Ancillary techniques Flashcards
Indications for immunohistochemistry
diagnosis/characterisation of neoplasms
infectious organisms
prognostic/predictive
Tissue requirements for IHC
Best if immediate fixation, for 12-48 hours
Antigen retrieval (usually heat-induced)
Unstained tissue on charged slide
Method for IHC
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
Principle of IHC
selectively detects tissue antigens via labelled antibodies (using peroxidase reaction for colour)
Components of IHC
- 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)
Advantages of IHC
Adv: sensitive and specific, can use routine materials, correlation with morphology, compatible with routine fixatives
False negatives in IHC
inappropriate antibody or wrong concentration
loss of antigen in tissue (eg autolysis, prolonged fixation, decal)
antigen below level of detection
False postives in IHC
cross-reactivity/nonspecific binding
endogenous peroxidase/biotin (esp hepatocytes)
entrapped normal tissues/pigments/proteins
What genes does the Mass Array test for?
EGFR, BRAF, KRAS, NRAS, CKIT (panel)
process for Mass Array
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
uses and technique for direct immunofluorensce
Used for: inflammatory skin and kidney biopsies
Process:
fresh tissue, frozen or in transport medium
fluorescent label on direct antibody
advantages/disadvantages of DIFL
adv: visual resolution high
disadv: temporary (can’t be stored), background staining, need special microscope, can’t use FFPE
Control tissue in IHC
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
Adv/disadv of automation in lab
adv: consistency, speed, less reagent used, less staff time
disadv: capital cost, ongoing cost, less flexible
principle of Next Gen Sequencing/massive parallel sequencing
Parallel sequencing on PCR-amplified fragmented DNA from a tumour, with complex bioinformatic quantification against published mutation databases.
principle of microarray
specific DNA probes attached to a solid surface which anneal to target (sample) DNA strands to simultaneously identify expression of multiple genes
uses of microarray
expression profiling
comparative genomic hybridisation
SNP detection (eg drug candidates, predisposition to disease, cancer/germline mutations)
how does flow cytometry work?
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)
cytogenetics - pros and cons
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)
Tissue requirements for cytogenetics
sample maximally viable tumour
transport in sterile culture medium with antibiotics
transport on ice (prevents autolysis and microbial growth)
Principle of FISH
tagged probes bound to chromosome-specific DNA sequences to allow structural and numeric analysis
advantages of FISH
many clinical uses (incl low prolif tumours)
can use interphase cells
can use air dried and fixed cells
detects numeric abnormalities
disadvantages/pitfalls of FISH
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)
HER2 IHC - false negatives
- Delay to formalin
- Tumour heterogeneity
- Antibody titration (concentration too low)
HER2 IHC - false positives
- 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
HER2 FISH - false negatives
- Pre-analytical factors also affects FISH (less than IHC)
- Insufficient protease treatment of tissue
- Including normal epithelium/ stroma/ lymphocytes
HER2 FISH - false positives
–Including DCIS (NB - mark your slide properly)
–Artifact (compare tumour cells to normal breast)
discordant HER2 IHC and FISH (what is considered positive?)
Consider any of these positive:
- HER2 IHC 3+
- HER2/CEP ratio ≥ 2.0
- HER2 copy number >6
equivocal HER2 FISH (why and what do you do?)
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
fusion vs break-apart FISH
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)
MSI testing - what kind of test is it and what problems are there?
PCR-based assay to detect increase in short tandem repeat (microsatellite) sequence
pitfalls include:
contaminating nonneoplastic tissue
MS marker used
how do you set up a new immuno?
pick correct clone or use kit
consult data sheet
select positive and negative controls
select antigen retrieval methods
carry out titration
principle of PCR
denaturation
annealing
extension
repeated
