Histological Techniques & Analysis Flashcards
What are biomaterials ?
- Exogenous materials which restore function to damaged tissue
- vascular graft, stent, pacemaker
What are some issues of biomaterials ?
- biocompatibility
- surface chemistry & topography
- materials escape from graft
Define Biocompatibility
- the ability of a biomaterial to perform its function, without eliciting any undesirable local or systemic effects
What are the main cells involved in dictating the biocompatibility of implanted material ?
neutrophils, macrophages, lymphocytes, fibroblasts, mast cells, tissue specific cells
Describe the role of neutrophils in biocompatibility
- 1st line of immunological defence
- produce wide range of molecules that can damage implants
- key for cell signalling that coordinates tissue healing
Describe the role of macrophages in biocompatibility
- clear debris from implant site
- secretome is key for dictating the way a tissue heals
- if left uncontrolled can damage implant & tissues
Describe the role of lymphocytes in biocompatibility
- chronic inflammatory cells
- remove foreign molecules persistent throughout the body
- potential role in material destruction & tissue healing
Describe the role of fibroblasts in biocompatibility
- ‘matrix secreting machines’ omnipresent in many tissues
- secrete abundant extra cellular matric to secure implant in place
Describe the role of mast cells in biocompatibility
- tissue resident cells which act as hubs to direct inflammation at the site of tissue injury through secretion of huge number of cytokines & inflammatory mediators
Describe the role of tissue specific cells in biocompatibility
- osteoblasts (bone), chondrocytes (cartilage), adipocytes (fat)
- all needed to perform regenerative roles in devicwe specific applications
Define Cell infiltration
the extent to which cells have gotten inside of an impant, and what sort of cells they are
Define Encapsulation
- the manner in which the body has ‘sealed’ the implant from the tissue environment around it
Define resorption/degeneration
how well the implant retains in structural integrity over time
Define Haemorrhage
- the presence of red cells which arent contained within the vasculature
Describe Foreign Body Giant cells
- specialised immune cells which engulf macroscopic materials
What are often found at implant sites ?
- polymorphonuclear leukocytes
Define Vascularisation
provides nutrient influx & waste efflux to/from an implant
Describe Oedema
- presence of excessive interstitial fluid withing a tissue space
What can long term persistance of Lymphocytes indicate?
- implant failure
Describe ISO10993
- set of standard for evaluation the biocompatibilty of medical devices before clinical trials
- typically presented as ‘pathologic index’
- scoring system 0-4 presence =4 abscence =0
Describe Napthol ASD Chloroacetate Esterase
- also known as ‘Leders stain’
- esterases found in leukocytes react with defined molecules to produce a coloured product
- naphthol couples with fast red violet LB to create a coloured deposit at site of enzyme activity
What is the most common histological method for identifying bone ?
- Von Kossa Staining
- it doesn’t directly interact with calcium
Describe Von Kossa Staining
- uses solution of silver nitrate to displace calcium ions from calcium containing compounds in the ECM
- silver compounds are then photochemically degraded into silver atoms by exposure to light
- result is a black precipitate where calcium was present
What diseases can Von Kossa indicate?
- demonstrate the broken-down bone trabeculae leading to brittleness we associate with osteoporosis
- can indicate rickets if collagenous material is amongst the black stained mineralised matrix
Describe Alizarin red stain
- also used to identify presence of bone
- interacts directly with calcium ions forming a chelate which remains on the section surface
- less specific than Von Kossa
- calcified ECM = deep red
Describe the protocol for alizarin red stain
- brings sections to distilled water via xylene & ethanol
- stain slides with 2% alizarin red for 2mins
- shake off excess dye & blot
- dehydrate in acetone, clear in xylene & mount in synthetic mounting medium
How is alizarin red quantified using a spectrophotometer ?
- stain is dissolved off the section and its absorbance measured
- greater the absorbance, the more bone there was in the section
What helps cartilage resist compressive forces?
- the extracellular matrix of cartilage contains abundant proteoglycan structures
What does Alcian Blue bind to ?
- proteoglycan structures
- turning them a blue/turquoise colour
Describe Alcian blue stain
- copper containing dye
- +ive charge of coppr allows interaction with -ive charged moieties present in glycosaminoglycans & mucopolysaccharides
- glcosylated proteins are abundant in ECM of cartilage
What is the protocol of Alcian blue stain?
- deparaffinize slides & hydrate to distilled water
- stain in alcian blue solution
- wash in running water 2mins
- rinse in distilled water
- counterstain in nuclear fast red solution for 5 mins
- wash in running tap for 1min
- dehydrate with ethanol gradient
- clear in xylene
- mount with DPX
What are the results of using Alcian Blue stain?
- strongly acidic/sulphated sugar substances = blue
- nuclei = pink/red
- cytoplasm = pale pink
What does combining alcian blue & PAS allow ?
visualisation a larger range of sugars in a tissue
What dye combination allow for the relationship between bone & cartilage ?
alcian blue & alizarin red
Why is fat commonly used ?
- reconstructive procedures
- similar mechanical properties to lots of soft tissue
Describe Oil Red O stain
- stains fat droplets present in cells a deep red colour
- mechanism of dye action is largely due to lipophilicity
- means it interacts with lipid compartments within cells & remain insoluble in aqeuos environment of normal cytoplasm
What is the Oil red O stain procedure?
- cut frozen sections, air dry
- fix in formalin, wash with running tap water
- rinse with 60% isopropanol
- stain with freshly prepared oil red o
- rinse with 60% iso…
- lightly stain nuclei with alum haematoxylin
- rinse with distilled water
- mount in glycerine jelly
What diease can oil red o help visulise ?
- visualises fat droplets which accumulate in hepatocytes during non-alcoholic fatty liver disease
