Centrifugation - PDF - University of Uyo

DEPARTMENT OF MEDICAL BIOCHEMISTRY FACULTY OF BASIC MEDICAL SCIENCES COLLEGE OF HEALTH SCIENCES UNIVERSITY OF UYO – UYO COURSE CODE: MBC 211 COURSE TITLE: PHYSICAL AND ANALYTICAL BIOCHEMISTRY SESSION: FIRST SEMESTER 2024/2025 CREDIT UNIT...

DEPARTMENT OF MEDICAL BIOCHEMISTRY FACULTY OF BASIC MEDICAL SCIENCES COLLEGE OF HEALTH SCIENCES UNIVERSITY OF UYO – UYO COURSE CODE: MBC 211 COURSE TITLE: PHYSICAL AND ANALYTICAL BIOCHEMISTRY SESSION: FIRST SEMESTER 2024/2025 CREDIT UNIT: (3 CH) LECTURER: DR GRACE EKPO COURSE CONTENTS:  Centrifugation: Definition; Principle; Types of Centrifuge; Types of Centrifugation; Applications of Centrifugation.  Cell Fraction; Methods of Cell Fractionation.  Structure, Function and Fractionation of Extracellular Organelles.  Overview of Instrumentation in Medical Biochemistry: Autoclave; Hot Plate; Pipette; Water Distiller; Bunsen Burner; Laboratory Refrigerator and Freezers; pH Meter; Balances; Dry Block Heater; Colorimeter; Dispensers; Flame Photometer; Fume Cupboard; Gas Analyser; Gas Detector; Petri Dish; Measuring Cylinder; Photometer; Reagent Bottle; Homogenizer; Penetrometer; Spectrophotometer; vortex etc.  Human Specimen Collection and Handling.  Reference Ranges.  Quality Assurance and Quality Control in the Laboratory.  Laboratory Practical. LESSON ONE 1(a) Centrifugation: Definition; Principle; Types of Centrifuge; Types of Centrifugation; Applications of Centrifugation. CENTRIFUGATION  Centrifugation is a technique of separating substances which involves the application of centrifugal force.  The particles are separated from a solution according to their size, shape, density, the viscosity of the medium and rotor speed. 1|Page  Principle of Centrifugation  In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles that are lighter than it floats to the top.  The greater the difference in density, the faster they move. If there is no difference in density (isopycnic conditions), the particles stay steady.  To take advantage of even tiny differences in density to separate various particles in a solution, gravity can be replaced with the much more powerful “centrifugal force” provided by a centrifuge.  A centrifuge is a piece of equipment that puts an object in rotation around a fixed axis (spins it in a circle), applying a potentially strong force perpendicular to the axis of spin (outward).  The centrifuge works using the sedimentation principle, where the centripetal acceleration causes denser substances and particles to move outward in the radial direction.  At the same time, objects that are less dense are displaced and move to the center.  In a laboratory centrifuge that uses sample tubes, the radial acceleration causes denser particles to settle to the bottom of the tube, while low- density substances rise to the top. 2|Page  TYPES OF CENTRIFUGE LOW-SPEED CENTRIFUGE Ts1) Most laboratories have a standard low-speed centrifuge used for routine sedimentation of heavy particles 2) The low-speed centrifuge has a maximum speed of 4000-5000rpm 3) These instruments usually operate at room temperatures with no means of temperature control. 4) Two types of rotors are used in it,  Fixed angle  Swinging bucket. 5) It is used for sedimentation of red blood cells until the particles are tightly packed into a pellet and supernatant is separated by decantation. HIGH-SPEED CENTRIFUGES 1. High-speed centrifuges are used in more sophisticated biochemical applications, higher speeds and temperature control of the rotor chamber are essential. 2. The high-speed centrifuge has a maximum speed of 15,000 – 20,000 RPM 3. The operator of this instrument can carefully control speed and temperature which is required for sensitive biological samples. 4. Three types of rotors are available for high-speed centrifugation-  Fixed angle 3|Page  Swinging bucket  Vertical rotors ULTRACENTRIFUGES 1. It is the most sophisticated instrument. 2. Ultracentrifuge has a maximum speed of 65,000 RPM (100,000’s x g). 3. Intense heat is generated due to high speed thus the spinning chambers must be refrigerated and kept at a high vacuum. 4. It is used for both preparative work and analytical work. TYPES OF CENTRIFUGATION 1. Differential Pelleting (differential centrifugation)  It is the most common type of centrifugation employed.  Tissue such as the liver is homogenized at 32 degrees in a sucrose solution that contains buffer.  The homogenate is then placed in a centrifuge and spun at constant centrifugal force at a constant temperature.  After some time a sediment forms at the bottom of a centrifuge called pellet and an overlying solution called supernatant.  The overlying solution is then placed in another centrifuge tube which is then rotated at higher speeds in progressing steps. 2. Density Gradient Centrifugation  This type of centrifugation is mainly used to purify viruses, ribosomes, membranes, etc.  A sucrose density gradient is created by gently overlaying lower concentrations of sucrose on higher concentrations in centrifuge tubes  The particles of interest are placed on top of the gradient and centrifuge in ultracentrifuges.  The particles travel through the gradient until they reach a point at which their density matches the density of surrounding sucrose.  The fraction is removed and analyzed. 3. Rate-Zonal Density-Gradient Centrifugation  Zonal centrifugation is also known as band or gradient centrifugation  It relies on the concept of sedimentation coefficient (i.e. movement of sediment through the liquid medium)  In this technique, a density gradient is created in a test tube with sucrose and high density at the bottom.  The sample of protein is placed on the top of the gradient and then centrifuged.  With centrifugation, faster-sedimenting particles in sample move ahead 4|Page  of slower ones i.e. sample separated as zones in the gradient.  The protein sediment according to their sedimentation coefficient and the fractions are collected by creating a hole at the bottom of the tube. 4. Isopynic Centrifugation  The sample is loaded into the tube with the gradient-forming solution (on top of or below pre-formed gradient, or mixed in with self-forming gradient)  The solution of the biological sample and cesium salt is uniformly distributed in a centrifuge tube and rotated in an ultracentrifuge.  Under the influence of centrifugal force, the cesium salts redistribute to form a density gradient from top to bottom.  Particles move to point where their buoyant density equals that part of gradient and form bands. This is to say the sample molecules move to the region where their density equals the density of gradient.  It is a “true” equilibrium procedure since depends on bouyant densities, not velocities Eg: CsCl, NaI gradients for macromolecules and nucleotides – “self- forming” gradients under centrifugal force. APPLICATIONS OF CENTRIFUGATION Centrifugation is a technique that uses centrifugal force to separate components of a mixture based on their density, size, and shape. Here are some key applications: 1. Biological Research: o Cell Separation: Isolating different cell types from blood or tissue samples. o Protein Purification: Separating proteins based on size or density, often used in biochemical research. 2. Clinical Diagnostics: o Blood Component Separation: Isolating plasma or serum from blood samples for testing. o Urine Analysis: Concentrating and separating components for diagnostic purposes. 3. Food Industry: o Cream Separation: Separating cream from milk in dairy processing. o Juice Clarification: Removing pulp and sediment from fruit juices. 4. Pharmaceuticals: 5|Page  o Drug Formulation: Purifying active ingredients and removing impurities. o Vaccine Production: Concentrating viral particles or antigens. 5. Environmental Science: o Water Treatment: Removing suspended solids and contaminants from water samples. o Soil Analysis: Separating soil particles for study of composition and contamination. 6. Industrial Applications: o Material Separation: In manufacturing processes, separating materials based on density (e.g., in recycling). o Chemical Reactions: Separating products from reaction mixtures. 7. Nanotechnology: o Nanoparticle Separation: Isolating nanoparticles for research and application in various fields. 8. Genetics: o DNA/RNA Purification: Isolating nucleic acids from cells or tissues for genetic analysis. LESSON ONE (1b): Cell Fraction; Methods of Cell Fractionation. Cell fractionation is a laboratory technique used to separate cellular components while preserving their individual functions. This process allows researchers to study specific organelles or cellular structures in detail. Here are some common methods of cell fractionation: 1. Differential Centrifugation  Description: This method involves sequentially spinning a cell homogenate at increasing speeds.  Process: o Cells are broken open (homogenized) to release their contents. o The homogenate is centrifuged at low speeds to pellet the largest components (e.g., nuclei). o The supernatant is then centrifuged at higher speeds to separate smaller organelles (e.g., mitochondria, microsomes).  Applications: Commonly used to isolate organelles for biochemical studies. 2. Density Gradient Centrifugation  Description: This technique uses a density gradient (usually created with a sugar or salt solution) to separate particles based on their density. 6|Page   Process: o A homogenate is layered on top of a pre-formed gradient in a centrifuge tube. o When spun, organelles will migrate to their specific density levels, forming distinct bands.  Applications: Often used to purify specific organelles like ribosomes or lipid membranes. 3. Isopycnic Centrifugation  Description: A specialized form of density gradient centrifugation where particles reach an equilibrium position at their buoyant density.  Process: o Similar to density gradient centrifugation, but the focus is on maintaining the density of the gradient throughout the centrifugation.  Applications: Used for separating nucleic acids or specific protein complexes. 4. Ultracentrifugation  Description: Involves spinning samples at extremely high speeds (up to 100,000 x g or more).  Process: o This method can effectively separate very small particles like viruses or ribosomes.  Applications: Used in molecular biology for studying macromolecules and subcellular structures. 5. Filtration and Size Exclusion  Description: Involves passing the homogenate through filters or gels that separate components based on size.  Process: o Larger components are retained while smaller ones pass through.  Applications: Useful for separating whole cells from cell debris or for isolating specific sizes of proteins. 6. Magnetic Bead Separation  Description: Uses magnetic beads coated with antibodies to isolate specific cell types or organelles.  Process: o Beads bind to target components, and a magnet is applied to separate the bound components from the rest. 7|Page   Applications: Frequently used in immunoprecipitation and cell sorting. 7. Gradient Centrifugation with Ultrafiltration  Description: Combines gradient centrifugation with ultrafiltration techniques.  Process: o After initial separation, ultrafiltration can refine the isolation of specific organelles.  Applications: Enhances purity and concentration of isolated components. LESSON TWO (2a): Structure, Function and Fractionation of Extracellular Organelles Extracellular organelles, such as exosomes and microvesicles, play critical roles in intercellular communication and various physiological processes. Here’s an overview of their structure, function, and methods for their fractionation. Structure 1. Exosomes: o Size: Typically 30-150 nm in diameter. o Origin: Formed within multivesicular bodies (MVBs) that bud from the endosomal membrane. o Membrane: Composed of a lipid bilayer enriched in specific proteins, lipids, and RNA. 2. Microvesicles: o Size: Larger than exosomes, ranging from 100-1,000 nm. o Origin: Bud directly from the plasma membrane of cells. o Membrane: Also composed of a lipid bilayer, containing proteins and lipids that reflect the parent cell’s characteristics. 3. Apoptotic Bodies: o Size: Generally larger than microvesicles, 1-5 µm in diameter. o Origin: Formed during the process of apoptosis (programmed cell death). o Composition: Contain cellular debris, including organelles and nuclear fragments. Function 1. Exosomes: o Cell Communication: Transfer proteins, lipids, and RNA between cells, influencing recipient cell behavior. 8|Page  o Immune Response: Modulate immune responses by presenting antigens or delivering signaling molecules. o Waste Management: Facilitate the removal of unwanted materials from cells. 2. Microvesicles: o Cell Signaling: Involved in local and systemic signaling processes by transferring bioactive molecules. o Homeostasis: Help maintain cellular homeostasis and respond to stress by exchanging components. o Tissue Repair: Play roles in wound healing and tissue regeneration. 3. Apoptotic Bodies: o Cell Clearance: Assist in the safe removal of dying cells by phagocytes, preventing inflammation. o Intercellular Signaling: May carry signals that influence surrounding cells in tissue repair and regeneration. Fractionation Methods 1. Differential Centrifugation: o Process: A series of centrifugation steps at increasing speeds to separate extracellular organelles based on size and density. o Application: Initially pelleting larger debris, followed by the isolation of microvesicles and exosomes. 2. Density Gradient Centrifugation: o Process: Samples are layered over a gradient (e.g., sucrose or iodixanol) and centrifuged, allowing separation based on buoyant density. o Application: Provides a more refined isolation of exosomes and microvesicles, yielding high-purity fractions. 3. Filtration: o Process: Utilizes filters with specific pore sizes to separate extracellular organelles from larger particles. o Application: Often used in conjunction with centrifugation to enrich for exosomes and microvesicles. 4. Ultrafiltration: o Process: Uses membranes that allow small molecules to pass while retaining larger extracellular organelles. o Application: Efficiently concentrates and purifies exosomes from culture media or biological fluids. 5. Magnetic Bead Isolation: o Process: Employs magnetic beads coated with antibodies 9|Page  targeting specific surface proteins of exosomes or microvesicles. o Application: Allows for targeted isolation of specific subsets of extracellular organelles based on their protein markers. LESSON (TWO) 2b: Overview of Instrumentation in Medical Biochemistry Instrumentation in medical biochemistry encompasses a variety of tools and techniques used for analyzing biological samples, diagnosing diseases, and monitoring health. These instruments collectively facilitate the various analytical, preparatory, and safety procedures essential in medical biochemistry, ensuring high-quality research and accurate diagnostics. Here’s an overview of key instruments and their applications: 1. Spectrophotometers  Function: Measure the intensity of light absorbed by a sample at specific wavelengths.  Applications: Used for quantifying biomolecules (like proteins, nucleic acids, and metabolites) and enzyme assays. 2. Chromatography Systems  Types: o High-Performance Liquid Chromatography (HPLC): Separates and quantifies compounds in a mixture. o Gas Chromatography (GC): Analyzes volatile compounds.  Applications: Used for drug analysis, metabolic profiling, and hormone measurement. 3. Mass Spectrometers  Function: Measures the mass-to-charge ratio of ions to identify and quantify molecules.  Applications: Used for identifying proteins, metabolites, and drugs in complex mixtures, as well as in proteomics and metabolomics studies. 4. Electrophoresis Equipment  Types: o Agarose Gel Electrophoresis: Separates DNA or RNA fragments. o SDS-PAGE: Separates proteins based on size.  Applications: Used in genetic analysis, protein purification, and 10 | P a g e  quality control. 5. Enzyme Immunoassays (EIAs) and ELISA Kits  Function: Utilize antibodies to detect and quantify specific antigens or antibodies in a sample.  Applications: Commonly used for diagnosing infectious diseases, measuring hormone levels, and screening for allergens. 6. Clinical Chemistry Analyzers  Function: Automated systems that perform a wide range of biochemical tests on blood and other body fluids.  Applications: Used for routine tests like glucose, cholesterol, liver function tests, and electrolyte panels. 7. Nuclear Magnetic Resonance (NMR) Spectroscopy  Function: Analyzes molecular structure and dynamics based on the magnetic properties of atomic nuclei.  Applications: Used in metabolomics and structural biology to study metabolites and macromolecules. 8. Polymerase Chain Reaction (PCR) Machines  Function: Amplifies specific DNA sequences through thermal cycling.  Applications: Widely used in genetic testing, pathogen detection, and research applications. 9. Flow Cytometers  Function: Analyze and sort cells based on their physical and chemical characteristics.  Applications: Used in immunology, cancer research, and diagnostics for analyzing cell populations. 10. Microarray Platforms  Function: Allow simultaneous analysis of thousands of genes or proteins.  Applications: Used in genomics and proteomics for gene expression profiling and disease marker discovery. 11. Autoclave  Purpose: Sterilizes equipment and media with high-pressure steam.  Application: Critical for eliminating microorganisms before experiments. 12. Hot Plate  Purpose: Provides a controlled heat source.  Application: Used for heating solutions and maintaining reaction temperatures. 13. Pipette 11 | P a g e   Purpose: Accurately measures and transfers small liquid volumes.  Application: Essential for sample preparation and assays. 14. Water Distiller  Purpose: Produces purified water through distillation.  Application: Supplies sterile water for experiments, reducing contamination risks. 15. Bunsen Burner  Purpose: Produces an open flame for heating and sterilization.  Application: Commonly used for sterilizing tools and heating samples. 16. Laboratory Refrigerator and Freezers  Purpose: Store samples at controlled low temperatures.  Application: Refrigerators for short-term, freezers for long-term preservation of biological materials. 17. pH Meter  Purpose: Measures the acidity or alkalinity of a solution.  Application: Essential for monitoring pH in various biochemical assays and processes. 18. Balances  Purpose: Accurately weighs samples and reagents.  Application: Critical for preparing precise concentrations in experiments. 19. Dry Block Heater  Purpose: Provides uniform heat without water.  Application: Used for incubating samples at specific temperatures. 20. Colorimeter  Purpose: Measures the absorbance or transmittance of light in a sample.  Application: Useful for quantitative analysis of colored solutions. 21. Dispensers  Purpose: Dispenses precise volumes of liquids.  Application: Facilitates the quick and accurate addition of reagents. 22. Flame Photometer  Purpose: Analyzes the concentration of certain metal ions by measuring emitted light from a flame.  Application: Commonly used for detecting sodium, potassium, and lithium. 23. Fume Cupboard  Purpose: Provides a ventilated workspace to safely handle hazardous 12 | P a g e  chemicals.  Application: Essential for protecting users from toxic fumes and vapors. 24. Gas Analyser  Purpose: Measures concentrations of gases in a sample.  Application: Used for respiratory gas analysis and environmental monitoring. 25. Gas Detector  Purpose: Detects the presence of harmful gases.  Application: Ensures safety in the laboratory by monitoring for leaks. 26. Petri Dish  Purpose: Cultivates microorganisms and cells.  Application: Used for microbiological assays and cell culture experiments. 27. Measuring Cylinder  Purpose: Measures liquid volumes accurately.  Application: Useful for preparing solutions in precise quantities. 28. Photometer  Purpose: Measures light intensity or concentration of light-absorbing substances.  Application: Employed in various biochemical assays for quantification. 29. Reagent Bottle  Purpose: Stores chemicals and reagents safely.  Application: Ensures proper labeling and storage conditions for laboratory use. 30. Homogenizer  Purpose: Mixes and breaks down samples to create uniform suspensions.  Application: Used for preparing tissue samples for analysis. 31. Penetrometer  Purpose: Measures the consistency of semi-solid substances.  Application: Commonly used in quality control of pharmaceutical formulations. 32. Vortex Mixer  Purpose: Mixes small volumes of liquid samples rapidly.  Application: Used to ensure uniformity in samples and reagents. LESSON (THREE) 3a: Human Specimen Collection and Handling Human specimen collection and handling are critical steps in medical 13 | P a g e diagnostics and research, ensuring the accuracy and reliability of results. Human specimen collection is a critical process in healthcare and research, involving the gathering of biological samples for diagnostic testing, monitoring, or research purposes. Here’s an overview of key aspects: Types of Specimens 1. Blood o Methods: Venipuncture (drawing blood from a vein), capillary collection (fingerstick). o Uses: Blood tests for chemistry, hematology, blood cultures, etc. 2. Urine o Types: Random, midstream clean catch, 24-hour collection. o Uses: Urinalysis, culture tests, drug testing. 3. Tissue o Methods: Biopsies (needle, excisional, or incisional). o Uses: Pathology examinations, histology. 4. Saliva o Methods: Swabs or collection containers. o Uses: Hormone testing, DNA analysis. 5. Sputum o Methods: Collected from the respiratory tract, often after deep coughing. o Uses: Microbiological cultures, cytology. 6. Other Specimens o Stool: For gastrointestinal tests. o Synovial Fluid: From joints for analysis. o CSF (Cerebrospinal Fluid): Collected via lumbar puncture for neurological testing. Collection Procedures 1. Preparation o Patient Identification: Confirm the patient’s identity and explain the procedure. o Informed Consent: Obtain consent from the patient for the collection process. 2. Aseptic Technique o Use sterile equipment and follow hygiene protocols to prevent contamination. 3. Collection Timing o Some tests require specific timing (e.g., fasting for glucose tests, timed urine collections). 14 | P a g e  4. Volume o Collect the correct volume to meet test requirements. Insufficient amounts can lead to inaccurate results. Handling and Storage 1. Labeling o Clearly label all specimens with patient details (name, ID, date, time of collection) and type of specimen. 2. Transport o Use appropriate containers and packaging to prevent leakage and contamination during transport to the laboratory. 3. Storage Conditions o Follow specific guidelines for storage:  Blood: Refrigerated or at room temperature, depending on the test.  Urine: Typically refrigerated; some tests may require immediate processing.  Tissue: Fixed in formalin or frozen based on the analysis requirements. Safety Considerations 1. Personal Protective Equipment (PPE) o Use gloves, masks, and gowns as needed to protect against exposure. 2. Biohazard Disposal o Properly dispose of sharps and other biohazardous materials in designated containers. Quality Control 1. Training o Ensure that all personnel involved in specimen collection are adequately trained in techniques and protocols. 2. Documentation o Maintain accurate records of all specimens collected, including any issues encountered during the process. LESSON (THREE) 3b: Reference Ranges Reference ranges, also known as normal ranges, are important benchmarks used in laboratory medicine to interpret test results. They represent the values typically found in a healthy population and serve as a guide for assessing individual patient results. 15 | P a g e  Here’s an overview: 1. Definition Reference ranges indicate the range of values expected for a healthy individual for a specific test. These ranges are determined through statistical analysis of samples from a healthy population. 2. Establishment of Reference Ranges  Population Selection: Reference ranges are established using a representative sample of a healthy population, considering age, sex, ethnicity, and other factors.  Statistical Methods: Typically, the range is defined as the mean ± 2 standard deviations (SD), encompassing about 95% of the healthy population.  Laboratory Variability: Reference ranges can vary between laboratories due to differences in equipment, reagents, and methodologies. 3. Factors Influencing Reference Ranges  Demographics: Age, gender, race, and geographic location can all affect normal values.  Physiological Conditions: Factors like pregnancy, diet, and medications can alter test results.  Circadian Rhythms: Some values fluctuate throughout the day (e.g., cortisol levels). 4. Common Tests and Their Reference Ranges  Blood Glucose: Normal fasting levels are typically between 70-100 mg/dL.  Complete Blood Count (CBC): o White Blood Cells (WBC): 4,500-11,000 cells/µL o Hemoglobin:  Men: 13.8-17.2 g/dL  Women: 12.1-15.1 g/dL  Lipid Profile: o Total Cholesterol: 50 mg/dL for women  Liver Function Tests: o Alanine Aminotransferase (ALT): 7-56 units/L o Aspartate Aminotransferase (AST): 10-40 units/L 5. Interpretation of Test Results  Above Normal Range: May indicate a potential health issue (e.g., diabetes, liver disease).  Below Normal Range: Can also signify problems (e.g., anemia, malnutrition).  Contextual Interpretation: Always consider results in conjunction with clinical symptoms and medical history. 6. Limitations of Reference Ranges  Not Absolute: Individual results may fall outside the reference range and still be clinically normal.  Dynamic Nature: Health status can change, affecting the relevance of the reference range over time. 7. Clinical Use Reference ranges are essential for:  Diagnosing conditions  Monitoring disease progression  Evaluating treatment efficacy LESSON (FOUR)4a: Quality Assurance and Quality Control in the Laboratory Quality Assurance (QA) and Quality Control (QC) are essential components of laboratory operations, ensuring the accuracy, reliability, and consistency of test results. Implementing robust QA and QC practices is essential for laboratories to maintain high standards, comply with regulations, and ensure the accuracy of diagnostic results. This leads to improved patient outcomes and trust in laboratory services. Here’s an overview of both concepts: Quality Assurance (QA) Definition: QA encompasses all systematic activities designed to ensure that the laboratory processes meet established standards and requirements. It is focused on preventing errors and ensuring high-quality outcomes. 17 | P a g e Key Components: 1. Standard Operating Procedures (SOPs): o Development and implementation of written procedures for all laboratory processes. o Ensures consistency and compliance with protocols. 2. Training and Competency: o Regular training for laboratory personnel to ensure they are knowledgeable about procedures and equipment. o Competency assessments to confirm staff can perform tests accurately. 3. Documentation: o Maintaining accurate records of all laboratory activities, including training, test results, and equipment maintenance. o Ensures traceability and accountability. 4. Internal Audits: o Regular evaluations of laboratory processes to identify areas for improvement and compliance with regulations. 5. Management Review: o Periodic reviews by management to assess the effectiveness of the QA program and implement necessary changes. 6. Compliance with Regulations: o Adhering to local and international standards, such as ISO 15189 for medical laboratories or CLIA regulations. Quality Control (QC) Definition: QC involves the operational techniques and activities used to monitor and control the quality of laboratory results. It focuses on identifying defects in the process. Key Components: 1. Control Samples: o Use of control materials (known samples) to verify the accuracy and precision of test results. o Control limits are established, and results are monitored against these limits. 2. Calibration: o Regular calibration of laboratory instruments to ensure they provide accurate measurements. o Calibration schedules should be adhered to based on manufacturer guidelines and regulatory requirements. 3. Proficiency Testing: o Participation in external proficiency testing programs to 18 | P a g e  evaluate laboratory performance against peers. o Helps identify areas needing improvement. 4. Statistical Process Control: o Application of statistical methods to monitor and control processes, helping to identify trends or shifts in data. o Charts and graphs are often used to visualize performance. 5. Corrective Actions: o Implementation of procedures for addressing any deviations from expected results or quality standards. o Documenting the root cause analysis and actions taken to prevent recurrence. Integration of QA and QC  Continuous Improvement: Both QA and QC are part of a continuous improvement cycle that aims to enhance laboratory performance.  Risk Management: Identifying potential risks in laboratory processes and implementing strategies to mitigate them.  Patient Safety: Ultimately, QA and QC contribute to better patient care by ensuring that test results are accurate and reliable. LESSON (FOUR) 4b: Laboratory practicals Laboratory practicals are hands-on sessions designed to teach students and professionals how to perform various experiments and procedures in a laboratory setting. They provide valuable experience in applying theoretical knowledge and developing practical skills. Laboratory practicals are an essential part of scientific education, providing the hands-on experience necessary to develop competent and confident laboratory professionals. Here’s an overview of what laboratory practicals typically involve: 1. Objectives of Laboratory Practicals  Skill Development: Enhance technical skills related to instrumentation, sample handling, and analytical techniques.  Application of Theory: Bridge the gap between theoretical knowledge and real-world applications.  Problem-Solving: Develop critical thinking and troubleshooting skills through practical experimentation.  Data Analysis: Learn to collect, analyze, and interpret data accurately. 2. Components of a Laboratory Practical  Preparation: o Pre-Lab Assignments: Review background material, safety 19 | P a g e  protocols, and procedures before the lab session. o Material and Equipment Setup: Ensure all necessary materials, reagents, and equipment are prepared and functional.  Conducting Experiments: o Following Protocols: Adhere to standard operating procedures (SOPs) for accuracy and safety. o Data Collection: Systematically record observations and measurements during the experiment.  Safety Practices: o Personal Protective Equipment (PPE): Use gloves, goggles, and lab coats as appropriate. o Emergency Procedures: Be familiar with the location of safety equipment (e.g., eyewash stations, fire extinguishers) and protocols for spills or accidents. 3. Types of Laboratory Practicals  Biochemical Analysis: Techniques such as spectrophotometry, chromatography, and electrophoresis.  Microbiological Techniques: Culturing microorganisms, performing sensitivity tests, and staining procedures.  Chemical Synthesis: Conducting reactions and purifying products.  Tissue Culture: Growing and manipulating cells in vitro for research purposes. 4. Data Analysis and Interpretation  Calculations: Performing necessary calculations (e.g., concentration, dilution factors).  Statistical Analysis: Applying statistical methods to assess data reliability and significance.  Reporting Results: Writing laboratory reports that summarize objectives, methods, results, and conclusions. 5. Common Challenges  Technical Difficulties: Equipment malfunction or unexpected results can arise; problem-solving skills are crucial.  Time Management: Balancing time effectively to complete all tasks within the allocated session.  Collaboration: Working effectively in teams, communicating findings, and dividing tasks. 6. Evaluation and Feedback  Peer Review: Engaging in discussions with peers about findings and methodologies. 20 | P a g e   Instructor Feedback: Receiving constructive criticism on technique and reporting to improve future performance. 7. Importance of Laboratory Practicals  Real-World Relevance: Prepares students and professionals for careers in healthcare, research, and industry.  Critical Thinking: Encourages independent thinking and the application of scientific principles.  Innovation and Discovery: Fosters creativity and curiosity, leading to new insights and advancements in science. TEXTBOOKS: (1) CROOK, MARTIN A. “CLINICAL BIOCHEMISTRY AND METABOLIC MEDICINE” (YEAR OF PUBLICATION 2018) EIGHT EDITION. (2) VASUDEVAN, P.M “TEXTBOOK OF BIOCHEMISTRY FOR MEDICAL STUDENTS” (YEAR OF PUBLICATION 2017) SIX EDITION. (3) CHATTERJEA, M.N “TEXTBOOK FOR MEDICAL BIOCHEMISTRY” (YEAR OF PUBLICATION 2018) THREE EDITION. (4) BHAGAVAN, N. “ESSENTIALS OF MEDICAL BIOCHEMISTRY” (YEAR OF PUBLICATION 2019) SEVEN EDITION. 21 | P a g e