Ancillary techniques

Question 1

Indications for immunohistochemistry

Answer 1

diagnosis/characterisation of neoplasms

infectious organisms

prognostic/predictive

Question 2

Tissue requirements for IHC

Answer 2

Best if immediate fixation, for 12-48 hours

Antigen retrieval (usually heat-induced)

Unstained tissue on charged slide

Question 3

Method for IHC

Answer 3

Primary antibody (Ig): polyclonal (more sensitive, less specific) or monoclonal

Diluted (to titrated amount for contrast between specificity and sensitivity) and applied

Detected:

direct or indirect methods

avidin-biotin conjugate methods

Question 4

Principle of IHC

Answer 4

selectively detects tissue antigens via labelled antibodies (using peroxidase reaction for colour)

Question 5

Components of IHC

Answer 5

• Primary antibody (Ig): polyclonal (more sensitive, less specific) or monoclonal
• Secondary antibody bound to:

avidin-biotin (A-B method) OR

Dextran polymer (in polymer-based method)

• enzyme (peroxidase or alk phos)
• DAB substrate (precipitates to brown colour)

Question 6

Advantages of IHC

Answer 6

Adv: sensitive and specific, can use routine materials, correlation with morphology, compatible with routine fixatives

Question 7

False negatives in IHC

Answer 7

inappropriate antibody or wrong concentration

loss of antigen in tissue (eg autolysis, prolonged fixation, decal)

antigen below level of detection

Question 8

False postives in IHC

Answer 8

cross-reactivity/nonspecific binding

endogenous peroxidase/biotin (esp hepatocytes)

entrapped normal tissues/pigments/proteins

Question 9

What genes does the Mass Array test for?

Answer 9

EGFR, BRAF, KRAS, NRAS, CKIT (panel)

Question 10

process for Mass Array

Answer 10

FFPE tissue from slides

DNA extracted and amplified via PCR

excess nucleotides removed (from PCR process)

desalt to remove ions (use resin)

run MALDI-TOF (mass spectrometry)

analysis

Question 11

uses and technique for direct immunofluorensce

Answer 11

Used for: inflammatory skin and kidney biopsies

Process:

fresh tissue, frozen or in transport medium

fluorescent label on direct antibody

Question 12

advantages/disadvantages of DIFL

Answer 12

adv: visual resolution high
disadv: temporary (can’t be stored), background staining, need special microscope, can’t use FFPE

Question 13

Control tissue in IHC

Answer 13

External control (best if on same slide): detects technical or reagent failure

Internal control: detects fixation/processing/storage problems

Negative control: detects endogenous biotin and peroxidase activity

Question 14

Adv/disadv of automation in lab

Answer 14

adv: consistency, speed, less reagent used, less staff time
disadv: capital cost, ongoing cost, less flexible

Question 15

principle of Next Gen Sequencing/massive parallel sequencing

Answer 15

Parallel sequencing on PCR-amplified fragmented DNA from a tumour, with complex bioinformatic quantification against published mutation databases.

Question 16

principle of microarray

Answer 16

specific DNA probes attached to a solid surface which anneal to target (sample) DNA strands to simultaneously identify expression of multiple genes

Question 17

uses of microarray

Answer 17

expression profiling

comparative genomic hybridisation

SNP detection (eg drug candidates, predisposition to disease, cancer/germline mutations)

Question 18

how does flow cytometry work?

Answer 18

single stream of suspended cells through a laser, with resulting scattered light measured by photodetectors.

For each cell calculates:

size (forward scattter)

complexity (side scatter)

surface labels (fluorescence via antibodies)

Question 19

cytogenetics - pros and cons

Answer 19

Allows whole genome analysis - eg diagnosis, or dieecting further testing

Limitations: needs viable tissue with proliferating cells. low resolution (only sees numerical and gross structural abnormalities)

Question 20

Tissue requirements for cytogenetics

Answer 20

sample maximally viable tumour

transport in sterile culture medium with antibiotics

transport on ice (prevents autolysis and microbial growth)

Question 21

Principle of FISH

Answer 21

tagged probes bound to chromosome-specific DNA sequences to allow structural and numeric analysis

Question 22

advantages of FISH

Answer 22

many clinical uses (incl low prolif tumours)

can use interphase cells

can use air dried and fixed cells

detects numeric abnormalities

Question 23

disadvantages/pitfalls of FISH

Answer 23

signal fading (need to take photos)

truncation artifact (ie missing DNA in cell analysed)

aneuploidy and polyploidy

autofluorescence

partial hybrisidisation failure

can’t see morphology/architecture at same time (esp for HER2)

Question 24

HER2 IHC - false negatives

Answer 24

• Delay to formalin
• Tumour heterogeneity
• Antibody titration (concentration too low)

Question 25

HER2 IHC - false positives

Answer 25

- Edge artifact (usually in core biopsies) - Cytoplasmic positivity obscuring membranes - Over-retrieval of the Ag - Antibody titration (concentration too high) - Including DCIS in the HER2 score

Question 26

HER2 FISH - false negatives

Answer 26

- Pre-analytical factors also affects FISH (less than IHC) - Insufficient protease treatment of tissue - Including normal epithelium/ stroma/ lymphocytes

Question 27

HER2 FISH - false positives

Answer 27

–Including DCIS (NB - mark your slide properly) –Artifact (compare tumour cells to normal breast)

Question 28

discordant HER2 IHC and FISH (what is considered positive?)

Answer 28

Consider any of these positive: ## Footnote - HER2 IHC 3+ - HER2/CEP ratio ≥ 2.0 - HER2 copy number \>6

Question 29

equivocal HER2 FISH (why and what do you do?)

Answer 29

due to: Polysomy, cen17 amplification, heterogenous HER2 expression Approach: * count more cells * second opinion from experienced FISH scientist * repeat FISH with different control probe * repeat IHC and FISH on different block

Question 30

fusion vs break-apart FISH

Answer 30

fusion: tags specific fusion genes (more specific, less sensitive) break-apart: tags either side of a breakpoint (less specific, more sensitive, fusion partner not known)

Question 31

MSI testing - what kind of test is it and what problems are there?

Answer 31

PCR-based assay to detect increase in short tandem repeat (microsatellite) sequence pitfalls include: contaminating nonneoplastic tissue MS marker used

Question 32

how do you set up a new immuno?

Answer 32

pick correct clone or use kit consult data sheet select positive and negative controls select antigen retrieval methods carry out titration

Question 33

principle of PCR

Answer 33

denaturation annealing extension repeated