Things I Forget

Question 1

What makes up an undifferentiated panel

Answer 1

Melanoma: S100, HMB45, Vimentin
Sarcoma: Desmin, Vimentin
Carcinoma: AE1/AE3, CAM 5.2, EMA
Lymphoma: CD45, Vimentin
Neuroendocrine: Neuron Specific Enolase

Question 2

What antibodies make up a breast cancer panel

Answer 2

ER
PR
Her2
P63/SMMS
Ki67

Question 3

What is in situ hybridisation

Answer 3

• A technique used to detect specific nucleic acid sequences of RNA or DNA within cells or a tissue.
  * Makes use of labelled probes to hybridise a known target mRNA or DNA sequence within the tissue.
  * The labelled probes are then detected using an antibody specific to the label
  Through this the probes can be used to detect the expression of a particular gene of interest as well as the location of its mRNA

Question 4

What is hybridisation?

Answer 4

Hybridisation is the process in which two complementary single-stranded DNA and/or RNA molecules bond together to form a double-stranded molecule

Question 5

What is EMA

Answer 5

Epithelial membrane antigen

Question 6

What reagents do you use to decalcify bone

Answer 6

RDC (Rapid decalcifer) - hydrochloric acid

Formic acid - weaker acid for smaller bones etc

Question 7

Write about the Melanoma markers

Answer 7

S100
▪ Positive in nearly all melanomas
HMB45
▪ A highly sensitive marker for melanoma cells
Vimentin
Tends to be positive in melanoma with elevated expression seen in invasive, migratory melanoma

Question 8

Write about the carcinoma markers

Answer 8

EMA
▪ Epithelial membrane antigen
▪ Expressed by nearly all epithelial cells
▪ Very useful in undifferentiated panel
AE1/AE3
▪ A cocktail of anti-cytokeratin antibodies
▪ Recognise multiple cytokeratins,
▪ Cytokeratins are keratin proteins found in the intracytoplasmic cytoskeleton of epithelial tissue
Cam 5.2
▪ Another cytokeratin ▪ Expressed by glandular epithelium
Of particular use in expressing colorectal cancer and pancreatic cancer

Question 9

Write about the sarcoma markers

Answer 9

Desmin
▪ A type of filament protein found in muscle cells
▪ Expressed tyipcaly in skeletal muscle skells and most smooth muscle cells
▪ Desmin is a specifiic marker for rhabdomyosarcomas

Vimentin
▪ Vimentin + Desmin upregulation is seen in sarcomas
Vimentin is a filament protein expressed in mesenchymal cells such as those of the cartilage, bone, bone marrow, skeletal tissues etc -> any cell that comes from the mesenchymal stem cell lineage

Question 10

List the silver stains
(6)

Answer 10

Grocotts methenamine silver
Gordon and sweets reticulin
Millers Elastin Van Gieson
Warthin and Starry
Masson Fontana (Melanin)
Jonne’s Methenamine silver (glomeruli basement membrane)

Question 11

Overview of frozen sections

Answer 11

Rapidly frozen in liquid nitrogen to -196 degrees
OCT medium attaches section to chuck
Cut on cryostat at -20
Rapid H&E, lipid staining or IHC

Question 12

Explain how an ABC based assay works

Answer 12

▪ Use of biotinylated secondary antibodies which link tissue-bound primary antibodies and an avidin-biotin-reported enzyme complex
▪ Avidin is tetravalent and can therefore form large complexes containing multiple copies of the biotinylated reporter enzyme – signal amplification
▪ Reporter enzyme is biotinylated -> this biotin facilitates the binding to the avidin complex which thus facilitates the binding to the biotin on the 2’ antibody (enzyme-biotin-avidin-biotin-2’)
This is then combined with a detection method either DAB or AP colorimetric IHC to visualise the reult

Question 13

Explain how colorimetric IHC works

Answer 13

• IHC whereby enzymes are used to convert soluble substrates into insoluble, chromogenic products (visible)
  Enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (AP) are most commonly used
  Enzyme conjugated secondary antibodies are usually used for indirect detection I.e. with the use of a primary antibody but they can also be used in direct detection of antibodies

Question 14

How does DAB work

Answer 14

• Secondary antibody is labelled with Horse Radish Peroxidase
• HSP acts on the substrate Hydrogen peroxide
  This forms a brown precipitate known as 3, 3-diaminobenzidine (DAB) at the site of the antigen

Question 15

How does AP work

Answer 15

• AP red detection kit
• Secondary antibody is labelled with Alkaline phosphatase
• AP acts on naphthol substrate + fast red
  This forms a red pigment, 3-amino-9-ethylcarbazole (AEC), at the site of the antigen

Question 16

What is DAB

Answer 16

• Secondary antibody is labelled with Horse Radish Peroxidase
• HSP acts on the substrate Hydrogen peroxide
• This forms a brown precipitate known as 3, 3-diaminobenzidine (DAB) at the site of the antigen

Question 17

What is AP

Answer 17

• AP red detection kit
• Secondary antibody is labelled with Alkaline phosphatase
• AP acts on naphthol substrate + fast red
  This forms a red pigment, 3-amino-9-ethylcarbazole (AEC), at the site of the antigen

Question 18

What does DAB stand form

Answer 18

3, 3-diaminobenzidine

Question 19

What does AEC stand for?

Answer 19

3-amino-8-ethylcarbazole

Question 20

What stains are part of the PAP stain

Answer 20

OG6
EA50
Haematoxylin to stain the nuclei

Question 21

What are the three components of the EA50 stain?

Answer 21

Eosin Y and Light Green SF mixed in Phosphotungstic acid (PTA).

Question 22

What is the OG6 component of the pap stain?

Answer 22

A cytoplasmic stain which stains keratin an orange colour