Principles of laboratory

• The enzyme is added to the substrate, which undergoes hydrolysis.
• Evaluation of biochemical components of tissues, organs and biological fluids from humans

  1. Confirm a health status

  2. Collaborate identifying a disease (diagnosis)

  3. Monitor the progress of a patient

  4. Monitor the efficacy of a treatment

  5. Provide a prognosis of a pathology
• The interpretation of clinical biochemical tests is usually based on dynamic assessments from data
• Aims of research


  • Methodology
  • Disease physiopathology
  • Clinical value of biochemical variables
• Ethical issues faced by practitioners in laboratory medicine

  • Confidentiality of genetic information
  • Confidentiality of patient medical information
  • Allocation of resources
  • Codes of conduct
  • Publishing issues
  • Conflict of interest
• Molecules that can be measured and have clinical applicability

  • Nucleic acids
  • Amino acids
  • Peptides and proteins
  • Carbohydrates
  • Lipids
  • Mineral salts, ions
  • Dissolved gases
  • Metabolic substrates
  • Final metabolite
  • Enzymes
  • Structural components of organs and tissues
  • Genome
  • Hormones (derived from peptides or not)
  • Vitamins
  • Medication and drugs
  • Antibodies
  • Cell signalling biomolecules
  • Metals (toxics; oligoelements)
  • Toxics derived from organics
• Methodologies in laboratory medicine

  • Spectrophotometry (atomic absorption spectrophotometry)
  • Colorimetry
  • Enzymatic methods
  • Fluorimetry
  • Turbidimetry
  • Nephelometry
  • Luminiscence
  • Atomic spectrometry: Emission, Absorption
  • Osmometry
  • Modes of separation: partition chromatography, electrophoresis, chromatography, centrifuge
  • Electrochemical: Potentiometry and ion-selective electrodes, Voltammetry/Amperometry
  • Immunochemical: Agglutination assays, Immunodiffusion, Immunofixation, Immunonephelometry, Immunoanalysis with labeled reagents: Isotopes, Enzymes, Fluoroprobes, Luminescence
• The usefulness of biochemical parameters for diagnostic purposes
• Organospecific markers


  • Useful to verify the release of tissue components to the blood flow to point that an organ or tissue is: Obstruction, Inflammation, Intoxication. The analytes to be considered will be enzymes or structural components: Excessive production, Low production
• Organospecific markers


  • Metabolic substrates, i.e. Glucose
  • Final or intermediate metabolite, i.e. Hyperphenylketonuria
  • Hormones
  • Genome analysis
  • Vitamins
  • Basic analytes, i.e. H2O, electrolytes (Na+, K+)
  • Intercellular signaling molecules, i.e. Cytokines
  • Specific biochemical components of blood: Proteins, Lipoproteins, coagulation factors
• Analytical process


  Starts with prescription and finish when the results are delivered to the physician. Stages: Preanalytical, Analytical, Postanalytical
• Preanalytical stage


  Patient preparation: Laboratory tests are affected by many factors such as exercise, diet, recent intake of food, time, other variables. Proper patient preparation is essential for test results to be meaningful so collection of samples is done in the morning, fasting. Specimen collection: Blood is extracted from veins located in the elbow or capillary blood from the heel. Problems: Prolonged tourniquet application: local anoxia to cells and excessive venous back pressure. Anoxia causes small solutes (K+) to leak from cells which leads to false values. Excessive venous pressure concentrates cells, proteins, substances bound to proteins (Ca2+). Searching for a vein with the needle. Hemolysis during and after collection alters the concentration
• Anoxia


  Causes small solutes (K+) to leak from cells which leads to false values
• Excessive venous pressure


  Concentrates cells, proteins, substances bound to proteins (Ca2+)
• Hemolysis during and after collection

  Alters the concentration of any analyte
• Separating blood components


  1. Adding anticoagulants: Plasma + blood cells

  2. Allowing coagulation: Serum (blood plasma without fibrinogen)
• Serum or plasma must be separated from blood cells as soon as possible (1-2 h)


  1. Red blood cell lysis

  2. Enzymes of the red blood cells + anaerobic glycolysis glucose consumption
• Samples are preserved at 4°C


  NEVER freeze the whole blood (erythrocyte lysis)
• EDTA (sodium, potassium salts)
• Oxalates (sodium, potassium, ammonium, lithium salts)
• Citrate (sodium salt)
• Acid citrate dextrose
• Sodium fluoride
• Iodoacetate (sodium salt)
• Heparin (sodium, potassium, ammonium, lithium salts)
• To obtain a precise and accurate analytical result
• Factors in the postanalytical stage
• Reference ranges for the different analytes instead of normal values
• Clinical decision limits
• False positives (FP)
• False negatives (FN)
• Joint Commission's definition of quality
• Institute of Medicine's definition of quality
• ISO 9000
• Features of a quality system
• Quality system


  • Organizational structure
  • Procedures
  • Processes
  • Media necessary to implement quality management
• Features of a quality system

  • Documentation: All activities from its early beginning until the end MUST be documented (define, measure, analyze, improve and control DMAIC) (SIX SIGMA)
  • Responsibility: The main quality responsible from an institution is who exerts the authority. There is a hierarchical organization in a laboratory.
  • Resources: Quality is expensive but the absence of quality is a negligence
• Reference values


  • Reference values obtained by observation or measurement of a particular type of quantity on an individual selected for comparison using defined criteria (reference individual)
  • Subject-based reference values: Previous values from the same individual, obtained when he or she was in a known state of health
  • Population-based reference values: Obtained from a group of well-defined reference individuals
• Requirements for the selection of reference individuals

  1. All groups of reference individuals should be clearly defined

  2. Inclusion criteria define factors required for the study, while exclusion criteria list factors rendering individuals inappropriate

  3. Selection criteria determine which individuals should be included in the group of reference individuals
• Longitudinal comparison


  An actual value is compared with values obtained from the same individual in a previous period characterized by a defined health state