lipids - tissue sampling, processing and staining -...
TRANSCRIPT
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Lipids
Dr Phil Bryant, Wales, UK
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Lipids
Lipids are structural components of cell membranes
Lipids store energy
Lipids form the basis of hormones
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Phosphatides (lecithin, cephalin, sphingomyelin) Cerebrosides (glycolipids & galacto-lipids such as kerasine and phrenosine)
Fatty acids (saturated and unsaturated) Alcohols (sterols such as cholesterol)
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Fat cells - adipocytes
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Two types of fat cell
White fat cell
Most common Unilocular - single, large lipid
droplet Large diameter (>100u)
Subcutaneous Omentum Mesentery
Brown fat cell
Less common Multilocular – many small
droplets Common in new-born
Around kidney Neck Mediastinum
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Fat cells are thought to develop from fibroblast- like cells
Fat droplets coalesce into a large droplet, leaving only a thin rim of cytoplasm
Nucleus is usually pushed to the side
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When tissue is fixed and stained, the single large lipid droplet is extracted and the cells look empty
Fat cells
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Identification of lipids
1. Solubility - differentiation of lipids by their solubility in various solvents
2. Examination by polarised light
3. Reduction of osmium tetroxide
4. Demonstration by fat soluble dyes
5. Histochemical methods
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1. Solubility
Keilig’s extraction using fresh human brain, extracted with 3 changes over 24 hours
Cold acetone – simple lipids, neutral lipids & cholesterol
Hot acetone – compound lipids such as cerebrosides
Hot ether – phosphatides such as lecithin and cephalin
Hot chloroform and methanol – all lipids
Following extraction, blocks hydrated through alcohol to water, frozens cut and stained with Sudan black
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2. Examination by polarised light
Three types of refractility:
Isotropic (monofringent) – neutral fats & fatty acids
Anisotropic (birefringent) – any crystalline lipid
Maltese cross (birefringent) – cholesterol esters
Also use phase contrast microscopy
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Polariser and phase contrast
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3. Reduction of osmium tetroxide Colourless OsO4 to black OsO2
Marchi’s for degenerate myelin
After normal myelin has been oxidised by chrome salts, it will not react with osmium tetroxide
Degenerate myelin contains oleic acid which is not oxidised by chrome salts and will reduce osmium tetroxide to black osmium dioxide
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4. Fat soluble dyes
These dyes have a high affinity for fats, lipids, lipoproteins and triglycerides
The fat soluble dyes are oil red O, Sudan II, Sudan III, Sudan IV and Sudan black B
Staining solutions are generally alcoholic
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Oil red O
Demonstrates neutral lipids and fatty acids
Oil red O is a fat-soluble dye
More soluble in fat than in the dye solvent
Solvent is usually isopropanol
Cut frozen sections, usually leave unfixed
Stain in oil red O and counterstain
Mount in aqueous media
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Oil red O
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Sudan IV and Sudan black B
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Bromine - Sudan black B
One section stained with Sudan black after bromination
Control section stained without bromination
Cholesterol, cholesterol esters and phospholipids stain blue / black but remain unstained in control
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5. Histochemical methods
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Nile blue sulphate
Demonstrates acidic and neutral fats
Nile blue sulphate contains 2 components
Red oxazone which dissolves in neutral fats
Blue oxazine reacts with fatty acids & phospholipids
Fix frozen sections in formol calcium for 1 hour
Method requires 2 solutions: Nile blue sulphate at 60oC for 30 minutes Methyl green solution for 5 minutes
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Nile blue sulphate
Neutral fats, oils and cholesterol esters – red Fatty acids and phospholipids - blue
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Perchloric acid-naphthaquinone (PAN)
Cholesterol and cholesterol esters stain grey/blue
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Other histochemical methods
Luxol fast blue – phospholipids (and myelin)
Baker’s acid haematein – phospholipids
Fischler’s method – fatty acids
Osmium tetroxide - α naphthylamine (OTAN) – phospholipids – useful for differentiating hydrophilic lipids (sphingomyelin, cerebrosides etc) from hydrophobic lipids (fatty acids & cholesterol esters)
Fluorescent dyes such as phosphine 3R and benzpyrene
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Why demonstrate fat
Fat may appear abnormally as a result of trauma such as bone fractures
Fat released into bloodstream can cause emboli
Identify fatty tumours such as liposarcoma
Identify fat in other pathological conditions
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Lipid pathology
1. Fatty degeneration
2. Lipoma
3. Liposarcoma
4. Atheroma
5. Thecoma
6. Tay-Sach’s disease
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1. Fatty degeneration
Also known as steatosis or fatty change
Abnormal retention of lipid within cells
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2. Lipoma
Benign tumour composed of adipose tissue (body fat)
Most common benign soft tissue tumour
Usually moveable and painless
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3. Liposarcoma
Rare, malignant tumour arising in fat cells deep in soft tissue such as the thigh
Large and bulky often with satellites outside of the main tumour
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Undifferentiated liposarcoma left side Differentiated liposarcoma on right side Benign fatty tissue in the centre (has fewer blood vessels)
3. Liposarcoma
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4. Atheroma
Swelling in arterial walls with macrophages that contain fatty acids and cholesterol (clefts)
Also contain calcium and fibrous connective tissue
Occurs in atherosclerosis
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5. Thecoma
Benign ovarian tumour composed of theca cells
Oestrogen producing
Occurs in older women, generally after the menopause
Tumour cells have abundant lipid-filled cytoplasm
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6. Tay Sach’s disease Known as gangliosidosis or
hexosaminidase A deficiency
Caused by gangliosides accumulating in nerve cells
Progressive mental deterioration in children with death usually by 4 years
Lipid stored as concentric, laminated bodies