Cell Path and Pigments

Question 1

Artefact

Answer 1

Deposits produces as a result of chemical reaction in tissue

Question 2

Endogenous (Haematogenous, Autogenous)

Answer 2

Produced within tissue and have a physiological function or are a by-product of normal process

Question 3

Exogenous

Answer 3

Gain access to body accidentally with no physiological function. Usually minerals, enter by inhalation or implantation in skin during industrial exposure

Question 4

Formalin pigment

Answer 4

Artefact Pigments

brown/black deposit following fixation in acid
formalin, especially seen in haemorrhagic tissue. Removed with picric acid

Question 5

Malarial pigment

Answer 5

Artefact Pigments

similar to formalin pigment, formed in/near RBC’s
with parasite.

Question 6

Mercury pigment

Answer 6

Artefact

black deposit formed with fixatives containing
Mercury. Removed with iodine + sodium thiosulphate

Question 7

Dichromate deposits

Answer 7

Artefact pigments

yellow/brown deposits after potassium
dichromate fixation and insufficient washing prior to dehydration. Removed
with acid alcohol

Question 8

Bile pigments

Answer 8

Endogenous Pigment

breakdown of RBC’s. Red/brown (bilirubin), green (biliverdin). Liver diseases or haemolytic disease.

May be due to cholestasis

Question 9

Lipofuscin

Answer 9

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.

Question 10

Melanin

Answer 10

Endogenous pigments

Product of melanocytes. Black/brown pigment. Eye, skin, hair, brain and melanoma. Demonstrated by Masson Fontana (black)

Question 11

Iron

Answer 11

Endogenous Pigment

Stored as haemosiderin in ferric state (Fe3+). Fine brown colour. Liver, spleen, marrow. Demonstrated by Perls’ Prussian Blue reaction

Question 12

Haemosiderin

Answer 12

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)

Question 13

Haemosiderin Diseases

Answer 13

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)

Question 14

Perls Method for Ferric Iron

Answer 14

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.

Question 15

Calcium

Answer 15

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.

Question 16

Copper

Answer 16

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)

Question 17

Uric Acid and Urates

Answer 17

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

Question 18

Principle of a polarising microscope

Answer 18

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

Question 19

Carbon

Answer 19

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

Question 20

Asbestos

Answer 20

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).

Question 21

Fats and Lipids

Answer 21

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

Question 22

Histochemistry

Answer 22

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

Question 23

Lysochrome

Answer 23

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

Question 24

Lipids

Answer 24

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

Question 25

ID Lipids

Answer 25

Solubility - Traditional wax sectioning techniques remove lipids. H&E staining gives negative artifact image

Solubility - Section cut unfixed, frozen tissue. Subsequent short fixation in formalin, neutral fat only preserved not fixed. No dehydration/clearing and sections mounted in aqueous mountants

Microscopic examination
Bright-field microscopy (H&E, Sudan-type stains, Oil Red O)
Polarised light microscopy (Oval fat bodies in urinary sediment examination - Lipiduria)

Osmium Tetroxide fixation - uncommon in light microscopy, blackening
lipids

Question 26

Lipid storage disorders (lipidosis)

Answer 26

Metabolic inherited disorders (rare)
Accumulation of lipids in cells (overproduction, decreased metabolism)
Varies organs but in particular lipid accumulation in CNS – developmental and mature neurodegeneration, mental retardation, death

Question 27

Fabry Disease

Answer 27

accumulation lipids in kidney