Investigations & applications
Light microscopy
The bulk of a pathologist's diagnostic work is performed on tissue sections
examined by routine light microscopy. The steps involved in light microscopy
are:
| 1. Preserving the tissue in a fixative (usually
formalin) |
 |
2.Selecting pieces of tissue to examine under
the microscope |
 |
3. Embedding the selected tissue in paraffin wax
| 4. Cutting 5m thick sections from the paraffin
wax block |
 |
5. Staining the sections with:
- Haematoxylin and Eosin
(H & E)
- Special stains to detect specific features as necessary
|
 |
The whole process takes about 24 hours.
As you have learnt by now, most major organs have mixtures of all four tissue
types, organised so that they have maximum functional capability. We will now
look at skin and colon to see how this is achieved.
Concentrating on skin and colon, we will now look at some examples of light microscopy
in practice
Case 1 - Skin Tumour
The patient is a 75 year old man who was a labourer before he retired. Over the
past year he had noticed a slowly growing lump on his right shin
Case 2 - Colonic Tumour
The patient is a 59 year old woman who presented with a 2 month history of weight
loss and bleeding per rectum. Investigations showed a lesion in the ascending
colon which was removed at operation
Immunohistochemistry
This widely-used method uses labelled antibodies to detect specific antigens in tissue sections. Antibodies can be polyclonal or monoclonal (antigen-specific). The steps involved in immunohistochemistry are:
- applying primary antibody to tissue section
- primary antibody binds to specific antigen
- localising primary antibody using second antibody directed against the
first
- second antibody is labelled with a coloured marker which can be detected
down the microscope
The main uses of immunohistochemistry are in:
- tumour pathology - to demonstrate histogenesis in undifferentiated tumours
by looking for antigens related to differentiation
- renal pathology - to demonstrate different types of immune complexes in
glomeruli, important in the diagnosis of glomerulonephritis
- suspected infective disease - to detect specific organisms
Examples of monoclonal antibodies in tumour pathology include:
|
Antibody
|
Target
|
Positivity indicates
|
| Epithelial membrane antigen |
Epithelial antigen |
Epithelial tumour |
| Vimentin |
Mesenchymal antigen |
Mesenchymal tumour |
| Leukocyte common antigen |
Lymphoid antigen |
Lymphoma |
| Desmin |
Muscle antigen |
Muscle tumour |
| S100 protein |
Melanocyte antigen |
Melanocytic tumour |
| Neurofilament |
Neural antigen |
Neural tumour |
We will now look at an example of immunohistochemistry in practice
Case 3 - Brain tumour
7. The patient is a 70 year old man who presented with a right hemiparesis.
Investigations showed a mass in the left side of his brain which was biopsied.
There are three photomicrographs of this lesion labelled A, B and C. A and B
are stained with H & E, C is stained with epithelial membrane antigen
Electron microscopy
With this technique, a beam of electrons is passed through ultrathin tissue sections and the images are focused by a column of electromagnetic lenses. The use of electrons allows much higher magnifications and better resolution than that obtained by light microscopy. Despite this, electron microscopy is not much used nowadays as it has been superseded by immunohistochemistry which has similar uses but is much cheaper and simpler to perform
The main uses of electron microscopy are in:
- tumour pathology - when immunohistochemistry has failed to provide a diagnosis
in an undifferentiated tumour, ultrastructural features related to differentiation
can be sought
- renal pathology - to demonstrate the distribution of immune complexes in
glomeruli, important in the diagnosis of glomerulonephritis
- identification of - viruses, amyloid and inorganic materials such as asbestos
Examples of ultrastructural features related to differentiation are:
| Feature |
Found in: |
| Tight junctions |
Glandular epithelium |
| Desmosomes |
Squamous epithelium |
| Short regular microvilli |
Glandular epithelium |
| Long irregular microvilli |
Mesothelioma |
| Thin & thick filaments |
Striated muscle cells |
Case 4 - Lung tumour
The patient is a 4 year old boy who presented with recurrent chest infections.
A chest X-ray showed a mass in the lower lobe which was resected.
Cytology
Cytology is the study of cells rather than tissues. The technique involves obtaining
a sample of cells which are then placed on a slide, stained and examined microscopically.
Cells can be obtained through:
- exfoliation : cells are shed or scraped from a mucosal surface. Examples
are:
- sputum
- fluids (pleural effusions, ascites)
- brushings (from bronchi, oesophagus)
- cervical smears
- fine needle aspiration : cells are sucked from a lump or lesion through
a fine gauge needle. Examples are:
- breast lumps
- thyroid lumps
The main uses of cytology are
- diagnosis of benign or malignant tumours
- diagnosis of specific infections
- screening for malignancy in at-risk populations
- follow-up of patients with previous malignancy
Advantages of cytology over histology include:
- procedure is relatively painless
- technique is simpler and can be performed anywhere
- results can be obtained more quickly, often "on-the-spot"
Disadvantages of cytology
- tissue architecture cannot be assessed
- difficult to distinguish between in-situ and invasive carcinomas
Case 5- Cervical smear
The patient is a 35 year old woman who underwent a routine cervical smear. Photo-micrograph
A shows a normal smear, photomicrograph B show the patient's smear
Case 6 - Breast aspirate
The patient is a 55 year old woman who presented with a 6 month history of a lump
in the upper outer quadrant of her left breast. Mammography showed a suspicious
lesion and a fine needle aspirate was carried out. Photomicrograph A is of an
aspirate from normal breast, photomicrograph B is from the patient's lump
Molecular biology
Much of the recent progress in medical science has resulted from spectacular advances
in molecular biology. Although the techniques are used infrequently in diagnostic
histopathology, they are used extensively in research. The principle underlying
all techniques in molecular biology is hybridisation between complimentary strands
of either DNA & DNA or DNA & RNA
The commonly used techniques are:
- Southern blotting
- Northern blotting
- in-situ hybridisation
- polymerase chain reaction (PCR)
- in-situ PCR
The main clinical uses of molecular biology are:
- detection of infectious agents
- diagnosis and prognosis of malignancy
- gene rearrangements
- gene translocations
- mutated oncogenes
- gene amplification
- detection of relapse
- definition of prognostic groups
- detection of hereditary disease
- forensic medicine : "genetic fingerprinting"
Case 7 - In-situ hybridisation
The photomicrograph has been taken from the lymph node of a patient with Hodgkin's
disease (a type of lymphoma). The tissue section has been treated with a probe
for Epstein-Barr virus.
B
B.
B.
