Biomarker test development and validation Flashcards
How is ‘dementia’ diagnosed?
- Cognitive and neuropsychological tests
- Neurological evaluation
- Psychiatric evaluation
- Brain scans (CT, MRI, PET)
- Laboratory tests
- Biofluid biomarkers
Which fluid biomarkers for AD are there currently?
The ATN framework:
- Amyloid (AB42/AB40 ratio)
- Tau (pTau)
- Neurodegeneration (tTau, NfL)
Fill in whether biomarkers in CSF or brain are increased or decreased.
In brain:
- AB
- NfL
- tTau
- pTau
In CSF:
- AB
- pTau
- tTau
- NfL
In brain:
- Increased → AB, NfL, tTau, pTau
- Decreased → x
In CSF:
- Increased → pTau, tTau, NfL
- Decreased → AB
What is the ideal biomarker?
- Easily accessible
- Disease specific
- Not influenced by other factors
- Reflects pathology
A newly identified biomarker for AD is acetylated Tau.
Why is there an interest in this biomarker?
- It slows tau turnover and promotes accumulation in vitro and in vivo
- Induces more severe neurodegenration compared to WT tau
- Worsens behavioral deficits including spatial learning and memory retention.
What is assay validation?
The process of proving that an analytical method is acceptable for its intended purpose.
Why is assay validation important?
- To minimize analytical and instrumental errors
- To give reliable and reproducible results
- To ensure the quality of the assay results
- To be assured of the correctness of results
- To confirm that the assay measures what it is intended to measure
Assay validation consists of two steps: technical and clinical validation.
What is important to measure for technical validation?
- Precision
- Sensitivity
- Linearity
- Recovery
- Parallelism
- Sample stability
- Selectivity / specificity
Explain in regard to the technical validation what the definition is of:
- precision
- sensitivity
- linearity
- recovery
- parallelism
- sample stability
- specificity
- selectivity
- precision → indicates how well a method gives the same result when a single sample is tested repeatedly.
- sensitivity → indicates the detection and quantification limits of an assay, whereby the highest and lowest concentrations of analyte have been measured with acceptable levels of precision and accuracy.
- linearity → indicates how well a sample with a high concentration can be diluted to a concentration within the working range and still give a reliable result
- recovery → investigates the matrix effect (→the sample matrix should not interfere with the measurement of your analyte of interest)
- parallelism → investigates if the binding characteristic of the endogenous biomarker to the antibodies is the same as for the standard.
- sample stability → tests the chemical stability of an analyte in a given matrix under specific condition for given time intervals.
- specificity → the ability to assess the exact component in a mixture
- selectivity → the ability to differentiate the components in a mixture from each other
What is the difference to intra-assay precision and inter-assay precision?
- Intra-assay precision → same sample measured within 1 assay plate/run
- Inter-assay precision → same sample measured between different assay plates/runes
What is the meaning of an upper limit of quantification (ULOQ) and lower limit of quantification (LLOQ)?
It denotes the (dynamic/working) range an assay can measure.
What is the hook effect in regard to linearity?
It gives falsely low results with certain immunoassays in the presence of excess amount of analyte of interest
What is the matrix effect in regard to recovery?
The method should be able to differentiate the biomarker of interest from endogenous, unrelated components in the sample matrix
In regard to parallelism, what does it mean when the binding characteristics are parallel or non-parallel?
- Parallel → binding characteristic of endogenous biomarker to the antibodies are the same as for the standard
- Non-parallel → binding characteristic of endogenous biomarker to the antibodies are different compared to the standard.
What is meant with clinical validation?
The proof that the biomarker identifies the concept of interest.
