Sample Preparation in Histopathology

Question 1

What is autolysis?

Answer 1

Breakdown of plant or animal tissue by the action of enzymes contained within the affected tissue.

Question 2

What is putrefaction?

Answer 2

Decomposition produced by the action of bacterial enzymes derived from the bowel. Fungal enzymes may also contribute.

Question 3

What is fixation?

Answer 3

Aims to maintain cells in as life-like state as possible following their removal from the body. Stabilises the proteins within a tissue and prevents the effects of putrefaction.

Question 4

What are the benefits of fixation?

Answer 4

Tissues become easier to cut and certain cellular components stain more easily.

Question 5

What is the purpose of adding salts to fixatives?

Answer 5

To produce an isotonic solution that does not result in cell shrinkage or swelling.

Question 6

What are the 2 types of fixatives?

Answer 6

1) Cross-linking (additive) fixatives

2) Precipitating (non-additive) fixatives

Question 7

What are cross-linking fixatives?

Answer 7

Bind amino acids in proteins to adjust their tertiary structure thus preventing autolysis and putrefaction.

Question 8

What are precipitating fixatives?

Answer 8

Remove water from the cellular matrix, thus disrupting the tertiary structure of proteins which results in precipitation.

Question 9

Give some examples of cross-linking additives.

Answer 9

Formaldehyde, gluteraldehyde, osmium tetroxide.

Question 10

Give some examples of precipitating additives.

Answer 10

Alcohol, methanol, acetone.

Question 11

Which factors affect fixation and how?

Answer 11

1) Temperature - increase in temperature leads to decreased fixation times
2) Specimen size - larger specimens take longer to fixate
3) Volume - increased fixative to tissue ration leads to decreased fixation time
4) pH - addition of buffers can slow fixation
5) Osmotic concentration
6) Concentration - stronger fixation means more rapid fixation
7) Duration - longer immersion in a fixative leads to more complete fixation

Question 12

What is formalin and how is it produced?

Answer 12

The most commonly used fixative in the UK.

Produced by dissolving formaldehyde in water. When dissolved, it forms methylene hydrate which then forms polymers.

Question 13

What is Bouins solution used for?

Answer 13

Testicular biopsies - enhances nuclear detail.

Question 14

What are some common decalcification agents?

Answer 14

Acids:

• Nitric acid (very fast)
• Hydrochloric acid (fast)
• Formic acid (slow)

Chelating agents:

• EDTA (very slow)
• Trisodium Citrate (very slow)

Question 15

When is decalcification complete?

Answer 15

1 - XRAY
2- Chemical end-point testing
3 - Manual Manipulation

Question 16

What processes are involved in Processing, Embedding and Microtomy?

Answer 16

• Macroscopic examination and block selection
• Tissue processing
• Tissue embedding
• Microtomy
• Cryotomy

Question 17

What does macroscopic examination and block selection involve?

Answer 17

Specimen is sent to the cut-up area and examined. Macroscopic detection is dictated by the attending pathologist who takes measurements and remarks on any interesting features.

Question 18

What does tissue processing involve?

Answer 18

• Tissue sections (5 micrometers) prepared
• The tissue is impregnated with a medium that is firm enough to support the tissue but soft enough to permit sectioning - paraffin wax is used for this.

Question 19

What must you do to the medium used for tissue processing, since water and paraffin wax do not mix?

Answer 19

Step 1 - dehydration in ascending concentrations of alcohol to remove the water: this aims to achieve 100% IMS. NOTE: PARAFFIN WAX IS NOT MISCIBLE WITH ALCOHOL

Step 2 - use of a clearing agent: xylene is miscible with both alcohol and paraffin wax. During this stage, all alcohol is removed.

Question 20

What is microtomy?

Answer 20

Use of a microtome to cut a thin section of a tissue.

Question 21

What is cryotomy?

Answer 21

Routine tissue sectioning can take too long therefore cryotomy is done to achieve quick results

The tissue is frozen in liquid nitrogen and fixed to a small cork disk using OCT reagent

Question 22

FFPE vs Cryotomy?

Answer 22

Frozen sectioning involves no tissue processing and no fixation (health and safety consideration)