W7L1 Part 2 - Introduction to Microscopy Flashcards
Decalcification
Bone and other calcified tissue such as calcified plaques, some tumours etc., must have calcium removed before sectioning
Bouin’s and Zenker’s will both fix and can decalcify tissues, although slowly
Other decalcifying agents cannot fix tissue
Small tissues are required and require 1-2 hrs to decalcify
Large calcified structures need to sectioned into smaller fragments
Prolonged decalcification will affect histologic detail and some antigenic sites especially ER, PR, p53 and Ki-67
Fixation of Bone - Specimen Handling
Tissue should be fixed for 12-24hrs in NBF, mercury containing fixatives e.g. Zenker’s
70% ethanol is used for samples suspicious for gout
X-ray of specimens can direct the investigation to allow bone sawing of appropriate representative material
Trim larger samples with a bone saw to smaller more manageable sizes around 3mm thick
Fix on bed of gauze or suspend to allow perfusion
Decalcification - Selection of Blocks
If radiograph images indicate a tumour involves cortical and cancellous bone, then sample the softer bone components to speed up the tissue processing
Femoral head with fractures - sample tissue around the fracture to determine the associated pathology
Decalcifying Agents - Acids
Organic acids - weak organic acids - buffered formic - most common at 10% aq solution - acetic and picric acids are slow but gentle for small specimens Mineral acids - HCl Acid - strong inorganic acids - time can be from 24hrs-days
Decalcifying Agents - Chelating Agents
React with metal ions to form a stable, water-soluble complex
Ethylendiaminetetra acetic acid (EDTA)
Acts in neutral pH 7 - 7.5
Very slow, but no tissue damage and preserves enzymes
Time: 6-8 weeks
Can be combined with acid to improve overall morphology preservation
Surface Decalcification
Used when the presence of calcium was not expected;
- in kidney, lung, prostate or skin tissue
- usually revealed during trimming where the surface may need to be decalcified prior to microtomy
- occasionally, bone that has been decalcified, may need more decal solution and surface decal may be used
- surface decalcification allows penetration of into the block to dissolve calcium
Process:
- expose the cut surface of the block to a decal fluid for 15mins e.g. 1% HCl or 10% Formic acid
Factors Influencing Decalcification - Concentration of Decal Agent
> concentration > likelihood of tissue damage
Depletion of acid or chelator by calcium
- avoid by changing fluid daily, using larger than 50X tissue volume of fluid
Endpoint test the reaction to avoid over decalcification
Factors Influencing Decalcification - Temperature
Increased temperature e.g. 60◦C, speeds up decalcification but can lead to maceration of tissue
RT is ideal
4◦C if planning of leaving the tissue over a weekend
Increases EDTA rate without damage but loss of enzymes
Other Factors Influencing Decalcification
Agitation
Suspension
Vacuum
Completion of Decalcification
Aim is to prevent over exposure to decal agent
X Ray method - cannot be used mercury fixatives
Physical method - probing with needle but may lead to artefact in tissue
Chemical method - detect free calcium released from the bone calcium oxalate test
Is Haematoxylin a Dye?
Haematoxylin (Hx) on its own is not a dye and will NOT stain tissues
It needs to be combined with a mordant
Mordants are usually metal cation, such as aluminium, iron or chromium
Hx in complex with aluminium salts is cationic and acts as a basic dye
It is +ve charged and can react with negatively charged basophilic cell components such as nucleic acids in the nucleus
For this reason, nuclei stain blue
Haematoxylins
Hx is colourless and without oxidation, it has little or no value as a biological stain
Haematein is the oxidation product from Hx, a natural dye responsible for the colour properties
- natural oxidation: exposure to light and air
- chemical oxidation: using sodium iodate (Mayer’s Hx) or mercuric oxide (Harris’s Hx)
The use of chemical oxidising dyes converts Hx to haematein almost instantaneously
Alum Hx
Most widely used in routine H&E stains
The mordant is aluminium in the form of aluminium potassium sulfate or aluminium ammonium sulfate
Stain the nuclei a blue-black colour when the tissue section is washed in a weak alkaline solution like STWS or lithium carbonate
Can be used;
- regressively (section is overstained)
- progressively (stained for predetermined time)
Disadvantage - cannot be used when staining with acidic solutions
- e.g. Van Gieson staining
Iron Hx
Iron salts are used both as the oxidising agent and mordant
Most common are ferric chloride and ferric ammonium sulfate
- Weigert’s Hx
- Heidenhains Hx
Over oxidation is the main disadvantage with these stains
- solutions are prepared separately and then mixed immediately before use
- more time consuming
Advantage - capable of demonstrating a wider range of tissue structures than the alum Hx
Other Hx
Tungsten Hx - used for the Mallory PTAH technique
- CNS material
Molybdenum Hx - uses molybdic acid as the mordant
- demonstration of collagen and coarse reticulin fibres
Lead Hx - incorporate lead for the demonstration of endocrine cells of the alimentary tract
