Instrumentation

Question 1

Benefits of a tissue processor

Answer 1

• decreases time and labour by implementing overnight runs and short cycles
• diagnosis can be made faster
• lab stuff have more time to carry out other technical work

Question 2

What factors influence the processing rate in the tissue processing phase

Answer 2

• Agitation : aids penetration and reduces overall processing time by up to 30%
• Heat : increases the rate of diffusion. 45 degrees reduces time by increases shrinkage
• Vacuum: necessary for impregnation with paraffin wax.

Question 3

What are the 2 types of tissue processors

Answer 3

Open system and closed system. Open system has the tissue being transferred from one solution to the next and the closed system has fluids pumped in and out of an enclosed chamber

Question 4

advantages and disadvantges of open system tissue processor

Answer 4

ADV - allows for selection of wider choice of fixatives and processing reagents. Vacuum can be available at all stations and heat at some

DIS: evaporation of reagent - health and fire hazard. In the case of a malfunction, the tissue would most likely dry out.

Question 5

Advantages of closed system tissue processor

Answer 5

ADV:
- Vacuum and heat is available at any stage,
- more rapid schedules,
- fluid spillage containment
- Less toxic fumes emitted
- specimens cannot dry out in the chamber in the event of a malfunction

Question 6

components of the embedding station

Answer 6

clean, filtered paraffin wax held at 2-4 degrees above its melting point, cold plate to rapidly cool the wax, and a supply of mould in which to embed the tissues.

Question 7

How are (a) elongated tissues, (b) tissues with walls eg cysts and (c) tissues with tubular structures eg appendix, embedded?

Answer 7

a - placed diagonally and not parallel across the block
b - embedded on edge
c - embedded in cross section

Question 8

How is the skin embedded in paraffin

Answer 8

Tissues are embedded from soft to hard areas, therefore the epidermis should be at the top of the block so that it will cut last.
Hairy or keratinized epithelia are oriented to face the blade diagonally

Question 9

How are large rectangular sections embedded

Answer 9

They are embedded at a slight angle to the knife edge so that the knife cuts through a small area first ad gradually introduced to freater surface area.

Question 10

After embedding in paraffin, rapid cooling is carried out. Why is it rapid?

Answer 10

It allows for the smallest paraffin crystal size possible to form.

Question 11

What is the clearance angle in a microtome

Answer 11

This is the angle between the block face and lower cutting facet of the knife. This is around 3-10 degrees. This is the only adjustable angle on a microtome.

Question 12

Two types of microtome blades

Answer 12

Low profile blades are general purpose and used routinely. They are generally sold with a coated edge and dimensionally smaller than a high profile knife.
High profile blades are used to cut thicker sections, can be coated or uncoated

Question 13

sectioning for electron microscopy

Answer 13

ultrathin sections of less than 100 nanometers are required. Samples are hardened by fast and deep freezing or by embedding in resin.
Knives used are glass or diamond

Question 14

micrometers of sections when cutting

Answer 14

15 micrometers when trimming wax, and 3-4 when fine cutting.

Question 15

temperature of the floatation bath

Answer 15

kept at 10 degrees below the melting temp of the wax

Question 16

what are some floatation artefacts

Answer 16

trapped air bubbles, folds, debris and parched earth effect which is artefactual separation of tissue components.

Question 17

When are adhesion slides used

Answer 17

• sections submitted to strong alkali solutions or high temperatures during staining such as in IHC
• crytostat sections
• tissues from the CNS
• Tissues containing blood clots

Question 18

what is the principle of frozen sections for microtomy

Answer 18

when the tissue is frozen, the water will turn to ice and act as the embedding medium to enable thin slicing of the tissue.

Question 19

How lower and higher temperatures affect frozen microtomy. At what temp is it most carried out, and what are the exceptions

Answer 19

Lower temp -> harder block
Higher temp -> softer block, softer tissue
regular temp is -20
Liver, brain, lymph node, spleen and uterine curettings are sectioned at -10
Fatty tissue required even colder temperatures due to having the lowest water content

Question 20

Most commonly used methods to freeze fresh tissue

Answer 20

• Liquid nitrogen at -190 (most rapid - snap freezing)
• Isopentane cooled in liquid nitrogen at -150 degrees (for skeletal muscle enzyme histochemistry and lymphocyte surface markers IHC)
• Dry ice, CO2 at -70
  Aersol sprays at -50 (for small blocks of tissue excluding muscle)

Question 21

What must be done to tissues snap frozen with liquid nitrogen

Answer 21

It must be allowed to equilibrate to the cryostat chamber temperature before sectioning

Question 22

Important factors to have high quality frozen sections

Answer 22

• good working microtome
• good clean sharp blade
• tissue and matrix fresh and not dehydrated
• properly adjusted anti roll plate
• optimum cutting temperature for tissue
• well trimmed sample
• knife angle between 30-50 degrees

Question 23

Cryostat maintenance

Answer 23

• defrosted, cleaned and disinfected frequently
• kept clean due to risk of infections and risk of cross contamination with an aberrant section
• dried before lubricating and re cooling
• refrigerant coils must be kept free of dust or the motor may overheat
• all routine cleaning and maintenance procedures

Question 24

How to have less freeze artifact

Answer 24

The lower the freezing solution temperature, the faster the freezing time and the less freeze artifact will occur.

Question 25

How are frozen tissues stored, including fixed and unfixed tissues

Answer 25

• normally stored at -70 degrees, in an airtight container.
• fixed cryostat sections stored in an air tight container at 4 degrees
• unfixed sections for IMF or IHC should be wrapped in foil and stored at -70.

Question 26

performing H&E on cryostat section

Answer 26

rapid H&E is carried out progressively on tissue immediatley fixed in formol-alcohol or 70% alcohol