Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Anatomical Pathology RCPA Viva > Ancillary techniques > Flashcards

Ancillary techniques Flashcards

1
Q

Indications for immunohistochemistry

A

diagnosis/characterisation of neoplasms

infectious organisms

prognostic/predictive

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Tissue requirements for IHC

A

Best if immediate fixation, for 12-48 hours

Antigen retrieval (usually heat-induced)

Unstained tissue on charged slide

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Method for IHC

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Principle of IHC

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Components of IHC

A
  • 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)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Advantages of IHC

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

False negatives in IHC

A

inappropriate antibody or wrong concentration

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

antigen below level of detection

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

False postives in IHC

A

cross-reactivity/nonspecific binding

endogenous peroxidase/biotin (esp hepatocytes)

entrapped normal tissues/pigments/proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What genes does the Mass Array test for?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

process for Mass Array

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

uses and technique for direct immunofluorensce

A

Used for: inflammatory skin and kidney biopsies

Process:

fresh tissue, frozen or in transport medium

fluorescent label on direct antibody

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

advantages/disadvantages of DIFL

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Control tissue in IHC

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Adv/disadv of automation in lab

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

principle of Next Gen Sequencing/massive parallel sequencing

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

principle of microarray

A

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

17
Q

uses of microarray

A

expression profiling

comparative genomic hybridisation

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

18
Q

how does flow cytometry work?

A

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)

19
Q

cytogenetics - pros and cons

A

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)

20
Q

Tissue requirements for cytogenetics

A

sample maximally viable tumour

transport in sterile culture medium with antibiotics

transport on ice (prevents autolysis and microbial growth)

21
Q

Principle of FISH

A

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

22
Q

advantages of FISH

A

many clinical uses (incl low prolif tumours)

can use interphase cells

can use air dried and fixed cells

detects numeric abnormalities

23
Q

disadvantages/pitfalls of FISH

A

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)

24
Q

HER2 IHC - false negatives

A
  • Delay to formalin
  • Tumour heterogeneity
  • Antibody titration (concentration too low)
25
Q

HER2 IHC - false positives

A
  • 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
26
Q

HER2 FISH - false negatives

A
  • Pre-analytical factors also affects FISH (less than IHC)
  • Insufficient protease treatment of tissue
  • Including normal epithelium/ stroma/ lymphocytes
27
Q

HER2 FISH - false positives

A

–Including DCIS (NB - mark your slide properly)

–Artifact (compare tumour cells to normal breast)

28
Q

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

A

Consider any of these positive:

  • HER2 IHC 3+
  • HER2/CEP ratio ≥ 2.0
  • HER2 copy number >6
29
Q

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

A

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
30
Q

fusion vs break-apart FISH

A

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)

31
Q

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

A

PCR-based assay to detect increase in short tandem repeat (microsatellite) sequence

pitfalls include:

contaminating nonneoplastic tissue

MS marker used

32
Q

how do you set up a new immuno?

A

pick correct clone or use kit

consult data sheet

select positive and negative controls

select antigen retrieval methods

carry out titration

33
Q

principle of PCR

A

denaturation

annealing

extension

repeated

Anatomical Pathology RCPA Viva (14 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions