1. Introduction to Pathology

Question 1

What is disease?

Answer 1

• A pathological condition of a body part, an organ, or a system characterised by an identifiable group of signs or symptoms
• Disease can be considered to be a consequence of failed homeostasis with consequent morphological and function disturbances
• These disturbances can be manifest at the level of the whole person, an organ, or a tissue. However, in all physical diseases, the cell is the central player.

Question 2

What is pathology?

Answer 2

• Study of suffering (pathos = suffering, logos = study)
• Branch of medicine concerned with disease and understanding the process of disease
• Attempts to explain why patients experience symptoms and guides treatment
• Often involves diagnosis

Question 3

what are The main branches of pathology?

Answer 3

• Medical Microbiology which includes:
o Virology
• Chemical Pathology (Clinical Biochemistry)
• Haematology
• Immunology
• Cellular Pathology (histopathology and cytopathology) which includes:
o Neuropathology
o Forensic Pathology
o Paediatric Pathology

Question 4

Explain chemical, haem, immuno and micro pathology discipline

Answer 4

Chemical- – Biochemical investigations of disease, e.g., endocrinology, diabetes, lipidology, thyroid disease, inborn errors of metabolism
Haematology- diseases of the blood( clotting, transduction and bone marrow transplantation)
Immunology- disease of the immune system e.g., allergy,
autoimmunity and immunodeficiency
Medical microbiology- disease causing microbes including advice on antibiotic usage

Question 5

What is the cellular discipline of pathology

Answer 5

histopathology/ cytopathology
– Examine organs, tissues and cells for diagnosis and to guide treatment, often cancer work
– Conduct autopsies

Question 6

Describe the 4 branches of cellular pathology

Answer 6

• Cytopathology – disaggregated cells rather than tissue
• Neuropathology – confined to brain, spinal cord, nerves and muscle
• Forensic Pathology - medicolegal investigation of suspicious or criminal deaths, attend crime scenes, perform detailed autopsies and act as expert witnesses in court
• Paediatric Pathology – tissue samples from children, undertake foetal, perinatal and paediatric autopsies

Question 7

What is the importance of a microscopic diagnosis?

Answer 7

• Definitive diagnosis
• Before major surgery to remove a lesion a microscopic diagnosis is required
– This guides the type and extent of surgery

Question 8

Compare histology and cytology

Answer 8

Histology involves looking at tissue whilst cytology involves looking at specific cells.
H:
• Often therapeutic as well as diagnostic
• Can assess architecture as well as cellular atypia
• Can differentiate invasive from in situ disease
• Can provide information on completeness of excision and more complete information on grading and staging
• Better for immunohistochemical and molecular testing

c:
• Faster and cheaper
• Non-invasive or minimally invasive and safe
• Can be used for cells in fluids
• Sometimes a preliminary test before other investigations or more tissue taken for histology
• Higher inadequate and error rates
• Generally used to confirm/exclude cancer/dysplasia and not to diagnose any other condition with accuracy

Question 9

Give examples of histology

Answer 9

Core biopsies, cancer resection specimens, excised skin lesions, endoscopic biopsies

Question 10

give examples of cytology

Answer 10

Fine needle aspirates of breast, thyroid, salivary glands, lung; effusions; cervical smears; sputum; urine

Question 11

How does a histopathologist arrive at a diagnosis?

Answer 11

• Pattern recognition
• This histopathologist asks her/himself:
- Is this normal or not?
- Is this inflammatory or neoplastic?
- Is this benign or malignant?
- Is this a primary tumour or a metastasis?`

Question 12

When a cancer has been identified what further information can a histopathologist tell us?

Answer 12

• Type of cancer
• Grade of cancer ( how ageresive the cancer will be)
• Stage of cancer (what further management is needed)
• Completeness of excision and if margins are involved,
which ones
• Likely efficacy of further treatments (molecular
pathology HER2, ER/PR, EGFR, PDL1 status) (whether certain drug treatments will be effective)

Question 13

where can serous carcinomas occur?

Answer 13

• Ovary
• Fallopian tube
• Uterus
• Cervix
• Peritoneum

Question 14

What is the TNM staging system?

Answer 14

It’s a system used to describe the amount and spread of cancer in a patient’s body, using TNM.

1. T describes the size of the tumor and any spread of cancer into nearby tissue.
2. N describes spread of cancer to nearby lymph nodes.
3. M describes metastasis (spread of cancer to other parts of the body).

Question 15

List the stages involved in tissue sample preparation

Answer 15

• Fixation
• Cut-up (trimming)
• Dehydration
• Embedding (processing)
• Blocking
• Microtomy
• Staining
• Mounting
• Microscopy
• Report writing

Question 16

What is fixation

Answer 16

• Hold tissue in ‘suspended animation’
• Usually use formalin (formaldehyde in water)
• Penetrates tissue at approximately 1mm/hr
• Usually fix for 24-48 hours

Question 17

What is autolysis and how do you overcome it?

Answer 17

It is self digestion which begins when the blood supply is cut off, it destroys the cells and tissue architecture
Block this via fixatives(inactive tissues, prevent bacterial growth, harden tissue)

Question 18

Describe what happens during trimming

Answer 18

• Samples are taken and placed into a cassette
– About the size of a stamp so they can be adequately infiltrated by chemicals
– May need to take 30 or more in complicated cases
– Cassettes have holes in
– They are placed in racks in formalin

Question 19

Describe what happens during dehydration

Answer 19

This process is used to remove water from tissue.
It involves using alcohol in a vacuum so that water is drawn out of the cells.
The alcohol is then replaced with xylene which can mix with wax.

Question 20

Describe what happens during embedding

Answer 20

• Molten parafffin wax is used for embedding.
• Tissue is taken out of the cassettes by hand and put into metal blocks.
• These are filled with molten paraffin wax and the body of the cassette is placed on top
• The wax is liquid when hot but dries rock hard when cool.
• The wax is allowed to harden and the metal tray is removed
• This process occurs overnight.

Question 21

Describe what happens during microtomy

Answer 21

• Very thin (3-4 microns) sections are cut from the block using a microtome
• Sections must be so thin that we can see through them with a microscope
• The thin wax sections are floated on a waterbath and picked up on a microscope slide

Question 22

Describe what happens during staining

Answer 22

• Colouring of the tissue specimen occurs so it can be seen under a microscope.
• H&E is often used.
• Haematoxylin stains nuclei purple.
• Eosin stains cytoplasm and connective tissue pink.

Question 23

Describe what happens during mounting

Answer 23

• Mounting medium is applied to the slide
• The coverslip is put on top
• The mounting medium dries and hardens, preserving
the tissue and attaching the coverslip.

Question 24

Describe what happens during microscopy

Answer 24

The tissue sample is observed under a microscope.

Question 25

How can immunohistochemistry be used to study tissue

Answer 25

• Demonstrates substances in/on cells by labelling them with specific antibodies • Usually the antibody is joined to an enzyme (e.g., peroxidase) that catalyses a colourproducing reaction • Highlights the substances usually with a brown colour • Any substance that is antigenic can be demonstrated: – Contractile protein actin – identifies smooth muscle cells – Cadherins – Hormone receptors, e.g., ER, PR – Her2 receptor – Microorganisms, e.g., CMV, HPV, herpes simplex

Question 26

How can cytokeratins be used to provide information about cancer sites

Answer 26

• Cytokeratins are a family of around 20 intracellular fibrous proteins which are present in almost all epithelia. • Their presence demonstrates epithelial differentiation and the different cytokeratins show tissue-specific distribution. • They can therefore be used to give information about the primary site of a carcinoma, particularly when used in combination, e.g.: o CK7+/CK20- : indicates lung, breast, endometrium, ovary or thyroid carcinomas o CK7-/CK20+ : indicates large bowel or some gastric carcinomas

Question 27

What is the HER2 receptor?

Answer 27

It's a growth factor receptor which predicts the response | of breast cancer to the drug Herceptin).

Question 28

What is molecular pathology?

Answer 28

Molecular pathology is the study of how diseases are caused by alterations in normal cellular molecular biology. This can be due to altered DNA, RNA or protein, but most often molecular pathology refers to changes in DNA.

Question 29

What do in situ molecular tests show? Give an example

Answer 29

In situ molecular tests show how DNA is altered in the context of the microscopic tissue structure. Examples include fluorescence in situ hybridisation (FISH) which tests for gains of additional copies of genes, such as HER2 in breast cancer, which, as with immunohistochemistry for the Her2 receptor, show if a particular tumour is likely to respond to anti-HER2 treatments like Herceptin. FISH tests can also show if genes have been translocated (i.e., broken and/or rearranged) or completely deleted from the genome, and if particular types of viral DNA are present.

Question 30

What can sequencing of DNA purified from tumour tissue show?

Answer 30

sequencing of DNA purified from tumour tissue can show if a particular point mutation (i.e., a single nucleotide change) is present in a particular gene. This is also very useful; for example, certain mutations in the EGFR gene regularly occur in lung cancers, and again these show that the case is likely to respond to anti-EGFR treatments like erlotinib.

Question 31

What can mRNA expression profiling methods show?

Answer 31

mRNA expression profiling methods can show how active a large number of genes are, at the RNA level. This is also useful diagnostically, as mRNA expression ‘signatures’ can predict how a given tumour is likely to behave, e.g., the risk of cancer spread/recurrence after surgery for breast cancer.

Question 32

What is a frozen section?

Answer 32

Frozen sections are histological preparations that, in view of time constraints, bypass the processes of formalin fixation and embedding in paraffin wax and instead involve rapidly freezing a small piece of fresh tissue on a cryostat. This piece of tissue can then be sliced thinly, stained, mounted and passed to a pathologist for microscopy. The whole process takes about 10 minutes from receipt of the tissue in the laboratory to provision of a result to a clinician.

Question 33

When are frozen section used?

Answer 33

Tissue for frozen section is taken during the course of an operation with the aim of establishing the presence and nature of a lesion whilst the patient is still under anaesthetic.

Question 34

Compare the quality of a frozen section to that of a routine paraffin wax embedded section

Answer 34

Frozen sections are harder to interpret than routine paraffin wax embedded sections as the cell morphology is less well preserved and not as easy to interpret. For this reason, errors, usually false negatives, can occur with frozen sections.