Principles of Analysis Flashcards
What is a definitive method?
A method of exceptional scientific accuracy suitable for certification of reference material
What is a reference method?
A method demonstrating small inaccuracies against definitive method
What is a Routine Method?
Method deemed sufficiently accurate for routine use against reference method and standard reference materials (SRM)
What are examples for Cholesterol?
Definitive Method: ID/GC/MS Isotope dilution/gas chromatography/mass spectrometry
Reference: Abell-Kendall method
- Hydrolysis of cholesterol esters with alcoholic KOH
- Extraction of total cholesterol with hexane for 15mins -> dried in vacumn
- Treated with acetic acid/acetic anhydride/sulphuric acid 30mins ->Abs 620nm
Routine Method: Enzymatic Method
- Cholesterol esters + cholesterol esterase → free cholesterol
- Cholesterol oxidase → cholestene-3-one + H2O2
- 4-aminoantipyrine + phenol → red quinoneimine dye Abs 540/600nm
What is a Primary Standard?
A substance of known chemical composition and high purity that can be accurately quantified and used for assigning values to materials and calibrating apparatus
What is a Standard Reference Material?
Reference material issued by an institute whose values are certified by a reference method which establishes traceability
What is the secondary standard?
A commercially produced standard for routine use calibrated against a primary standard or reference material
What is the internal standard?
- A substance not normally present in the sample. Added to both standard and sample to correct for variation in conditions between different samples run – e.g. HPLC, GC, MS.
- The internal standard is also used to verify instrument response and retention time stability.
What are Calibrator Requirements?
- Prepared from pure substance
- Stable and homogenous material
- Matrix similar to assay matrix e.g. serum
- No chemical interferences
- If possible should be obtained commercially to minimise error
What are calibrator values?
Commercially available calibrators have a concentration with a particular value:
- Stated value – no certification
- Assigned value – given arbitrarily or derived using a non-reference method
- Certified value – certification of value by particular institute or body
- Standard Reference Method TM value – derived used a reference method
What is Traceability?
An unbroken chain of comparisons of measurements leading to a reference value
What makes a Good method?
- Analytical Accuracy: Measure of agreement between a measured quantity and true value
- Analytical precision: Measure of agreement between replicates
Why is accuracy and precision required for result to be effective clinically?
- Precision is important for following course of disease/monitoring therapy i.e. reproducibility
- Accuracy is important for diagnosis since measured value is compared to reference range
- For screening precision and accuracy are important to avoid false positives and false negatives
What is verification and validation?
- Verification – confirmation, through provision of objective evidence, that the specified requirements have been fulfilled.
- Validation – confirmation, through provision of objective evidence, that the requirements for a specific intended use or application have been fulfilled.
What guidelines are used in the assessment of assay performance?
ISO 15189 : 2012 Medical laboratories – Requirements for quality and competence
What is trueness?
- Difference between true and measured value
- The measure of trueness is systematic error or bias
- The idea is that a measurement is true when it is aimed squarely at the centre
- Trueness and precision are independent of one another
How is Trueness measured?
- Repeat analysis of multiple levels of certified reference materials.
- Results are compared with the assigned value
- Recovery experiments
- Comparison with results from “fresh” EQA material
- Correlation with a current/accepted method using patient samples (comparability)
What is Precision?
- Results are clustered
- The degree of precision can be measured by quantifying the overall effect of all random errors
How is Precision measured?
- Repeatability: Minimum 20 results obtained from repeat analysis of IQC and patient samples on the same run Intermediate
- Precision: Minimum 20 results obtained from repeat analysis of IQC and patient samples from runs on different days
What is the equation for Precision?
Coefficient of variation (CV)= [SD/M] x 100%
- Advantage of being unit-less
- Allows comparison between results from different analytes in a way standard deviation cannot be Ideal CV <5% and no worse than 10% except at low levels
What is Accuracy?
- Accuracy is a combination of trueness and precision
- Accuracy therefore involves systematic and random error
How is Accuracy measured?
Total Error
Estimate the TE and therefore accuracy by combining:
- Estimate of bias from method comparison / EQA
- Estimate of precision from replication studies (intermediate precision)
TE = bias + 2 SD
What is measurement uncertainty?
- A parameter associated with the result of a measurement, that characterises the dispersion of the values that could be reasonably attributed to the measurand.
How is Measurement uncertainty assessed?
- The basic parameter of Measurement Uncertainty is standard deviation
- Best estimate of the “true value” ± measurement uncertainty (2xSD from intermediate precision)
What is interference?
The effect on the analytical measurement of a particular component by a second component
What are types of Interferences?
Analytical
- Cross-reactivity with other compounds
- Interference in methodology e.g. increase in absorbance due to lipaemia
Physiological
- Drugs e.g. prolactin increased by antipsychotics
What is Analytical Specificity and Sensitivity?
- Analytical Specificity: Measure of a method to determine only the analyte of interest i.e. cross-reactivity
- Analytical sensitivity: Ability of a method to detect small concentrations
What is the Limit of Blank?
- Highest measurement result that is likely to be observed (with a stated probability) for a blank sample
What is Limit of Detection?
- Lowest amount of analyte in a sample that can be detected with (stated) probability, although perhaps not quantified as an exact value
What is Limit of Quantification?
- Lowest amount of analyte in a sample that can be quantitatively determined with stated acceptable precision and trueness under stated experimental conditions
- Functional sensitivity is the analyte concentration at which the method CV = 20% or some other pre-determined CV
What is Functional Sensitivity?
Functional sensitivity is defined as the lowest concentration that can be measured with acceptable intermediate precision [CV]
What is the measuring interval?
- The interval between the upper and lower concentration of analyte in the sample for which it has been demonstrated that the method has suitable levels of precision, accuracy and linearity
- Range between the LOQ and the highest concentration studied during verification/ validation. Determined by linearity
What is linearity?
- An assessment of the difference between an individual’s measurements and that of a known standard over the full range of expected values
- Usually performed by comparing results from a series of dilutions of known standard across assay range
How is reference range determined?
- If the concentration of a particular analyte is measured in samples from a normal healthy population (n>120) then a range of results is obtained
- If plotted as a frequency plot a Gaussian distribution is usually observed (may be normal or skewed)
If you measure an analyte in 20 people how many will be outside of the reference range? 1 in 20 or 5%
How is reference range calculated?
- Parametric method: Assume Gaussian distribution of data or transformed (log) data. Determine reference limits (percentiles) as +/- 2SD from mean
- Non-parametric method: Makes no assumption about type of distribution. Results ranked and cut-off taken at x% of values in each tail
- Target driven reference range: e.g. cholesterol: reference range cannot be derived from “healthy” population. JBS2 targets for treatment of hyperlipidaemia
What are exclusion criteria for reference ranges?
- Diseases
- Risk factors
- Obesity
- Hypertension
- Genetically or environmental
- Intake of pharmacological agents
- Medication
- Oral contraceptive
- Drug abuse
- Alcohol
- Tobacco
- Physiological states
- Pregnancy
- Stress
- Excessive exercise
What is Biological Variation?
- Results used to derive a reference range contain elements of both analytical and biological variation
- Biological variation introduced by physiological factors, diet, fluid intake, exercise
What is the equation for biological variation?
- CV (total) = √ (CV^2analytical + CV^2biological)
What is clinical sensitivity and clinical specificity?
- Clinical Sensitivity – The ability of a TEST to correctly identify those who HAVE the disease A highly sensitive tests has few false negatives
- Clinical Specificity – The ability of a TEST to correctly identify those who do NOT HAVE the disease A highly specific test has few false positives
What is Predictive value and diagnostic efficiency?
- Predictive value (+/-) = measure of the ability of test to correctly assign individual to either disease or non-diseased group
- Diagnostic efficiency = proportion of true results
What is an equation for Sensitivity?
- Sensitivity = True Positives / (True Positives + False Negative)
What is an equation for Specificity?
- Specifity = True Negatives / (True Negatives + False Positives)
What is Positive Predictive Values and Negative Predictive Values?
- Positive Predictive Value (PPV): The ability of a test to detect the presence of disease
- Negative Predictive Value (NPV): The ability of a test to detect the absence of disease
What can affect predictive values?
- Predictive Values ARE affected by prevalence
- As disease prevalence increases the PPV also increases
- As disease prevalence decreases the NPV increases
What is the equation for Positive Predictive Value?
Positive Predictive Value = True Positive / (True Postive + False Positive):
What is the equation for Negative Predictive Value?
- Negative Predictive Value = True Negative / (True Negative + False Negative)
What are Cut-Off Values?
- Often diagnostic test results and associated clinical outcome are NOT naturally binary (either positive or negative) but continuous
- For test results that are continuous variables a clinical threshold/cut-off values have to be applied to classify test results as being positive or negative
