Clinical Biochemistry

• Veterinary Diagnostics Strategies


  Methods of analysis: Physical, chemical, biochemical and immunoassay methods
• Course level ILO


  Describe the composition and importance of chemical and cellular components of blood and tissue and the diagnostic use of blood and tissue analysis to study normal biological processes as well as pathological disorders in animals
• Lecture level ILOs


  • Describe the samples of use in clinical biochemistry and relevant analytes
  • Understand the use of Beer-Lambert's Law in spectroscopy
  • Describe the basis of biochemical & chemical analysis
  • Describe the use of enzymatic analysis
  • Understand the importance of calibration of assays
  • Describe how antibodies function as reagents in immunoassay
• See Google Scholar for Institute for Biodiversity Animal Health and Comparative Medicine
• Applications of Veterinary Clinical Biochemistry

  • Companion Animal - Quantitative diagnosis & prognosis, Monitoring of treatment
  • Competitive Animal - Monitoring training, Fitness for racing
  • Production Animal - Metabolic profile – nutritional status, Herd health, Food safety – ante mortem inspection
• Types of samples


  • Biological Fluid - Blood, Plasma, Serum, Cells, Urine, Cerebrospinal fluid, Peritoneal fluid, Synovial fluid, Saliva
• Biochemistry Analytes


  • Ions
  • Substrates - Urea, glucose, creatinine
  • Proteins - Enzymes, hormones, antibody, specific proteins (eg albumin)
• Urinalysis (Analysis of Urine)


  • Cells
  • Stones
  • Specific gravity
  • bilirubin
  • Glucose
  • Ketones
  • pH
  • Blood
  • Haemoglobin
  • Total protein content
  • Albumin
  • Bence Jones (IgG light chain)
  • Urine dipstick tests
• Biochemical Assays


  1. Sample

  2. Signal

  3. Raw data

  4. Computation

  5. Result
• Biochemistry Analysers


  Running ~400 tests per hour, Sample, Reagent & Cuvette segments
• Clinical biochemistry contributes to diagnostic information

  • quantitative
  • routine tests shown
  • many other special tests
  • reference ranges
  • Units
  • H and L flags
• Units


  • SI units - Europe, Asia, S. America
  • Gravimetric - USA
  • Analyte - Glucose, Urea, Total protein, Sodium
• Enzyme Units


  International Units (of activity) = IU, 1 IU = amount of enzyme able to transform 1 μmol of substrate (or produce 1 μmol of product) per minute under specified (standard) conditions, For diagnostic analysis unit is IU/litre (IU/L), Temperature dependent
• Standard Conditions


  • Temperature (37°C, 30°C, 25°C)
  • Buffer and pH
  • Substrate concentration >> Michaelis Constant (Km*)
  • Co-factor concentration (eg Mg2+, NADH)
  • Substrate formula/source
  • Monitoring wavelength (λ) depends on colour produced
• Stability of Analyte


  Useful analytes must be stable in serum until analysed, Glucose - Metabolised by blood cells, Ammonia - Evaporates rapidly, Progesterone - Metabolised in milk by somatic cells
• Spectroscopy


  Many test procedures measure absorption or change in absorption of light in UV or visible spectrum, Absorption (A) & change in absorption (ΔA) easily measured by a spectrophotometer with sample in a cuvette, Absorption is proportional to concentration of analyte
• Beer-Lamberts Law


  A = ε lc, where A = absorption, ε = molar absorption coefficient, l = path length, c = concentration
• Types of analysis


  • Electrolytes - Na+, Cl-, Ca2+
  • Substrates - Urea, glucose
  • Protein - Total protein, albumin, globulin
  • Enzymes - Alk Phos, ALT, CK
  • Hormones - Thyroxine, cortisol
• Electrolytes


  Hydrogen ion electrode – pH meter, Ion selective electrode, flame photometry
• Element Analysis


  Atomic Absorption Spectrometry - Elements absorb light at specific wavelengths, Different lamps used for different elements, Ca & Mg also determined by chemical reactions
• Enzyme analysis - Alkaline phosphatase

  1. p nitrophenol phosphate (colourless) -> p-nitrophenol + phosphate (yellow colour, absorbs at 410 nm)

  2. Light source -> Spectrophotometer -> Measures light absorbed
• Standard Conditions for Alk Phos

  • Temperature 37°C
  • Buffer and pH Diethanolamine, pH 9.8
  • Substrate concentration >> Km 10 mmol/L
  • Co-factor concentration Mg2+ at 0.5 mmol/L
  • Substrate p-nitrophenol phosphate
  • Monitoring wavelength λ 410 nm
• Enzyme Activity


  Initial velocity (V0) of reaction determined, Converted from μmol of product per minute per ml to International Unit per litre (IU/L), V0 = reaction rate before substrate depletion or product inhibition
• Enzyme analysis - Lactate Dehydrogenase


  Lactate + NAD+ -> Pyruvate + NADH + H+, No absorbance at 340nm, Absorbs at 340nm, LDH in sample, Enzyme activity in sample is directly proportional to increase in absorbance at 340nm
• Linked Enzyme Analysis - Alanine Transaminase (ALT)


  Alanine + α-ketoglutarate -> glutamate + pyruvate, ALT in sample, + NADH + H+ -> Lactate + NAD+, Spectrophotometer measures reduction in absorbance of NADH at 340nm, ALT activity is directly proportional to rate of reduction in A340
• Enzymes for Glucose Assay

  Glucose + ATP -> Glucose-6-phosphate + ADP, Hexokinase as reagent, Glucose-6-phosphate + NAD+ -> Gluconate-6-phos + NADH + H+, G6P-DH as reagent, Measure at 340nm
• Chemical Reaction for Analysis

  Protein + biuret -> biuret reaction, Creatinine + picric acid -> creatinine - picric - acid - complex (yellow/red 492nm)
• Calibration of Assays


  • By standard - Single standard, Multiple standards – standard curve
  • By reaction rate - Automated/computerised calculation
• Single Point Calibration


  Linear relationship between analyte and signal (ie absorbance), Allows for variation in analytical system, Commercially available multi-analyte calibrator solution provides calibration of many assays
• Multiple Point Calibration


  Where non-linear relation between analyte concentration and signal, Immunoassays
• Immunoassay


  Assays using antibody as a reagent, Detection & quantification of Antibody <====> Antigen reaction, Ab=Ag Highly specific interaction, High sensitivity assays (low Limit of Detection), High specificity assays, Specific antiserum required, Many formats
• Immunoassay Formats


  • Immunoprecipitation
  • Immunoagglutination
  • Immunoturbidimetry
  • Radioimmunoassay
  • Enzyme Linked Immunosorbent Assay
  • Chemi-luminescence immunoassay
  • Lateral flow immunoassay
  • Multiplex immunoassay - Protein arrays
• Applications of Immunoassay


  • Specific protein/peptides - CRP, troponin, insulin
  • Infectious disease - Virus detection
  • Specific antibody responses - Serology, antibody response to pathogen
  • Allergy testing
  • Low Mr biochemicals of low concentration
  • Hormones - Thyroxine, cortisol, progesterone
  • Drugs (therapeutic and illegal)
• Endocrine Assays


  • Thyroid function tests - Total thyroxine, Free thyroxine, TSH stimulation
  • Hyperadrenocorticism (Cushing's syndrome) - Cortisol, ACTH stimulation test, Dexamethasone suppression test
  • Progesterone assay - Pregnancy detection
• Antibody


  Evolved to bind to protein/chemicals of non-self e.g. pathogens, Opsonise bacteria, activate complement, Can be produced to react with 'any' molecule, For immunoassay polyclonal Ab in rabbits, sheep/goats, chicken, Raise monoclonal in mice & immortalise Ab production
• Structure and Function of Antibodies

  Antigen binding sites, Two sites per antibody
• Polyclonal antibody raised by immunization of rabbit, sheep, donkey with the
• Immunoassay Formats


  • Immunoprecipitation
  • Immunoagglutination
  • Immunoturbidimetry
  • Radioimmunoassay
  • Enzyme Linked Immunosorbent Assay
  • Chemi-luminescence immunoassay
  • Lateral flow immunoassay
  • Multiplex immunoassay - Protein arrays
• Applications of Immunoassay


  • Specific protein/peptides
  • Infectious disease
  • Specific antibody responses
  • Allergy testing
  • Low Mr biochemicals of low concentration
  • Hormones
  • Drugs (therapeutic and illegal)
• Endocrine Assays


  • Thyroid function tests
  • Hyperadrenocorticism (Cushing's syndrome)
  • Progesterone assay