MAID 2.1-2.8 Flashcards
OR calculation?
(TP/FN)/(FP/TN)
At what EGFR is contrast CT probably a bad idea?
<45/30, although not absoulte
Key danger groups for ionising radiation?
Children that are likely to need high lifetime dose e.g. Crohns, and pregnancy (more due to risk of breast cancer then fetal harm; V/Q more likely to harm fetus). However, if need test then get test.
Risk with contrast MRI?
If using gadolinium-based contrast, can get NSF. Get fibrosis of skin, joints, eyes, lungs/heart/liver. Usually have ESRD.
Imaging for gallstones?
All stones will show up on US/MRCP; non-calcified will not be seen on AXR/CT. ERCP may be needed to actually clear (MRCP is not therapeutic).
Which infections may need radiological biopsy?
Thinks like discitis and osteomyelitis, as blood cultures are fairly useless and must be certain about diagnosis because antibiotics courses are prolonged
Likelihood ratio definition?
The probability of a finding in those with the disease/same probability in those without disease. 1 means exactly as likely in each.
What may interfere with analytical specificity?
Related molecules e.g. metabolites, matrix effects, heterophilic or antireagent antibodies
What are heterophilic antibodies?
Weak antibodies against poorly defined antigens; get broad reactivity and so can interfere with assays
Significance of high accuracy and low precision?
Not a systematic bias as mean is good, but total error is significant
Use of high precision and low accuracy?
Can be useful if use reference ranges/diagnostic rule out for that particular assay, but has no value between assays
Assays and tests?
For any test e.g. FBC a whole range of assays is available; precision and error are relevant to assays, sens and spec are intrinsic to a test (although relate to the assay)
Principles of spectophotometry?
Reaction must either produce or consume a substance that absorbs light at a certain wavelength; progress affects proportion which affects absorption. Used for many common tests
Pros and cons of spectophotometry?
\+ = fully automatable, fast, cheap - = affected by haemolysis/icterus/lipaemia (get interference because some breakdown products e.g. Hb have very broad absorption), not available for many analytes
Polyclonal vs monoclonal antibody diagnostics?
- Polyclonal uses mixture of antibodies, isolated from animal serum, low cost, recognise multiple epitopes. High affinity (may work even if an epitope is masked) BUT have between-batch variation.
- Monoclonal uses single Ab, isolated from single cell line. High cost, recognises SINGLE epitope. Minimal batch variability and high specificity.
Immunoassay (1): sandwich?
Used for large molecules e.g. peptides. Have capture Ab anchored to solid support; binds to analyte to fix it. Then add signal Ab with radiolabel, binds to different epitope on analyte. Wash away remainder; signal proportional to analyte concentration.
Immunassay (2): competitive?
Used for small molecules. Capture Ab on solid support; add sample Ag and labelled version; compete with each other. Signal inversely proportional to analyte.
When would you use sandwich versus competitive immunoassays?
Sandwich uses two binding sites (epitopes) per antigen; better for larger molecules e.g. proteins. Competitive only one so better for smaller.
Pros and cons of immunoassays?
\+ = often automatable, wide range of analytes, fast, can be highly sensitive. - = manual for some analytes, CROSS-REACTIVITY (binding of Ab to other epitopes) can be expensive, heterophilic antibodies in sample can interfere.
Examples of cross-reactivity in immunoassays?
Measuring cortisol; get significant cross-reactivity to some compounds e.g. corticosterone, very minor to prednisolone. Only matters in specific cases e.g. CAH, metyrapone therapy. The opposite is true in pseudo-Cushing’s
How does IHC work?
Tend to use indirect immunostaining (amplifies signal). Add primary Ab; binds to tissue Ag. Secondary Ab added; binds to constant region of primary with one ‘arm’ and to stain complex with other ‘arm’
Clinical applications of IHC?
Diagnosis of primary malignant tumours (especially when poorly differentiated), likely site of met origin, categorising malignancies, detecting molecules with prog/ther significance e.g. HER2, detection of minimal disease (small numbers of tumour cells), used alongside FNA cytology, used for semi-quantitative proliferation index
Limitations of IHC?
- Epitope masking (protein cross-linking during fixation; can reverse with heat/enzymes).
- Background staining (non-specific binding of 1/2ndary Abs, or endogenous signal enzyme)
- Ab selection/performance (select carefully, validate).
- Standardisation of IHC tests is challenging (wide range of variables)
Immunoassay microarrays?
(Mostly academic). Take sample, biotinylate, conjugate protein to Ab array, then add dye (binds it biotin). Allows many different protein to be detected and conc. determined at once.
Pros and cons of immunoassay microarrays?
\+ = multiplex detection (100s-1000s of Ag), cost-effective compared to single, powerful technique for investigating new biomarkers/drugs - = imprecise compared to traditional immunoassays (semi-quant), perhaps not robust enough for clinical practice, standardisation/calibration issues
Pros and cons of mass spec?
\+ = can be highly specific/sensitive, applicable to a wide range of analytes, low cost consumables and can multiplex. - = expensive equipment (high one-off cost), high level of expertise, standardisation. Isobaric interference/ion suppression.
What does the metabolome consist of?
Sugars, nucleotides, amino acids, lipids (lipidome).
Technology driving proteomics?
2D gels, mass spec, microarray immunoassays etc. 2D gels involves electophoresis of disease sample overlaid to control, then compare. MALDI-TOF mass spec. creates peptide ‘fingerprint’ and compares to control; allows identification of potential biomarkers.
Uses of metabolomics?
Biomarker discovery (finding metabolites that discriminate benign vs carcinoma e.g bile/urine/saliva), personalised medicine (test every metabolite at once for rapid diagnosis). Could be used in toxicology when testing drugs; typical metabolites of liver/renal injury could be detected early
Limits of “-omics”?
Huge amount of data, hard to interpret, need statistical significance to be useful clinically, multi-step process so hard to standardise, biomarker validation is extremely lengthy process.
Examples of when personalised cancer medicine is diagnostic?
BCR-ABL in CML, JAK2 in myeloproliferative disorders
Examples of when personalised cancer medicine is predictive?
EGFR in NSCLC; predicts TKI response. HER2 in CaBreast; amplification predicts reponse to Herceptin
Examples of when personalised cancer medicine is prognostic (different to predictive)?
TP53 in CLL = bad outcomes, BRAF in CRC = bad outcomes.
Examples of when personalised cancer medicine is used for disease monitoring?
BRC-ABL1 in CML used for minimal residual disease detection (as is only found in leukaemia cells i.e. not germline!
Type of DNA tests for very big mutations? (aneuploidy/large deletions/translocations?)
Cytogenetic e.g. karyotyping, FISH. Cytogenetics means how chromosomes relate to cell function. FISH is quicker than karotype.
Types of DNA tests for microdeletions?
Can used FISH again, or arrayCGH (compare labelled patient DNA to labelled reference DNA; get different colour ratios in duplications/deletions). aCGH also counts as cytogenetics
Two main uses of cytogenetics in children?
- Does this child have X syndrome?
- Does this child have an identifiable chromosome problem? (quite large scale).
e. g. Downs, Williams (large deletion found in FISH/aCGH)
Problems with aCGH?
If find duplications/deletions without specific linked syndromes, or find multiple changes, can be hard to ascertain clinical significance i.e. findings are often “unique”.
Specificity of karyotyping?
Usually ~100% i.e. no child with trisomy 21 will NOT have Down’s.
Use of single BASE sequencing?
Used to see if known family mutation is passed on, or look for specific ‘driver’ mutation in a tumour e.g. BRAF; will need to extract tumour DNA first. Use MALDI/TOF as different base fragments will be revealed; cheaper and easier than sequencing.
Use of single GENE sequencing?
Used for specific rare diseases linked to single genes e.g. Marfans, NF1, ADPKD, DMD, CF. Use next-gen sequencing.
Use of gene PANEL sequencing?
Specific rare disease diagnosis if multiple genes implicated (heterogeneity) e.g. familial breast cancer (~5 genes), familial HCM (30 genes). Use next-gen sequencing.
Use of whole EXOME sequencing?
Sequence all exons. Can add “virtual” panels. 30 megabases (30,000,000 BP). Use high-throughput DNA sequencing; aim is to find varients that alter protein sequences @ much lower cost than whole genome. Especially effective in study of rare Mendelian disease, as all genetic variants may be relevant. Aim to find the variants that are only present in very small numbers of people.
Whole exome vs whole genome?
Exome process good for rare Mendelian disease, because assumes that any severe causing variants are much more likely to be in protein coding sequence (1% of whole genome).
When is whole exome sequencing better than SNP array?
Better if variant is extremely rare; only have SNP probes for fairly common abnormalities i.e. recognised single nucleotide polymorphisms.
Why do you need two probes in SNP arrays?
Detects both alleles; otherwise failure would be indistinguishable form homozygosity of non-probed allele.
Main use of SNP arrays?
Used to map SNPs to assocations with specific diseases to determine susceptibility; involves looking for KNOWN variants rather than extremely rare mutations.
Whole GENOME sequencing?
All genes (introns and exons) and everything in between. Huge potential for in silico analysis, proven effectiveness in rare inherited disease. Will eventually become the standard DNA test and replace all current cytogenetics tests.
Applications for whole genome sequencing?
- Establish mutation frequency for whole genomes (= ~70 per generation from parent to child). Much higher in cancer.
- Genomic-wide associating studies
- Diagnostic use (very rare conditions)
