GSR Flashcards
What is the _____ of Gordon and Sweets Reticulin
Classification
Target tissue component
Mechanism of staining
Principle of staining
Positive controls
Classification - connective tissue stain
Target tissue component : reticulin fibers
Mechanism of staining: metallic
impregnation
Principle of staining: argyrophilic (tissue staining that is stained with silver needs an external reducer) metallic impregnation and metallic substitution
Positive controls: liver, spleen, lymph node and adrenal gland
What is the ______ of reticulin fibers
Source
classification
composition
distribution
properties
Source: produced by fibroblasts as tropcollagen
classification: Type 3 and 4 collagen
composition : Carbohydrate coating (glycoprotein and polysacc containing proteoglycans)
distribution : found in basement membranes, as meshwork support in hemopoietic tissues, precollagenous reticulin in the dermis
properties : not elastic, no tensile strength, weak birefringent , not demonstratable by dyes
What are the steps for silver staining and the reagents that are used
1.Oxidation - KMnO4 acidified
2.Bleaching - oxalic acid
3.Sensitization - Ferric ammonium sulphate - iron alum
4. Silver impreg- ammoniacal silver solution
5.reduction - 10% formalin
6. toning - gold chloride
7. fixation - sodium thiosulphate
8.counterstain - nuclear acid red
What is the first step in silver staining
oxidation
when glycol group hexose sugars are oxidized to aldehydes
-needed for fibre impregnation
-aldehyde groups react indirectly with silver
oxidizers
KMnO4 - produces reactive aldehyde groups this reagent is acidified with H2SO4 which suppresses impregnation of the nuclei making it more specific for the reticulin fibers
PMA and HIO 4
What is the 2nd step in silver impregnation
Bleaching
-removes any undesired effects from oxidizing agent
-oxalic acid is used to remove discoloration from KMNO4
What do we do to help the silver be more reactive
use iron first and then go in with silver
What is the 3rd step
sensitization
-metallic impregnation step
-reticulin has low affinity for silver
Sensitizers like : ferric ammonium sulphate (IRON ALUM), uranyl nitrate and dilute silver nitrate are used
- we need to use a metallic salt like ferric ammonium sulphate so it can react with aldehyde groups on reticulin to form aldehyde-iron complexes
-impregnation occurs when metallic salts are deposited ON or AROUND but NOT in tissue component being demonstrated
-this forms metal - organic compound with reticulin fibers
-metal is then replaced by silver
IRON ALUM = bisulphate salts
what is step 4
Silver impregnation
-put ammoniacal or diamine silver AgNH32 on the tissue
-this causes 4 atoms of silver to be deposited on the site of reactive sugars in the reticulin
-Aldehyde groups will reduce the diamine silver to metallic silver
-this group under goes ion exchange or metallic substitution with the iron bound to aldehyde
-silver will be deposited where iron was and iron will replace the silver in solution
-dont wash too much because it can reduce the restaining but if you wash too little it can increase background staining
-silver ions are still not visible under the microscope so
problem that can arise is that there are not enough silver deposits to provide proper visibility
What is step 5
known as developing step
-use of external reducer - 10% formalin or 3-4% formaldehyde
-any diamine ions that havent been washed off are reduced to metallic silver
-causes high visibility of precipitate on reticulin fibers
What is step 6
Toning
-bound metallic silver is treated with gold chloride
-allows for better contrast and clarity
when overtoning occurs the reticulin fibers look red instead of grey
What is step 7
fixation
removing unreduced silver, this prevents non specific blackening with light exposure
-using sodium thiosulphate (hypo)
What is step 8
Counterstain
this is optional
-light green which stains acidophilic tissue parts : muscles and RBCs
-any red stain (cationic dyes like nuclear fast red or neutral red) which reacts with phosporyl groups (-) in the nuclei that were suppressed by HMnO4 during oxidation
staining due to ionic bonding
steps to prep ammoniacal silver
- be sure to chemically clean all glassware and rinse with distilled water
-tap cant be used because there is metal in tap water from the pipes that interferes with staining
-end point is dirty water appearance
-NaOh helps the solution darken
-add NH4OH , you need to add drop by drop until it looks like dirty water
-top up with dh2o until you get to your end volume
-filter before use to prevent silver deposits
-dont add too much over titrate NH4OH the solution will become clean this can cause lack of sensitivity, weak or lack of impregnation of reticulin fibers
-dont discard in the sink because the unstable silver salt will break down into silver azide or silver nitride
What happens when you over titrate
back titrate with 10 AhNo3
Why should be solution look like dirty water when preparing ammoniacal silver
clear water = too much ammonia which can cause a decrease in sensitivity = incomplete impregnation of reticular fibers
why do you need to rinse between silver solution and formalin in reducing step
prolonging wash causes reduced reticulin staining and insufficient wash causes excessive background
What type of forceps are used
plast or wax coated only
What is sensitizer
iron alum
double sulphate or bipsulphate
why do you need to wash slide after staining NFR
inadequate rinsing causes cloudiness
why do we need to use fresh ammnonia
NH4OH can become weak with ammonia loss in solution
-increased NH4OH in titration means decreased strength
-reticulin seen as gray black not sharp black
how do you store ammonical silver
store in the fridge and bring to RT before use because the solution ages
-is explosive
what type of pattern should we be seeing on the reticulin
silver deposition should be linear - black
-should not be granular - repeat if so
-nuclei and cytoplasm should be stained as per counter stain
all slides must be what before being picked up
they must be charged because of the high pH of the solution
how to discard ammoniacal silver
-must be neutralized first with bit of salt or saline producing a white precipitate
-leave for 5 mins and then pour in labeled waste container
-rinse acid washed glassware 5x with dH2O
What is this stain used for
differential diagnosis of tumors
-loss of meshwork if there is a tumor
light stain if you over titrate need to use a normal slide to compare
