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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
Q

Immunohistochemistry use in breast cancer

A

Staining for her Herceptin and oestrogen receptors

26
Q

Limitations of immunohistochemistry

A

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
Q

Immunoassay microassay mechanism

A

Protein biotynilation of sample, addition of antibodies, add dye-streptaverdin and observe

28
Q

Benefits of immunoassay microassay

A

Lots of antigens at once
More cost effective than single analyses
Good for developing new bio markers or therapeutic targets

29
Q

Disadvantages of immunoassay microassay

A

Imprecise - semiquantitive
Not currently robust enough for clinical practice
Standardisation/calibration issues

30
Q

Mass spectrometry mechanism

A

Target detection of specific molecular fragmentation patterns

31
Q

Advantages of mass spectrometry

A

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
Q

Disadvantages of mass spectrometry

A

Isobaric interference (similar compounds producing fragments with same mass charge ratio) and ion suppression
Expensive equipment
High expertise needed
Standardisation hard

33
Q

Proteomics definition

A

Study of the full set of proteins encoded by the human genome(30000 genes, 250000/300000 human proteins)

34
Q

2d electrophoresis

A

2 dimensional separation of proteins found in sample- compare disease to healthy identify potential markers

35
Q

MALDI-TOF Mass spectrometry

A

Produces peptide fingerprint by measuring compounds across wide mass range
Overlay and compare to normal to find potential biomarkers

36
Q

Metabolomics definition

A

The study of all the small molecules in human fluids cells or tissues (3000-4000)
Use for steroid profile already

37
Q

Limitations of ‘omics’

A

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
Q

Difference between genetics and genomics

A

Genetics is looking at genes whereas genomics is looking at the whole genome

39
Q

Germline vs somatic mutations

A

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
Q

Reasons to look at germline DNA

A

Inherited disease (mendelian or mitochondrial), disease risk (e.g cancer) and pharmacogenomics (drug dosing and safety)

41
Q

Use of somatic DNA - personalised cancer medicine

A

Looking at cancers- diagnosis, prediction, prognosis, therapy, prevention and ongoing monitoring after treatment

42
Q

What is cell free DNA testing? Uses?

A

Looking for DNA shed by cancer in patients plasma - use for screening, diagnosis and monitoring after treatment

43
Q

What is a genomic test?

A

A test that uses nucleic acids (DNA and RNA) to answer diagnostic or prognostic questions

44
Q

Cytogenetics definition and methods

A

The study of inheritance related to the structure and function of chromosomes. Useful for syndromes and chromosomal abnormality identification - karyotyping, FISH and aCGH

45
Q

What is karyotyping?

A

Genome scale test which visualizes whole chromosomes - useful for identifying correct number of chromosomes

46
Q

What is Fluorescent in situ hybridisation (FISH)?

A

Using labelled DNA sections to visualise either whole chromosomes or deletions/duplications of small sections

47
Q

What is array comparative genome hybridisation (aCGH)?

A

Comparing patients DNA to a reference in an array to check whether present in the same proportions to look for deletions and duplications

48
Q

What is DNA sequencing?

A

Looking the base sequence and comparing to normal

49
Q

When would you do single base testing?

A

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
Q

When would you test a single gene?

A

Specific rare disease caused by 1 gene diagnosis - e.g Marfan, NF-1, duchenne, CF etc.

51
Q

When would you test a panel of genes?

A

Specific rare disease that can be caused by multiple genes diagnosis - e.g familial hypertrophic cardiomyopathy/ familial breast cancer

52
Q

What is exome sequencing?

A

Sequencing all exons (180,000 exons, 30 megabases)

53
Q

What is genome sequencing?

A

Sequencing the whole genome (exons and introns) - 3 billion bases

54
Q

Uses of whole genome sequencing

A

Identifying rare genetic diseases, drug sensitivities and testing tumour against germline to identify targets

55
Q

Problems with whole genome sequencing

A

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
Q

What is a VUS?

A

Variation of uncertain significiance

57
Q

Process of whole genome sequencing in undiagnosed syndrome

A

Identify variants, look at family history, narrow down potential causes and decide which variations most likely to cause

58
Q

DNA test variations: polymorphism

A

Change in base sequence but same amino coded for so normal

59
Q

DNA test variations: Uncertain

A

Codes for different but similar amino acid so unsure of effect on protein structure

60
Q

DNA test variations: mutation

A

Different non-similar amino acid coded for so protein structure effected

61
Q

Problems with variations of uncertain significance

A

Limited data, unreported, found in controls, effects unknown so phenotype can’t be attributed to it

CSIM (5 decks)

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