practical 2- carbohydrates and PAS staining Flashcards
what are carbohydrates
sugars and their derivatives
in preserved (fixed) cells and tissues, which carbohydrates are available for demo
polysaccharides, proteoglycans and glycoproteins (mucosubstances)
what is meant by mucosubstances
macromolecular compounds composed of only carbs or partially of carbs
what do polysaccharides consist of, give 2 examples
entirely of carbohydrates
glycogen and cellulose
what do proteoglycans consist of, give 3 examples
long polysaccharide chains attaches to smaller protein core
decorin, heparin and heparin in sulphate proteoglycans
what do glycoproteins consist of, give 3 examples
proteins bearing numerous covalently bound oligosaccharide chains
serum proteins, collagen, aggrecan
why must the tissue remain intact when exploring methods to test for carbohydrates
because interest is in the precise location of the mucosubstance in the tissue/cell
what are the 3 different methods to test for carbohydrates
direct staining using cationic dyes (e.g. alcian blue)
direct staining using chemical tests (e.g. periodic acid and schiffs stain)
use of lectins (e.g. concanavalin A)
how does the periodic acid (PA) test work
PA oxidises glycols to aldehydes by breaking up the vicinial diols into monosaccharides with a pair of aldehydes at the 2 free ends of the monosaccharide ring
what is schiffs (S) reagent
mixture of pararosaniline and sodium metabisulphate
what is the schiffs (S) test
s reagent is mixed with the dialdehydes which produces an insoluble magenta compound
(because the S reagent produced a pararosaniline adduct which stains glycol-containing cellular elements pink to red)
what is the schiffs reagent used for
to identify specific tissue structures or pathological alterations depending on tissue content
what does PAS stand for
periodic acid-schiff
what is the experimental procedure used to identify mucosubstances
counterstain using haematoxylin to identify cell nuclei
in PAS staining, how is the section dewaxed
using histoclear
in PAS staining, how is the sample hydrated
immerse in decreasing concentrations of alcohol
absolute (100%) alcohol
90% alcohol
70% alcohol
in PAS staining, how is the sample oxidised
in 1% periodic acid
in PAS staining, how is the sample actually stained
immerse in schiffs reagent
in PAS staining, how is the nuclei stained
use mayers haematoxylin
in PAS staining, how is the sample dehydrated
immerse in increasing concentrations of alcohol
70% alcohol
90% alcohol
absolute (100%) alcohol
in PAS staining, what are the final 2 steps
clear in histoclear
mount in DePeX and dry in fume cupboard
in PAS staining, what colour would neutral mucosubstances appear
red
what 4 cells are found in seminiferous tubules
spermatogonia
spermatids
spermatozoa
sertoli cells
in the test booklet for practical 2, label A and B on the diagram for cells in rat testis
A- spermatids
B- spermatogonium, sertoli cells (primary and secondary spermatocytes)
name the interstitial cells in rat testis
leydig cells
what is the function of the leydig and where is it located in a cell
production of testosterone
nuclei towards the bottom of the cell
what does the haematoxylin stain identify
DNA/nucleus
which of the following would not be positively stained by PAS
a. polynucleotides
b. polysaccharides
c. proteoglycans
d. glycoproteins
a. polynucleotides
in the testes, where are the leydig cells found
a. adjacent to the basement membrane of the seminiferous tubule
b. centre of seminiferous tubule
c. adjacent to ST
d. in the wall of the ST
c. adjacent to ST
which of the following is the correct sequence for PAS staining with Haematoxylin counter stain
a. rehydrate, periodic acid, schiffs, haematoxylin, dehydrate
b. rehydrate, schiffs, haematoxylin, periodic acid, dehydrate
c. rehydrate, schiffs, periodic acid, haematoxylin, dehydrate
d. rehydrate, periodic acid, haematoxylin, schiffs, dehydrate
a. rehydrate, periodic acid, schiffs, haematoxylin, dehydrate
what is HER2
epidermal growth factor (EGR)
plasma membrane receptor
where is HER2 coded
on proto-oncogene on chromosome 17
human EGR receptor 2 is involved in signalling of what
embryogenesis
cell growth
cell differentiation
apoptosis
where is HER2 located
in epithelium of breast, lung, bladder and ovary cells
why is HER2 important in breast cancer
HER2 is over expressed in 10-20% of breast cancers
over expression can either due to increased gene amplification or altered status
over expression of HER2 indicates poor prognosis
what is herceptin
humanised mouse monoclonal antibody, which targets HER2
when is herceptin used
when a patient shows an over expression of HER2
how does HER2 grading of samples take place
formalin fixed paraffin sections are used for immunocytochemical and FISH assessment
what 2 things is HER2 grading dependent on
intensity
completeness of staining cell membrane
in HER2 grading, what is the staining like in grade 0 samples
no staining/very slight partial membrane staining in <10% tumour cells
in HER2 grading, what is the staining like in 1+ grade samples
faint barely perceptive membrane in >10% tumour cell, cells stained in part of the membrane
in HER2 grading, what is the staining like in 2+ grade samples
weak to moderate complete membrane staining in >10% TC
in HER2 grading, what is the staining like in 3+ grade samples
strong complete membrane staining >30% TC
in HER2 staining, state whether treatment is used in each grade
0 and 1+ no treatment used
2+ referred for FISH (fluorescent in situ hybridisation)
3+ herceptin treatment is used
when taking breast specimen samples, how does a needle core biopsy work
needle inserted into lump to draw sample fluid and tissue
may be difficult if needle deflects
when taking breast specimen samples, how does a lympectomy/open biopsy work
surgical procedure to remove all/part of a lump
tissue surrounding lump is also removed
when taking breast specimen samples, how does a mastectomy work
removal of breast tissue and axillary lymph nodes
when taking breast specimen samples, how does a sentinel lymph node biopsy work
sentinel lymph nodes are removed
why may a HER IHC profile alter between different methods of obtaining a breast specimen
may be result of:
exhibiting an altered genetic/protein profile during tumour development
preoperative chemotherapy
time of specimen to fixative
time of specimen in fixative
specimen processing type and schedule
how are tissue samples fixed
using buffered formalin and paraffin wax
what is meant by invasive breast cancer
cancer grows into healthy tissue
what is meant by in situ breast cancer
cancer stays within milk ducts/lobules
why would a DCIS (ductal carcinoma in situ) not be treated with herceptin
because it is located within the milk ducts
what is FISH
fluorescent in situ hybridisation
how does FISH work
centromeric region of Ch17 is marked with green fluorescent signal
HER2 is marked with red
assess 20-60 cells, ratio of red: green is calculated
diagnosis is dependent on ratio of gene: chromosome
positive = >2.2 negative = <1.8 equivocal = 1.8-2.2 (in this case assess more cells and IHC)
what is CISH
chromogenic in situ hybridisation
similar to FISH but allows chromogenic visualisation of HER2 on Ch17
either reported as direct count of signal cluster
what are the 3 main tests to identify carbohydrates
direct staining using cationic dyes (e.g. alcian blue)
direct staining using chemical tests (e.g. periodic acid schiffs stain)
use of lectins (e.g. concanavalin)
