Viva Qs

Question 1

Microorganism stains
(4)

Answer 1

Ziehl Neelson
Gram Tworts
Warthin and Starry
Grocotts methenamine silver stain

Question 2

Pigment stains
(4)

Answer 2

Haemosiderin/Iron -> Perl’s prussian blue
Amyloid -> Congo Red
Melanin -> masson fontana
Calcium -> Von Kossa

Question 3

Silver stains
(4)

Answer 3

Grocotts methenamine silver stain for fungi
Masson fontana for melanin
Warthrin and Starry for Spirochetes e.g. Helicobacter
Gordon and Sweets Reticulin Stain

Question 4

Trichrome stains

Answer 4

Masson Trichrome for muscle + CT
Gomori Trichrome for muscle + CT
Martius Scarlet Blue for fibrin

Question 5

Carbohydrate stains
(4)

Answer 5

Periodic Acid Shiff -> carbohydrates + glycogen
Diastase periodic acid-schiff -> carbohydrates - glycogen
Alcian blue -> acidic mucins
Alcian Blue/PAS

Question 6

Step by step of processing

Answer 6

Formalin fixation

Alcohol dehydration

Clearing in xylene

Impregnation with parafin wax

Question 7

What would you do if your QC for specials failed but test worked?

Answer 7

Check if correct material has been used
Check if block has been cut through
Check processing of control block
Repeat test

Question 8

What would you do if QC for specials failed and test failed

Answer 8

Check reagents
Check method -> correct incubation etc
Check processing
Check if correct control used
Check if block cut through
Repeat

Question 9

Undifferentiated panel

Answer 9

Melanoma: S100, HMB45, Vimentin
Lymphoma: CD45, Vimentin
Sarcoma: Desmin, Vimentin
Carcinoma: EMA, AE1/AE3, CAM5.2
NE: Neuron Specific Enolase

Question 10

Breast Cancer Panel

Answer 10

Estrogen Receptor
Progesterone Receptor
Her2

Ki67
PDL-1

DCIS: p63/SMMS

Question 11

What would you do if IHC control failed?

Answer 11

Check reagents -> have they been stored correctly, freeze/thaw cycles for antibodies etc

Might need longer incubation of primary antibody/higher concentration

Insufficient/incorrect form of antigen retrieval

Incorrect antibodies used

Primary and secondary antibodies not compatible

Antibody denatured

Secondary antibody exposed to light

Insufficient deparaffinisation

PBS contaminated with bacteria -> damage the phosphate groups on the protein of interest -> use fresh PBS

Question 12

What would a lot of background staining in IHC mean

Answer 12

Insufficient blocking -> endogenous antigen present

Primary antibody concentration too high

Non specific binding of secondary antibody

Too much amplification

Too much substrate applied

Question 13

What is antibody optomisation for IHC

Answer 13

ascertain which, if any, form of antigen retrieval is necessary to unmask the antigen (heat or enzyme mediated)

Ascertains which dilution gives appropriate staining

Ascertains incubation time and temperatures

Ascertains which is the most suitable clone for IHC

Question 14

Peroxidase blocking

Answer 14

▪ Some enzymes used in chromogenic detection methods such as peroxidase are also naturally occurring in some tissues -> if not blocked this will cause interference
▪ Peroxidase activity can be seen in tissues such as the kidney, liver and tissue containing rbcs such as vasculature
▪ Can test the tissue for endogenous peroxidase by incubating it in DAB -> if a brown colour is observed then a blocking step is required
Blocking involves incubating the tissue with Hydrogen peroxide

Question 15

EMAR vs HIER

Answer 15

EMAR = proteinase K proteolytic enzyme

HIER = heat for epitope retrieval in microwave

Question 16

% Receptor expression for breast cancer

Answer 16

• Estrogen receptors are highly expressed in over 70% of breast cancer
  Her2 is over expressed in over 20% of breast cancers

Question 17

DDISH

Answer 17

○ Dual-colour dual-hapten in-situ hybridisation
○ Used for the quantitative detection of amplification of the Her-2 gene (human epidermal growth factor receptor 2) using a dual colour chromogenic CISH as well as SISH
Looks at the Her2 and C17 genes
Used for breast cancer and gastric cancer patients for whom Herceptin treatment is being considered
○ Carried out using a cocktail of probes – a HER2 probe and a control chromosome 17 probe
This allows for the enumeration of both Her2 positive tumour nuclei and normal cells

Question 18

ISH

Answer 18

• 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 19

Types of ISH

Answer 19

FISH -> fluorescent
CISH -> chromogenic
SISH -> silver
DDISH -> dual-colour dual-hapten

Question 20

Examples of ISH

Answer 20

DDISH is done for any Her-2 IHCs which are 2+

FISH is done for EBV -> linked to sporadic Burkitt lymphoma in HIV patients

Looking at getting ISHf or Kappa and Lamdba chains

Question 21

Molecular testing

Answer 21

BRAF = melanoma
MSI = colorectal
NRAS = melanoma + colorectal
KRAS = lung, colorectal + pancreatic

Question 22

Cytology stains:

Answer 22

PAP and MGG

Question 23

Cytology samples

Answer 23

Mostly CSFs, BALs and cyst fluid

CSF (looking for tumour mets)
FNAs
Bronchial aspirates, washings, lavages
EBUS
Cyst fluid
Thyroid aspirates (from ?HIV in infectious disease clinic)
Joint fluid -> query goitre
Ascites, pleural etc

Question 24

Thin Prep

Answer 24

Creates a monolayer of cells for PAP stain
ThinPrep 2000
Sample mixed -> laser determines concentration of cells -> quantifies how much of the sample is needed -> sample taken up through filter -> any cells present don’t make it through filter -> filter pressed to charged slide -> cells transferred to slide -> slide placed in IMS for staining

Question 25

Cell blocks

Answer 25

○ Clot block -> made from clumps/clots present in the cytology sample -> can be processed as normal in neutral buffered formalin
Matrix block -> uses a gel/agar matrix to suspend cells in solution in a matrix to form a semi-solid structure suitable for microtomy/staining etc -> cannot be processed as normal -> must be processed in non-buffered formalin -> use programme 7 in processing

Question 26

Cytospin

Answer 26

Done for MGG
Condensed cells for MGG

Question 27

Cytology fixation

Answer 27

Cytolyt (ethanol) preserved sample for PAP

Fresh samples for MGG

Question 28

Components of a good H&E

Answer 28

Contrast between nuclear and background staining

Variability of eosin intensity staining