Histopathology

Question 1

What is a histopathologist

Answer 1

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 is a cytopathologist

Answer 2

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.

Question 3

What 4 samples do histopathologist deal with

Answer 3

Biopsies, Resection specimens, frozen sections and post-mortems

Question 4

How are biopsies formed

Answer 4

1) Tissue removed from patient is placed in formalin solution which preserves tissues by cross-linking proteins
2) Then embedded in paraffin wax to allow very thin sections (2-3um)
3) cut by an instrument known as a microtome
4) Mounted on a glass microscope slide for further preparation prior to analysis

Question 5

What questions can biopsies answer

Answer 5

• Is tissue normal?
• Is the tissue inflamed and what is the likely cause?
• Is the tissue cancerous and what type is it?
• Biopsies are primarily used to make diagnoses e.g. is there a need for surgery?

Question 6

What do Haemotoxylin and Eosin do?

Answer 6

Identify the nuclei and cytoplasmic granules of leukocytes

Question 7

What do Zieh-Neelsen stain do?

Answer 7

Stain acid-fast bacteria red which aids the diagnosis of tubercolosis .

*Acid-fast bacteria (also known as acid-fast bacilli or AFB) are microorganisms resistant to decolorization by an acid, hence, the term acid-fast. Acid fastness is a unique characteristic of M. tuberculosis

Question 8

What does Oil Red O stain do?

Answer 8

stains lipids

Question 9

what does congo red do?

Answer 9

stains collagen and elastin (ECM components)

Question 10

How are resections occupied?

Answer 10

Taken from tissue that has been removed as part of a surgical procedure and can be processed as for a biopsy.

Question 11

What are resections used to look at?

Answer 11

Primarily look at the stage of the disease, eg.

How far is the cancer progressing?
Has the cancer penetrated the bowel wall, spread to the lymph nodes or liver?

Has all the cancerous tissue been removed or is chemo needed?

Question 12

Frozen sections

Answer 12

Taken during surgical procedures and are examined by pathologists in real time while the patient is being operated upon.
The procedure can give a rapid diagnosis in minutes which can be relayed back to the surgeon to inform the surgery.

Question 13

Hoe is a frozen section obtained?

Answer 13

The freshly taken tissue is frozen by a machine known as a cryostat

Cut

Then mounted on glass slides and stained as for biopsies.

Tissues need to be fresh and free of preservatives like formalin

Question 14

Questions that can be answered with a frozen section

Answer 14

Is the tissue cancerous?
Has all the cancerous tissue been removed?
Is there another pathological process going on?

Question 15

Timescales of biopsies, resections and frozen sections

Answer 15

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

Question 16

How are fine needle aspirates obtained?

Answer 16

a fine needle is used to get into lesion and suck out (aspirate) the cells which can then be analysed as smear.

Powerful technique as the needle can access relatively inaccessible tissues such as thyroid nodule and assess the suspect mass without need for surgery

Question 17

Disadvantage of fine needle aspirates

Answer 17

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

Question 18

What is immunohistochemistry?

Answer 18

It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.

Question 19

What can high levels of circulating antibodies recognising double stranded DNA indicate?

Answer 19

systemic lupus erythematosus (SLE)

Sjögren’s syndrome

Rheumatoid arthritis.

Degree of number of antibodies there can be used to indicate the number/levels of antibodies circulating in blood plasma

Question 20

what can attachments include

Answer 20

enzymes
fluorescent probes
magnetic beads
drugs

Question 21

how do enzymes attached to antibodies work

Answer 21

e.g. peroxidase, alkaline phosphatase. Used with a colourless substrate to give a coloured product. Also detection of oestrogen receptors on breast cancer tissue biopsies.

Question 22

how do fluorescent probes attached to enzymes work

Answer 22

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

Question 23

how do magnetic beads attached to antibodies work

Answer 23

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

Question 24

how do drugs attached to antibodies work

Answer 24

e.g. the biological Kadcyla, an anti-HER2 antibody linked to the cytotoxic chemical emtansine. 

HER2 is overexpressed in around 30% of breast cancers and Kadcyla has NICE (National Institute for Health and Care Excellence) approval for the treatment of advanced metastatic breast cancers.

Question 25

why are antibodies used as good diagnostic tools

Answer 25

The unique specificity of antibodies for their target antigens is the basis of many diagnostic tests.

Antibodies are relatively easy to generate and can be used to detect not only proteins but other biological molecules such as carbohydrates and lipids.

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

How can manufactured antibodies be used in different scenarios?

Answer 26

• Blood group serology
• Immunoassays e.g. detection of hormones, circulating antibodies/antigens
• immunodiagnosis e.g. infectious diseases- presence of circulating anti-HIV antibodies suggests infection with HIV. Similarly, high circulating levels of antibody might be suggestive of a myeloma , or, if the antibodies are of the IgE class, suggestive of an allergic phenotype

Question 27

How does an ELISA assay work?

Answer 27

I​n 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.

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

flow cytometry

Answer 28

allows the detection of specific cells, notably lymphocyte subpopulations, using different coloured fluorescently conjugated specific antibodies. .

Question 29

how does flow cytometry work

Answer 29

T​he 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.