HC4: Pathology of normal immune response

Techniques to visualize diseased tissue

Pathology always starts with tissue:

• Biopsies
• Resections
• Autopsies

3 techniques can be combined to make a final diagnosis:

• Immunohistochemical techniques
  • Possible to make a diagnosis in an hour
  • Tissue is frozen
• Light microscopy
  • Most important
  • A tissue specimen goes into a paraffine block to make very thin slices → the slices are stained
    • Glomeruli of a healthy kidney have silver staining
    • In case of chronic inflammation, blue dots are present → lymfocytes
• Electron microscopy
  • Used to look for details
    • For example podocytes

Myocardial infarction:

Normal light microscopy of the heart shows that all cells have a nucleus (the heart is a muscle). Many blood vessels and few lymphocytes are present. A myocardial infarction is an ischemic injury, usually caused by a thrombus in a coronary artery:

1. Cell death: necrosis of myocytes
   • Hours after the incident
   • Cardiomyocytes lose their nucleus
   • Neutrophils are the first leukocytes to arrive to clean up the necrotic tissue
2. Inflammatory reaction with neutrophilic granulocytes
   • Days after the incident
   • Ischemic tissue is infiltrated massively by cells with lobulated nuclei → neutrophilic granulocytes
3. Fibroblast proliferation: remodeling of connective tissue through collagen disposition
   • Weeks after the incident
   • Fibrotic tissue is starting to replace the dead cells
4. Scar formation
   • Months after the incident

This is a general mechanism that can be applied in all tissues → the same process can happen in inflamed skin or lung tissue.

Histomorphology of inflammatory cells and their markers

Inflammatory cells have a distinct histomorphology:

• Granulocytes
  • Indicate an acute infection or inflammatory process
  • Polymorphonuclear cells
    • Neutrophils
      • Most frequent
    • Eosinophils
      • Less frequent
    • Basophils
      • Very rare
  • Marker: MPO
• Lymphocytes
  • Are associated with chronic processes
  • Dark nuclei
  • Small cytoplasm
  • Mononuclear cells
  • Markers
    • CD3: entire population
    • CD8: cytotoxic T-cells
    • CD4: T-helper cells
• Plasma cells
  • Produce antibodies
  • Pericentric nucleus
  • B-cells
  • Marker: CD20
• Macrophages
  • Large cells
  • Present to clean up the mess → digest all sorts of things
  • May turn into multinucleated cells
  • Marker: CD68
• Other cells
  • Dendritic cells
  • Fibroblasts
  • Histiocytes

Visualizing what one cannot directly see

Some questions cannot be answered by "just" looking at light microscopy:

• How can B-cells be distinguished from T-cells?
• Is the complement system involved?
• Are these large cells indeed macrophages or are they tumor-cells?
• Is there a SARS-CoV2 related protein in this cell?
• Are immune complexes involved?

Immunohistochemistry:

Immunohistochemistry is a technique which makes it possible to visualize proteins in tissue. Proteins are stained with a color. This is done by picking an antibody that fits nicely into the protein and then adding color to it.

This technique can be used to visualize B- and T-cells in cases like Hashimoto's thyroiditis:

• Normal thyroid tissue: hormones are made in the pink areas:
• Hashimoto's thyroiditis: many blue dots are visible → these are lymphocytes destructing the thyroid tissue
  • By staining a specific antibody in B-cells or T-cells, it becomes possible to see where there are B-cells and where there are T-cells

Immunohistochemistry is also useful in cases of maternal cytomegalovirus infection. This can cause intra-uterine death. The virus, a viral protein, goes through the placental wall. Such a case looks relatively normal under the microscope, but when the CMV-protein is stained, many CMV-infected cells within the placenta that cause the intra-uterine death become visible.

Direct immunofluorescence:

Direct immunofluorescence is another technique to visualize what one can't see. In direct immunofluorescence, an antibody with an immunofluorescent signal is incubated onto a fresh frozen tissue slide, for example against C3 which appears to be present in a glomerulus. This gives a fluorescent signal as result.

Limitation

The main limitation of pathological tissue examination is that a biopsy is always just a snapshot of a dynamic biological process. The pathologist always has to interpret this snapshot in relation to the clinical context.