TISSUE PROCESSING Flashcards
- optional step
- decalcification is needed
- Bones, teeth, calcified specimens
- Process whereby calcium or lime salts are removed from tissues, following fixation
DECALCIFICATION
Acid decalcifying agents?
- nitric acid
- hydrochloric acid
- formic acid
- trichloroacetic acid
- sulfurous acid
- chromic acid
- citric acid
Histopathologic techniques
- numbering
- fixation
[decalcification] - dehydration
- clearing
- wax impregmentation
- embedding
- blocking
- trimming
- sectioning
- staining
- mounting
- labelling
6 main factors involved in fixation?
- hydrogen ion concentration
- temperature
- tissue section thickness
- osmolality
- concentration of fixative
- duration of fixation
hydrogen ion concentrations should be ph of ____ to be satisfactory…
6-8
temperature of fixatives in manual methods is?
room temperature
the ideal size for light microscopy in fixatives of tissue section thickness is
2 cm^2
osmolality should be ____ so that it cannot alter the morphology of the cell…
slightly hypertonic [400-450 mOsm]
concentration of fixative is?
low
duration of fixation is ____ hrs. once specimen is received…
commonly used reagent/chemical in duration of fixation?
2-6
Neutral buffered formalin
Types of Aldehyde Fixatives:
- formaldehyde [formalin]
- 10% formol saline
- 10% neutral buffered formalin
- formol corrosive [formol sublimate]
- glutaraldehyde
- commercially available as 40% PURE STOCK SOLUTION
- pure stock solution are not ideal for routine fixation
- commonly used @ 4% solution, making a 10% formalin for fixation
Formaldehyde [formalin]
advantages of formaldehyde?
- cheap
- readily available
- stable [no storage requirement]
- easy to prepare
- compatible w/ many routine stains
disadvantages of formaldehyde?
- irritating fumes
- produce shrinkage if prolonged fixation occurs
- produce quality of cytologic staining if unbuffered
- Saturated Formaldehyde diluted to 10% with sodium chloride
- Common diluent used: distilled water
10% formol saline
- saturated formaldehyde diluted to 10% & buffered w/ sodium dihydrogen phosphate & disodium hydrogen phosphate
- Common diluent used: distilled water
- Buffering a solution makes sure that it will reduce shrinkage from happening
10% neutral buffered formalin
- saturated formaldehyde diluted to 10% using saturated aqeous mercuric chloride [makes this fixative acidic]
formol corrosive [formol sublimate]
- Made up of two formaldehyde residues linked by three carbon chains
1. ____ solution for small tissues & needle biopsies [2-4 hrs]
2. ____ solution for large tissues (less than 4 mm thick) [6-8 hrs]
Glutaraldehyde
1. 2.5%
2. 4%
advantages of glutaraldehyde:
- more stable effect on tissues
- preserves plasma proteins
disadvantages of glutaraldehyde:
- more expensive
- less stable as a reagent
- Most common metallic fixative (Aq. Solution 5-7%)
- Used as a secondary fixative
- Included with many compound fixatives
- Mercury deposits are removed before staining (0.5% iodine solution in 70% ethanol for 5-10 mins)
component of mixture used to reduce detrimental effect of this fixative.
Mercuric chloride
Glacial acetic acid
Advantages of Mercuric chloride:
-
Nuclear components are shown in
fine detail
Disadvantages of Mercuric chloride
- Causes marked shrinkage of cells
- Cause precipitation of cells; mercury that will cause confusion in microscopy
Example of Chromate fixatives?
- chromic acid
- potassium dichromate
- 1-2% Aqueous Solution
- Used as a component in compound fixatives
Chromic Acid
- 3% Aqueous solution
- Preserves lipids
- In formalin or formaldehyde, lipids are not preserved well
Potassium Dichromate
Alcohol dehydrating agents?
- ethanol
- methyl alcohol
- butyl alcohol
- Recommended for routine dehydration of tissues
- “best dehydrating agent”
- works the fastest & the least hazardous
Ethanol
Used for blood and tissue films for smear preparations
Methyl Alcohol
- For plant and animal micro-techniques
- works the slowest
Butyl alcohol
Starting point ng babaran sa alcohol?
70-75%
Common clearing agents?
- Xylene
- Toluene
- Most commonly used as a clearing agent (1/2-1 hr)
- Ideally used on tissue blocks with less than 5 mm thickness
Xylene
Advantages of Xylene?
- most rapid clearing agents
- miscible w/ alcohol & paraffin wax
- cheap
Disadvantages of xylene?
- Makes tissues excessively hard and brittle if used for long periods (>3 hrs)
- Not suitable for nervous tissues and lymph nodes
clearing agent used for nervous tissues & lymph nodes?
chloroform
- 1-2 hours clearing time
- Suitable substitute for xylene or benzene
benzene - clearing agent but more toxic
Toluene
Advantages of toluene?
- Tissues do not become excessively hard or brittle
- Not carcinogenic
Disadvantages of toluene?
- More expensive
- Reacts relatively slower than xylene or benzene
Melts at 54-58 C (Laboratory temperature must be taken in consideration)
Paraffin wax
Advantages of Paraffin wax?
- Individual sections of the tissue block may be cut with ease
- Allows for rapid process (24 hrs)
- Tissue blocks may be stored immediately
Disdvantages of Paraffin wax?
- Overheated paraffin makes specimen brittle
- Prolonged impregnation causes shrinkage of cells
- Not recommended for fatty tissues
- Utilizes micro-anatomical studies of tissue
- It is a regressive staining method
HAEMATOXYLIN AND EOSIN STAINING
is used to stain nuclear components
Haematoxylin
used for cytoplasmic components
Eosin
Nuclei?
blue to black
Karyosome?
dark blue
Karyosome?
dark blue
Cytoplasm?
light pink
Cartilage?
pale pink
Calcified bone?
purplish blue
Decalcified bone matrix, collagen, and osteoid?
pink
muscle fibers?
deep pink
