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Tutorial MLS2001

Accuracy & Precision

Accuracy

• Definition: The accuracy of a test refers to its ability to yield the TRUE value of an analyte in a sample, which is crucial for diagnostic precision.

• Measurement Insight: When an analyte is measured 20 times, the resulting values create a distribution around the true value, revealing possible errors due to the measurement process.

• Calculation of the TRUE Value: The TRUE value is defined as the mean of all collected measurements, representing the ideal benchmark against which accuracy is assessed.

Precision

• Definition: Precision is defined as the capacity of a test to reproduce the SAME result consistently when analyzing the same specimen under identical conditions.

• Variability Representation: This consistency is typically evaluated through the Standard Deviation (SD), where lower SD indicates higher precision.

Accuracy & Precision: Improvement Strategies

Enhancing Accuracy

1. Standardized Procedures: Implementing rigorously standardized lab procedures minimizes operational variances and enhances result reliability.

2. Statistical Comparisons: Making statistically valid comparisons with established reference methods ensures the credibility of test results.

3. Control Samples: Utilizing control samples with predetermined values assists in calibrating the test outcomes.

4. Proficiency Testing: Engagement in external proficiency testing programs helps in benchmarking performance against peer laboratories.

Ensuring Precision

1. Standards and Reference Samples: Proper inclusion of standards and reference samples in testing workflows fosters consistency in results.

2. Replicate Determinations: Conducting statistically valid replicate determinations for each sample enhances the reliability of findings.

3. Control Samples Monitoring: Day-to-day and between-run precision must be monitored through quality control samples to ensure ongoing accuracy in results.

Calibration

• Definition: Calibration entails aligning instruments to operate cohesively or adjusting them to precise measurements.

Purpose of Calibration

• Calibration is vital for ensuring measurements are both accurate and precise, contributing to comparable results across different instruments and diminishing biases in readings throughout varying ranges.

How Calibration is Performed

1. Reference Standards: Calibration involves using established reference standards with known values across the requisite measurement range.

2. Calibration Curves: Calibration curves are established to create functional relationships between standard values and measured values, essential for understanding instrument behavior.

Calibration Frequency

• Calibration should occur:

  • Before major critical measurements to ensure high accuracy.

  • After significant measurements where data reliability may be questioned or when instruments have been in operation for extended periods.

  • Following any incidents that might compromise equipment functionality.

  • Whenever observation results appear questionable or inconsistent, suggesting potential instrumental errors.

  • As per specific analytical requirements, which may mandate calibration certificates.

Quality Control (QC)

Importance of QC

• Quality Control is integral to ensuring the accuracy and reproducibility of laboratory tests, critical for maintaining standards of healthcare diagnostics.

• QC is essential for identifying potential error sources, estimating their magnitudes, and alerting personnel to emerging quality issues.

Goals of Quality Control

• The main objectives include achieving accuracy, ensuring precision, and reducing the total chemical method error to align with industry standards.

Properties of Quality Control Materials

1. Quality control materials mimic human biological fluids (e.g., serum, plasma) for practicability and relevance in testing.

2. They should be stable for extended periods without the need for preservatives, enhancing usability.

3. QC materials must be free from communicable diseases, ensuring safety in laboratory workflows.

4. Knowing the concentrations of analytes in QC samples is vital for benchmark comparisons.

5. They should be simple to store and dispense, while being cost-effective.

QC Objectives

• Maintain continuous monitoring of accuracy in testing.

• Promptly detect accuracy-related issues.

• Enable the comparison of test results over time for trend analysis.

Quality Control vs Quality Assurance (QA)

• Inconsistent performance in QC can lead to misdiagnosis, delayed treatment, and significantly increased costs in patient care.

• It’s noteworthy that pooled sera may exhibit variability and present a higher risk of infection relative to the employment of third-party controls, emphasizing the significance of established QC protocols.

Purpose of Quality Control

• To preserve a continuous record of precision in laboratory practices.

• Acts as an early warning system for detecting trends in control measurements.

• Facilitates valid assessments of accuracy when compared against known sera standards.

• Essential for supporting automated analyzers to maintain testing regularity and integrity.

• Monitors methods for accuracy and enhances the skills evaluation of laboratory technologists.

• Crucially identifies and prevents the reporting of incorrect patient values, which can have profound impacts on treatment outcomes.

Spectrophotometer & Its Components

Purpose

• A spectrophotometer measures the light transmitted by a solution to determine the concentration of a light-absorbing substance, providing insights vital for numerous biochemical analyses.

Components

1. Light Source: Provides the light necessary for measurement.

2. Monochromators: Allows selection of specific wavelengths of light for analysis.

3. Sample Cell (Cuvette): Holds the sample being measured.

4. Photodetectors: Detects the transmitted light after passing through the sample.

Sample Cell

• Cuvettes can be either round or square; it is paramount that the light path remains constant to ensure absorbance accuracy.

• Square cuvettes are generally preferred as they tend to produce less error from lens effects.

• It’s critical that cuvettes be free from scratches and are of quartz material in cases of UV applications.

Measurement Options

• Qualitative Analysis: Using spectral data to identify different classes of compounds in biological samples based on UV absorption patterns.

• Quantitative Analysis: Commonly examines concentrations of substances such as urea, creatinine, lipid profiles, DNA, and proteins.

Spectrophotometer QC Maintenance

Routine Checks

• Routine maintenance checks include washing components, blanking procedures, and verifying water and temperature stability.

• It involves aligning components, assessing bulb longevity, and performing contamination checks to maintain optimal instrument function.

Spectrophotometer QA

• Conduct monthly checks that incorporate testing wavelength accuracy with standard solutions, as well as stray light assessments and evaluations of linearity and calibration curve performance.

Fluorescence Spectroscopy

• Fluorescence spectroscopy measures emitted light when a molecule returns to a lower electronic state post-excitation, widely applied in various biochemical assays.

Fluorometer QC

Ideal Characteristics

• Constant photon output across all measured wavelengths to ensure reliable readings.

• Efficient monochromators and detection systems are critical for optimizing data quality.

Need for Calibration in Fluorometry

• Standard lamps must be used for adjusting emission wavelengths ensuring accurate results. Routine experiments should also include blank scans to check for impurities in assays.

Factors Affecting Fluorescence

• Critical factors include pH levels, temperature fluctuations, contamination, purity of reagents, turbidity, and the presence of air bubbles, all of which can impact data integrity.

Advantages of Fluorescence

• High sensitivity and specificity offer significant advantages for detecting analytes at low concentrations.

• Offers flexibility due to numerous filters that cater to various experimental requirements.

Multiplexing

• Multiplexing allows for the simultaneous detection of multiple targets using various fluorophores tailored for diverse applications.

Applications of Fluorophores

1. Immunofluorescence: Techniques applied in cellular studies.

2. Cytogenetics (FISH): Used for visualizing chromosomal abnormalities.

3. Flow Cytometry: Enables analysis and sorting of cells based on fluorescence characteristics.

4. Fluorescent Immunoassays: Combines immunoassay principles with fluorescence for improved sensitivity.

Multiplexing Tips

• When performing multiplexing, utilize antibodies from various host species and ensure optimization of detection methods before actual experimentation.

Photobleaching

• Photobleaching refers to the damage or loss of fluorescence signal in compounds due to prolonged exposure to excitation light, presenting a challenge in experimental design.

Disadvantages of Fluorescence

• Considerations include susceptibility to environmental changes, contamination risks, quenching effects, and the ongoing maintenance requirements needed to ensure analytical accuracy.

Electrophoresis Principle

• Electrophoresis is based on the principle that molecules migrate toward respective electrodes depending on their charge, a process widely utilized in bioscience.

Types of Electrophoresis

1. Zone Electrophoresis: Traditional method separating molecules by charge differences.

2. Thin Layer Electrophoresis: Utilizes a thin layer of gel for separating samples efficiently.

3. Capillary Electrophoresis: Employs narrow capillaries for high-resolution separation.

4. Immunoelectrophoresis: Merges electrophoresis with immunodiffusion, increasingly used for serum protein analysis.

Immunoelectrophoresis Procedure

• This technique combines electrophoresis with immunodiffusion in a gel matrix, allowing for qualitative and quantitative assessments of serum proteins.

Applications of Electrophoresis

• Primarily applied in studying protein alterations associated with diseases and diagnosing immunoglobulin deficiencies.

Isoelectric Focusing (IEF)

• A technique that evaluates the mobility of molecules based on pH differences within a gradient, allowing for sharper band resolutions compared to traditional methods.

Why Use Atomic Absorption Spectroscopy (AAS)?

• AAS is instrumental in health spheres for monitoring essential trace metals and detecting toxic metals across various industrial applications, ensuring compliance with safety standards.

Limitations of AAS

• AAS is affected by several types of interference including chemical, ionization effects, matrix differences, and various spectral interferences, which must be acknowledged during analysis.

Standard Addition Technique

• This technique adds standards directly to the samples being tested to reduce discrepancies caused by matrix interference, enhancing analytical validity.

Immunoassays Overview

• Immunoassays utilize the specific binding interactions between antibodies and antigens for the detection and quantification of analytes, a widely adopted methodology in medicine and research.

Polyclonal vs. Monoclonal Antibodies

1. Polyclonal Antibodies: Characterized by a mixture of antibodies derived from different immune cells; they are typically less expensive and produced more quickly.

2. Monoclonal Antibodies: Recognize a single epitope, offer greater specificity, are uniform in production, but come at a higher cost.

Turbidimetry

• Measures transmitted light with respect to particle concentration and size, often utilized in various biochemistry applications.

Nephelometry

• This technique measures scattered light and is more sensitive than turbidimetry, making it suitable for low-concentration samples.

Heterogeneous vs. Homogeneous Assays

• Heterogeneous assays require separation methods post-reaction, while homogeneous assays can be conducted in a single solution, facilitating easier workflows.

Interferences Affecting Immunoassay Performance

• Various types of interferences can impact analyte detection in immunoassays; common interferences include cross-reactivity of antibodies and pre-analytical factors like sample handling and storage conditions.

Applications of Ion Selective Electrode (ISE)

• ISEs are valuable tools for measuring pH levels, electrolyte concentrations, and blood gases, playing a significant role in clinical diagnostics.

Direct vs. Indirect ISE

• Direct ISE: Provides accurate measurements of ion activities directly.

• Indirect ISE: Involves a dilution step before measurement which may lead to inaccuracies in certain sample conditions due to effects of matrix complexity.

Automation in Laboratories

• Automation in laboratories is vital for enhancing test efficiency, minimizing variations between runs, and significantly improving error management protocols, facilitating advanced research and clinical capabilities.