Histopathology

Question 1

What is histopathology?

Answer 1

Deals with tissues, examining the architecture of the tissue; observations used in diagnosis, as well as towards informing the efficacy of a particular treatment

Question 2

What are tissue samples?

Answer 2

Biopsies, resection specimens, frozen sections, and post-mortems

Question 3

What are biopsies?

Answer 3

Concerns small sections of tissues removed from the patient; immersed in a formalin solution that preserves the tissues by cross-linking proteins.

Question 4

What is formalin?

Answer 4

Preserves tissues by cross-linking proteins, samples are embedded in paraffin wax, enabling thin sections to be cut by a microtome.
Sample mounted on a microscope slide for preparation prior to analysis

Question 5

What is the main function of a biopsy?

Answer 5

As a diagnostic tool, e.g. is there a need for surgery?

Question 6

Which chemical stains are used for cells in the biopsy section?

Answer 6

Haematoxylin and Eosin staining used to identify nuclei and cytoplasmic granules of leukocytes

Question 7

Which type of stain will stain acid-fast bacteria red, aiding in diagnosis for TB infection?

Answer 7

Ziehl-Neelsen stain

Question 8

What are resection specimens?

Answer 8

Sourced from tissue removed from surgical procedure, processed as for a biopsy

Question 9

What are resections used for?

Answer 9

To identify the stage and progression of the disease (cancers, diagnostic too for further treatment)

Question 10

Why are tissues donated to biobanks?

Answer 10

Used to inform genomic studies of the disease process

Question 11

Which sample is taken during surgical procedures and examined by pathologists in real time?

Answer 11

Frozen sections

Question 12

How are sections frozen?

Answer 12

By cryostat, cut then mounted on glass slide and stained for biopsies

Question 13

Why are frozen sections used?

Answer 13

Rapid diagnosis, relayed back to the surgeon to inform the surgery
Used to see if there are cancerous tissues and the number, as well as identifying other pathological processes
Fresh tissue sample w/o formalin

Question 14

How long are frozen sections stored and used for? (result to reach clinician)

Answer 14

30 minutes

Question 15

What is the timescale for biopsies? (result to reach clinician)

Answer 15

2-3 days

Question 16

What is the timescale for resection specimens? (result to reach clinician)

Answer 16

5-7 days

Question 17

What is cytopathology?

Answer 17

The study of disease at a cellular level.
Cytopathologists identify cells, collected and smeared onto a microscope slide
The slide is stained and examined.

Question 18

What is used to analyse for a smear when inserted into a lesion?

Answer 18

Fine needle aspirates. The fine needle sucks out (aspirates) cells

Question 19

Why are fine needle aspirates used?

Answer 19

The needle penetrates inaccessible tissue (thyroid nodule), and assess the suspect mass without the surgical requirement

Question 20

What is the disadvantage with fine needle aspirates?

Answer 20

Cytology concerns individual cells, and thereby unable to determine the architecture of the tissue and cellular influences on adjacent cells.

Question 21

What does brown coloration indicate during a skin biopsy?

Answer 21

Antibody recognizing the endothelial cell marker CD31 certifies the biopsy as an endothelial cell tumor.
Combining biopsies with analysis of fine needle aspirate taken from an enlarged lymph node, a diagnosis of reactive lymphadenopathy can be established due to mixed cell population observed

Question 22

What is used in immunohistochemistry?

Answer 22

Antibodies can be used to specifically detect molecules in the process of immunochemistry.

Question 23

Which region of the antibody is used to attach with markers?

Answer 23

Fc region.

Question 24

What are the four main attachments to antibody Fc regions?

Answer 24

Enzymes
Fluorescent probes
Magnetic beads
Drugs

Question 25

Which enzymes are attached to Fc regions of antibodies?

Answer 25

Peroxidases, and alkaline phosphatase used in conjunction with a colourless substrate to produce a coloured product- CD31 stain

Also allows the detection of oestrogen receptors on breast cancer tissue biopsies.

Question 26

Why are fluorescent probes used in antibody diagnostics?

Answer 26

Rapid measurement of the levels of molecules within a sample

Multiplexing allows a measure of several molecules in a single clinical sample

Question 27

What is multiplexing?

Answer 27

Several antibodies with different fluorescence

Question 28

Why are magnetic beads used in antibody diagnostics?

Answer 28

Purifies cells from other cells

E.g. anti-CD3 antibodies used to deplete bone marrow of T cells prior to use in bone marrow grafts

Question 29

When are antibody diagnostics used?

Answer 29

Blood group serology

Immunoassays (detection of hormones) circulating antibodies/antigens

Immunodiagnosis - presence of circulating anti-HIV antibodies suggests infection with HIV
High circulating levels of antibody suggest myeloma
IgE classes suggestive of the allergic phenotype

Question 30

What does ELISA stand for?

Answer 30

Enzyme-linked immunosorbent assay

Question 31

What is ELISA?

Answer 31

Serum samples allowed to adhere to a plastic plate; probed with a specific antibody raised against the molecule of interest

Antibody conjugated with an enzyme catalyses colourless substrate into a colour product

Reference to a standard curve, relatively absorbance of the solution can be used to determine precise levels of the molecule being studied

Question 32

What is the first stage of ELISA?

Answer 32

Antibody coating: Specific capture antibody is immobilized on high-protein binding plates, incubated
Plates are blocked with irrelevant protein (alubmin)

Question 33

What is protein capture during ELISA?

Answer 33

Samples and standard dilutions are added to the wells, and will be captured by the bound antibodies.

Question 34

What is a detection antibody during ELISA?

Answer 34

Specific biotinylated detection antibody inserted to wells, enables detection of the captured protein, Wash unbound detection antibodies.

Question 35

Which enzyme is conjugated with the wells?

Answer 35

Strepavidin-enzyme conjugate with alkaline phosphates is added to the wells and will bind to the biotinylated antibody

Question 36

What happens upon addition of substrate to ELISA?

Answer 36

Colorimetric substrate is added to the wells, and will form a coloured solution when catalysed by the enzyme

Question 37

How is the absorbance measured during ELISA?

Answer 37

By ELISA reader = amount of protein established.

Question 38

What is flow cytometry?

Answer 38

Allows detection of specific cells using fluorescently conjugated specific antibodies that associate to CD specific receptor sites on the lymphocyte cell surface membrane

Antibodies have different colours (different fluorophores)

Question 39

How does flow cytometry work?

Answer 39

Cells labelled with differently conjugated antibodies, run as a stream of single cells through a laser beam. Colour of emitted light and forward/side scatter denotes the identity of the cell surface molecules expressed, the size and granularity of cells.

Question 40

What questions can microscopic examinations of biopsies answer?

Answer 40

Is the tissue normal?
Is the tissue inflamed and, if it is, what is the likely cause?
Is the tissue cancerous and, if it is, what type of cancer is it?

Question 41

How are the two ways that antibodies can be used as a diagnostic tool?

Answer 41

Through direct (conjugate on the antibody itself that recognises antigen- primary antibody) and indirect detection (secondary antibody with conjugate attaches to primary antibody that is bound to antigen)