Lecture 2/28

• • Immunofixation
  • Starts with same sample placed in all parts of electrophoresis gel
  • Same sample in all five lanes
  • Attach antibodies to five lanes 
  • Complex will form if IgG, IgM, IgA, kappa or lambda are present
• Immunofixation

  • normal: IgG, IgM to be present
  • may or may not be present: IgA
  • abnormal: Kappa or lambda chains
• kappa or lambda chains will be produced in an immune disorder in which excess antibodies are produced (e.g. multiple myeloma)
• Immunofixation

  • will have positive controls at the bottom (in circles)
  • antisera placed on top of controls
• • Rocket electrophoresis
  • Antibody is impregnated in agar itself
  • pH tightly controlled
• • Rocket electrophoresis
  • Use standards, controls, and patient samples (3)
  • If antigen we are looking for is present, precipitate forms and starts at edges
• Rocket electrophoresis

  • precipitate forming at edges "locks" specimen in place
  • allows more specimen to proceed outward
  • looks rocket-like
• Rocket electrophoresis

  • the higher or longer the rocket, the more antigen in patient sample
  • height of the rocket is proportional to the amount of antigen in the sample
• • Isoelectric focusing
  • Gel with pH gradient in it 
  • Sample applied at neutral pH
• Isoelectric focusing

  • Protein will migrate to its isoelectric point
  • From negative side to positive until no net charge
• • Urine - normally, no protein should be in urine
  • Can be used to detect glomerular filtration issues/renal disease/tubular disorders (3)
• urine

  • 24 hour total protein allows for variation in circadian rhythms
• urine

  • Microalbumin is an early indicator of glomerular damage
  • Urine dipstick will not pick up microalbumin, as it is not sensitive enough for these trace amounts
• urine

  Tubular proteinuria - tubules are unable to reabsorb even small amounts of protein
• Doctors may order protein electrophoresis and immunofixation to see what’s going on for protein positive urines
• • Cerebrospinal fluids - chem on CSF is always stat
• cerebrospinal fluid

  In house, test spinal fluids for glucose and total protein
• cerebrospinal fluid

  Normal amount of protein in spinal fluid: 15-45 mg/dL
• cerebrospinal fluid

  • Proteins will be increased in viral and bacterial meningitis and in multiple sclerosis
• cerebrospinal fluid

  • False increase in protein in traumatic taps
  • Dramatic increase will only be in tube 1
• traumatic tap - capillary or venule hit during spinal tap; blood gets into sample
• cerebrospinal fluids

  • Oligoclonal banding - type of electrophoresis
  • Trying to detect multiple distinct bands in gamma region
• oligoclonal banding must be performed concurrently with a serum protein electrophoresis
• oligonal banding is Positive for multiple sclerosis, severe infections, and inflammation (3)
• • Synovial fluid (joints) and pleural fluid - protein testing usually isn’t useful 
• Amniotic fluid - protein most commonly performed is a-fetoprotein
• • Myoglobin - oxygen carrying pigment found in muscle
  • “Hemoglobin of muscle”
• myoglobin is found in both skeletal and cardiac muscle
• myoglobin is not useful for cardiac events
• myoglobin is most often ordered for muscle diseases

  • Muscle wasting diseases
  • Elevated in trauma
  • Rhabdomyolysis
• • Rhabdomyolysis is a disease in which muscle is atrophied, diseased, damaged
• for diagnosis of Rhabdomyolysis, most often urine protein is ordered
• Troponin is a cardiac enzyme
• • Fibronectin - glycoprotein that can be formed in liver, peritoneal macrophages, endothelial cells, and fibroblasts (4)
• fibronectin exists in multiple forms
• fibronectin function

  • cell adhesion, tissue differentiation, and growth and wound healing (4)
• • Fetal fibronectin - indication or predictor for preterm delivery 
• fetal fibronectin is also called fFn
• an increased in fFn can be found in cervical and vaginal secretions
• • Amyloid 
  • Protein polysaccharide complex deposited in tissues during some chronic infections, malignancies, and rheumatic disorders (3)