practical 2- carbohydrates and PAS staining

Question 1

what are carbohydrates

Answer 1

sugars and their derivatives

Question 2

in preserved (fixed) cells and tissues, which carbohydrates are available for demo

Answer 2

polysaccharides, proteoglycans and glycoproteins (mucosubstances)

Question 3

what is meant by mucosubstances

Answer 3

macromolecular compounds composed of only carbs or partially of carbs

Question 4

what do polysaccharides consist of, give 2 examples

Answer 4

entirely of carbohydrates

glycogen and cellulose

Question 5

what do proteoglycans consist of, give 3 examples

Answer 5

long polysaccharide chains attaches to smaller protein core

decorin, heparin and heparin in sulphate proteoglycans

Question 6

what do glycoproteins consist of, give 3 examples

Answer 6

proteins bearing numerous covalently bound oligosaccharide chains

serum proteins, collagen, aggrecan

Question 7

why must the tissue remain intact when exploring methods to test for carbohydrates

Answer 7

because interest is in the precise location of the mucosubstance in the tissue/cell

Question 8

what are the 3 different methods to test for carbohydrates

Answer 8

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)

Question 9

how does the periodic acid (PA) test work

Answer 9

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

Question 10

what is schiffs (S) reagent

Answer 10

mixture of pararosaniline and sodium metabisulphate

Question 11

what is the schiffs (S) test

Answer 11

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)

Question 12

what is the schiffs reagent used for

Answer 12

to identify specific tissue structures or pathological alterations depending on tissue content

Question 13

what does PAS stand for

Answer 13

periodic acid-schiff

Question 14

what is the experimental procedure used to identify mucosubstances

Answer 14

counterstain using haematoxylin to identify cell nuclei

Question 15

in PAS staining, how is the section dewaxed

Answer 15

using histoclear

Question 16

in PAS staining, how is the sample hydrated

Answer 16

immerse in decreasing concentrations of alcohol

absolute (100%) alcohol
90% alcohol
70% alcohol

Question 17

in PAS staining, how is the sample oxidised

Answer 17

in 1% periodic acid

Question 18

in PAS staining, how is the sample actually stained

Answer 18

immerse in schiffs reagent

Question 19

in PAS staining, how is the nuclei stained

Answer 19

use mayers haematoxylin

Question 20

in PAS staining, how is the sample dehydrated

Answer 20

immerse in increasing concentrations of alcohol

70% alcohol
90% alcohol
absolute (100%) alcohol

Question 21

in PAS staining, what are the final 2 steps

Answer 21

clear in histoclear

mount in DePeX and dry in fume cupboard

Question 22

in PAS staining, what colour would neutral mucosubstances appear

Answer 22

red

Question 23

what 4 cells are found in seminiferous tubules

Answer 23

spermatogonia
spermatids
spermatozoa
sertoli cells

Question 24

in the test booklet for practical 2, label A and B on the diagram for cells in rat testis

Answer 24

A- spermatids

B- spermatogonium, sertoli cells (primary and secondary spermatocytes)

Question 25

name the interstitial cells in rat testis

Answer 25

leydig cells

Question 26

what is the function of the leydig and where is it located in a cell

Answer 26

production of testosterone nuclei towards the bottom of the cell

Question 27

what does the haematoxylin stain identify

Answer 27

DNA/nucleus

Question 28

which of the following would not be positively stained by PAS a. polynucleotides b. polysaccharides c. proteoglycans d. glycoproteins

Answer 28

a. polynucleotides

Question 29

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

Answer 29

c. adjacent to ST

Question 30

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

Answer 30

a. rehydrate, periodic acid, schiffs, haematoxylin, dehydrate

Question 31

what is HER2

Answer 31

epidermal growth factor (EGR) plasma membrane receptor

Question 32

where is HER2 coded

Answer 32

on proto-oncogene on chromosome 17

Question 33

human EGR receptor 2 is involved in signalling of what

Answer 33

embryogenesis cell growth cell differentiation apoptosis

Question 34

where is HER2 located

Answer 34

in epithelium of breast, lung, bladder and ovary cells

Question 35

why is HER2 important in breast cancer

Answer 35

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

Question 36

what is herceptin

Answer 36

humanised mouse monoclonal antibody, which targets HER2

Question 37

when is herceptin used

Answer 37

when a patient shows an over expression of HER2

Question 38

how does HER2 grading of samples take place

Answer 38

formalin fixed paraffin sections are used for immunocytochemical and FISH assessment

Question 39

what 2 things is HER2 grading dependent on

Answer 39

intensity completeness of staining cell membrane

Question 40

in HER2 grading, what is the staining like in grade 0 samples

Answer 40

no staining/very slight partial membrane staining in <10% tumour cells

Question 41

in HER2 grading, what is the staining like in 1+ grade samples

Answer 41

faint barely perceptive membrane in >10% tumour cell, cells stained in part of the membrane

Question 42

in HER2 grading, what is the staining like in 2+ grade samples

Answer 42

weak to moderate complete membrane staining in >10% TC

Question 43

in HER2 grading, what is the staining like in 3+ grade samples

Answer 43

strong complete membrane staining >30% TC

Question 44

in HER2 staining, state whether treatment is used in each grade

Answer 44

0 and 1+ no treatment used 2+ referred for FISH (fluorescent in situ hybridisation) 3+ herceptin treatment is used

Question 45

when taking breast specimen samples, how does a needle core biopsy work

Answer 45

needle inserted into lump to draw sample fluid and tissue may be difficult if needle deflects

Question 46

when taking breast specimen samples, how does a lympectomy/open biopsy work

Answer 46

surgical procedure to remove all/part of a lump tissue surrounding lump is also removed

Question 47

when taking breast specimen samples, how does a mastectomy work

Answer 47

removal of breast tissue and axillary lymph nodes

Question 48

when taking breast specimen samples, how does a sentinel lymph node biopsy work

Answer 48

sentinel lymph nodes are removed

Question 49

why may a HER IHC profile alter between different methods of obtaining a breast specimen

Answer 49

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

Question 50

how are tissue samples fixed

Answer 50

using buffered formalin and paraffin wax

Question 51

what is meant by invasive breast cancer

Answer 51

cancer grows into healthy tissue

Question 52

what is meant by in situ breast cancer

Answer 52

cancer stays within milk ducts/lobules

Question 53

why would a DCIS (ductal carcinoma in situ) not be treated with herceptin

Answer 53

because it is located within the milk ducts

Question 54

what is FISH

Answer 54

fluorescent in situ hybridisation

Question 55

how does FISH work

Answer 55

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) ```

Question 56

what is CISH

Answer 56

chromogenic in situ hybridisation similar to FISH but allows chromogenic visualisation of HER2 on Ch17 either reported as direct count of signal cluster

Question 57

what are the 3 main tests to identify carbohydrates

Answer 57

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)