LAB-PRELIM-MOLECULAR-BIOLOGY

Page 1: Quality Assurance & Quality Control in Molecular Biology Laboratory

Molecular Analysis

• Molecular analysis involves studying biomolecules (DNA/RNA, proteins, nucleic acids).

• Goals include diagnostics and investigations, especially in crime and unidentified causes.

• Molecular testing requires a request from a physician for diagnosis, treatment, and prognosis.

• Key aims:

  • Select appropriate tests for diagnosis and treatment.

  • Generate accurate, clinically relevant results to improve patient care.

Pre-Analytical Process

• Involves activities before specimen analysis, such as:

  • Patient identification and preparation.

  • Sample types include:

    • Solid tissue (best for analysis)

    • Blood

    • Body fluids (saliva, cerebrospinal fluid, stool, urine)

• General Policies for Specimen Collection:

  • All requests must include:

    • Relevant patient ID

    • Type of specimen

    • Ordered test

    • Date/time of collection

    • Reason for testing

    • Contact info of physician

  • Specimens must be received in acceptable conditions, i.e., not tampered, hemolyzed, or degraded.

Pre-Analytical Errors

• Caused by issues in specimen handling:

  • Examples include mislabeling, sample collection errors, or improper transportation.

  • Avoid errors through proper sampling and handling, e.g., fresh freezing methods like snap freezing.

Specimen Processing

• Care for different sample preparations:

  • Paraffin embedded produces lower quality of nucleic acids compared to fresh samples.

  • White blood cells (WBCs) should be isolated for nucleic acid extraction, as red blood cells (RBCs) lack nuclei.

Page 2: Procedures for Molecular Amplification

Aliquoting

• Definition: Dividing a larger sample into smaller portions for analysis.

• Purpose:

  • Avoid unnecessary handling of samples.

  • Preserve original sample integrity.

  • Create back-up samples.

Collection Tubes & Special Requirements

• Use of appropriate collection tubes is vital, including the correct labeling of specimens.

• Additional details may include:

  • Forensic documentation (Chain of Custody)

  • Parentage testing requirements:

    • Consent forms, ethnicity consideration, ID verification.

Standard Operating Procedures (SOPs)

• Documentation for specimen handling.

• Accession Records:

  • Tracking all specimens, documenting date of receipt, identifier, and patient information.

• Sample Unacceptability:

  • Outlined conditions leading to unacceptable specimens and how they are recorded.

Page 3: Analytical Process - Test Performance and Safety

Analytical Hazards

• Biological Hazards:

  • Includes samples with pathogens or genetic materials leading to potential infections.

  • Precautions to avoid exposure include:

    • Avoid mouth pipetting and consuming food in labs.

Chemical Hazards

• Safe handling and storage of chemicals required, with emphasis on:

  • Labeling, avoiding reactions, and adhering to safety protocols.

• Proper use of decontaminated agents is crucial for maintaining a safe work environment.

DNA/RNA Testing

• Stages include:

  • Extraction of nucleic acids from biological samples.

  • Quantification to assess concentration and contamination presence.

Page 4: Test Validation and Proficiency Testing

Key Terms

• Sensitivity: Ability to correctly identify true positives.

• Specificity: Ability to identify true negatives.

• Calibration and Quality Assurance: Essential to ensure accurate results.

  • Regular assessments must meet standards established by regulatory bodies like the FDA.

Test Results Interpretation

• Four potential outcomes for test results:

  • True Positive (TP), True Negative (TN), False Positive (FP), False Negative (FN).

Page 5: Quality Assurance Post-Analytical Process

Activities Involved

• Ensuring high accuracy/proficiency levels after testing.

• Activities include:

  • Documentation of results and standard operating procedures (SOPs).

  • Regular training and assessments of laboratory personnel.

  • Maintenance and calibration of laboratory instruments.

Content in Report

• Must include:

  • Patient ID, test performance details, possibility of false results, and responsible individual's contact information.

Page 6: Microscopy

Overview

• Microscopy is used to view objects that are too small for the naked eye.

  • Historical timeline notes: First microscope by Zachariah Janssen (1590) and developments by Robert Hooke (1665) and Antonie van Leeuwenhoek (1676).

Types of Microscopes

• Brightfield, Darkfield, Phase Contrast, Inverted, Fluorescence, Confocal, Polarized, Electron.

• Each type serves different functions (e.g., visualization methods, magnification levels).

Page 7: Microscope Operation

Care and Use

• Proper radiation and care during use are outlined, including:

  • Secure slides and start with lower magnifications.

Slide Preparation Types

• Impression, Smear, and Covered Slides.

  • Techniques vary based on specimen type.

Page 8: Analytical Devices

Spectrometry

• Main methods include Spectrophotometry, Fluorometry, Turbidimetry, and Nephelometry.

• Adheres to the Beer-Lambert Law for concentration determination.

Components of Measurement

• Light Source, Monochromators, Cuvettes, Photodetectors.

  • Ensure accurate and continuous light paths for readings.

Page 9: Fluorometry

Components

• Light sources and filters play critical roles in excitation and emission processes.

  • Fluorophore properties assessed.

Sample Holder and Measurement Systems

• Single and double beam systems allow for varied approaches in measuring concentrations effectively.

Page 10: Luminescence Techniques

Applications

• Methods like Chemiluminescence and Bioluminescence are addressed for their applications in molecular biology.

• Detection methods in biomolecules and environmental assays.

Page 11: Other Analytical Techniques

Advanced Techniques

• Flame Tests, Atomic Absorption, and Laser Spectrometry.

  • Each possesses unique principles for analyzing specific compounds.

• Collection methods for various measurements include ion-selective and pH electrodes, emphasizing their significance in quantitative testing.