Introduction To Techniques Flashcards
Give an overview of the process a specimen will go through in routine histopathology.
Specimen reception»_space;> fixation»_space;> tissue selection (dissection)»_space;> processing»_space;> embedding»_space;> microtomy»_space;> demonstration techniques»_space;> quality control»_space;> pathologist.
What problems need to be overcome in order for the pathology department to provide microscopic material for diagnosis by pathologist and material to archive?
Human tissue degrades, Human tissues are mostly water, Light transmission through human tissue, Whatever you do to human tissue will affect its appearance, Cost, History and habit, Human driven systems aren't perfect.
What takes place in specimen reception?
Specimen reception receives and logs samples and ensures that specimens are fully tracked.
They check materials being received into the lab are correct and ensures the paperwork matches.
Samples are designed a unique number.
What is the purpose of specimen fixation?
Specimen fixation preserves human tissue.
It prevents cellular breakdown due to auto lysis and bacteria.
It preserves physical structure, chemical structure and tissue components.
What factors influence chemical fixation?
Type of fixative, Composition of the tissue, The form of the fixative (liquid, gas, etc), pH, Vehicle (chemicals that allow fixative to penetrate more easily), Temperature, Duration, Volume and concentration of fixative.
How have fixatives historically been classified?
Aldehydes,
Oxidising agents,
Protein-denaturing agents,
Unknown mechanisms.
How do most fixatives act?
Most fixatives act on proteins within the tissues by anchoring and stabilising the proteins and thus the tissue components.
What is a more modern way of classifying fixatives?
To look at the way they work on tissue themselves and which tissue elements they fix best (i.e. Proteins, fats, carbohydrates, nucleic acids) or what tissue they fix.
What fixative fixes proteins best?
Formaldehyde.
What fixative would be best for fixing fats?
Mercuric acid.
What fixative would be best for fixing carbohydrates?
Alcohol based fixative.
What is the best fixative for fixing nucleic acids?
Formaldehyde based fixatives.
What artefacts may occur as a result of fixation?
Pigments (e.g. Brown formaldehyde pigment),
Volume changes (e.g. Glomerulus shrinkage),
Alteration of tissue chemistry,
Loss of tissue components.
What is the standard fixative used in the cytology lab?
Formaldehyde - usually at a concentration of 4% known as formalin.
What is one problem with using formalin as a fixative and how may this be overcome?
Formaldehyde based fixatives don’t penetrate very well and are most effective if used in larger volumes. Because we only use formaldehyde as a 4% formalin solution we need very large volumes.
We can however incise the tissue to penetrate better, but this does risk damaging it. We make margins using Indian ink so that we don’t lose track of the margins of excision.
What is a good fixative to use for sperm?
Bouins alcohol based fixative is good for sperm and for nuclear detail.
What type of fixative should be used for nucleic acids?
Alcohol based (carnoys).
What is a good fixative to use for electron microscopy?
Glutaraldehyde. It isn’t as safe as formaldehyde. It makes tissue very hard and rigid and is therefore good for electron microscopy.
Why do tissues need to be dissected in the histology department?
Tissue selection is required because we need smaller samples because they are easier to examine and fix. It is a very skilled process.
We physically can’t store whole specimen taken from patients.
Who carries out tissue dissection?
Either a pathologist or a trained scientist carries out dissection on a ventilated bench.
Describe the general process of tissue dissection.
The process of dissection is a pivotal point on the whole pathology process.
Margins are marked. This is very important in cases where the margins of the tissue are not easily defined in order to ensure that the whole of a tumour is removed.
Small pieces are put in to labelled and numbered cassettes and then put back into fixative.
The dissector will also provide a description of the sample.
Health and safety and cleanliness are very important.
What is involved in tissue processing on the histology lab?
Processing is the treatment of the tissue necessary to impregnate it with a solid medium to facilitate the production of microscope slides.
Processing involves a wide range of chemicals to dehydrate the tissue and can be hazardous.
The impregnation medium of choice is usually paraffin wax with some other agents added to improve density. It has a melting range of 58-63 degrees Celsius.
1) . Complete fixation in formalin.
2) . Dehydration in increasing concentration of alcohols (avoids tissue shock).
3) . Clearing of dehydrating agent in xylene.
4) . Impregnation with wax.
5) . Embedding
What factors may affect tissue processing?
Composition of tissue (density),
Agitation makes take up wax,
Temperature (increased temperature speeds up the process),
Fluid viscosity (more fluid agents are faster acting),
Vacuum can pull fluid into tissue.
What artefacts might arise as a result of tissue processing?
Alteration of tissue chemistry,
Volume changes,
Loss of tissue components.
What are the advantages of tissue processing automation?
Tissue processing can be fully automated. This is advantages as it can take up to 23 hours manually. Automated tissue processing is carried out in a tissue processor. Automation saves man power and allow the process to be enhanced by agitation and the use of vacuum chambers. Chemicals are pumped in and out of the vacuum chamber. Molten paraffin wax is then introduced after the final xylene step.
What step takes place after tissue processing?
Embedding.
What does embedding involve?
After the processing the wax blocks need surrounding to make sure that they are fully rigid and supported. This involves an embedding machine which has a cold plate and a molten wax chamber.
The blocks are taken from the tissue processor and then placed in the molten wax in the embedding machine chamber. The samples are then removed from their cassettes and placed into an appropriate sized mould. The specimen need to be orientate correctly within the mould to ensure we can see an edge. The tissue processing cassette is then placed on top of the mould and it is topped up with wax.
What would be a good embedding material for electro microscopy samples?
Resin or plastic.
What embedding material could we use when the processing required for paraffin wax would destroy the tissue components?
Agar or gelatine as they don’t require the use of alcohol.
What is the microtomy step achieve?
It makes the slides from the paraffin wax blocks.
What factors may influence the microtomy step?
Density and rigidity of impregnation and embedding medium and tissue.
The presence of solid material.
The type of microtome used (usually a rotary microtome now).
Describe the process of microtomy.
1) . Chill the paraffin blocks to make them more rigid ensuring nice thin sections.
2) . Place the block in the chuck. Turn the microtome handle so that the chuck moves up and down against the blade creating 2-3 um thick sections (can cut between 0.5 and 10um).
3) . The sections are then taken and floated out on a water bath and picked up on a slide.
When are frozen sections and what are they used for?
A frozen section is a thin slice of tissue cut from a frozen specimen. They are often used to make rapid diagnosis (e.g. in surgery).
Frozen sections may also be used when fixation and processing may destroy what you want to look at (e.g. fat assessment).
Describe how frozen sections may be made.
1) . Sample received into department.
2) . Tissue is selected and placed onto a cork disk with moutant.
3) . Tissue is frozen and then a slice is taken using a cryostat which is then picked up using a slide.
Why isn’t frozen sectioning used routinely?
Frozen sectioning is not routinely used for a number of reasons.
The tissue needs freezing down to -30 degrees Celsius in some cases and it is difficult to subsequently store.
It is very useful when we need a very quick answer (20 mins) such as intraoperatively.
Frozen sectioning is backed up by normal processing to get a more storeable and useable sample.
What type of chemicals are organic dyes based on?
Benzene ring type chemicals.
List 3 types of histochemical demonstration techniques.
1) . Organic dyes
2) . Colourless stains that produce colour by interacting with tissue elements (e.g. Schiff reagent reacting with aldehyde groups to give pink colour).
3) . Metallic pigments that stick to tissue components.
Most stains are aqueous based. What difficulties does this pose?
Wax is hydrophobic and so the wax must be removed using xylene and then alcohol and then water before staining is able to take place.
What are the basic principles and interactions by which stains work?
The primary action of any chemical stain is due to a chemical or electrostatic reaction with tissue components.
Why do some tissue elements stain better than others?
The primary action of any chemical stain is due to a chemical or electrostatic reaction with tissue components.
Some things stain better than others because of repelling forces, the size of staining molecules, the speed of the staining reaction (the quicker the reaction the less likely we can remove it before it has reacted with all of the tissue components - i.e. we can’t evict the stain from certain tissue compartments).
The strength of the bonds is why tissues stay stained, however, the stains will fade over time. They are not stable indefinitely.
How else can stains be classified other than by their mechanism of action?
We can also classify stains by what they demonstrate.
I.e.
Demonstration of tissue structure,
Demonstration of tissue components,
Demonstration of material that does not appear on normal tissue,
Demonstration of infectious agents.
What is the Masson trichrome stain?
It is a 3 stain process in which different densities of tissue stain differently.
What does Alcian blue demonstrate?
Alcian blue demonstrates acidic mucins.
What does PAS demonstrate and how does it work?
PAS demonstrates neutral mucins.
PA is used to produce aldehyde groups on mucins and then Schiffs reagent is used to react with them.
What can the Oil Red O stain be used to demonstrate?
The Oil Red O stain can be used to demonstrate fat on frozen section material.
What can the Masson Fontana stain be used for?
The Massin Fontana stain can be used to demonstrate melanin by enhancing its staining.
What stain can be used to demonstrate amyloid?
Congo Red stain can be used to demonstrate amyloid.
What does Pearle’s stain demonstrate?
Pearle’s stain demonstrates iron from red blood cell break down.
What stains may be used to demonstrate helicobacter?
Toluidine blue and silver methods.
Why do most labs limit their regularly performed special stains to about 20 different types even though there are hundreds available?
It keeps costs down and is more consistent.
There is a lot of redundancy in special staining methods.
Stains are of used on their own. True or false?
False. Staines are rarely used on their own. Timing is critical. Stains are used in conjunction with other chemical reagents to adjust the uptake and distribution of stains.
What is haematoxylin?
Haematoxylin is a nuclear stain. It can give considerable detail about the nuclear details of cells (i.e. doesn’t simply demonstrate them).
What does eosin do in the H and E stain?
Eosin acts as a counter stain to demonstrate all the tissue components other than the nucleus. It also gives information about tissue structure. It can demonstrate cell membranes, fibrous tissue etc.
H and E staining is good at identifying cells that have large and active nuclei (i.e. rapidly dividing cells). What does this make the H and E staining procedure particularly good at examining?
Tumour cells.
How is the H and E stain usually carried out these days?
On automated machines that stain hundreds of slides per day. They are stained in one machine and then cover slipped in another.
What is the aim of quality control in the histology lab?
To ensure the ID and link between samples and patients and to ensure satisfactory test quality.
What 2 steps does most quality control on the histology lab consist of?
1) . Tracking of patient info.
2) . Microscopic examination of the slide.
What will happen before a slide leaves the histology department?
Before a slide leaves the department and goes out to a pathologist a scientist will assess that slide and make sure that the fixation is adequate, no damage has occurred due to microtomy, the staining itself is demonstrating the correct tissue components and equally and importantly actually identifying the tissue type is correct.
Identifying the tissue type and cross referencing with the patient record is an important quality control method.
No material will leave the histology department without quality control.
What is the very basic principle of IHC?
IHC uses antibodies to detect antigens in cells or tissues.
True or false? IHC can demonstrate the presence and localisation of antigen.
True.
IHC can detect as few as ____ per cell.
IHC can detect as few as 100 molecules per cell.
True or false? Multiple labelling can be used to detect different antigens simultaneously in IHC.
True.
List some of the diagnostic uses of IHC.
Differentiation of tumours, Tumour sub-typing, Confirm or refute H and E diagnosis, Secretory products, Lymphoma/leukaemia characterisation, Identification of metasteses, Prognostic markers.
What are the main 2 antibodies used for IHC? Briefly describe these antibodies.
1) . IgG - is Y shaped and has two identical binding sites for antigens.
2) . IgM - is a larger molecule and consists of 5 igG like molecules bound together to form a pentamer.
Antibodies may be polyclonal or monoclonal.
