amyloidosis (ad cyto)1

• Amyloidosis


  A disease in which abnormal proteins (amyloid) are resistant to being broken down and accumulate in the body's tissues
• Amyloid proteins


  • They are resistant to proteolysis (being broken down)
  • They accumulate in extracellular spaces
• If amyloid builds up in organs


  It causes those organs to function poorly
• Typical symptoms of amyloidosis

  • Unexplained weight loss
  • Fatigue
  • Shortness of breath
  • Foamy urine
  • Swelling in the ankles and legs
  • Numbness and tingling in the hands and feet
• Predisposition to form abnormal proteins

  Can be inherited from parents or arise from DNA mutations acquired during lifetime
• Types of amyloidosis


  • AL (amyloid light chain)
  • AA (amyloid associated)
  • Aβ (β amyloid)
  • Aβ2m (β2 microglobulin)
  • IAPP (islet amyloid polypeptide)
• AL (amyloid light chain) amyloidosis

  Caused by too many unassembled, misfolded light chains that cannot be broken down efficiently and form amyloid fibrils
• Reactive (secondary) amyloidosis (AA)


  Caused by increased levels of the circulating serum amyloid A protein, which elevates in response to infection and inflammation
• Familial (hereditary) amyloidosis


  Caused by a mutant transthyretin (TTR) protein produced in the liver
• Amyloid β2M- Dialysis-associated amyloidosis

  Caused by accumulation of intact and modified β2-microglobulins in patients on chronic hemodialysis or peritoneal dialysis
• Pathological diagnosis of amyloidosis

  1. Congo red staining shows pink color and apple-green birefringence under polarized light

  2. Immunohistochemistry can identify AA amyloidosis
• Modified alkaline Congo red staining for amyloid

  1. Prepare Congo red stock and working solutions

  2. Stain tissue sections, rinse, counterstain with hematoxylin, mount
• Congophilic amyloid plaques cause apple-green birefringence when viewed through crossed polarimetric filters
• Hematoxylin and eosin staining is used to quench nonspecific staining by Congo red
• Micrograph showing amyloid deposits (pink) in small bowel

  • H&E stain
• Modern antibody technology and immunohistochemistry can cause trouble in amyloidosis diagnosis because epitopes can be concealed in the amyloid fold
• Slide preparation


  Sections should be heat-fixed (60ºC to 100ºC) to the slides for a minimum of 2-5 minutes prior to staining, preferably at the time the sections are mounted on the slides
• Congophilic amyloid plaques


  Generally cause apple-green birefringence when viewed through crossed polarimetric filters
• Hematoxylin and eosin stain


  Used to quench (satisfy) the dyes' activity in other places such as the nucleus where the dye might bind, to avoid nonspecific staining
• Amyloid deposits (pink) in small bowel

  • H&E stain
• Epitopes


  Can be concealed (hidden) in the amyloid fold; an amyloid protein structure is generally a different conformation from that which the antibody recognizes
• Gastric biopsy specimen of a patient with primary amyloidosis

  • Shows plasma cells with expression of kappa light chain (Immunoperoxidase, orig. ×400)
• Alveolar walls near the pleura and interlobular septa


  • Showed patchy, slight widening and a homogeneous, pink material suggesting amyloid
• Crystal violet stain


  Shows a violet metachromasia typical of, but not specific for, amyloid
• Congo red positivity


  Remains the gold standard for diagnosis of amyloidosis, attributed to the environmental change as these dyes intercalate between beta-strands
• Small bowel duodenum with amyloid deposition

  • Congo red 10X
• Artifact can be defined as unrelated, self-colored artificial features found in tissue sections
• Artifacts


  • They occur in tissue sections before fixation, during fixation, grossing of specimen, tissue processing, sectioning, staining and preservation of tissue section
  • Some are easily distinguishable from normal or diseased tissue components while some are difficult to distinguish
• Pre-Fixation Artifacts


  • Excision margins of fresh surgical specimens marked with coloring agents
  • Forceps artifacts or crush/squeeze artifacts
  • Starch artifact due to contamination of specimen with starch powder used as lubricant in surgical glove
  • Floaters – Pieces of tissue from one case transferred to the next case
• Artifact due to contaminations can be prevented by careful manipulation of specimen at every step of preparation and cleaning dissecting board after treating each specimen
• Fixation Artifacts


  • Brown-Black granular and yellow stains distributed randomly throughout the tissues due to formalin, mercuric chloride and picric acid
  • Pseudo calcification due to calcium acetate used as buffer
  • Destruction of tissue due to inadequate removal of picric acid after Bouin's fixative
• Treat sections with saturated alcoholic picric acid solution or prevent it by fixing in buffered formalin
• Tissue Processing Artifacts


  • Over dehydration makes the tissue hard, brittle and shrunken
  • Under or Incomplete dehydration results in improper infiltration of paraffin
  • Over and Under clearing of tissue causing excessive hardening, and thus obstruct paraffin impregnation of tissue making it difficult to cut during sectioning
  • Air Bubble Entrapment and "Parched Earth (crackes)" effect due to increase in temperature of water bath
• Staining Artifacts


  • Contamination by microorganisms/foreign particle/expired solution seen a deposition on the section
  • Undissolved and precipitated stain will lead to deposition on the sections
• Mounting Artifacts


  • Air bubble entrapment, residual water and excessive use of mounting media
• Formalin pigment, Retraction artifact, Failure To Stain Because Of Residual Wax On The Section