final review Flashcards
whats orthochromic
orthochromic is when tissue stains the same color as the dye
whats leuco compound
in schiff reagent when the quinoid ring loses it color it’s called leuco compund which is colorless
then reacts with aldehydes to form a new color
why do we hydrate tissues
to remove parrafin & make tissue more miscible with stain ( stain is water based)
when would you not hydrate tissue
when stain is alcoholic stop at 70 %
why do we dehydrate
to prepare for resinous mounting media
when would you not dehydrate
when using a water based mounting medium
ex, in oil red O we use glycerol as mounting media
do you DCM frozen sections
we DCM frozen sections, we dont hydrate( bc no parrafin) but we still need to mount
how to ripen hematoxylin
naturally : sunlight & dela field
artificailly : sodium iodine
aluminium mordant vs iron mordant
aluminum ( preferred) - longer shelf life
iron mordant - shorter shelf life
Accentuators
what does chloral hydrate do
prevents scum in hematoxylin( don’t need to filter)
why is bluing so important
goes from red ( soluble ) to blue ( insoluble )
nuclear stain is less likely to be removed
ex. scotts tap water = bluing agent
H&E - white spots following deparaffinization
clearing agent + water = cloudy or white spots
nuclear stain not crisp
fixation not complete
oveheated
poor processing
Pale nuclei
not enough dye or poor quality dye in the section
not enough section in dye
overexposure to acid
thin section
overstained or dark nuceli
too much dye in section
too much section in dye ( too thick)
red or brown nuclei
over oxidized hematoxylin
not enough bluing
pale cytoplasmic staining
pH
not enough dye in section
not enough section in dye
cytoplasm overstained
too much dye
too much section
eosin not 3 different shades
fixation
dehydrate & clear well
70% best for differentiation
pH 4.6-5 recommended
blue - black precipitate on top
hematoxylin was not filtered & metallic sheen was picked up on sections
hazy or milky water on slides
clearing agent & water have come together
not miscible
water bubbles seen in microscopically
incomplete dehydration
remove coverslip with xylene ( in chemical hood ), dehydrate, return to xylene, coverslip
change solutions of clearite 3 and alcohol
check for hazy milky slides before mounting ( saves time )
uneven H& E staining
contamination of wax in closed processor with water or fiaxative
dark staining on edge
laser or electrocautery
cannot fix burned tissue
poor contrast nuclei vs cytoplasm
poor nuclear staining
excessive cytoplasmic staining
good technique for H&E
checking slides after each staining step
do not allow sections to dry during procedure ( gloossy black nuceli & brown stippling)
keep solutions covered when not in use
how to make & store schiff reagent
treat basic fuchsin( parosaniline) with surlfuric acid ( breaks quinoid ring ) to form colorless compund ( leucofuchsin)
combine with aldehyde will form color bright red) only after WASHING to remove surfuric acid
store in fridge but bring to room temp before use
test with formaldehyde bc aldehydes form with schiff reagent
smells like sulfur bc of surluric acid
another name for PAS
McManus method
how to make PAS more specific for glycogen
what fixative is preferred for glycogen
amylase or diastase = more speciific
alcohol prevents glycogen streaming
whats a good control for glycogen
- liver
- endo/ecto cervix
whats a good control for basement membrane
kidney
metachromatic stains
stain of tissue is a different color than the original color of dye
change of color is due to polymerization of the dye molecules by the tissue chromatropes
true metachromatic dyes
- toludine blue
- methylene blue
alcian blue
stains acid mucin
at pH2.5 bith sulfated & carboxylated mucin is stained
at pH 1.0 only sulfated mucin stained
Alcian blue -PAS allows us to be able to diagnose
Barretts esphagus
alkaline congo red stains
amyloids
- builds up in conditons such as amyloidosis and eventually so much that it overtakes normal function of tissue and patient dies
how to confirm that we see amyloid and not stain deposit
birefringent ( set up scope for polarization )
what is used to remove unreduced silver in silver stains
hypo
sodium thiosulfate
why do we reduce silver stains & example
reduces residual silver deposited at reaction site
ex. formaldehyde or hydroquinine
what causes over staining in silver
if toning is carried out too long the backgroun dof the stained slide mat be violet to red color
in masson trichchrome what do we post mordant in
bouins - to make more brilliant
masson trichrome colors/ size
RBC : yellow
muscles: red
collagen : blue/green
what does heteropolyacid do
Phosphomolybdic acid (PMA) and/or
Phosphotungstic acid (PTA)
“Colorless” anionic dyes
binds to tissue commponents
Collagen binds lots of PMA / PTA,
Heterpolyacid fits through collagen pores
and replaces the small anionic dye so collagen appears unstained
Large molecule dye bonds with the
heterpolyacid and stains collagen
what is GMS used to stain
fungus* gold standard fungus stain
can also be used to stain urates
where is fouchets reagent
bile stain
made up of ferris chloride and trichloracetic acid
components of fouchets reagent
ferric chloride in trichloralacetic acid
exogenous and endogenous pigments
endogenous
exogenous
lipofuchin & bile pigment how to differentiate
lipofuschin is a wear & tear pigmnent
negative with bile stain fouchets
bile may accumulate when there is an obstruction
positive with bile fouchets stain ( green )
melanin
how is it removed
how to stain
reducer or not
is an argentaffin substance thst doesnt require an extrsneous reducer so it it schmorl positive
removed by bleaching
not removed by weak acids
can use fontana masson to stain
pearls prussian blue vs turnball
pearls prussian blue
- for ferric iron
- uses potassium ferrocyanide
turnball
- for ferrous iron ( rare)
- used in schmorl reaction
- uses ferricyanide to produce ferrous ferricyanide
what stain can we use for melain and how to make it more sepcific
fontana masson
to make more specific
- make 2 slides and bleach one using potassium promagonate or hydrogen peroxide
what methods are used for apud cells that are arrgarophilic
Grimelius & Churukin
both stains use hydroquinone as reducer & counterstained with nuclear fast red
IHC is more accurate means of ID
exogenous pigments
carbon
abestos - birefringent
- coated with iron so can stain with pearls prussian blue
calcium staining
stains blue/ purple in H& E
found abnormally in necrotic tissue
stain with
- von kossa * gold standard/ classic method ( uses light reduction )
- alizarin red
urates what fixative should be avoided
water & lithium fixatives should be avoided
only use alcohol or frozen section
birefringent
GMS used to stain
copper is associated with what disease and methods used
wilsons disease
rhodanine: more sensitive. less specific
rubeanic acid : less sensitive, more specific
IHC least sensitive to most sensitive
direct (used for kidney or skin biopsies- use frozen sections )
indirect ( add patient serum to know antigen; looking for antinuclear antibodies )
- 2 step & 3 step
PAP( secondary & tertiary )
ABC & LSAB ( most specific )
fixation of gynecological specimens
fixed immediately
ethanol
methanol can be used causes less shrinkage
dont use formalin
nongyneological specimens
dont fix- bring to lab immeditaely
excess blood can obscure details
acetic acid in carnoys an clarkes lysis RBC- making it easier to ibserve nuclear detail
liwuid based cytology two system
both produce high qulity monolayer slides
1. thin prep- uses filtration method
2. sure path liquid based pap test - monolayer preparations by sedimentation
heparin
added to some body fluids to prevent clotting
nickle method
breast/ nipple discharge
CSf fluid
can deteriorate rapidly, if acant bring to lab immediately
use prefixative golding solution
- dilute specimen with an amount of alcoholic saline or saccomanno fluid equal to the volum eof the fluid
urine for cytology
first morning not reccomennded
pap stain 5 dyes 3 solutions
know these !
- hematoxylin ( nuclear stain)
- ORange G-6 first counterstain ( keratinized cells )
- EA36 ( polychrome cytoplasmic stain)
- light green
- eosin Y
- Bismark brown
cytology stain color results
chromatin - blue
keratin- orange
superficial squamous cells- variable shades of pink
nucleoli,cilia, RBCs - cells variable shades of pink
all metabolic cell cytoplasm cells- varibale shades of
