Hispathology

Question 1

What does a hispathologist do?

Answer 1

• A histopathologist deals with tissues.
• She/he will examine sections, noting the architecture of the tissue and asking what it tells us about a particular condition.

Question 2

What does a cytopathologist do?

Answer 2

• A cytopathologist deals with cells.
• They are often are the individuals responsible for taking the cells from the patient, preparing them for examination and then delivering their expert diagnosis on the cell sample
• Cytopathologists work with cellswhich are collected and then smeared onto a microscope slide. The slide can then be stained and examined

Question 3

What does a hispathologist deal with?

Answer 3

1. Biopsies
2. Resection specimens
3. Frozen sections
4. Post-mortems
   - There are often taken by surgeons and examined by the pathologist in realtime, to inform the surgical process

Question 4

What are biopsies?

Answer 4

1. Biopsies are small sections of tissues that are removed from the patient and typically placed in a formalin solution which preserves the tissues by cross-linking proteins
2. They are then embedded in paraffin wax to allow very thin sections (2-3μm thick) to be cut by an instrument known as a microtome
3. These are mounted on a glass microscope slide for further preparation prior to analysis.

Question 5

What can microscopic rumination of biopsy answer?

Answer 5

• Is the tissue normal?
• Is the tissue inflamed and, if it is, what is the likely cause? (Lecture 9)
• Is the tissue cancerous and, if it is, what type of cancer is it? (Lecture 13)
Biopsies are primarily used to make diagnoses e.g. is there a need for surgery?

Question 6

Are chemical stains used? What are examples?

Answer 6

• Chemical stains are often employed with the tissue sections to aid the identification of cells within the biopsy section.
• For example, Haemotoxylin and Eosin (H&E) staining can be used to identify the nuclei and cytoplasmic granules of leukocytes within tissues (Practical 2). Similarly, the Ziehl-Neelsen stain will stain acid-fast bacteria red, aiding in the diagnosis of tuberculosis infection (Practical 4).

Question 7

When are resection specimens taken?

Answer 7

-Resection specimens are taken from tissue that has been removed as part of a surgical procedure and can be processed as for a biopsy.

Question 8

What are resections specimens used to look at?

Answer 8

• Resections are used primarily to look at the stage the disease (Lecture 13). For example, it is known that the patient has a cancer, but how far is the disease progressing? For example has the cancer penetrated the bowel wall, spread to the lymph nodes or the liver? Has all the cancerous tissue been removed or is chemotherapy needed?
• Tissue from resection specimens can also be donated to biobanks and used to inform genomic studies of the disease process, with the input of other medical professionals such as immunologists and microbiologists.

Question 9

When are frozen sections taken?

Answer 9

-Frozen sections are taken during surgical procedures are are examined by pathologistsin real time while the patient is being operatedupon.

Question 10

How is a frozen section taken?

Answer 10

-The freshly taken tissue is frozen by a machine known as a cryostat, cut then mounted on glass slides and stained as for biopsies.

Question 11

What can frozen sections give an answer for?

Answer 11

-The procedure can give a rapid diagnosis in minutes which can be relayed back to the surgeon to inform the surgery. For example:
• Is the tissue cancerous?
• Has all the cancerous tissue been removed?
Is there another pathological process going on

Question 12

What are the timescales for a result form the histopathology lab to reach clinician?

Answer 12

Frozen section: 30 minutes
Biopsies: 2-3 days
Resection specimen: 5-7 days

Question 13

What is fine needle aspirates?

Answer 13

• A fine needle can also used to get into a lesion and suck out (aspirate) the cells which can then be analysed as for a smear
• This is a very powerful technique as the needle can penetrate relatively inaccesable tissues e.g. a thyroid nodule and assess the suspect mass without the need for surgery.

Question 14

What Is the downside of fine needle aspirates?

Answer 14

-the cytopathologist is only looking at cells and is unable to comment upon the likely architecture of the tissue

Question 15

What is a powerful combo?

Answer 15

Histopathology and cytopathology

Question 16

What is CD31?

Answer 16

• An antibody recognising the endothelical cell marker CD31, typical of endothelial cells allows us to definitely define this biopsy as being of an endothelial cell tumour (above figure).
• If these biopsies are then combined with analysis of a fine needle aspirate taken from the patient’s enlarged lymph nodes (shown below), then a diagnosis of reactive lymphadenopathy can be given due to the mixed cell population observed.

Question 17

What can be seen in a Skin biopsy taken from an individual with Kaposi’s sarcoma?

Answer 17

Spindly looking cells can be seen to penetrate the collagen fibres. Immunohistochemistry can also be used to identify key molecules within the tissues.

Question 18

How can antibodies help in diagnosis?

Answer 18

1. Antibodies are extremely useful molecules which can aid in the diagnosis of disease.
2. The immunology labs offer 86 different tests of which 53 are used to detect the levels of specific antibodies circulating in patients.
3. For example, high levels of circulating antibodies recognising double stranded DNA are assoaciated with systemic lupus erythematosus(SLE), Sjögren’s syndrome and rheumatoid arthritis.

Question 19

What are attachments?

Answer 19

1. Manufactured antibodies can be used to specifically detect molecules in the process of immunohistochemistry.
2. Attachments (also known as conjugations) to the Fc region of the antibody (PoM primer) can be made, permitting a wide variety of uses.

Question 20

What are different attachments?

Answer 20

1. Enzymes
2. Fluorescent probes
3. Magnetic beads
4. Drugs

Question 21

How can enzymes be an attachment?

Answer 21

1. Enzymes: e.g. peroxidase, alkaline phosphatase.
   2Used with a colourless substrate to give a coloured product, e.g. the CD31 staining we looked at on the previous page.
2. Also detection of oestrogen receptors on breast cancer tissue biopsies.

Question 22

How can fluorescent probe be used as an attachment?

Answer 22

1. These can allow the rapid measurement of the levels of molecules within a sample 2.Multiplexing (using several antibodies with different fluorescence) can allow the measurement of several molecules in a single precious clinical sample.

Question 23

How can magnetic beads be used as an attachment?

Answer 23

1. e.g. the purification of cell types. With the use of a magnet, cells can be depleted of contaminents e.g. anti-CD3 antibodies used to deplete bone marrow of T cells prior to use in bone marrow grafts.

Question 24

How can drugs be used as an attachment?

Answer 24

1. e.g. the biological Kadcyla, an anti-HER2 antibody linked to the cytotoxic chemical emtansine. HER2 isoverexpressed in around 30% of breast cancers andKadcyla has NICE (N ational Institute for Health and Care Excellence)approval for the treatment of advanced metastatic breast cancers.

Question 25

How can antibodies be used as a diagnostic tool?

Answer 25

1. The unique specificity of antibodiesfor their target antigens is the basis of many diagnostic tests.
2. Antibodies are relatively easy to generate and can be used to detect not only proteins but other biological molecules such as carbohydrates and lipids. 3.This can either be direct detection of the antigen or via the detection of an unlabelled antibody by a second antibody conjugated with a fluorescent probe or enzyme.

Question 26

What are different scenarios that antibodies can be manufactured?

Answer 26

1. Blood group serology (ALS 4).
2. Immunoassayse.g. detection of hormones, circulating antibodies/antigens.
   3 .Immunodiagnosis e.g. infections diseases (presence of circulating anti-HIV antibodies suggests infection with HIV. Similarly, high circulating levels of antibody might be suggestive of a myeloma (Lecture 14) or, if the antibodies are of the IgE class, suggestive of an allergic phenotype (Tutorial 2, ALS 5).

Question 27

What happens in Elsa?

Answer 27

1. In an ELISA (Enzyme linked immunosorbent assay), clinical samples (typically serum samples) are allowed to adhere to a plastic plate, after which they are probed with an specific antibody raised against the molecule of interest
2. The antibody is conjugated with an enzyme which can turn a colourless substrate a particular colour.
   - With reference to a standard curve, the relative absorbance of the solution (Practical 1) can be used to determine precise levels of the molecule being studied.

Question 28

What is flow cytometry?

Answer 28

The technique of flow cytometry allows the detection of specific cells, notably lymphocyte subpopulations, using fluorescently conjugated specific antibodies

Question 29

What are these are typically molecules expressed on the cell surface and may include?

Answer 29

• anti-CD3+ T cells – pan T cell marker
• anti-CD4+ T cells – T helper/cells
• anti–CD8+ T cells – cytotoxic T cells
• anti–CD19+ B cells
• anti–CD56+ Natural Killer (NK) Cells
• This may be highly informative as to the nature of a particular inflammatory reaction (lecture 9).

Question 30

How are the cells labelled?

Answer 30

• The cells are labelled with differently conjugated antibodies then run as a stream of single cells through a laser beam (s)
• The colour of the light emitted and the forward or side scatter of the laser beam can denote the identity of the cell surface molecules expressed and the size and granularity of the cells.