8.5 cm) in approximately 30 minutes.

• Includes sample loading guides for accurate placement of samples.

• Features a leak-free gel casting system for reliable gel preparation and electrophoresis.