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CSIM > Diagnostics 2 (Lab and genomic) > Flashcards

Diagnostics 2 (Lab and genomic) Flashcards

1
Q

High diagnostic specificity

A

Minimal false positives

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2
Q

High diagnostic sensitivity

A

Minimal false negatives

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3
Q

High analytical specificity

A

Measures only analyte of interest

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4
Q

High analytical sensitivity

A

Capable of measuring low concentrations

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5
Q

Limit of detection

A

Smallest concentration that can be determined from zero

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6
Q

Limit of quantification

A

Smallest concentration that can be measured with acceptable precision

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7
Q

Precision

A

Repeatability of experiment expressed as coefficient of variation or std deviation

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8
Q

Coefficient of variation

A

Ratio of std deviation to the mean - low CV = high precision

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9
Q

Accuracy

A

How close the result is to the true value

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10
Q

Total error

A

Precision and accuracy both low

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11
Q

Spectrophotometry

A

Using a reaction that produces/consumes a substance absorbing uv/visible light at a certain wavelength. Degree of conductance of light is used to quantify the substance.

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12
Q

Advantages of photospectometry (3)

A

Fully automatable
Fast
Cheap

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13
Q

Disadvantages of photospectometry (3)

A

Haemolysis, lipaemia, icterus all affect
Interference
Not possible for many chemicals

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14
Q

Polyclonal antibodies characteristics (6)

A 
Mixture of abs
From animal serum
Cheap
Recognise multiple epitopes
Varies between batches
High affinity (multiple epitopes targeted)
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15
Q

Monoclonal characteristics (6)

A 
Single ab
Isolated from hybridoma cell line
Expensive
Single epitope targeted
Little batch variability
High specificity
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16
Q

Immunoassay - sandwich format mechanism

A

Sample containing analyte added to mixture with capture antibody. After incubation, excess sample washed away and signal antibody with label added. Signal strength directly proportional to amount of analyte.

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17
Q

Immunoassay sandwich format use

A

Larger molecules such as peptides or proteins

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18
Q

Competitive assay immunoassay use

A

Smaller molecules e.g. Steroids

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19
Q

Competitive assay immunoassay mechanism

A

Capture antibody starts bound to labelled molecule. Sample added and analyte displaces the labelled molecule. Wash away remains. Signal inversely proportional to amount of analyte.

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20
Q

Advantages of immunoassay

A

Often automatable
Automated assays are fast
High sensitivity
Applicable to wide range of analytes

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21
Q

Disadvantages of immunoassay

A

Cross-reactivity (antibodies bind with to multiple epitopes)
Not all are automatable
Expensive
Heterophilic antibodies ( some patients have antibodies which can bind to the receptors)

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22
Q

Immunohistochemistry definition

A

Identification of specific molecules in tissue using labelled antibodies - used in parallel with traditional histology (not a replacement)

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23
Q

Immunohistochemistry mechanism

A

Indirect immunostaining to amplify signal-

Primary antibody binds to both antigen and a secondary attached to a signal molecule e.g. peroxidase anti peroxide

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24
Q

Uses of immunohistochemistry

A

Diagnosis of primary malignant tumours esp. when poorly differentiated
Determining likely origin of metastasis
Categorising malignancies (eg leukaemia/lymphoma)
Detection of molecules with prognostic/therapeutic significance (her2 and ER)
Detection minimal disease (few tumour cells)
Used with fna (primary or mets)
Estimation of semi-quantitive proliferation

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25
Immunohistochemistry use in breast cancer
Staining for her Herceptin and oestrogen receptors
26
Limitations of immunohistochemistry
Epitopes masking- protein crosslinking due to formaldehyde fixation (antigen retrieval methods improve this) Background staining- nonspecific binding of primary and secondary antibodies and endogenous signal enzyme (protein/enzyme blocking improve this) Antibody selection and performance (must be carefully selected and validated) Standardisation difficult
27
Immunoassay microassay mechanism
Protein biotynilation of sample, addition of antibodies, add dye-streptaverdin and observe
28
Benefits of immunoassay microassay
Lots of antigens at once More cost effective than single analyses Good for developing new bio markers or therapeutic targets
29
Disadvantages of immunoassay microassay
Imprecise - semiquantitive Not currently robust enough for clinical practice Standardisation/calibration issues
30
Mass spectrometry mechanism
Target detection of specific molecular fragmentation patterns
31
Advantages of mass spectrometry
Highly specific and sensitive Applicable to a wide range of analytes (usually low Mw in labs) Cheap consumables Allows multiplexing - analyse panels of markers simultaneously
32
Disadvantages of mass spectrometry
Isobaric interference (similar compounds producing fragments with same mass charge ratio) and ion suppression Expensive equipment High expertise needed Standardisation hard
33
Proteomics definition
Study of the full set of proteins encoded by the human genome(30000 genes, 250000/300000 human proteins)
34
2d electrophoresis
2 dimensional separation of proteins found in sample- compare disease to healthy identify potential markers
35
MALDI-TOF Mass spectrometry
Produces peptide fingerprint by measuring compounds across wide mass range Overlay and compare to normal to find potential biomarkers
36
Metabolomics definition
The study of all the small molecules in human fluids cells or tissues (3000-4000) Use for steroid profile already
37
Limitations of 'omics'
Huge data hard to analyse Statistical significance doesn't equal diagnostic performance Multiplex technologies potentially not robust enough for clinical practice Standardisation issues Biomarker validation long and difficult
38
Difference between genetics and genomics
Genetics is looking at genes whereas genomics is looking at the whole genome
39
Germline vs somatic mutations
Germline mutations are inherited from the parents and in all cells whereas somatic happen in that person so are only present in that cell and its descendants
40
Reasons to look at germline DNA
Inherited disease (mendelian or mitochondrial), disease risk (e.g cancer) and pharmacogenomics (drug dosing and safety)
41
Use of somatic DNA - personalised cancer medicine
Looking at cancers- diagnosis, prediction, prognosis, therapy, prevention and ongoing monitoring after treatment
42
What is cell free DNA testing? Uses?
Looking for DNA shed by cancer in patients plasma - use for screening, diagnosis and monitoring after treatment
43
What is a genomic test?
A test that uses nucleic acids (DNA and RNA) to answer diagnostic or prognostic questions
44
Cytogenetics definition and methods
The study of inheritance related to the structure and function of chromosomes. Useful for syndromes and chromosomal abnormality identification - karyotyping, FISH and aCGH
45
What is karyotyping?
Genome scale test which visualizes whole chromosomes - useful for identifying correct number of chromosomes
46
What is Fluorescent in situ hybridisation (FISH)?
Using labelled DNA sections to visualise either whole chromosomes or deletions/duplications of small sections
47
What is array comparative genome hybridisation (aCGH)?
Comparing patients DNA to a reference in an array to check whether present in the same proportions to look for deletions and duplications
48
What is DNA sequencing?
Looking the base sequence and comparing to normal
49
When would you do single base testing?
Known mutation in a family (e.g BRCA) or specific driver mutation in a tumour (e.g BRAF) - can use MALDI-TOF to test
50
When would you test a single gene?
Specific rare disease caused by 1 gene diagnosis - e.g Marfan, NF-1, duchenne, CF etc.
51
When would you test a panel of genes?
Specific rare disease that can be caused by multiple genes diagnosis - e.g familial hypertrophic cardiomyopathy/ familial breast cancer
52
What is exome sequencing?
Sequencing all exons (180,000 exons, 30 megabases)
53
What is genome sequencing?
Sequencing the whole genome (exons and introns) - 3 billion bases
54
Uses of whole genome sequencing
Identifying rare genetic diseases, drug sensitivities and testing tumour against germline to identify targets
55
Problems with whole genome sequencing
Everyone has >3million variants, 2500 are non-synonymous (would change base- some likely to be damaging) and 150 loss of function variants - unkown what many of these do
56
What is a VUS?
Variation of uncertain significiance
57
Process of whole genome sequencing in undiagnosed syndrome
Identify variants, look at family history, narrow down potential causes and decide which variations most likely to cause
58
DNA test variations: polymorphism
Change in base sequence but same amino coded for so normal
59
DNA test variations: Uncertain
Codes for different but similar amino acid so unsure of effect on protein structure
60
DNA test variations: mutation
Different non-similar amino acid coded for so protein structure effected
61
Problems with variations of uncertain significance
Limited data, unreported, found in controls, effects unknown so phenotype can't be attributed to it

CSIM (5 decks)

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