2 - Histology

Question 1

Histology

Answer 1

the study of the structure of animal and plant tissues as visualised

Question 2

Cytology

Answer 2

study of the microscopic appearance of cells under the microscope

Question 3

3 important variables of microscopy

Answer 3

1. Magnification
2. Optical resolution
3. Contrast

Question 4

Light vs electron microscope

Answer 4

x1000 magnification vs x 1 x 106 magnification
Resolution down to 200nm vs Resolution down to 0.2nm
Brightfield vs Scanning EM
Phase-contrast +Fluorescent vs Transmission EM

Question 5

Resolution

Answer 5

The separation distance at which 2 objects in an image can be apart and still distinguished as separate

Question 6

Electron microscopy

Answer 6

Transmission EM
• electron beam
• 2D images 
Scanning EM
• electrons scattered from surface of sample
• lower resolution than TEM
• 3D images

Question 7

Fluorescent

Answer 7

Shows locations of specific molecules in the cell.

Fluorescent substances absorb short- wavelength UV radiation, and emit longer-wavelength visible light.

Question 8

Confocal microscope

Answer 8

Uses lasers and special optics for optical sectioning.

Only regions with a narrow depth of focus are imaged, regions above and below the plane appear black

Question 8

Brightfield microscopy

Answer 8

Stain with dyes to help distinguish cells
• Fix cells/tissue
• Embed
• Cut into thin sections
New problems:
- processing alters cell structure/molecules = may produce artefacts
– only gives snapshot of dead cells
– 2D image of a 3D structure
New methods to monitor living tissue cultures

Question 9

Fixation

Answer 9

• Common fixatives
-formaldehyde (10% formalin) and glutaraldehyde
- alcohol
• Arrests biological activity
• Prevents tissue degradation (autolytic + bacteria)
• Render the cells more amenable to staining

Question 10

Fixation and embedding of tissues

Answer 10

• Fixed tissues require dehydration prior to embedding – remove water to prevent tissue damage
• Support the tissue by embedding in a hard medium such as paraffin wax (LM) or freezing (LM) or plastic resin (EM)
• Steps in tissue processing all cause artefacts (distortions in cell/tissue architecture e.g. shrinkage)

Question 11

Tissue sectioning maachines

Answer 11

Cryostat
Microtome
Vibratome
Immobilised tissues are held in place and sectioned thinly using a sharp blade

Question 12

Cryostat

Answer 12

• tissues are frozen (-10-20C)
• section thickness ~10-40 mm
• tissue collected onto slides or “free-floating”

Question 13

Microtome

Answer 13

• tissues are embedded in wax (room temp)
• section thickness ~5-40 mm
• tissue collected onto slides

Question 14

Vibratome

Answer 14

• tissues are glued to holder (room temp or chilled)
• section thickness ~40-400 mm
• tissue collected onto slides or “free-floating”

Question 15

Histology – start to finish

Answer 15

Specimen dissected
Fixation
Dehydration
Embedding
Sectioning
Staining
Pathologist review and report

Question 16

Haematoxylin and Eosin (H&E)

Answer 16

Haematoxylin (basic dye)
• stains acidic structures a purplish blue
• nuclei, ribosomes and RER i.e. high DNA/RNA content
Eosin (acidic dye)
• stains basic structures red/pink
• e.g. cytoplasmic proteins

Question 17

Giemsa

Answer 17

• standard method for staining blood cells

* Nuclei stain dark blue to violet and cytoplasm pale blue

Question 18

Toluidine blue

Answer 18

• basic stain that stains acidic components various shades of blue/purple

Question 19

Masson’s trichrome

Answer 19

• stains connective tissue
• nuclei/basophilic structures stain blue
• collagen stains green or blue
• cytoplasm bright red

Question 20

Periodic acid-Schiff rxn

Answer 20

• stains complex carbohydrates purple/magenta

* e.g. Mucin in goblet cells in the intestine

Question 21

Immunohistochemistry

Answer 21

• Use antibodies or other methods to label a specific protein or cell status
• Positive stain = brown or black

Question 22

Simple columnar epithelium

Answer 22

absorptive / secretory surfaces

Question 23

Stratified epithelia

Answer 23

• protective function, e.g. skin

Question 24

Brain tissue

Answer 24

thin axon for rapid cell-cell communication

Question 25

Smooth muscle tissue

Answer 25

elongated cells to maximise contractile properties

Question 26

Breast tissue

Answer 26

• Breast cancer usually forms in the ducts and lobules of the breast
• Alterations in lobule cells and invasion of adjacent adipose tissue = Malignant tumour

Question 27

Carcinoma

Answer 27

neoplasm derived from epithelial cells of the skin or lining of internal organs

Question 28

Normal vs. dysplasic colon

Answer 28

• villiform change of the epithelium
• crypt budding, branching, and crowding
• irregularity of crypt contour
• increased angiogenesis

Question 29

Prostate cancer

Answer 29

• Cancer is characterised by luminal hyperproliferation, loss of the basal layer, breakdown of basement membrane, immune cell infiltration and stromal reactivity.
• Luminal cells make up > 99% of tumours, basal cells constitute < 0.1% of tumour epithelial cells.

Question 30

Benign tumours

Answer 30

• arise with high frequency but pose little risk because they are localised and small
Tumour has clear boundaries and has not grown into surrounding tissue

Question 31

Human Tissue Act (2004)

Answer 31

• Regulates activities - removal, storage, use and disposal of human tissue.
• Consent - fundamental principle of the legislation and underpins the lawful removal, storage and use of body parts, organs and tissue.
• DNA - unlawful to have human tissue with the intention of its DNA being analysed, without the consent of the person from whom the tissue came.
• Tissue removed and stored for diagnosis does not fall under the Act