Cell path practicals Flashcards
what is the purpose of differentiation
removing excess stain - we regressively stain with haematoxylin (over stain) to remove later
(removing background)
why do sections need bluing after haematoxylin staining
ammonia (bluing solution) changes the red-purple haem to a blue purple stain so is more distinguishable and clear to identify from the eosin colours
The blueing solution also has an alkaline pH. This re forms a mordant-dye lake in the tissue which makes the blue dye more permanent and insoluble compared to the purple one
what conclusions are drawn from H&E staining
staining of normal tissue sections
Haem - basic dye which stains DNA -
chromosomes- deep blue
Eosin- acidic dye which stains proteins -
muscle- deep pink
collagen- pale pink
RBC- orange
What cell type contains the iron stain
macrophages
what is the principle of PERLs staining
- HCL splits off the bounded proteins allowing potassium Ferro cyanide to combine with Ferric ions producing a Prussian Blue colour.
- Staining time vary depending on the amount of ferrous ions present.
what are tissue deposits containing ferric ions called
hemosiderin deposits?
what is the difference between haemosiderosis and haemochromatosis
haemosiderosis - focal deposition of iron - no tissue damage
haemochromatosis - iron overload - typically systematic process in which iron deposits cause tissue damage
what is the purpose of perls Prussian blue
demonstrate ferric ions location
collagen - red
muscle - yellow
nuclei - red
what counterstain is used in perls Prussian blue
van gieson - collagen - red
muscle - yellow
neutal red - nuclei - red
Perls: what tissue is used for positive control?
Sections containing Ferric ions e.g Spleen, Liver, Bone Marrow
What are Perls Reagents?
HCL acid and Potassium Ferrocyanide
explain the function of HCL in Perls Stain
HCL splits off the bounded proteins allowing to stain blue
What is the purpose of a Pigment Special Stain - Formalin Pigment?
to compare the presence of formalin pigment (appearing as dark brown, black granules) around red blood cell
why does formalin pigmentation occur?
- deposits formed by the action of acidic (pH<5.6) fixatives on Haemoglobin.
- tissues stored for long times
What is the principle of Formalin Pigment Removal?
Alcoholic Picric Acid is used to remove formalin pigment then stained using routine H&E to see if the pigment is still present.
what does PAS stain
hepatocytes - glycogen mucins mucoprotein glycoprotein fungi
what is the counterstain in PAS
haematoxylin
what is the principle of PAS staining
exposes tissue to peroic acid which oxidises compounds with free OH groups, resulting in dialdehydes = Schiffs create magenta coloured complex formation.
what type of cells contain the lipofuscin pigment
liver, kidney, heart muscle, retina, adrenals, nerve cells, ganglion cells
where in the cell is the lipofuscin located
in cytoplasm around the nucleus
what cells contain lipochromes
aging post mitotic cells
what other histochemical stains identify Lipofuscin apart from PAS
sudan black B
long Ziehl-neelson acid fast
what is lipofuscin also known as
the wear and tear pigment
how do cells produce lipofuscin
byproduct of failed intracellular catabolism
what is it believed about excessive lipofuscin?
causes cell death - not just a bystander
what colours and their organelles are shown in PAS staining
PAS - red/magenta - glycogen, basement membranes, reticular fibres, cartilage. or superficial fungal elements if candida infection
what does PAS demonstrate
carbohydrates in tissues (glycogen)
basic principles of PAS staining?
periodic acid oxidises tissue carbs, producing aldehyde groups, Schiffs reagent condenses these an forms bright red colouration.
conditions necessary for a PAS positive reaction
- must contain the 1-2 glycol grouping, an equivalent amino or alkylamino grouping, or the oxidation product CHOH-CO
- must not diffuse away after fixation
- must give an oxidation product that doesn’t diffuse away
- must be present in sufficient concentration to produce detectable final colour
