Introduction to Histology

Question 1

What is histology?

Answer 1

The study of cells and tissues by microscopy

Question 2

What is histopathology?

Answer 2

The study of diseased tissue by microscopy

Question 3

What is the ABCDE for determining whether a mole is normal or cancerous?

Answer 3

Asymmetry

Border

Colour

Diameter

Evolving

Question 4

What is meant by asymmetry?

Answer 4

If you draw a line through the middle of the mole, the halves of a melanoma will not match in size

Question 5

What is meant by border?

Answer 5

The edges of a melanoma tend to be uneven, crusty or notched

Question 6

What is meant by colour?

Answer 6

Healthy moles are uniform in colour

A variety of colours, especially white and/or blue is bad

Question 7

What is meant by diameter?

Answer 7

Melanomas are usually larger in diameter than a pencil eraser

Question 8

What is meant by evolving?

Answer 8

When a mole changes in size, shape or colour or begins to bleed or scab, this is dangerous

Question 9

What is the extent of the spread of a tumour called?

Answer 9

Staging

Determining how far a tumour has spread

Question 10

What are the 4 stages in the clinical applications of histology?

Answer 10

1. make a diagnosis
2. determine prognosis
3. plan and confirm treatment
4. predict/confirm response to drugs

Question 11

Why is histology important?

Answer 11

It allows ‘normal’ to be defined

This allows comparisons and abnormal characteristics to be identified

Question 12

What is the first stage in histological preparation?

Answer 12

Tissue sampling

This involves taking a tissue sample by surgical excision

Question 13

What is the second stage in histological preparation?

Why is it needed?

Answer 13

Fixation

The tissue is fixed by placing it in a chemical to preserve it

The tissue will begin to degrade if it doesn’t have a blood supply

Question 14

In which 3 ways does fixation preserve tissues?

Answer 14

1. stopping intrinsic autolytic enzyme action (autolysis) which involves the tissue breaking itself down
2. prevention of bacterial contamination (putrefaction)
3. increasing mechanical strength to preserve structure and morphology

Question 15

How do fixatives work?

Answer 15

They link molecules together so that they no longer function

They also kill any bacterial contaminants

Question 16

What types of bonds are formed by aldehyde and alcohol fixatives?

Answer 16

Aldehyde fixatives form protein covalent cross-links

Alcohol fixatives denature proteins and cause aggregation/fixation

Question 17

What types of bond are formed by oxidising fixatives?

Answer 17

Protein covalent cross-links via oxidation

Question 18

Why is freezing not often used as a method of fixation?

Answer 18

It is a quick solution but results in poor morphology

Question 19

How does formalin (formaldehyde solution) work?

Answer 19

It forms protein covalent cross-links

Joining together complex molecules within the tissue means they no longer function and the tissue becomes rigid

Question 20

What are the pros of using formalin?

Answer 20

1. it has good penetration and mechanical strength

2. good tissue morphology preservation

Question 21

What are the cons of using formalin?

Answer 21

1. it is poor at preserving DNA and RNA

2. it needs to be quite warm in order to work

Question 22

How does glutaraldehyde work?

Answer 22

It is similar to formalin but is a larger molecule so forms protein covalent cross-links

Question 23

What are the pros of using glutaraldehyde?

Answer 23

1. it works well at low temperatures

2. it is used for electron microscopy

Question 24

What are the cons of using glutaraldehyde?

Answer 24

1. it only works on smaller tissue samples

Question 25

How does ethanol work as a fixative?

Answer 25

It fixes by precipitation It reduces protein solubility so that they form precipitates

Question 26

When is ethanol used as a fixative and why?

Answer 26

1. cytology smears 2. nucleic acid research It doesn't cross-link so it doesn't damage DNA or RNA

Question 27

What is the third stage in histological preparation?

Answer 27

Block selection This involves slicing the sample and placing areas of interest in a plastic cassette

Question 28

What is the fourth stage in histological preparation?

Answer 28

Tissue processing This involves slicing the tissue thinly, placing it on a slide and staining it to look under a microscope

Question 29

What must the properties of the tissue be like to allow it to be sliced thinly? How is this overcome?

Answer 29

It must be stiff and resistant to mechanical trauma It is placed in wax to allow thin sections to be cut

Question 30

What are the 4 stages involved in placing a tissue in wax?

Answer 30

1. dehydration - removing water from the tissues using alcohol 2. clearing - replacing alcohol with xylene 3. wax infiltration - replacing xylene with paraffin wax 4. embedding - orientate the tissue to form a paraffin block

Question 31

Why must water be removed from the tissues before they are placed in wax?

Answer 31

Wax is hydrophobic

Question 32

What is the fifth stage in histological preparation?

Answer 32

Section cutting and mounting

Question 33

What are the stages involved in section cutting and mounting?

Answer 33

1. making a thin slice of tissue from the wax block 2. place the block in a slicer to shave off bits of tissue 3. ribbon of paraffin placed in water 4. ribbon of paraffin picked up by slide

Question 34

What is the sixth stage in histological preparation? Why is it needed?

Answer 34

Section staining An unstained tissue section is translucent to light, so dyes are added to make it visible

Question 35

What is the purple component of haemotoxylin and eosin stain? What does this stain and why?

Answer 35

Haemotoxylin is purple It is a basic dye that is attracted to acidic structures The nuclei (DNA) are stained purple

Question 36

What is the pink component of haemotoxylin and eosin stain? What does this stain and why?

Answer 36

Eosin is pink It is an acidic dye that will stain basic structures pink Proteins in the cytoplasm are dyed pink

Question 37

What is periodic acid schiff (PAS) stain used to detect?

Answer 37

1. mucin and mucopolysaccharides 2. fungal organisms 3. visualisation of basement membranes

Question 38

What is a mucin?

Answer 38

A high molecular weight, heavily glycosylated protein produced by epithelial tissues

Question 39

What is the drawback of using PAS stain?

Answer 39

Glycogen is PAS positive Glycogen will stain a deep magenta colour

Question 40

What is DPAS and what is it used for?

Answer 40

PAS is combined with diastase Diastase in an enzyme which removes glycogen and helps to distinguish it from other PAS positive elements

Question 41

What is Gram stain used for?

Answer 41

To identify bacteria based on the constituents of their cell walls

Question 42

How are Gram positive and Gram negative bacteria distinguished from each other?

Answer 42

Gram positive stain blue Gram negative stain red

Question 43

What is Giesma stain used for detecting?

Answer 43

H. pylori bacteria in the stomach which are involved with inflammation

Question 44

What colour will Giesma stain colour the bacteria and human cells?

Answer 44

Human cells are purple Bacterial cells are pink

Question 45

What is Grocott's methenamine silver stain (GMS) used for?

Answer 45

Detecting fungi It shows fungal cell walls as black

Question 46

What is Oil red O stain used to detect?

Answer 46

It is used to stain fat

Question 47

Why can Oil red O only be used on frozen tissue and not processed tissue?

Answer 47

Xylene removes all the oil from the tissues This means there are no fats in processed tissue

Question 48

What is Orcein stain used to detect?

Answer 48

1. copper-associated proteins 2. elastic fibres 3. hepatitis B sAg

Question 49

What disease results in abnormal retention of copper? Which stain can be used to detect this?

Answer 49

Wilkinson's disease leads to an abnormal retention of copper Orcein stain

Question 50

Which organ is Orcein stain most commonly used in?

Answer 50

Liver

Question 51

What is Perl's stain used for?

Answer 51

It is used in lung tissue to look for asbestos bodies

Question 52

What colour will Perl's stain dye components?

Answer 52

it dyes iron blue It also dyes ferruginous (asbestos) bodies

Question 53

What is Ziehl Neelsen stain used for?

Answer 53

Identifying mycobacterium They contain mycolic acid and large amounts of fatty acids, waxes and complex lipids

Question 54

What is the downside of using tinctorial stains, like H&E?

Answer 54

They are not specific

Question 55

What is immunohistochemistry?

Answer 55

Using antibodies against a specific protein target It is not a stain, but a way of locating a specific molecule

Question 56

What is the first stage in immunohistochemistry?

Answer 56

A solution containing primary antibody is added to formalin-fixed paraffin embedded tissue The primary antibody is allowed some time to find the target complementary antigen

Question 57

What happens after the primary antibody is added in immunohistochemistry?

Answer 57

Unbound and surplus antibodies are washed away and a secondary antibody is added

Question 58

What happens once the secondary antibody is added in immunohistochemistry? What is special about the secondary antibody?

Answer 58

The secondary antibody is given time to bind to the primary antibody The secondary antibody carries a linker molecule with HRP enzymes

Question 59

What is added after the secondary antibody in immunohistochemistry and why?

Answer 59

DAB is added HRP enzymes transform DAB into a coloured precipitate This gives a visual representation of where the primary antibody first bound to its target

Question 60

What is the major benefit of using immunohistochemistry?

Answer 60

The antibody stain allows you to see if a specific protein is present in the tissue This allows it to be used in diagnosis

Question 61

How can immunohistochemistry be used in prognosis?

Answer 61

Through mismatch repair If a tumour has lost expression of its mismatch repair protein, it will no longer be brown