1A - Pathology, Microtomy, Frozen Sections, Immunohistochemistry Flashcards
What is disease? (2)
- pathological condition of body part/ organ/ system with identifiable group of signs/ symptoms
- consequence of failed homeostasis with morphological and function disturbances
What is pathology? (4)
- the ‘study of suffering’
- disease, process of disease, cellular dysfunction
- why patients experience symptoms
- guides treatment, diagnosis
Types of pathology and brief description: (5 [+3 +1])
- Chemical pathology(clinical biochemistry): endocrinology, lipidology, diabetes, metabolism
- Haematology: leukaemias, clotting, transfusions, bone marrow
- Immunology: allergy, autoimmunity, immunodeficiency
- Medical Microbiology: antibiotic use, microbes -> Virology
- Cellular Pathology (Histopathology & Cytopathology: disaggregated cells rather than tissues) —> Neuropathology: brain, spinal cord, nerves, muscles. Forensic Pathology: suspicious/ criminal deaths, medicolegal, autopsy, expert witness in court. Paediatric Pathology: foetal, perinatal and paediatric autopsy
Histology vs Cytology (with examples):
HISTOLOGY:
- e.g. core biopsy; endoscopic biopsy; excised skin lesions
- therapeutic and diagnostic
- differentiates invasive from in situ disease
- better for immunohistochemical and molecular testing
CYTOLOGY:
- e.g. fine needle as pirates of breast, thyroid, lung; effusions; smears; sputum; urine
- faster and cheaper
- used for cells in fluids
- higher inadequate and error rates
Serous carcinoma can occur in: (5)
- ovary
- Fallopian tube
- uterus
- cervix
- peritoneum
Questions to consider when Histopathologist is making a diagnosis: (4)
1) normal or not?
2) inflammatory or neoplastic?
3) benign or malignant?
4) primary tumour or metastasis?
When it is cancer, we need to know the: (5)
- type of cancer
- grade of cancer
- stage of cancer
- completeness of excision( Mohs surgery), and if margins are involved then which ones
- likely efficacy of treatment (ER and Her2 receptors)
Stages of cancer: (3)
Tumour - 0,1,2,3
Nodes - 0,1,2
Metastasis - 0,1,X
Using microscope to look at tissue - the steps: (8)
1) Autolysis and fixation
2) Choosing correct bits of tissue
3) Hardening the tissue, so can be thinly sliced
4) Tissue into wax, so can be cut
5) Cutting very thin sections
6) Colouring the tissue, so can see under microscope
7) Preserving and protecting sections of tissue
8) Looking at slides and making a diagnosis
When does autolysis (self-digestion) begin?
When the blood supply is cut off
What does autolysis do, in terms of cell and tissue?
It destroys the cell and tissue architecture (everything needed to make a diagnosis)
We can block the biochemical process of autolysis with fixatives - what do fixatives do?
- inactivate issue enzymes and denature proteins
- prevent bacterial growth
- harden the tissue
Describe fixation and what is used for it:
- It holds tissue in ‘suspended animation’
- Gives the tissue the appearance of ‘cooked meat’
- It uses FORMALIN (formaldehyde in water) which penetrates tissue at 1mm/hr and is fixed for 24-48 hours
What happens to the samples after fixation? Describe:
Samples are placed into cassettes, which are the size of a stamp (in order to be adequately infiltrated by chemicals), there are 30+ samples, there are holes in cassettes, cassettes are placed in racks in formalin
In order to harden the tissue, which substances are used? Describe:
- Tissue is impregnated with hardening agent: paraffin wax
- But must remove water first:
… dehydration using alcohol in vacuum (water drawn out of cell)
… replace alcohol with xylene…mixes with water
… replace xylene with molten paraffin wax…will be inside cells also
What piece of equipment is used for Embedding?
Processors
Usually overnight
Describe what happens when tissue needs to be put in the wax so that it could be cut later on:
- Manually take tissue out of cassette and out in METAL BLOCKS
- Fill with MOLTEN PARAFFIN WAX and place body of cassette on top
- Wax is allowed to HARDEN, and metal tray is removed
- ‘Blocking’
Cutting very thin slices of tissue:
- 3-4 microns
- using a microtome
Why does tissue need to be cut into very thin slices?
So pathologist can see through them with a microscope
Microtomy involves:
Thin wax sections floating on water bath and being picked up on microscope slide
What is the tissue stained with? Which structures are seen as which colour? What do other stains demonstrate?
Haematoxylin (‘bloodwood tree’) stains nucleus purple/ blue
Eosin stains cytoplasm and connective tissue pink
Other stains demonstrate different substances, structures, microorganisms
Describe the process of preserving and protecting the slices:
MOUNTING:
- mounting medium applied to slide
- coverslip put on top
- mounting medium dries and hardens…this preserves the tissue and attaches the coverslip
What is immunohistochemistry?
- It demonstrates substances in/on cells by LABELLING them with specific antibodies
- ANTIBODY joined to ENZYME e.g. Peroxidase, that catalysts a colour-producing reaction
- It highlights the substances with a BROWN colour
Any substance that is antigenic, can be demonstrated in immunohistochemistry - examples include:
- Contractile protein actin (smooth muscle cells)
- Cadherins - cell adhesion molecules, deficient in carcinomas e.g. Lobular breast carcinoma
- Hormone receptors e.g. ER, PR
- Her2 receptors - growth factor receptor, predicts response of breast cancer to Herceptin
- Microorganisms e.g. CMV, HPV, herpes simplex
What are cytokinins, where are they present, how many are known, what is their function (CK+/-)?
- Intracellular fibrous proteins
- present in almost all epithelial
- 20+ known
- markers for epithelial differentiation - show tissue-specific distribution in epithelia
- can give info about primary site of carcinoma especially when used in combination…
…CK7+/CK20- (lung, breast, endometrium, ovary, thyroid)
…CK7-/CK20+ (large bowel, some gastric carcinomas)
What is Molecular Pathology?
Study of how diseases are caused by alterations in normal cellular molecular biology; can be due to altered DNA, RNA or protein
What do in situ tests show? What is an example of this type of test?
In situ tests show show DNA is altered in tissues prepared for Microscopy
e.g. FISH to test for gains of additional copies of Her2 gene in breast cancer
What does sequencing of DNA purified from tumour tissue show? Give an example (with drug name)
Sequencing of DNA purified from tumour tissue can show if mutation is present in particular gene
e.g. If a certain mutation in EGFR gene is present in lung cancer, the. It is likely to respond to anti-EGFR treatment e.g. erlotinib
What does next generation sequencing allow?
Many genes being t stem simultaneously for mutations
What do mRNA expression profiling methods demonstrate? What can mRNA ‘signatures’ predict? Give an example
mRNA expression profiling methods demonstrate the level of activity of large number of genes simultaneously
mRNA signatures can predict how the tumour is likely to behave
e.g. Risk of breast cancer spread/recurrence after surgery (oncotypeDX breast cancer assay)
Describe FROZEN SECTIONS: what it entails, how often it is used, its purpose, what it establishes, its accuracy
- urgent histopathology
- hardening tissue quickly
- not routinely used - morphology is worse than in paraffin sections
- intra-operative (during surgery) … takes 10 mins in lab (from receiving specimen to giving a result)
- establishes presence and nature of lesion and INFLUENCES course of OPERATION
- 96% accuracy (not very high) … misinterpretation, absence of diagnostic tissue in frozen section
Simplified steps involved in making FROZEN SECTIONS:
1) fresh specimen arrival
2) cut-up
3) freezing the section
4) microtomy
5) finished slide
