Cell Path and Pigments Flashcards
Artefact
Deposits produces as a result of chemical reaction in tissue
Endogenous (Haematogenous, Autogenous)
Produced within tissue and have a physiological function or are a by-product of normal process
Exogenous
Gain access to body accidentally with no physiological function. Usually minerals, enter by inhalation or implantation in skin during industrial exposure
Formalin pigment
Artefact Pigments
brown/black deposit following fixation in acid
formalin, especially seen in haemorrhagic tissue. Removed with picric acid
Malarial pigment
Artefact Pigments
similar to formalin pigment, formed in/near RBC’s
with parasite.
Mercury pigment
Artefact
black deposit formed with fixatives containing
Mercury. Removed with iodine + sodium thiosulphate
Dichromate deposits
Artefact pigments
yellow/brown deposits after potassium
dichromate fixation and insufficient washing prior to dehydration. Removed
with acid alcohol
Bile pigments
Endogenous Pigment
breakdown of RBC’s. Red/brown (bilirubin), green (biliverdin). Liver diseases or haemolytic disease.
May be due to cholestasis
Lipofuscin
Endogenous pigments
‘wear and tear’ pigment due to lipid oxidation. Near nucleus. Normal process but excess accumulation linked to PNS/CNS diseases. Brown pigment. Lipochrome pigment. Stained by Sudan black, Periodic Acid-Schiff (PAS), Schorl’s, Long Ziehl-Neelsen technique. Found in heart muscle, liver and brain.
Melanin
Endogenous pigments
Product of melanocytes. Black/brown pigment. Eye, skin, hair, brain and melanoma. Demonstrated by Masson Fontana (black)
Iron
Endogenous Pigment
Stored as haemosiderin in ferric state (Fe3+). Fine brown colour. Liver, spleen, marrow. Demonstrated by Perls’ Prussian Blue reaction
Haemosiderin
iron-binding proteins (Fe3+). Destruction of old red blood cells. Particularly found in macrophages of the spleen and liver. Poorly available. Perls’ histochemistry: hydrochloric acid (unmasking), potassium ferrocyanide (chromogen - blue) (1:1 mix)
Haemosiderin Diseases
Haemochromatosis. Autosomal recessive disorder. Excessive absorption of iron. Multi-organ disorder. Liver (cirrhosis), heart (heart myopathy), pancreas (diabetes mellitus)
Haemosiderosis. Sometimes referred to as secondary haemochromatosis. None genetic cause. Focal deposits. Alcoholism or thalassaemia (blood transfusions)
Perls Method for Ferric Iron
Treat with solution 20% HCl w 10% K Ferrocyanide
Protein is split off by hydrochloric acid
Potassium ferrocyanide combines with
ferric iron (= Prussian blue Reaction
– blue)
1% of Neutral Red as a general stain in histology, as a counterstain in combination with other dyes, and for many staining methods.
Calcium
Endogenous pigments
Absorbed in GIT from food (vitamin D). Demonstrated by Von Kossa (Silver impregnation, not specific to calcium, black) and Alizarin (red). Teeth/bone (hydroxyapatite), osteoporosis.
Copper
Endogenous pigments
Important in metabolism (oxidase enzymes). Normally undetectable in histochemistry. Wilson’s disease (accumulation). Demonstrated by Shikata Orcein (brown), Rubeanic acid (green/black), rhodanine stain (red, picture)
Uric Acid and Urates
Endogenous pigments
Breakdown of purine nucleotides. High uric acids (kidney diseases, overweight, diabetes). Crystallisation in joints (Gout). Urate crystals are birefringent (polarising microscope)
Uric acid crystals in joints
Principle of a polarising microscope
Light source (unpolarised light)
Condenser (light is focussed)
Polariser 1: light is polarised
Only if specimen is birefringent or contains birefringent structures: light twisted by 90° (red line)
Objective (magnification)
Polariser 2: in crossed position compared to polariser 1 (only light that passed birefringent material is allowed to pass)
Light structures against dark background
Carbon
Exogenous pigments
Most common of the pigments, seen in lungs of urban dwellers, smokers. Absorbed by phagocytes. No histological methods, easy to identify (site), may be confused with melanin
Asbestos
Exogenous pigments
Long beaded fibres (silica), cause fibrosis, may lead to asbestosis and mesothelioma. Fibres become coated in protein sheaths containing haemosiderin, demonstrated by Perl’s Prussian Blue (asbestos bodies).
Fats and Lipids
Defined by their solubility in fat solvents and their insolubility in water. Conjugated lipids: Neutral fats Waxes Cholesterol esters Phosphoglycerides Sphingomyelins Ceramides Glycolipids
Unconjugated lipids:
Fatty acids
Steroids
Histochemistry
Traditional wax sectioning techniques remove the lipids
Special preservation and sectioning of tissue therefore important
Fixation is difficult for lipids. All but 2 fixatives can be used (osmium tetroxide, potassium dichromate). Osmium tetroxide fixes lipids but also blackens them. Neither fixatives are commonly used in histochemistry.
Best! Lipid histochemistry on unfixed lipid; frozen sections from cryostat
Option of short fixation after cutting/staining
Lysochrome
Lysochrome staining relies on using a dye that is very soluble in lipids but relatively insoluble in aqueous solvent.
No charges are involved
Dye prepared in complex aqueous solution (e.g. acetone and alcohol; 60% isopropanol solution).
Solvent use the dye close to saturation.
Colouring of lipids based on elective solubility; proteins and nucleic acids remain unstained
Removal of excess dye, keeps background clean.
Techniques using the lysochrome method:
Sudan-type dyes (Sudan I-IV or Sudan black)
Oil Red O
Nile Blue
Lipids
Biopsy - Divide into 2; snap frozen and formol-calcium fixation
Then apply - Sudan Black B, Oil Red O, Polarised Light (birefringence) and
other methods as appropriate.
Limitations of techniques:
Physical properties of lipids – change from “norm” of reaction (pure lipids stain differently from mixtures)
Melting points 370C = fluid in body, solid in section, therefore may or may not stain or react with reagents
