Histopathology

Question 1

What is the role of a histopathologist?

Answer 1

Deals with tissues

Examine sections - noting the architecture of the tissue and what it tells us about a particular condition

Question 2

Why is the information the histopathologist provides, useful?

Answer 2

Diagnosis

Efficacy of particular treatment

Question 3

Which 4 main tissue samples does the histopathologist work with?

Answer 3

Biopsies
Resection specimens
Frozen sections
Post-mortems

Question 4

What is a biopsy and what is it primarily used for?

Answer 4

Small section of tissue removed from the patient

Make a diagnosis

Question 5

How is the biopsy prepared?

Answer 5

Biopsy correctly labelled (i.e. hospital number, patient)
Typically placed in a formalin solution which preserves the tissues
Then embedded in paraffin wax to allow very thin sections (2-3μm thick) to be cut by an instrument known as a microtome
Mounted on a glass microscope slide for further preparation prior to analysis

Question 6

What information can a microscopic examination of a biopsy present?

Answer 6

Normal tissue?
Inflamed tissue? (And possible causes)
Cancer? (And type of cancer)

Question 7

What are chemical stains used for in a biopsy?

Answer 7

Aid the identification of cells within the biopsy section

Question 8

What is the chemical stain for leukocytes and what does it specifically stain?

Answer 8

Haemotoxylin and Eosin (H&E)

Nuclei and cytoplasmic granules

Question 9

What is the chemical stain for acid-fast bacteria and what can it be used for specifically?

Answer 9

Ziehl-Neelsen stain (stains acid fast bacteria red)

Diagnosing TB

Question 10

How does formalin preserve the tissues in a biopsy?

Answer 10

By cross-linking proteins

Question 11

What are resection specimens and what are they primarily used for?

Answer 11

Sample taken from tissue that was removed during a surgical procedure
Can be processed using the same method as for a biopsy
Primarily used to look at the stage of a disease e.g. cancer and how far it has spread

Question 12

What else can resection specimens be used for?

Answer 12

Donated to biobanks and used to inform genomic studies of the disease process- analysed by other medical professionals such as immunologists and microbiologists

Question 12

What are frozen sections and what are they primarily used for?

Answer 12

Taken during surgical procedures and are examined by pathologistsin real time while the patient is being operatedu​pon
Rapid diagnosis in minutes which can be relayed back to the surgeon to inform the surgery

Question 13

How is the frozen section prepared?

Answer 13

Freshly taken tissue is frozen by a machine known as a cryostat
After, cut then mounted on glass slides and stained as for biopsies

Note: MUST be fresh sample and not preserved in chemicals such as formalin

Question 14

What information can the frozen section present?

Answer 14

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

Question 15

What are the relative times for the histopathology lab’s results to reach the clinician?

Answer 15

F rozen section: 30 minutes
B​iopsies: 2-3 days
Re​section specimen: 5-7 days

Question 17

What is the role of a cytopathologist?

Answer 17

Deals with cells
Takes cells from the patient and prepares them for examination
Delivers their expert diagnosis on the cell sample

Question 18

How do cytopathologists prepare their microscopic slide?

Answer 18

Collected cells are smeared onto a microscope slide, which can then be stained and examined

Question 19

What is a method to collect cell sample from patients?

Answer 19

Fine needle aspiration - A fine needle can also used to get into a lesion and suck out (aspirate) the cells

Question 20

Why is this technique useful (fine needle aspiration)?

Answer 20

Can penetrate relatively inaccesable tissues e.g. a thyroid nodule
Minimally invasive - assess the suspect mass without the need for surgery

Question 21

What is a downside to this technique (fine needle aspiration)?

Answer 21

Only looking at cells- unable to comment upon the likely architecture of the tissue

Question 22

Why can histopathology and cytopathology together be a powerful combination? (use an example)

Answer 22

Aids diagnosis and treatment options
E.g. Biopsy of a patient’s sarcoma can look at spread and invasion, then fine needle aspirate taken from the patient’s enlarged lymph nodes can help specify the diagnosis further

Question 23

What else can Abs be used for other than as part of the immune response?

Answer 23

As a part of a diagnostic tool - e.g. synthesised antibodies detect molecules used in the process of immunohistochemistry
To target specific cells for treatment e.g. deliver drugs to specific cells or to deplete contaminents
Flow cytometry

Question 24

How can Abs carry out these various tasks?

Answer 24

They have attachments (called conjugations) on the Fc region (heavy chains / tail of the ‘Y’ that allows Ab to interact with cell surface receptors of other molecules)

Question 25

What are the 4 main attachments / conjugations on an Ab and some examples of how they can be used?

Answer 25

1. Enzymes - generally used with a colourless substrate to give a coloured product e.g. Abs recognising the endothelial cell marker CD31 2. Fluorescent probes - 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 sample 3. Magnetic beads - for purification of a cell's contaminants with the use of a magnet, e.g. anti-CD3 antibodies used to deplete bone marrow of T cells prior to use in bone marrow grafts 4. Drugs - Kadcyla, an anti-HER2 antibody linked to the cytotoxic chemical emtansine. HER2 isoverexpressed in around 30% of breast cancers and Kadcyla is used for the treatment of advanced metastatic breast cancers

Question 26

What features of Abs make them ideal to use as a diagnostic tool?

Answer 26

Unique specificity for their target antigens Easy to generate / manufacture Can be used to detect other biological molecules e.g. carbohydrates and lipids (not just proteins)

Question 27

What are some examples where Abs are used for diagnosis?

Answer 27

Blood group serology Immunoassays e.g. detection of hormones, circulating antibodies/antigens Immunodiagnosis e.g. infections diseases e.g. 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 28

What does the ELISA test stand for and what is it used for?

Answer 28

Enzyme linked immunosorbent assay | To detect precise levels of a molecule in a sample

Question 29

How does the ELISA test work?

Answer 29

Clinical sample taken from patient and allowed to adhere to the bottom of a plastic plate Abs attached to an enzyme, that are complementary to the specific molecule being tested for, are added to the solution If molecule is present, the Abs that are linked to enzymes will attach, the solution is then washed away to remove any unattached Abs A colourless substrate is added, if the Abs linked to the enzyme are present, the substrate will change colour Depending on the number of Abs linked to enzymes present, the colour change will be fast or slow / intense or pale With reference to a standard curve, the relative absorbance of the solution can be used to find the precise levels of a molecule in a sample

Question 30

What is flow cytometry?

Answer 30

A technique that allows for the detection of specific cells Notably lymphocyte subpopulations Using fluorescently conjugated specific antibodies Typically molecules expressed on the cell surface

Question 31

Some examples of the Abs used to detect cell markers on specific cells?

Answer 31

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

Question 32

What information does flow cytometry give to a clinician?

Answer 32

Nature of a particular inflammatory reaction

Question 33

How does flow cytometry work?

Answer 33

Cells are mixed with and labelled with differently conjugated, fluorescent Abs This fluid mixture is placed into a flow cytometer The sample is focused to flow one cell at a time through a laser beam The forward scatter and side scatter are recorded The scatter characteristics of the cell can denote the identity of the cell surface molecules expressed and the size and granularity of the cells