Anatomical Pathology - lecture 2Aetiology: the cause of disease Pathogenesis: the course of disease

• Focal death: one cell dies
• Infarct: many cells die (mainly due to the blockage of blood)
• 2 types of cell death

1. apoptosis: an involution of an individual cell in living organ (programmed cell death)
2. necrosis: death of cells while still in the living body

• classification of necrosis

1. liquefactive also known as colliquative, includes suppurative necrosis is most common
2. coagulative
3. caseous: soapy looking
4. fat : enzymic & traumatic
5. fibrinoid: little fibre, fibrin like

• other terms of necrosis: gangrenous, gummatous
• Suffix –it is indicates the inflammatory nature of a lesion eg hepatitis – inflamed liver. Exceptions pleuracy – inflamed pleura & pneumonia – inflamed lung
• Inflammatory response duration

1. Acute -        days to few weeks
2. Chronic-        weeks to month

The inflammatory response is generally the same irrespective of the nature of the injury. The manifestations are either local or generalized (systematic)

• Inflammation: local manifestations 5 cardinal signs

1. heat: only superficial injury due to an increase of local flood flow
2. pain- 2 components i) increase in tissue tension (oedema, pus or blood) ii) release of cellular chemicals (bradykinin, serotonin and prostaglandins)
3. swelling- local oedema as a result of increased vascular permeability, minimal effect by extravascular accumulation of leucocytes & haemorrhage
4. redness due to an increase in blood circulation in already dilated blood vessels
5. variable loss of function-2 components i) reflex inhibition of muscular movement as a result of pain ii) limitation of movement due to swelling

• oedema (Edema): the accumulation of abnormal amounts of fluid in tissues, especially in the interstitial connective tissue.
• Chemistry of dyes can be classified into natural and synthetic. Most synthetic dyes are organic

1. Aromatic series a derivative of the hydrocarbon benzene
2. Alternating double bonds are of great importance in forming coloured compounds
3. Some coloured compounds are dyes

• Benzene ring in dyes is known as quinone ring (C6H4 O2) also known as quinoid or quinonoid compounds. Oxygen atoms, each having a valency of 2 replace hydrogens in either the ortho or para position simultaneously
• Chromophore: atomic grouping are associated with colour are part of the dye molecule eg C=C, C=O, C=S, C=N, N=N, N=O. NO2
• Colour theory

1. The more chromophores that occur in a compound the more pronounced the colour
2. Colour is caused by the selective absorption of certain wavelengths of light (light which is transmitted or reflected from a substance lacks the absorbed wavelengths of light from the visible spectrum and so appears coloured)

• Chromogens: aromatic compounds containing chromophore radicals. It acts as solvent dyes. It coloured oils, fats and waxes by dissolving in them.
• Auxochrome: a chemical group in a dye molecule which ionize, this enhances the action of the chromophore and intensifies the colour of that dye. The type of auxochrome group in a dye determines how the dye will act as acidic (carbonxyl -COOH), basic (amino group –NH2) and what tissue elements it will bind to.
• Interactions of molecular groups in dyes

1. Chromogens require auxochromic groups to bind
2. Binding is either electrostatic or covalent with tissue end-groups, this stains tissues or textiles
3. The binding of the auxochromic group with tissue elements occurs either directly or via di- or trivalent metal ions (chelate or otherwise bind with the auxochrome and the tissue end-group)

• Requirements of biological dyes include

1. Reveal detail sought in the tissue which is suitable colour intensity and good affinity to bind to the component
2. Chemically reliable as its adequate solubility in chosen solvent, stability of the dye solution, purity, consistency of dye for reproducible results (consistent within and between batches of the dye) and fastness or resistance to changes in colour and intensity from light, extreme pH and changes and leaching by the mountant.

• Dye purity is determines by Colour Index (CI) number

1. Set up by industrial dyers in 1971
2. Certification by the US biological staining commission
3. Is a confirmation of the dye’s composition and it being a formulary preparation
4. Necessary for quality control and consistency in staining with each batch of dye which is produced

• Function of staining

1. Differentiates the cell cytoplasm from the nucleus
2. Distinguishes between types of tissue
3. Nature of the dye gives specificity to particular tissue elements & intensity of staining

Degrees affinity of the dye to specific tissue sites rather than other tissue components relates to intensity of staining (number of binding sties) or type and intensity of attractive forces acting.

• Dye to tissue binding includes both chemical and physical forces eg: hydrophobic bonding, Van der Waals’s forces, Coulombic attraction, hydrogen bonds and covalent bonds. Where solvent-solvent, dye-solvent, dye-dye and dye-tissue interactions are all occurring simultaneously and during the staining process
• Dye to tissue binding also relies on other factors: rate of penetration (tissue lattice structure and molecular size of dye), rates of uptake/reaction and rates of loss especially during differentiation
• Orthochromasia: tissue staining the same colour and shade as the one dye but of varying intensity eg. Methylene blue
• Metachromasia: tissue staining the same & different shades and colours as one dye. Eg. Toluidine blue (binds to a highly charged tissue elements  which alters the absorbed wavelength of visible light, altering the colour of the bound dye)
• Polychromasia: tissue staining many colours and shades as the staining solution is made up from compound of dyes eg. Any of the romanowsky type dyes used for staining of blood films or bond marrow smears (Leishman’s, Giemsa, Wright’s stains)
• most dyes are prepared as salts, auxochromes on a dye can be classed as either acidic or basic
• Basic dyes (basic auxochromes) eg. Methylene blue

1. dye can act as a cation (basic)
2. binds to form salts with negatively charged elements (eg nucleic acids known as basophilic substances)
3. stains best at basic pH because their ionisation is maintained

• acid dyes (acidic auxochromes) eg. Eosin Y and Biebrich Scarlet

1. the dye can act as a anion (acidic)
2. it binds to form salts with positively charged metal ions or associated with tissue elements (known as acidophilic substances)
3. acid dyes stain best at acidic pH because their ionisation is maintained

• amphoteric: some dyes posses both positively and negatively charged groups. These dye function as acid or basic depending on the pH of the solution. Eg. Haematein active component of haematoxylin
• basic radicals (NH+) of acid dyes and acid radicals (COOH-) combined with basic dyes are often colourless
• Tissue proteins are also amphoteric, their charge will vary with the pH of the staining solution so that proteins can bind either acid or basic dyes. At the protein’s isoelectric point the molecule is neutral and will bind with bother acid or basic dyes.
• a combination of acid and basic dyes in correct proportions form water-insoluble neutral compounds
• compounded dyes are made up as alcoholic solutions

1. they ionise when solution is diluted with water during staining procedure, selectively staining tissue elements which are oppositely charged
2. give polychromasiz
3. often used for blood films

• routine staining is Haematoxylin and Eosin Y staining universally
• Haematoxylin dye is a natural dye which extracted from the bark of the central American logwood tree, the active staining element is haematein
• Haematoxylin stain

1. Requires a mordant (metal ion) to act as a bridge between dye and tissue site
2. This metal ion binding of haematein molecules is known as a “co-ordination complex”
3. The bond is stable and fixed when alkaline

• Type of metal mordant strongly influences the tissue elements that haematoxylin solution will stain. Typical metal mordants for haematoxylin are Al, Fe, Pb, Tungsten and molybdenum
• Haematoxylin commonly use Al mordants, but they have the disadvantage of not being resistant to acid counterstains where a counterstain is acid in nature, so different mordanted haematoxylins are used usually iron haematoxylins
• Fe haematoxylins though resistant to acids are less stable and oxidize quickly, so need to be monitored carefully.
• Haematein: oxidized form of haematoxylin, is the staining agent in a solution of the haematoxylin dye known as “ripened haematoxylin”
• Ripening to Haematein

1. Done by exposing haematoxylin to oxygen in the presence of light (solution that continues to ripen as it ages and is stable for a longer time)
2. Or can be done chemically by adding sodium iodate or mercuric oxide (most of the haematoxylin is converted to haematein and has a shorter shelf life)
3. Ultimately the dye will breakdown with continued exposure to light and air and the dye will lose its staining properties

• Haematoxylin staining can be either progressive (does not need differentiation, as  is not overstained) eg. Gill’s, Mayer’s recipes or regressive (needs differentiation as there is overstaining). Eg. Mostly aluminium mordanted, harris’s, Heidenhain’s recipes.  It all depends on haematoxylin formulation or recipe
• Differentiation is the removal of excess dye from over-stained tissue; it’s done to see details of cellular structure. The bond between the basic haematoxylin dye and the mordant is acidified with acid alcohol. This detaches the dye so that excess can be washed away and haematein is amphoteric when acidified it has a red bias
• Bluing

1. To re-establishes the co-ordination complex once differentiation of haematoxylin is attained
2. Re-attaches the dye to the tissue the section needs to be made alkaline by washing copiously with wate

OR

3. Blued with Scott’s bluing solution
4. Strengthens the haematein/mordant bond precipitates the dye and prevents further loss
5. Haematein when alkaline it has a blue bias

• Eosin dyes is an xanthene dye, there are at least 3 common types Y, B & S. eosin Y is the most widely used
• Eosin Y dye

1. Used as an acid (negatively charged) counterstain a dye
2. Contrasts well with the primary staining dye
3. Highlights and discriminates between the primary staining and different tissue elements
4. Binds to positively charged (cationic) tissue site such as cytoplasmic proteins

• Eosin Y

1. Often made up as an alcoholic solution
2. Solubility in water is great and so would remain in solution not preferentially bind to tissue sites
3. Staining intensity is a matter of preference
4. Water solubility is of importance. Avoid rinsing off stained slides with water or even low concentration alcohols; it will be washed out of the tissue section. Therefore water differentiates eosin.