Lecture 1: 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?
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
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 tracebility
What is a Secondary Standard?
Commercially produced standard for routine use calibrated agaist a primary standard or reference material
What is an 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
- 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
- No chemical interferences
- If possible should be obtained commercially to minimise error
What are definitions of calibrator values?
- Stated value: no certifcation
- Assigned value: given arbitrarily or derived using a non-reference method
- Certified value: certification of value by particular institute or body
- Standard reference methodTM value: derived using a reference method
What is traceabillity?
An unbroken chan of comparisons of measurements leading to a reference value
What is Analytical Accuracy and Analytical Precision?
- Analytical Accuracy: Measure of agreement between a measured quantity and true value
- Analytical Precision: Measure of agreement between replicates
Good method is precise and accurate
What are clinical uses of results?
- Precision is important for following course of disease/monitoring therapy i.e. repoducibility
- Accuracy is important for diagnosis since measured value is compared to reference range
Why is precision and accuracy important for screening?
Screening is 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 specified intended use or application have been fulfilled
What is accuracy?
Improving accuracy is a combination of improving trueness and improving precision
What is Trueness?
Measure of trueness is systematic error or bias.
- The idea is that a measurement is true when it is aimed squarely at the centre of the target.
- Trueness and precision are independent of one another
How is trueness measured?
- Repeat analysis of multiple levels of certified reference materials. Results are comapred to assigned value
- Recovery experiments
- Comparison with result from fresh EQA material. Correlation with a current/accepted method using patient samples (comparability)
How is Precision measured?
Degree of precision can be measured by quantifying the overall effect of all random errors.
2 Forms:
- Repeatability
- Intermediate precision
What is repeatability?
Minimum 20 results obtained from repeat analysis of IQC and patient samples on the same run
What is intermediate precision?
Minimum 20 results obtained from repeat analysis of IQC and patient samples from runs on different days
What is the equation for measurement of precision?
Coeffiecient of variation (CV) = [SD/M] x 100%
- Advantage of being unit-less
- Allows cmparison between result from different analytes in a way standard deviation cannot be
- Ideal CV <5% and now worse than 10% except at low levels
How is Accuracy measured?
Accuracy involves systematic and random error. Estimate the total error by combining:
- Estimate of bias from method comparison/EQA
- Estimate of precision from replication studies (intermediate precision)
TE = Bias + 2SD
What is inteference?
The effect on the analytical measurement of a particular component by a second component
- 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 analytical sensitivity?
- Analytical Specificity: Measure of a method to determine only the analyte of interest
- Analytical Sensitivity: Ability of a method to detect small concentrations
What is Limit of Blank?
Highest measurement result that is likely to be observed (with a stated probability) for a blank sample
What is the 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 the 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
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
What is a reference range?
- 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)
- Values outside -2SD and +2SD are outside the normal range. Reference range lies with 95% of population
How are reference ranges 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
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Target driven reference range
- e.g. cholesterol: reference range cannot be derived from “healthy” population
What are some exclusion criteria from 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 and the equation for it?
- 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
CV (total) = √ CV2analytical + CV2biological
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?
Predictive value (+/-) = Measure of the ability of test to correctly assign individual to either disease or non-diseased group
What is diagnositic efficiency?
Diagnostic efficiency = proportion of true results
